annals of animal science - Instytut Zootechniki PIB

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ANNALS
OF ANIMAL SCIENCE
NATIONAL RESEARCH INSTITUTE OF ANIMAL PRODUCTION
Vol. 5
KRAKÓW 2005
No. 1
EDITORIAL BOARD
Je˛drzej Krupiński (Chairman) — Kraków-Balice,
Hermann Bogner — München,
Franciszek Brzóska — Kraków-Balice,
Hanna Czaja-Bogner— Kraków-Balice,
Clas Elwinger — Uppsala,
Tibor Gere — Gyöngyös,
Ingemar Gustavsson — Uppsala,
Eugeniusz Herbut — Kraków-Balice,
Dymitr Kaliszewicz — Olsztyn,
Jolanta Kurył — Jastrze˛biec,
Andrzej Potkański — Poznań,
Eligiusz Rokicki — Warszawa,
Marian Różycki — Kraków-Balice,
Yasuo Shioya — Ibaraki,
Zdzisław Smora˛g — Kraków-Balice,
Vasyl Vlizlo — Lviv,
Stanisław We˛żyk — Kraków-Balice
EDITORIAL STAFF
Editor-in-Chief — Ewa Słota
Deputy Editors-in-Chief — Marian Duniec, Mariusz Pietras
Secretary — Halina Lach
Editing — Danuta Dobrowolska, Halina Lach, Jerzy Pilawski
Cover design — Beata Barszczewska-Wojda
Address of Editorial Office — Instytut Zootechniki
ul. Sarego 2, 31-047 Kraków, Poland
The ‘‘Annals of Animal Science” are derived from the journal
‘‘Roczniki Naukowe Zootechniki” which has been published since 1974
This publication was supported by the Ministry of Science
and Information Society Technologies
 Copyright by National Research Institute of Animal Production
PL ISSN 1642-3402
Ann. Anim. Sci., Vol. 5, No. 1 (2005)
CONTENT
Genetics and farm animal breeding
1. A. Kozubska-Sobocińska, E. Słota, A. Pieńkowska-Schelling, C. Schelling — Comparative
hybridization of the Y chromosome in selected species of Bovidae . . . . . . . .
2. M. Wnuk, Z. Kotylak, M. Bugno, E. Słota — Karyotype analysis in pigs — hybrids of
European wild boar (Sus scrofa scrofa) and domestic pig (Sus scrofa domestica) . . .
3. B. Orzechowska — Predicted progress in improvement of carcass muscling and cuts with
indirect selection based on backfat thickness and loin eye area . . . . . . . . .
4. P. Paraponiak — Laboratory and sensory assessment of lamb muscle tissue with regard
to multidimensional analysis of the discriminatory function . . . . . . . . . . .
5. L. Czaja, K. Dziadek, E. Gornowicz — Quality of table eggs as influenced by laying hen
origin . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. A. Mazanowski, Z. Bernacki, T. Kisiel — Comparing the structure and chemical composition of duck eggs . . . . . . . . . . . . . . . . . . . . . . . .
7. A. Mazanowski, T. Kisiel, M. Adamski — Evaluation of some regional varieties of geese
for reproductive traits, egg structure and egg chemical composition . . . . . . . .
Biology, physiology and animal reproduction
8. J. Wieczorek, M. Cegła, W. Kareta — Factors affecting the oocyte recovery rate from
ovarian follicles of gilts . . . . . . . . . . . . . . . . . . . . . .
9. P. Gogol — Iron-induced luminescence of boar spermatozoa cells . . . . . . . .
10. M. Skrzyszowska, M. Samiec, Z. Smora˛g — Effect of in vitro culture conditions on the
developmental potential of porcine nuclear transfer embryos reconstructed with cumulus
cells analysed on apoptosis . . . . . . . . . . . . . . . . . . . . .
Animal nutrition and feedstuffs
11. F. Brzóska — Effect of soybean meal protected with Ca salts of fatty acids on cows’ yield,
protein and fat components in milk and blood . . . . . . . . . . . . . . .
12. B. Niwińska, J. Strzetelski — Effects of type and frequency of liquid feed on rumen
development and rearing performance of calves . . . . . . . . . . . . . .
13. B. Szymczyk — Effects of conjugated linoleic acid (CLA) on pig performance, carcass
quality, chemical composition of meat and serum lipid profile . . . . . . . . . .
14. B. Szymczyk — Effects of conjugated linoleic acid (CLA) on fatty acid composition, lipid
oxidation and quality of pork meat . . . . . . . . . . . . . . . . . . .
15. B. Borys., A. Borys — Effect of the form of rapeseed and linseed in lamb diets on
some health quality parameters of meat . . . . . . . . . . . . . . . . .
16. P. Strzetelski, F. Brzóska, W. Brzeziński — Iodine content of green fodder and cereal
crops in Poland . . . . . . . . . . . . . . . . . . . . . . . . .
17. R. Ga˛sior, K. Ślusarczyk, M. Szczypuła — Validation of a method for determining amino
acids in acid hydrolysates of feeds . . . . . . . . . . . . . . . . . . .
5
11
21
29
41
53
67
85
91
99
111
125
135
145
159
171
181
4
Environment, hygiene and animal production technology
18. A. Cisek, A. Balicka-Ramisz, B. Pilarczyk — Prevalence and control of gastrointestinal
and pulmonary nematodes in deer in a closed breeding population . . . . . . . .
Economics and organization of animal production
19. S. Okularczyk — Optimum scale of pig fattening on Polish family farms in light of economic
studies . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20. Z. Sokołowicz, J. Krawczyk — Economic efficiency of lengthening the productive life
of laying hens through moulting . . . . . . . . . . . . . . . . . . .
199
207
215
Ann. Anim. Sci., Vol. 5, No. 1 (2005) 5 – 9
COMPARATIVE HYBRIDIZATION OF THE Y CHROMOSOME
IN SELECTED SPECIES OF BOVIDAE*
A n n a K o z u b s k a - S o b o c i ń s k a 1, E w a S ł o t a 1, A l d o n a P i e ń k o w s k a - S c h e l l i n g 2,
C l a u d e S c h e l l i n g 3, 4
1
Department of Animal Immuno- and Cytogenetics, National Research Institute of Animal Production,
32-083 Balice n. Kraków, Poland
2
Department of Genetics and Animal Breeding, August Cieszkowski Agricultural University,
Wołyńska 33, 60-637 Poznań, Poland
3
Swiss Federal Institute of Technology, Zürich, Switzerland
4
Faculty of Veterinary Medicine, University of Zürich, Switzerland
Abstract
The syntenic and conservative nature of some chromosomes enables the use of several molecular
probes obtained from one species of animals to detect homologous DNA segments in another
species. This possibility was used to hybridize a biotin-labelled probe obtained by microdissection
of the bovine Y heterosome with ovine and caprine chromosomes. In metaphase plates of each
species we detected a clear yellow hybridization signal corresponding to the Y chromosome and
covering whole p and q arms. The syntenic and conservative nature of the Y chromosome in cattle,
sheep and goats enables the bovine Y chromosome painting probe to be used to diagnose karyotype
changes such as leukocyte chimerism or XXY trisomy in Bovidae species.
Key words: Bovidae, genetic conservatism, chromosome painting, Y chromosome, FISH technique
Genetic conservatism makes it possible to compare genomes of different
species at the level of nucleotide sequences (Cheng et al., 2001), chromosome
banding patterns (Słota et al., 2001) and groups of linked or syntenic genes that are
often in the same relationships even in taxonomically distant species (Solinas-Toldo
et al., 1995).
Comparisons of different species provided the basis for distinguishing three
categories of genes: those found in syntenic groups in all species of animals, genes
that are almost always syntenic, and those not belonging to genetically conserved
* This work was conducted as part of the research project no. 3 P06D 039 24, financed by the State
Committee for Scientific Research.
6
A. Kozubska-Sobocińska et al.
groups (Echard, 1989; Świtoński, 1992). This syntenic and conservative nature
of some chromosomes makes it possible to use a number of molecular probes
obtained by microdissection or chromosome sorting in one species of animals, for
FISH chromosome painting in another species (Chowdhary et al., 1996; Ponce de
Leon et al., 1996).
This study was designed to use a bovine molecular probe, specific to the
Y heterosome, to identify this chromosome on metaphase plates obtained after
lymphocyte culture from blood samples of rams and bucks.
Material and methods
FISH technique was used to analyse cytogenetic preparations obtained from
5 bulls, 10 rams and 5 bucks. Blood was taken from jugular vein into sterile lithium
heparin tubes. Lymphocyte cultures were performed according to method described
by Arakaki and Sparkes (1963) with minor modifications. After 72-h culture, fixed
leukocyte suspensions were spread on microscope slides. The metaphase chromosome preparations obtained from bulls, rams and bucks were hybridized according
to the protocols prepared by Pinkel et al. (1986) and Solinas-Toldo et al. (1995)
with a molecular probe specific to the bovine Y chromosome.
A chromosome-specific probe was prepared at the Swiss Federal Institute of
Technology (ETH, Zürich). The probe was obtained by microdissection of about
20 Y chromosomes from bovine metaphase plates, which were then amplified and
labelled by DOP-PCR, using biotinylated nucleotide bio-16-UTP (Goldammer et
al., 1996 a). Hybridization signals on metaphase chromosomes were revealed using
Avidin-FITC. Image analysis was performed under a fluorescent microscope
OPTON-Axiophot, using triple attenuation filters DAPI/FITC/Texas Red and the
computer image analysis system LUCIA-FISH (Laboratory Imaging Ltd, Prague,
Czech Republic).
Results
Using FISH technique, an attempt was made to apply the molecular probe
obtained with the microdissection of metaphase Y chromosomes of Simmental
cattle to identify this sex chromosome in sheep and goats, which belong to the
Bovidae family together with cattle. In the first stage, in which the efficiency of the
construct made was verified, the Y chromosome was painted in the chromosome
preparations of the bulls of breeds (Polish Red, Red-and-White) other than
Simmentals, which were the donor of the sex chromosome used for the microdissection. In both cases clear hybridization signals, which covered the whole
Y chromosome, were detected (Figure 1). Then, while maintaining previous
conditions of hybridization time (24 hours), the probe was used for interspecific
hybridizations. As a result, one intensely fluorescent signal corresponding to
Comparative hybridization of the Y chromosome
7
the Y chromosome was identified in each cell with 54,XY karyotype obtained
from rams (Figure 2) and on each 60,XY metaphase plate obtained from bucks
(Figure 3).
Discussion
Karyotype conservatism in Bovidae at the level of banding patterns, which
reveals homologies between chromosomes or their fragments (Słota et al., 2001),
stimulated a further search for the similarities among Bovidae species. The studies
concerned groups of syntenic or linked genes and the molecular structure of genes
and anonymous nucleotide sequences (Cheng et al., 2001; Edwards et al., 2000).
First attempts to show conservatism between chromosomes of different species
involved chromosome painting by in situ hybridization using probes specific to
human chromosomes (ZOO-FISH). The 24 chromosome-specific probes from the
human genome, used by Hayes (1995), showed complete homology in 18 cases and
painted only one bovine chromosome (including the Y chromosome), while the
others painted 2 or 3 autosomes. The author attributed the absence of signals in the
Y chromosome to the high content of repeat sequences and to the short segments of
DNA sequences that were hard to visualize. Also Chowdhary et al. (1996) detected
46 homologous segments in cattle and 4 in sheep using human chromosomespecific plasmid libraries. In both species, they observed clear signals on
the X chromosomes and absence of hybridization with the Y chromosome. Using
ZOO-FISH technique, Solinas-Toldo et al. (1995) detected 56 homologous fragments between human and bovine chromosomes and found complete conservatism
of the X chromosomes in both species and only one ZFY fragment hybridizing with
the Y chromosome. Studies on the PARX and PARY segments showed that the
sequences homologous to the human pseudoautosomal genes CSF2RA and ANT3
are identified on the X and Y chromosomes of the sheep and dog (Toder et al.,
1997).
Similarities between chromosomes of different Bovidae species were shown
many times using probes obtained from bovine chromosomes. These probes were
obtained by microdissection of whole chromosomes or their fragments or by PCR
from genomic DNA using appropriate starters. One application of a probe obtained
by microdissection of the Yp12 fragment is the identification of a complementary
sequence in the XY bivalent at the metaphase I stage and comparative hybridization
with the Yq12.1-12.6 probe from Bos indicus of a relevant segment on the q arm of
the Y chromosome in Bos taurus (Goldammer et al., 1996 b). The probe specific to
the Yp12 segment, produced by PCR, was also used to identify the Y chromosome
on metaphase plates and in spermatozoa (Révay et al., 2000).
It is worth noting that the Y chromosome is the most diverse chromosome in
terms of total length and the arm length ratio (Genest, 1981). Of the polymorphic
variants in cattle, a ‘‘large” submetacentric is characteristic of Simmental bulls and
a much smaller one, with metacentric morphology, is specific to Polish Red cattle
8
A. Kozubska-Sobocińska et al.
(Kozubska-Sobocińska and Słota, 2002). These breed specific differences were
accounted for in the present study during hybridization that verified the efficiency
of the probe that was obtained based on the material from Simmental bulls and gave
clear fluorescent signals on the metacentric Y chromosome characteristic of the
Polish Red breed (Figure 1).
The bovine painting probe, due to the small size of the Y chromosome, finds
little use for analysis of its evolutionary rearrangements in the Bovidae family, as
was the case for the X chromosome (Ponce de Leon et al., 1996; Prakash et al.,
1997). The syntenic and conservative nature of the Y chromosome in cattle, sheep
and goats enables the bovine Y chromosome painting probe to be used to diagnose
karyotype changes such as leukocyte chimerism (Kozubska-Sobocińska et al.,
2003), XXY trisomy (Słota et al., 2003) or identification of heterosomes in
spermatozoa (Di Berardino et al., 2004) in Bovidae species.
References
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C h e n g H., S h i H., Z h o u R., G u o Y., L i u L., L i u J., J i a n g Y., K u d o T., S u t o u S. (2001).
Characterization of Bovidae sex-determining gene SRY. Genet. Sel. Evol., 33: 687 – 694.
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aries L.), goat (Capra hircus L.), and cattle spermatozoa by double color FISH using bovine (Bos
taurus L.) X- and Y-painting probes. Mol. Reprod. Dev., 67: 108 – 115.
E c h a r d G. (1989). Domestic animal gene mapping: a comparative map of species investigated. In:
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E d w a r d s C.J., G a i l l a r d C., B r a d l e y D.G., M a c H u g h D.E. (2000). Y-specific microsatellite
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G o l d a m m e r T., W e i k a r d R., B r u n n e r R.M., S c h w e r i n M. (1996 a). Generation of chromosome fragment specific DNA sequences by microdissection and DOP-PCR. Mamm. Genome, 7:
291 – 296.
G o l d a m m e r T., B r u r m e r R.M., W e i k a r d R., S c h w e r i n M. (1996 b). Generation and use of
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H a y e s H. (1995). Chromosome painting with human chromosome-specific DNA libraries reveals the
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K o z u b s k a - S o b o c i ń s k a A., S ł o t a E. (2002). Y chromosome polymorphism in some species of
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K o z u b s k a - S o b o c i ń s k a A., S ł o t a E., P i e ń k o w s k a A. (2003). Zastosowanie techniki FISH do
diagnozy chimeryzmu leukocytarnego u owiec. Med. Wet., 59: 987 – 989.
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P o n c e d e L e o n F.A., A m b a d y S., H a w k i n s G.A., K a p p e s S.M., B i s h o p M.D., R o b l J.M.,
B e a t t i e C.W. (1996). Development of a bovine X chromosome linkage group and painting probes
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bovine cosmids to cattle, goat, sheep and buffalo X chromosomes. Hereditas, 126: 115 – 119.
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a synthetic Y-probe. Proc. 14th Europ. Colloq. Cytogenet. Domest. Anim., Abstr., Brno; p. 29.
S ł o t a E., K o z u b s k a - S o b o c i ń s k a A., B u g n o M., G i e m z a - M a r e k A., K u l i g B. (2001).
Comparison between the G-banded karyotype of the aoudad (Ammotragus lervia) and sheep (Ovis
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S ł o t a E., K o z u b s k a - S o b o c i ń s k a A., K o ś c i e l n y M., D a n i e l a k - C z e c h B., R e j d u c h B.
(2003). Detection of the XXY trisomy in a bull by using sex chromosome painting probes. J. Appl.
Genet., 44: 379 – 382.
S o l i n a s - T o l d o S., L e n g a u e r C., F r i e s R. (1995). Comparative genome map of human and cattle.
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Ś w i t o ń s k i M. (1992). Mapowanie genów u zwierza˛t gospodarskich. Med. Wet., 48: 131 – 134.
T o d e r R., G l ä s e r B., S c h i e b e l K., W i l c o x S.A., R a p p o l d G., G r a v e s J.A.M., S c h e m p p W.
(1997). Genes located in and near the human pseudoautosomal region are located in the X-Y pairing
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Accepted for printing 15 III 2005
ANNA KOZUBSKA-SOBOCIŃSKA, EWA SŁOTA, ALDONA PIEŃKOWSKA-SCHELLING,
CLAUDE SCHELLING
Porównawcza hybrydyzacja chromosomu Y u wybranych gatunków Bovidae
STRESZCZENIE
Synteniczno-konserwatywny charakter niektórych chromosomów pozwala na zastosowanie szeregu
maluja˛cych sond molekularnych, uzyskanych z chromosomów jednego gatunku zwierza˛t, do identyfikacji homologicznych odcinków DNA u innych gatunków. Wykorzystuja˛c te˛ możliwość podje˛to
próbe˛ hybrydyzacji sondy otrzymanej metoda˛ mikrodysekcji heterosomu Y bydła (znakowanej biotyna˛)
z chromosomami owiec i kóz. W płytkach metafazowych każdego z gatunków otrzymano wyraźny żółty
sygnał hybrydyzacyjny odpowiadaja˛cy chromosomowi Y obejmuja˛cy całe ramiona p i q. Wykazany
synteniczno konserwatywny charakter chromosomu Y u bydła, owiec i kóz pozwala na zastosowanie
bydle˛cej sondy maluja˛cej heterosom Y do diagnozowania zmian kariotypowych, typu chimeryzm
leukocytarny czy trisomia XXY, u gatunków należa˛cych do Bovidae.
KARYOTYPE ANALYSIS IN PIGS — HYBRIDS OF EUROPEAN WILD
BOAR (SUS SCROFA SCROFA) AND DOMESTIC PIG (SUS SCROFA
DOMESTICA)
M a c i e j W n u k 1, Z b i g n i e w K o t y l a k 1, M o n i k a B u g n o 2, E w a S ł o t a 2
1
Department of Genetics, Institute of Biotechnology, University of Rzeszów, Sokołowska 26,
36-100 Kolbuszowa, Poland
2
Department of Animal Immuno- and Cytogenetics, National Research Institute of Animal Production,
Abstract
A cytogenetic survey was carried out on crossbreds between wild boar (Sus scrofa scrofa) and
Duroc domestic pig (Sus scrofa domestica). GTG-banding technique was applied for identification
of particular pairs of chromosomes. Twenty-seven animals of the F1 generation were examined.
The whole F1 generation had the same father. All piglets of the F1 generation were characterized by
37,XX,t.rob15;17 or 37,XY,t.rob15;17 karyotype. Because of this karyotype formula their father
has to be a homozygous carrier for the 15;17 Robertsonian translocation. The mothers of
investigated piglets were described as 38,XX. Additionally, a single litter (four piglets) derived
from mating between a Sus scrofa scrofa boar (father of F1 generation piglets) and a sow descended
from F1 (37,XX,t.rob15;17) was analysed. The progeny of this backcross were three individuals
with 37 chromosomes and one with 36 chromosomes. The present investigation confirmed that
Robertsonian translocation carriers transmitted this aberration to the offspring in accordance
with Mendelian segregation. Because the boar carried the translocation in homozygous form, there
was no influence of this translocation on the litter size. F2 generation represents too small a sample
(one litter with four piglets), but the litter was evidently smaller than normal. This problem requires
further analysis. The present study suggested the need in Poland for programmes for genetic and
reproductive improvement incorporating chromosomal analysis of pigs of high genetic value.
Key words: pig, wild boar, karyotype, Robertsonian translocation
The normal karyotype of the domestic pig (Sus scrofa domestica) comprises
2n = 38 chromosomes. The diploid complement contains five pairs of submetacentric, two pairs of subtelocentric, five pairs of metacentric, six pairs of acrocentric
autosomes and two XX or XY heterosomes. Chromosome X is metacentric, similar
in size to pair no. 9 and Y is the smallest metacentric (Committee for the
Standardized Karyotype of the Domestic Pig, 1988). When the karyotype of the
domestic pig is compared to that of the wild boar (Sus scrofa scrofa) it can be
12
M. Wnuk et al.
concluded that both karyotypes are almost identical in animals having a 2n = 38
chromosome number (Gustavsson et al., 1973). However, especially in Sus scrofa
scrofa, this chromosome number is not constant as a result of Robertsonian
translocations, which may lead to a reduction of the chromosome number.
Robertsonian translocations are one of the most frequent chromosome rearrangements in mammals. They involve fusion of two telo- or acrocentric chromosomes
into one meta- or submetacentric chromosome. Robertsonian translocations change
the chromosome number, but not their arm number (NF), which is characteristic of
a given species (Long, 1988). This type of aberration has been found among others
in cattle, sheep, goat, horse, donkey, dog and mice (Gustavsson, 1969; Chandley et
al., 1975; Hanada and Muramatsu, 1988; Dai et al., 1994; Underkoffler et al., 2002;
Schifferli et al., 2003; Świtoński et al., 2003).
Centric fusion is also a widely distributed translocation type in the wild boar of
Central Asia (Sus scrofa nigripes) and Europe (Sus scrofa scrofa). S. s. nigripes
subspecies is characterized by fusion of chromosomes 16 and 17 and S. s. scrofa by
fusion of chromosomes 15 and 17 (Troshina et al., 1985). In these two subspecies
chromosome rearrangements induce variation in chromosome number (2n = 38, 37
or 36) causing karyotype polymorphism (Mc Fee et al., 1966; Živkovič et al., 1971;
Gustavsson et al., 1973; Bosma, 1976; Sysa, 1980; Rejduch et al., 2003 a).
In the domestic pig only one type of Robertsonian translocation identified as
13;17 has been diagnosed so far (Schwerin et al., 1986; Golish et al., 1986; Zhang et
al., 1991). However, centric fusion of chromosomes 13 and 17 is infrequent in
contrast to the 15,17 translocation in the European wild boar (Słota, 1998).
The influence of Robertsonian translocation on pig reproduction is still
a question of debate.
The aim of the present study was karyotype evaluation in the progeny of
European wild boar and domestic pig with regard to Robertsonian translocation
diagnosis.
Cytogenetic analyses were performed in a pig herd from the Experimental
Station in Werynia (south-east Poland), belonging to the University of Rzeszów.
Twenty-seven offspring derived from mating between a wild boar (Sus scrofa
scrofa) and 4 Duroc sows (Sus scrofa domestica), and 4 offspring derived from
mating of the same wild boar and one F1 generation sow were chosen for karyotype
analysis.
Blood samples were taken from the jugular vein into sterile tubes containing
heparin. Lymphocytes were cultured according to the method of Arakaki and
Sparkes (1963) with minor modifications. The chromosome preparations (10 – 20
from each animal) were obtained according to the routine protocol.
The metaphase spreads were stained by both conventional Giemsa staining and
GTG-technique (Wang and Fedoroff, 1972).
Karyotype analysis in pigs
13
Metaphase plates were observed under the light microscope using the MultiScan
computer image analysis system. The database comprised the pictures of metaphase
spreads with G-banded chromosomes, 2 – 5 for each investigated pig. The karyograms were prepared according to the recommendations of the Committee for the
Standardized Karyotype of the Domestic Pig (1988).
Results
Microscope analysis of Giemsa stained metaphase spreads revealed that all
investigated pigs of the F1 generation were characterized by diploid chromosome
number 2n = 37, of which 25 were meta- and submetacentric chromosomes,
10 acrocentric autosomes and two heterosomes — XX or XY.
Based on G-banded metaphase spreads and Standard Karyotype of Sus scrofa
domestica it was diagnosed that the additional submetacentric chromosome resulted
from a Robertsonian translocation between two acrocentric chromosomes. The
p arm of the fusion chromosome was identified by its banding pattern as
chromosome pair 17 and q arm as chromosome pair 15 (Figure 1 and 2).
Figure 1. Karyotype of a female crossbred between wild boar (Sus scrofa scrofa) and Duroc
domestic pig (Sus scrofa domestica) — 15;17 Robertsonian translocation carrier (heterozygous form) —
GTG technique
14
M. Wnuk et al.
Figure 2. Karyotype of a male crossbred between wild boar (Sus scrofa scrofa) and Duroc domestic
pig (Sus scrofa domestica) — 15;17 Robertsonian translocation carrier (heterozygous form) — GTG
technique
Figure 3. Pedigree of mating between Sus scrofa scrofa boar (father of F1 piglets) and sow descended
from F1 (37,XX,t.rob15;17)
15
Pedigree records of F1 generation pigs showed that the mean litter size is 10.
Cytogenetic analysis of one litter from mating between a wild boar (father of F1
generation) and a sow descended from F1 (37,XX,t.rob15;17) showed three animals
with 2n = 37 and one with 36 chromosomes in the offspring. This result confirms
that the boar (common father of both generations) was a homozygous carrier for
Robertsonian translocation and its karyotype formula was 36,XY,t.rob15;17. The
pedigree analysis in F2 generation, concerning Robertsonian translocation is shown
in Figure 3. The final litter (4 piglets) was evidently smaller and in weaker
condition than three litters of the F1 generation. However, F2 generation represented
a small sample, therefore no final conclusions can be made.
Discussion
Cytogenetic investigations of chromosome number polymorphism which have
been carried out for more than 40 years on several species of mammals showed that
variation in chromosome number is caused mainly by Robertsonian translocations
(Gustavsson and Sundt, 1967; McFee et al., 1966).
Among farm animals these translocations are particularly widely distributed in
cattle and more than 60 different Robertsonian translocations have been reported
(Rejduch et al., 1994).
The examinations carried out in domestic pigs and wild boars from Europe and
Asia showed variation in the diploid chromosome number — 2n = 36, 37 or 38.
Each subspecies was characterized by a different chromosome fusion — a 15;17
translocation was observed in S. s. scrofa L., 15;16 in S. s. nigripes and 13;17 in
S. s. domestica L.
In the present study, using GTG-banding technique, a 15;17 Robertsonian
translocation was identified in the crossbred between wild boar (S. s. scrofa) and
Duroc domestic pig (S. s. domestica). All pigs of the F1 generation were
heterozygous for the translocation. In the 19 animals karyotype formula was
established as 37,XX,t.rob15;17 and in remaining 8 as 37,XY,t.rob15;17. This
finding confirms the results reported by Sysa et al. (1984), who described two gilts
as 37,XX,t.rob15;17. They were the offspring of wild boar and domestic pig.
Analysis of a single litter derived from mating between a wild boar (father of F1
progeny) and a sow derived from F1 (2n = 37,XX,t.rob15;17) allowed diagnosing
one piglet with monozygous form of Robertsonian translocation and karyotype
formula 2n = 36,XY,t.rob15;17, and three animals heterozygous for Robertsonian
translocation.
Because F1 and F2 generation piglets had the same father (wild boar) and all
mothers had the 38,XX karyotype, based on offspring karyotype it was established
that the wild boar was a homozygous carrier of the 15;17 Robertsonian translocation.
The translocated chromosome 15;17 is transmitted to the offspring in accordance with Mendelian segregation. Therefore the crossing between animals having
16
M. Wnuk et al.
2n = 38 and 2n = 36 gave offspring with 2n = 37 chromosomes. The parents with
2n = 36 crossed with 2n = 37 produced offspring polymorphic with regard to
chromosome number: 2n = 36 and 2n = 37. The results presented in this paper
confirm those obtained by McFee and Banner (1969).
The influence of centric fusion on fertility and viability could be interesting for
breeders of wild boar and domestic pig crosses. Because the heterozygous
translocation carriers show reduced fertility the karyotype analysis can be a useful
tool for selection of reproductive material.
It is well known that in meiosis of heterozygous carriers for Robertsonian
translocation typical trivalent configurations are observed, which rarely associate
with the sex chromosomes bivalent. Therefore, mis-segregation of the trivalent at
anaphase I can concern gametogenesis by production of chromosomally unbalanced
gametes (Świtoński and Stranzinger, 1998).
Rejduch et al. (2003 b) observed in spermatocytes I from a wild boar with
37,XY,t.rob15;17 karyotype seventeen bivalents and a completely paired trivalent,
but did not observe any associations between trivalent and sex bivalent.
Heterozygous carriers of Robertsonian translocation are suspected to produce
normal gametes (n), balanced gametes (n-1) with translocated chromosome as well
as monosomic and trisomic gametes. In sheep data indicate a segregation pattern
favouring chromosomally balanced meiotic products and chromosomally abnormal
gametes are eliminated before maturation (Long, 1988; Dai et al., 1994). But so far
there is no information about similar prezygotic selection for gametes in pigs.
The negative effect of Robertsonian translocation on a single animal is rather
small and easily hidden by other factors influencing fertility (Gustavsson, 2004).
For example, the 1;29 translocation in cattle reduces their fertility by approximately
5 – 7% (Gustavsson, 1971). Studies on the influence of the 13;17 Robertsonian
translocation in domestic pig showed that this aberration reduced fertility by approximately 10 – 22% (Golish, 1989; McFeely et al., 1988). The progeny of the 13;17
translocation carriers were characterized by approximately 9% lower daily gain.
The information about the influence of the 15;17 or 16;17 Robertsonian
translocation on fertility of wild boar is limited because of the limited access to wild
animals. The results of the studies on crossbreds between wild boar and domestic
pig are still controversial. Tikhonov and Troshina (1980) described a pig crossbred — double (15;17 and 16;17) and Robertsonian translocation carriers showed
normal fertility (8 – 10 piglets per litter). In the present study the negative effect of
the 15;17 translocation on the carrier’s fertility was not observed in the F1
generation (average litter size — 10 piglets), probably because the boar was
a homozygous carrier of this translocation. In F2 generation only one litter was
investigated and this litter was evidently smaller (4 piglets). The parents of these
piglets were characterized by different chromosome number: – 2n = 36, 15;17
(translocation homozygous carrier) and – 2n = 37, 15;17 (translocation heterozygous carrier). However, only one litter represented too small a sample for clear
conclusions. Sysa et al. (1984) described the progeny of two crossbreds: wild
boar × (domestic pig × wild boar). The litter size was 8 and 6 piglets, respectively.
17
According to the information on http://www.republika.pl/dzik.htm the litter
size of the wild boar is usually between 6 – 9 piglets.
The molecular background of Robertsonian translocations has not yet been
clarified. Probably detailed investigations on Ac2 centromeric DNA satellite family
present in acrocentric chromosomes (Janzen et al., 1999) should solve this problem.
The analysis of Robertsonian translocations in Graomys griseoflavus indicates that
the presence of long sequences of satellite DNA located in centromeric regions of
non homologous acrocentric chromosomes can cause chromosomal rearrangement
of the centric fusion type (Zambelli et al., 2003).
The present report suggests once more that in Poland the programme of
cytogenetic control in pigs, including crossbreds, is urgently needed and can be
very useful for selection of parental material.
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M a r s h W., A n d e r s o n G.R. (1991). Two different types of chromosomal translocations found in
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Accepted for printing 6 I 2005
19
MACIEJ WNUK, ZBIGNIEW KOTYLAK, MONIKA BUGNO, EWA SŁOTA
Analiza kariotypu u świń — hybryd dzika europejskiego (Sus scrofa scrofa) i świni domowej
(Sus scrofa domestica)
STRESZCZENIE
Celem badań była cytogenetyczna ocena mieszańców dzika europejskiego (Sus scrofa scrofa)
i świni domowej (Sus scrofa domestica). Do identyfikacji poszczególnych par chromosomów
zastosowano technike˛ GTG. Zbadano 27 zwierza˛t pokolenia F1. Całe pokolenie F1 miało tego samego
ojca. Wszystkie prosie˛ta pokolenia F1 charakteryzowały sie˛ kariotypem 37,XX,t.rob15;17 lub
37,XY,t.rob15;17. Ten wzór kariotypu wskazuje, że ojciec jest homozygotycznym nosicielem translokacji robertsonowskiej 15;17. Matki badanych prosia˛t miały kariotyp 38,XX. Analizowano również
pojedynczy miot czterech prosia˛t pochodza˛cych z krzyżowania dzika europejskiego (ojca prosia˛t
pokolenia F1) i lochy F1 (37,XX,t.rob15;17). Potomstwem tej krzyżówki wstecznej były trzy osobniki
z 37 chromosomami i jeden z 36 chromosomami. Obecne badania potwierdziły, że nosiciele translokacji
robertsonowskiej przekazywali te˛ aberracje˛ potomstwu zgodnie z segregacja˛ mendlowska˛. Ponieważ
translokacja u knura wysta˛piła w postaci homozygotycznej, nie miała ona wpływu na wielkość miotu.
Pokolenie F2 stanowi zbyt mała˛ próbe˛ (jeden miot czterech prosia˛t), niemniej jednak sam miot był
mniejszy niż zwykle. Problem ten wymaga dalszej analizy. Przeprowadzone badania wskazuja˛ na
potrzebe˛ wprowadzenia programów doskonalenia genetycznego i rozpłodowego obejmuja˛cych chromosomowa˛ analize˛ u świń o wysokiej wartości genetycznej.
PREDICTED PROGRESS IN IMPROVEMENT OF CARCASS MUSCLING
AND CUTS WITH INDIRECT SELECTION BASED ON BACKFAT
THICKNESS AND LOIN EYE AREA
Barbara Orzechowska
Department of Animal Genetics and Breeding, National Research Institute of Animal Production,
Abstract
This study was aimed to show what progress in the muscling of carcass or cuts can be expected
when carrying out indirect selection on backfat thickness or loin eye area. Data on Polish Large
White and Polish Landrace gilts, performance tested at Pig Testing Stations were investigated.
Statistical analysis included the estimation of genetic and phenotypic parameters for slaughter
traits, i.e. backfat thickness measured over the shoulder, on the back, at the sacrum points I, II and
III, and weight of meat (in neck, shoulder, ham, leg, loin, belly, ribs, and side). Breeding progress
for traits correlated with traits under selection was determined according to a formula given by
Duniec. The present results demonstrate that certain improvements in weight of meat in the
carcass or carcass cuts can be made when carrying out indirect selection. Best results were shown
when carrying out selection for backfat thickness measured at the sacrum point I. Selection based
on this measurement improves carcass muscling by approximately 54% compared to direct
selection, in which total weight of meat from all carcass cuts would be accounted for. The
application of indirect selection based on this measurement increases the weight of meat in carcass
cuts, e.g. an improvement of 81% will be obtained for the neck (in relation to direct selection for
weight of meat in the neck).
Key words: pig, indirect selection, carcass, primary cut
The most common parameters used for assessing carcass quality are backfat
thickness and loin eye area. These parameters are accounted for both in the
regression equation used to estimate carcass meat percentage on a live animal
(Eckert and Żak, 2004) and in testing stations to evaluate the slaughter value of
animals postmortem (Różycki and Tyra, 2004). In testing stations, a selection index
is calculated for each animal tested. The index contains slaughter value traits such
as loin eye area, weight of rear ham without backfat and skin, and average backfat
thickness from five measurements.
22
B. Orzechowska
The present study was designed to show what progress in improvement of
muscling of carcass and individual cuts can be expected when carrying out indirect
selection for backfat thickness or loin eye area.
Polish Large White and Polish Landrace gilts, tested at the Pig Testing Stations
were investigated. For statistical calculations, 1330 gilts which were derived from
full matings and had at least 2 paternal half-sibs from 1 sow, were chosen. The
following traits were analysed: loin eye area, backfat thickness (over the shoulder,
on the back, at the sacrum points I, II and III), average backfat thickness from
5 measurements, weight of meat (neck, shoulder, ham, leg, loin, belly, ribs, side).
For all analysed traits, genetic parameters (heritability, correlations) were
calculated according to the method given by Becker (1967). The expected breeding
progress for traits correlated with the traits being selected was estimated according
to the formula:
∆Gj =
where:
∆Gj —
percent of
rGij —
hi —
hj —
hi rGij .
100
hj
progress in the trait j with indirect selection for the trait i, expressed as
progress achieved in direct selection for the trait j,
genetic correlation between the two traits i and j,
the root of heritability index for the trait i,
the root of heritability index for the trait j.
Results
Data on the characteristics of the analysed material are given in Tables 1 and 2.
Average weight of the analysed pigs was 86.4 kg, showing slight deviations for
individual animals (s = 1.5 kg). The animals achieved this weight by 186.3 days of
age on average, with daily gain during the test (30 – 80 kg) of 681 g on average.
After cooling, the carcasses weighed an average of 32.7 kg, with slightly higher
variation than for slaughter weight, which may point to differences in slaughter
yield.
Backfat measured on the side was thickest over the shoulder (3.3 cm) and
thinnest at the sacrum point II (1.7 cm) and on the back (1.8 cm). Individual
measurements showed a similar standard deviation. A similar standard deviation
was displayed by average backfat thickness from 5 measurements (2.3 cm). Loin
eye area was 33.3 cm2 and showed lower variation than backfat thickness, as
indicated by the correlation coefficient for this trait.
23
Predicted progress in improvement of carcass muscling
Table 1. Arithmetic means (x̄), standard deviations (δ) and variation coefficients (v) for fattening traits
and linear measurements of backfat and loin eye area
Trait
Body weight before slaughter (kg)
Age at slaughter (days)
Weight of cold side (kg)
Daily gain (g)
Backfat thickness (cm):
over the shoulder
on the back
at the sacrum point I
at the sacrum point II
at the sacrum point III
Average backfat thickness from 5
measurements (cm)
Loin eye area (cm2)
Arithmetic
means
(x̄)
Standard
deviations
(δ)
Coefficients of
variation
(v)
86.4
186.3
32.7
681
1.5
14.8
1.2
72
1.7
7.9
3.7
10.7
3.3
1.8
2.3
1.7
2.4
0.5
0.4
0.5
0.4
0.5
15.7
21.6
21.0
24.9
21.8
2.3
33.3
0.4
3.7
16.5
11.2
Table 2. Arithmetic means (x̄), standard deviations (δ) and variation coefficients (v) for weight of meat
of side and cuts
Kind of meat
Neck
Shoulder
Ham
Leg
Loin
Belly
Ribs
Weight of side without head
and feet
x̄
Weight of cuts
δ
v
x̄
Meat
δ
v
4.186
4.308
6.711
1.131
6.810
4.416
0.933
0.366
0.377
0.398
0.109
0.550
0.488
0.125
8.8
8.7
5.9
9.6
8.1
11.1
13.4
2.372
2.583
4.588
0.579
3.444
1.876
0.593
0.278
0.369
0.402
0.078
0.389
0.277
0.108
11.7
14.3
8.8
13.5
11.3
14.8
18.2
28.493
1.100
3.9
16.035
1.172
7.3
Table 2 gives arithmetic means, standard deviations and variation coefficients
for weight of meat, the right side and its cuts. The lowest variation of meat weight
was observed for ham, and the highest for ribs.
Heritability coefficients for weight of meat on the side and its cuts are shown in
Table 3. Tabular data indicate that the highest h2 for meat weight was characteristic
of ribs (0.605) and belly (0.600), and the lowest of shoulder (0.362).
Coefficients of phenotypic and genetic correlations among weight of meat on
the side, its cuts, loin eye area and backfat thickness are presented in Tables 4 a
and b. The coefficients of correlation with loin eye area indicate a close relationship
of this parameter with weight of meat of all cuts except belly (Table 4 b). For the
other two cuts, the correlation did not exceed the double value of error, and for
others (shoulder, ham, leg and loin) it was higher than the triple value of error.
24
B. Orzechowska
Table 3. Heritability coefficients for some slaughter traits
Trait
Backfat thickness:
over the shoulder
on the back
at the sacrum point I
at the sacrum point II
at the sacrum point III
Average backfat thickness from 5 measurements
Loin eye area
Weight of meat in:
neck
shoulder
ham
leg
loin
belly
ribs
primal cuts
Coefficients of
heritability
(h2)
Coefficient errors
(sh)
0.335
0.372
0.628
0.438
0.443
0.529
0.226
0.029
0.029
0.034
0.032
0.031
0.033
0.029
0.466
0.362
0.467
0.495
0.393
0.600
0.605
0.562
0.032
0.030
0.032
0.031
0.029
0.032
0.032
0.034
Table 4 a. Coefficients of phenotypic correlation between selected slaughter traits of pigs
Kind
of meat
Neck
Shoulder
Ham
Leg
Loin
Belly
Ribs
Side
Loin eye
area
Average
backfat
thickness
from 5
measurements
0.266*
0.380**
0.558**
0.309**
0.578**
0.352**
0.150
0.631**
− 0.247*
− 0.211*
− 0.275**
− 0.194
− 0.214*
− 0.191
− 0.166
− 0.338**
Backfat thickness
over the
shoulder
on the
back
at the
sacrum
point I
at the
sacrum
point II
at the
sacrum
point III
− 0.098
− 0.141
− 0.205*
− 0.140
− 0.155
− 0.110
− 0.072
− 0.215**
− 0.167
− 0.133
− 0.127
− 0.069
− 0.100
− 0.117
− 0.107
− 0.186
− 0.246*
− 0.173
− 0.221*
− 0.166
− 0.180
− 0.176
− 0.167
− 0.295**
− 0.224*
− 0.226*
− 0.283**
− 0.229*
− 0.225*
− 0.217*
− 0.190
− 0.354**
− 0.243*
− 0.169
− 0.241*
− 0.154
− 0.171
− 0.140
− 0.138
− 0.285**
* Significant at P = 0.05.
** Significant at P = 0.01.
Negative coefficients were found for the correlation between average backfat
thickness and meat content of the neck, ham, belly and ribs. Similar relationships
were shown by backfat thickness measured at three points on the sacrum. Backfat
thickness over the shoulder and backfat thickness on the back showed similar
relationships only in a few cases.
25
Table 4 b. Coefficients of genetic correlation between selected slaughter traits of pigs
Kind
of meat
Neck
Shoulder
Ham
Leg
Loin
Belly
Ribs
Side
Loin eye
area
Average
backfat
thickness
from 5
measurements
0.207
0.660
0.625
0.861
0.574
0.083
0.449
0.628
− 0.652
− 0.199
− 0.354
− 0.265
− 0.158
− 0.342
− 0.579
− 0.477
Backfat thickness
over the
shoulder
on the
back
at the
sacrum
point I
at the
sacrum
point II
at the
sacrum
point III
− 0.087
− 0.065
− 0.226
− 0.104
− 0.172
0.40
− 0.266
− 0.165
− 0.539
− 0.026
− 0.068
0.035
− 0.045
− 0.249
− 0.432
− 0.247
− 0.695
− 0.330
− 0.335
− 0.326
− 0.032
− 0.431
− 0.678
− 0.574
− 0.705
− 0.156
− 0.378
− 0.356
− 0.242
− 0.326
− 0.472
− 0.496
− 0.738
− 0.246
− 0.497
− 0.376
− 0.215
− 0.444
− 0.602
− 0.598
Coefficients higher than double error (rG > 2se) are single underlined.
Coefficients higher than triple error (rG > 3se) are double underlined.
Table 5. Percentage progress in improvement of muscling of cuts with selection based on backfat
thickness and loin eye area
Traits under selection
Response of
traits to
selection
Weight of meat in:
neck
shoulder
ham
leg
loin
belly
ribs
primal cuts
Backfat thickness
over the
shoulder
average
backfat
thickness
from 5
measurements
on the back
at the
sacrum
point I
at the
sacrum
point II
at the
sacrum
point III
48
81
68
72
69
39
37
36
33
48
36
38
44
69
54
28
40
44
38
51
53
32
54
46
34
loin
eye
area
52
43
58
43
27
40
The expected progress in changes in the weight of meat of cuts and side, with
selection for backfat thickness at different points on the back and loin eye is given
in Table 5. The expected improvement in meatiness was illustrated based on the
percentage of progress which will be obtained with indirect selection compared to
direct selection for the weight of meat of a particular cut. Where no significant
genetic correlation was found between pairs of traits mentioned in Table 4, no
estimation was made for the expected progress in muscling for a given pair of traits
(as indicated by a line).
26
B. Orzechowska
Discussion
Carcass meatiness is estimated using certain parameters, most often backfat
thickness measured at different locations of the carcass and loin eye area (the
cross-sectioned area of the longissimus muscle behind the last rib). The usefulness
of these parameters for determination of carcass muscling and carcass cuts was also
determined in the present study.
The correlation coefficients estimated between average backfat thickness from
five measurements and carcass muscling indicate a phenotypic (r = –0.338) and
genetic correlation (rG = –0.477). Also for loin eye area, the coefficient of
phenotypic correlation was estimated to be rG = 0.631, and that of the genetic
correlation was rG = 0.628. The correlation coefficients of these parameters with
weight of meat of individual cuts are of similar value. Lee and Yen (1991) report
that meat percentage in ham was negatively correlated with backfat thickness
(–0.53 to –0.61). Duniec (1960) showed that the phenotypic correlation between
backfat thickness from 5 measurements and amount of meat in carcass was
r = –0.595 (ranging for different measurement points from r = –0.407 to
r = –0.550), and the genetic correlation was rG = –0.524 (range of rG = –0.289 to
–0.651). Loin eye area in the studies of Duniec showed a higher correlation with the
meat of primal cuts (r = 0.568, rG = 0.860). In the studies performed 30 years later
using a slightly different method developed by Borzuta et al. (1996), similar
coefficients of phenotypic correlation were obtained between measurements of
backfat thickness, loin eye and side meat percentage.
The present results concerning the use of loin eye for estimation of carcass
muscling are convergent with the findings of other authors. Festerling et al. (1983)
report that the phenotypic correlation between loin eye area and carcass meat
weight ranged from 0.7 to 0.85. Similar values were obtained by Desmoulin and
Hackmann-Voltz (1981), Oster et al. (1987), Schirmer (1985) and Schmitten
(1985).
The present results indicate that selection for backfat thickness (mean from
5 measurements) results in little progress in carcass muscling (46% compared to
direct selection in which total weight of meat from all the cuts would be accounted
for). A similar result in terms of improved carcass muscling will be obtained
with selection on measurement of backfat thickness at the sacrum point II. Better
results will be achieved for selection based on backfat thickness at the sacrum point
I or III. When discussing the usefulness of individual measurements of carcass
thickness for increased meatiness of cuts, it is concluded that the best parameter is
measurement of backfat thickness at the sacrum point I. This measurement will
improve neck muscling by about 81% in relation to direct selection for meat weight
in the neck, and for ribs by 69% in relation to direct selection for weight of meat of
ribs.
27
References
B o r z u t a K., P i e c h o c k i T., S t r z e l e c k i ?., D i m k e W., G r z e ś k o w i a k E. (1996). Metoda
elektroniczno-liniowa jako nowy prosty sposób oceny mie˛sności tusz wieprzowych. Gosp. Mie˛s.,
z. 7.
D e s m o u l i n B., H a c k m a n n - V o l t z B. (1981). La conformation des carcasses et l’appreciation de
composition corporelle. Station de recherches sur I’élevage des porcs saint-gilles, 35590
I’hermitage. Proc. 32nd Annual Meeting of the European Association for Production.
D u n i e c H. (1960). Korelacje fenotypowe i genetyczne mie˛dzy niektórymi cechami użytkowymi oraz
ich wskaźniki odziedziczalności u świń typu mie˛snego. Wyd. własne IZ, nr 117.
E c k e r t R., Ż a k G. (2004). Stan hodowli i wyniki oceny świń. Wyd. własne IZ, XXII: 47 – 58.
F e s t e r l i n g A., B a h l m a n n A., S c h m i t t e n P. (1983). Zusammenhänge zwischen Schinkenform
und Fleischanteil beim Schlachtschwein. Vortragstagung der Deutschen Gesellschaft für Züchtungskunde e. V und der Gesellschaft für Tierzuchtwissenschaft, 22 und 23 September 1983,
Weihenstephon.
L e e D., Y e n H.T. (1991). Prediction of the percentage of lean in the ham in pig carcasses of both sexes.
J. Chinese Soc. Anim. Sci., 20 (4): 481 – 489.
O s t e r A., F e w s o n D., K o m e n d e r P., B r o n s c h e i d W., S a c k E. (1987). Schätzung des
Muskelgewebeanteiles beim Schwein autgrund der Forchheimer Teilstückzerlegung sowie üblicher
schlachtkörpermasse. Züchtungskunde, 59, 4: s. 281.
R ó ż y c k i M., T y r a M. (2004). Stan hodowli i wyniki oceny świń. Wyd. własne IZ, XXII: 59 – 74.
S c h i r m e r G. (1985). Schätzung populationsgenetischer Parameter für Merkmale der Fleischleistung
der Deutschen Landrasse in Baden-Württenberg. Züchtungskunde, 57, 1: s. 26.
S c h m i t t e n F. (1985). Bewertung der Schachtkörperqualität beim Schwein in der Schlachtkette.
Züchtungskunde, 57, 6: s. 412.
Accepted for printing 22 IX 2004
BARBARA ORZECHOWSKA
Spodziewany poste˛p w poprawie umie˛śnienia tuszy i wyre˛bów przy prowadzeniu selekcji
pośredniej w oparciu o grubość słoniny grzbietowej i powierzchnie˛ oka pole˛dwicy
STRESZCZENIE
Badania miały na celu wykazanie, jakiego poste˛pu w umie˛śnieniu tusz czy wyre˛bów należy
oczekiwać przy prowadzeniu selekcji pośredniej na grubość słoniny grzbietowej lub powierzchnie˛ oka
pole˛dwicy. W badaniach wykorzystano dane loszek ras wbp i pbz ocenianych w Stacjach Kontroli
Użytkowości Rzeźnej Trzody Chlewnej. Analiza statystyczna obejmowała szacowanie parametrów
genetycznych i fenotypowych dla cech rzeźnych, tj. grubości słoniny mierzonej: nad łopatka˛, na
grzbiecie, na krzyżu w punkcie I, II i III; masy mie˛sa (w karkówce, łopatce, szynce właściwej, golonce,
pole˛dwicy, boczku, żeberkach i półtuszy) i powierzchni oka pole˛dwicy. Wielkość poste˛pu hodowlanego
dla cech skorelowanych z cechami, na które prowadzono selekcje˛, określano za pomoca˛ wzoru podanego
przez Duńca. Rezultaty badań wskazuja˛ na możliwość uzyskania pewnych efektów w zwie˛kszeniu masy
mie˛sa w tuszy lub wyre˛bach przy prowadzeniu selekcji pośredniej. Wykazano, że najlepsze efekty
uzyskuje sie˛ przy prowadzeniu selekcji na grubość słoniny mierzonej na krzyżu w punkcie I. Selekcja
prowadzona w oparciu o ten pomiar daje poprawe˛ w umie˛śnieniu tuszy o około 54% w porównaniu do
28
B. Orzechowska
selekcji bezpośredniej, to znaczy takiej, w której uwzgle˛dniłoby sie˛ ła˛czna˛ mase˛ mie˛sa z wszystkich
wyre˛bów. Zastosowanie selekcji pośredniej w oparciu o ten pomiar prowadzi do zwie˛kszenia mie˛sa
w wyre˛bach, np. dla karkówki uzyska sie˛ poprawe˛ o 81% (w stosunku do selekcji bezpośredniej na mase˛
mie˛sa w karkówce).
LABORATORY AND SENSORY ASSESSMENT OF LAMB MUSCLE
TISSUE WITH REGARD TO MULTIDIMENSIONAL ANALYSIS
OF THE DISCRIMINATORY FUNCTION*
Paweł Paraponiak
Department of Technology, Ecology and Economics of Animal Production, National Research Institute
of Animal Production, 32-083 Balice n. Kraków, Poland
Abstract
This experiment was designed to determine physico-chemical quality and sensory parameters of meat
of pasture-reared purebred rams and their crosses with regard to multidimensional analysis of the
discriminatory function. Analysis was made on the musculus longissimus dorsi and musculus
semimembranosus obtained from 80 Polish Mountain Sheep (PMS) and Bergschaf (BF) ram lambs and
F1 and F2 crosses of PMS ewes × BF rams, 20 ram lambs per group (experiment — 10 animals,
replication — 10 animals). Muscles lowest in fat were characterized by the highest content of protein
(BF × PMS — fat content 1.82%, protein content 20.39%). Mean pH values of the analysed meat
ranged from 5.55 in F1 crosses to 5.69 in F2 crosses. Meat colour lightness was the highest (14.06%) in
BF × (BF × PMS) ram lambs and their meat had the lowest content of pigments (93.05 ppm). Water
holding capacity and heat losses of the meat were significantly differentiated by the experimental
group, but the results obtained were at the desired level. All meat sensory traits of the ram lambs
ranged from 4.29 to 4.79 points. The highest values of aroma intensity and quality were characteristic
of the meat of F1 rams (4.79 and 4.75 points, respectively). Meat tenderness was the highest in BF
× PMS rams (4.63 points) and the lowest in PMS lambs (4.30 points). High scores for palatability and
juiciness and the most favourable overall sensory score (multidimensional analysis of the discriminatory function) were given to the meat of BF × (BF × PMS) ram lambs. Chemical composition,
physical traits and the results of sensory assessment are all indicative of good technological suitability
and high quality of meat of the lambs reared under natural conditions.
Key words: ram lamb, meat, physico-chemical traits, sensory parameters
Reasons for the extensification of agricultural production in the European Union
should be sought in long-term overproduction in this sector. The scarcity of lamb meat
in the inner EU market and the growing consumer interest in organic products have
forced breeders to study lamb meat quality and health properties (Anderson, 2001).
* This work was conducted as part of NRIAP statutory activity, project no. 1435.3.
30
P. Paraponiak
Lamb meat as a dietetic product is valued for its unique taste. Efforts aimed at
improving the profitability of sheep production by crossing native breeds with
imported high-producing sheep must not detract from the quality and processing
suitability of the products (Ke˛dzior, 1995). The concurrent use in meat quality
evaluation of alternative statistical methods may make the results much easier to
interpret, showing similarities and dissimilarities between the analysed groups, as
determined by a certain group of qualitative traits rather than a single trait.
The aim of the present study was to determine physico-chemical quality and
sensory parameters of meat of purebred ram lambs and their crosses, reared on
pasture, with regard to multidimensional analysis of the discriminatory function.
The experiment was carried out in 1999 – 2002 under production conditions of
the Sheep Breeding Centre in Piorunka near Krynica, and the lambs were reared in
a uniform pasture feeding system. Slaughter at age of 200 days was carried out on
a total of 80 Polish Mountain Sheep (PMS) and Bergschaf (BF) ram lambs and F1
and F2 crosses of PMS ewes with BF rams, 20 ram lambs per group (experiment —
10 animals, replication — 10 animals; mean body weights of the ram lambs after
fasting were 30.9, 39.1, 33.7 and 35.6 kg, respectively).
Laboratory analysis of the meat was made on the musculus longissimus dorsi
and the musculus semimembranosus.
Analysis of chemical composition and physical properties of meat (musculus
longissimus dorsi) included: determination of water content by the drying method
(PN, 1973), protein content according to Kjeldahl (PN, 1975), fat content by Soxhlet
(PN, 1973) and calculations of relative meat quality parameters: the water to protein
ratio (W/P) and the fat to protein ratio (F/P). Measurements were made on pH after
24-hour cooling with Radelkis-type pH-meter (PN, 1977), total pigment content
following the method of Hornsey (Bunning and Hamm, 1970), colour lightness
using a three-colour colorimeter, water holding capacity according to Grau-Hamm
(forced drip; Tyszkiewicz, 1969), as well as thermal losses, i.e. the loss of meat
juices during preparation of the meat for sensory assessment (cooking the sample
until internal temperature of 85°C). Sensory assessment of meat aroma, juiciness,
tenderness and palatability (musculus semimembranosus, 5-point scale) was carried
out by a team of certified sensory sensitivity (Baryłko-Pikielna, 1975).
The results were analysed statistically with multivariate analysis of variance, taking
experimental group, year and replication as the differentiating factor. In all the analysed
traits no significant differences wee found between years and replications, while statistical
differences between the groups are described in tables. The F test value was compared
with the threshold value at significance level of α = 0.05 (when Fobl > Fgr, the zero
hypothesis about the equality of means in the experimental groups was rejected). Post-hoc
analysis was made with the HSD (honestly significant difference) test. Results of this
analysis are shown in tables using mean values followed by letters (Ostasiewicz, 2001).
31
Laboratory and sensory assessment of lamb muscle tissue
Additionally, the results of the organoleptic test of meat were analysed using
multidimensional analysis of the discriminatory function. Its first stage was
performed using the standard method, assuming a model in which the experimental
group was the grouping variable and measured sensory parameters of meat the
classifying variables. This was used to develop the configuration maps. In the
second stage, canonical analysis was performed to calculate mean values of two
canonical variables which illustrate the situation of individual experimental groups
in a two-coordinate (root) system (Jajuga, 1993).
All the calculations were performed with the ‘‘Anova/Manova” and ‘‘Discriminatory Analysis” procedures of the Statistica packet (Statistica for Windows,
StatSoft Inc., 1997).
Results
Basic chemical composition of lamb meat is given in Table 1. Water content of
the m. longissimus dorsi ranged from 76.58% in the Polish Mountain Sheep to
77.04% in Bergschaf and showed no significant differences. The lowest content of
dry matter (22.96%) was found in the m. longissimus dorsi muscle of BF rams. Fat
percentage varied from 1.82% in PMS ewe × BF ram crosses to 2.16% in F2 rams,
the difference being not significant. The highest protein percentage (20.39%) was
found in the meat of BF × PMS crosses and the lowest (19.81%) in the BF ×
(BF × PMS) group, with no statistically significant differences between the groups
(Table 1).
Table 1. Chemical composition of lamb meat
Trait
PMS
x̄
n
Water (%)
Dry matter (%)
Fat (%)
Protein (%)
Water to protein ratio (W/P)
Fat to protein ratio (FPB)
BF
SD
20
76.58
23.42
1.84
20.32
3.77
0.09
x̄
Groups
BF × PMS
SD
20
1.33
1.33
0.19
0.95
0.10
0.01
77.04
22.96
1.83
20.30
3.79
0.09
x̄
SD
20
1.20
1.20
0.42
0.89
0.11
0.01
76.68
23.32
1.82
20.39
3.76
0.09
BF (BF × PMS)
x̄
SD
20
0.69
0.69
0.50
0.89
0.08
0.01
76.59
23.41
2.16
19.81
3.76
0.11
0.63
0.63
0.52
0.85
0.09
0.01
Based on the calculated concentrations of water, fat and protein in the
m. longissimus dorsi, relative parameters of meat quality, i.e. the water to protein ratio
(W/P) and the fat to protein ratio (F/P; Table 1) were calculated. The highest W/P
ratio was found in the experimental group BF (3.79) and the lowest (3.76) in the
groups of F1 and F2 rams with 50 and 75% of BF ancestry (P > 0.05). The highest
F/P ratio (0.11) occurred in BF × (BF × PMS) F2 rams, compared to 0.09 in all the
other groups (P > 0.05; Table 1).
32
P. Paraponiak
Further laboratory analysis included measurements of the physico-chemical
properties of slaughter material (Table 2). In all the groups, they were made on the
m. longissimus dorsi except thermal losses (prior to sensory assessment), which
were determined on the m. semimembranosus.
The highest content of pigments (116.17 ppm) was found in the muscle of PMS
rams. It differed significantly in relation to the mean values of this trait, which were
determined in all the other experimental groups. The pigment content in Bergschaf
meat and in the meat of F1 and F2 BF crosses assumed similar values of 98.57, 96.64
and 93.05 ppm respectively (P > 0.05; Table 2).
Table 2. Physical properties of meat
Groups
Trait
PMS
x̄
n
Pigment content (ppm)
Colour lightness (%)
Water holding capacity (%)
Thermal loss (%)
pH
SD
x̄
20
116.17
11.50
26.07
41.85
5.57
b
a
b
b
a
BF × PMS
BF
SD
20
21.05
0.98
2.79
2.72
0.15
98.57
12.01
24.95
42.06
5.63
x̄
SD
20
BF (BF × PMS)
x̄
SD
20
a 23.94 96.64 a 15.72 93.05 a 13.68
a 1.51 12.11 a 1.22 14.06 b 1.87
b 4.22 25.42 b 3.05 19.11 a 1.84
b 3.29 42.39 b 3.27 37.62 a 5.82
b 0.16 5.55 a 0.09 5.69 b 0.09
a, b — arithmetic means marked with the same letter indicate classification into a uniform group based on post-hoc
analysis at α = 0.05.
The greatest colour lightness (14.06%) was found in the m. longissimus dorsi of
BF × (BF × PMS) F2 crosses, with statistically significant differences between the
above mean value and the respective values in the other experimental group.
Darkest colour (11.50%) of the m. longissimus dorsi appeared in the Polish
Mountain Sheep (Table 2). Water holding capacity, or the ability of meat to hold
water under pressure, ranged from 19.11% in BF × (BF × PMS) rams to 26.07% in
PMS rams (P ≤ 0.05). WHC values in the other groups were similar to those noted in
the PMS group and did not show significant differences. Thermal losses followed
a similar pattern to WHC values. Significantly the lowest values (37.62%) of this
trait were noted for the m. semitendinosus of F2 crosses of BF × (BF × PMS).
Thermal losses of meat in all the other experimental groups ranged from 41.85%
(PMS) to 42.39% (BF × PMS; P > 0.05). Lower pH24 was shown by the meat of F1
and PMS rams (5.55 and 5.57 respectively), and significantly higher values of this
measurement occurred in the groups BF and BF × (BF × PMS) F2 (5.63 and 5.69
respectively; Table 2).
After thermal treatment, the m. semitendinosus was subjected to sensory
assessment by a panel of judges, i.e. a group of experts with proven gustatory and
olfactory sensibility. The results are shown in Table 3.
33
Aroma intensity ranged from 4.54 points in the meat of F2 crosses of BF × (BF
× PMS) to 4.79 points in F1 crosses of BF × PMS. Differences between these
extreme values proved statistically significant. Aroma intensity of cooked meat,
originated from purebred rams of Polish Mountain Sheep and Bergschaf, was 4.65
and 4.72 points, respectively. These values showed no significant differences
between one another and compared to the values obtained in all the other
experimental groups. The lowest score for aroma quality was awarded to the Polish
Mountain Sheep (4.47 pts.) and slightly higher to F2 crosses (4.52 pts.). Significantly the most favourable aroma (4.75 pts.) was characteristic of the cooked meat of F1
rams with 50% BF ancestry (Table 3). Similar to aroma quality, meat tenderness
was the highest (4.63 pts.) in BF × PMS F1 rams. The lowest scores for tenderness
(4.30 pts.) were given to the meat of the Polish Mountain Sheep. The above extreme
values showed statistically significant differences. The meat of 75% Bergschaf and
25% PMS crosses was the most juicy (4.58 pts.), while the meat of F1 and
purebred BF rams was not much worse (4.46 and 4.40 pts., respectively). The
lowest score (4.31 pts.) for this trait was obtained by PMS rams, being significantly
different from the BF × (BF × PMS) group (Table 3).
Table 3. Sensory characteristics of lamb meat (points)
Groups
Trait
PMS
x̄
n
Aroma intensity
Aroma quality
Tenderness
Juiciness
Taste intensity
Taste quality
BF
SD
x̄
20
4.65
4.47
4.30
4.31
4.31
4.29
ab
a
a
a
a
a
SD
BF × PMS
x̄
SD
BF (BF × PMS)
x̄
SD
20
20
20
0.20
0.25
0.39
0.33
0.31
0.33
4.72
4.62
4.43
4.40
4.44
4.43
ab
ab
ab
ab
ab
ab
0.28
0.35
0.37
0.36
0.41
0.45
4.79
4.75
4.63
4.46
4.49
4.50
b
b
b
ab
ab
ab
0.15
0.19
0.28
0.26
0.32
0.32
4.54
4.52
4.53
4.58
4.54
4.54
a
a
ab
b
b
b
0.21
0.14
0.23
0.21
0.20
0.21
a, b — arithmetic means marked with the same letter indicate classification into a uniform group based on post-hoc
analysis at α = 0.05.
Of all the sensory traits of lamb meat, by far the most important are taste
intensity and quality. The lowest score for palatability (4.31 pts.) was given to the
meat of PMS rams and the highest (4.54 pts.) to the meat of F2 crosses of BF × (BF
× PMS), with statistically significant differences between these groups. The results
of taste quality assumed similar values to the results of taste intensity. In this case
too, the meat of PMS rams showed significantly poorer taste quality (4.29 pts.)
compared to F2 crosses (4.54 pts.). Taste quality scores for the meat of BF rams and
BF × PMS crosses (4.43 and 4.50 pts., respectively) did not show any significant
differences in relation to the other experimental groups (Table 3).
34
P. Paraponiak
PMS
BF
BF × PMS
BF × (BF × PMS)
Figure 1. Configuration of sensory traits of meat of the experimental lambs
Figure 1 shows the configuration of meat of experimental rams based on its
sensory characteristics. The perception map shown projects the situation of all the
rams, determined in 6-dimensional space by the scores of all other meat sensory
traits. Distances between individual points on the surface reflect their distances in
the 6-dimensional space. It is stated that the points in space corresponding to the F2
crosses are situated in a different region (moved towards negative values of root 1)
from the points representing purebred and F1 rams. Points that determine BF × PMS
rams tend to be situated at the level of negative values of the second root axis (Y
axis), points representing PMS rams are mostly located in the region of positive
values of this axis, while the distribution of respective values of purebred BF rams,
despite a relatively large scattering, resembles most the symmetric distribution in
relation to the X axis. Despite some differences in location, the above three areas
largely overlap.
Discussion
In this study no significant differences were found between the experimental
groups in basic chemical composition of meat, expressed in the content of water, fat
and protein. Many studies investigating the effect of breed and crossbreeding on
chemical composition of meat pointed to both stable (Ke˛dzior, 1991; PieniakLendzion et al., 1996; Kaczor et al., 2000; Niedziółka et al., 2000) and varying
content of protein (Gruszecki and Borkowska, 1996; Lipecka et al., 2000;
Roborzyński et al., 2000).
Genetic propensities for overfat lamb meat vary according to breed, the
crossbreeding scheme and slaughter age of the lambs. In muscle with the lowest fat
content the highest percentage of protein was found (BF × PMS — fat content
35
1.82%, protein content 20.39%). The highest fat content accompanied by the lowest
protein content was noted in the meat of F2 crosses of BF × (BF × PMS) (2.16 and
19.81%, respectively).
The content of protein in the meat of PMS, BF and F1 BF × PMS crosses, noted
in the present study, was higher than the results obtained by Ciuruś et al. (1996) in
the experiment conducted with the meat of two-breed crosses (Friesian rams
× Polish Mountain Sheep) and three-breed crosses (Friesian rams × (Blackheaded
Meat rams × Polish Mountain Sheep) (20.10 and 19.99% respectively). The content
of protein in the meat of BF × (BF × PMS) rams was lower than the values obtained
in the present work.
The relatively low content of intermuscular fat, ascertained in the present
experiment, is in agreement with Roborzyński et al. (2000), although the findings of
some authors point to higher overfatness of lamb meat (Roborzyński, 1992;
Pieniak-Lendzion, 1994; Krełowska-Kułas et al., 1995 b; Drożdż and Ciuruś, 1996)
than in the present study.
The water to protein ratio ranged from 3.76 to 3.79 and was close to the results
reported by Ke˛dzior (1995). Feder number ranges from 3.3 to 3.9 in adult slaughter
animals (Prost, 1985) and equals 4.1 in very young animals (Pieniak-Lendzion et
al., 2000). The level of this trait in the present study allows a conclusion that the
analysed meat came from animals of normal somatic maturity.
The F/P index ranged from 0.09 to 0.11, and its level was reflected in the stable
content of fat and protein in the muscles of the experimental rams. Covington et al.
(1970) showed that the lower degree of physiological maturity of muscles is related
to their high content of water and low content of intermuscular fat.
A very good and objective indicator of meat maturity and its processing
suitability is pH (Dowierciał et al., 1976), especially pH measured 24 h postmortem
(Tyszkiewicz, 1972). The mean pH values obtained here, despite the statistically
significant differences, were typical of normal meat (5.3-5.8; Hoffman, 1987).
These results are close to the values reported by other authors (Freudenreich et al.,
1985; Borys et al., 1986; Ke˛dzior and Paja˛k, 1994).
It was observed that in BF × (BF × PMS) F2 rams, greater acidity was
accompanied by slightly higher water holding capacity and lower heat losses of meat.
A similar relationship for the meat of F2 crosses [Blackheaded Meat rams × (Friesian
rams × Polish Mountain Sheep)] was also shown by Krełowska-Kułas et al. (1995 a).
Muscle tissue structure is above all the function of pH value and is most
important to meat colour. Bright colour is related to the meat of low pH, while dark
colour is related to the high pH values. The high pH value causes changes to the
muscle structure and inhibits oxygen permeation, thus increasing the content of
myoglobin. As a result of these processes, meat has a darker colour (Gajdosik et al.,
1993; Jedlicka, 1988). More relevant to this notion of colour is the effect of muscle
pigments (pigment content) than the general impression of meat colour (colour
lightness) (Hamm, 1972). The above observations are not reflected in the present
results, and the reason for the differences can be sought in the relatively small
differences in pH value and pigment content.
36
P. Paraponiak
The brightest colour was achieved by the meat of BF × (BF × PMS) F2 rams,
and the differences shown in relation to the other groups were statistically
significant. Considerable differences in the meat colour of PMS rams and F1 crosses
of Suffolk × PMS were also found by Drożdż and Ciuruś (1996). It is suggested that
the brighter colour of meat in the group of F2 crosses of BF × (BF × PMS) resulted
from the higher content of intermuscular fat in this meat. This relationship was also
noticed by Ke˛dzior and Paja˛k (1994).
The meat of rams from individual groups differed significantly in terms of water
holding capacity (ability to hold water under pressure) and thermal losses taking
place during the preparation of meat for sensory assessment, and the values
obtained were at the desired level (25 and 40% respectively; Hamm, 1972). The
meat of F2 crosses was characterized by a significantly higher water holding
capacity and thus it was more suitable for processing. Good water holding capacity is
usually linked to high scores for juiciness (Pezacki, 1981), as confirmed by the
present results. Most of the studies failed to show the effect of crossbreeding on
water holding capacity (Jackowska et al., 1984; Lirette et al., 1984; Ke˛dzior, 1991),
although some authors reported such differences (Korzeniowski et al., 1986;
Ke˛dzior et al., 1999).
Based on the results of physico-chemical analysis it is concluded that the meat
of lambs from all experimental groups had the desired level of the parameters
analysed, especially the ultimate pH of meat. As a result, no adverse signs of
accelerated or slowed glycolysis in meat (DFD or PSE meat) were found (Pezacki,
1981).
Results of sensory meat analysis exceeded 4 points in all the groups and were
higher than those in the study of Pieniak-Lendzion et al. (2000), in which rams were
fed a complete mixture CJ. This may be indicative of the beneficial effect of pasture
feeding on sensory quality of meat, although this conclusion is not obvious from the
available literature.
Aroma, which is often considered the most important determinant of lamb meat
palatability, may be responsible for its low consumption, resulting from lack of
acceptance (Ke˛dzior, 1995). The highest values of aroma intensity and quality were
characteristic of the meat of BF × PMS F1 rams. Meat aroma in all the experimental
rams was considered favourable, clearly tangible with flavour characteristics of
cooked meat.
The meat of BF × PMS rams was considered the most tender and that of PMS
lambs obtained the lowest scores. The high tenderness scores in all the experimental
groups may be associated with pasture feeding of the rams. Doroszewska et al.
(1968) showed that 11-month-old wethers fed the same green forages had more
tender meat than wethers fed dry feeds supplemented with green forage. However,
most of the studies failed to show the effect of breed and crossbreeding on meat
tenderness (Freudenreich et al., 1985; Borys et al., 1986; Ke˛dzior et al., 1999)
except the experiment of Hawkins et al. (1985).
Juiciness next to tenderness is the main characteristic of meat structure (Prost,
1985). Some authors reported a relationship between high juiciness and fat content
37
of meat (Dyren and Marchello, 1970; Cross et al., 1972). The results of the present
study did show this relationship: the relatively high fat content in meat of BF × (BF
× PMS) F2 crosses was accompanied by the highest juiciness. What is more this
meat, in addition to the highest scores for juiciness and the highest pH values, was
characterized by significantly highest water holding capacity. This relationship is
reflected in the results of other authors (Gajdosik et al., 1993), who demonstrated
that higher pH value and thus higher water holding capacity increase the juiciness
of meat.
Taste intensity and quality are related to other sensory traits of lamb meat,
especially aroma quality. If desirable, it has a major effect on meat palatability
(Ke˛dzior, 1995). This relationship was not confirmed by the results of the present
study, in which the highest palatability scores given to the meat of F2 crosses were
accompanied by the lowest scores for aroma intensity and quality.
Discriminatory analysis, based on all sensory traits of the meat of experimental
lambs, showed the greatest differences between F2 rams and all the other
experimental groups. It can therefore be concluded that the meat of these crossbreds
differed in sensory quality. This finding is reflected in Table 3, in which the meat of
the above rams, despite the lowest scores for aroma intensity and quality, was
characterized by the highest scores for juiciness, especially taste intensity and
quality. This goes to show that the least favourable meat aroma scores of F2 rams
did not affect its highest palatability, and as a result its more beneficial overall
sensory score (analysis of the discriminatory function).
In summing up the sensory scores of the meat of experimental lambs, it is
concluded that all the scores (4.29 to 4.79 points) were favourable, indicating the
high eating value of meat.
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Accepted for printing 31 V 2004
PAWEŁ PARAPONIAK
Ocena laboratoryjna i sensoryczna tkanki mie˛śniowej jagnia˛t z uwzgle˛dnieniem
wielowymiarowej analizy funkcji dyskryminacyjnej
STRESZCZENIE
Celem badań było określenie jakości fizykochemicznej oraz parametrów sensorycznych mie˛sa
tryczków czystych ras a także ich mieszańców, odchowywanych na pastwisku, z uwzgle˛dnieniem
wielowymiarowej analizy funkcji dyskryminacyjnej.
Badania przeprowadzono na mie˛śniu najdłuższym grzbietu (musculus longissimus dorsi) i półbłoniastym uda (musculus semimembranosus) pozyskanych od 80 tryczków ras: polska owca górska
(pog) i Bergschaf (BF) oraz mieszańców F1 i F2 pochodza˛cych z krzyżowania maciorek pog z trykami
BF, po 20 tryczków w każdej grupie (doświadczenie — 10 sztuk, powtórzenie — 10 sztuk).
Mie˛śnie o najmniejszej zawartości tłuszczu charakteryzowały sie˛ najwie˛ksza˛ zawartościa˛ białka
(BF × pog — zawartość tłuszczu — 1,82%, zawartość białka — 20,39%). Średnie wartości pH badanego
mie˛sa wahały sie˛ od 5,55 w grupie mieszańców F1 do 5,69 w grupie mieszańców pokolenia F2. Jasność
barwy mie˛sa tryczków osia˛gne˛ła najwyższa˛ wartość w grupie BF × (BF × pog) — 14,06%, przy
równocześnie najmniejszej zawartości barwników — 93,05 ppm. W grupie doświadczalnej w sposób
istotny zróżnicowana była wodochłonność i ubytki cieplne mie˛sa, a uzyskane wartości kształtowały sie˛
na poża˛danym poziomie. Wartości wszystkich cech sensorycznych mie˛sa tryczków doświadczalnych
mieściły sie˛ w granicach od 4,29 do 4,79 punktu. Najwyższymi wartościami nate˛żenia i jakości zapachu
odznaczało sie˛ mie˛so tryczków pokolenia F1 (odpowiednio: 4,79 i 4,75 pkt.). Za najbardziej kruche
uznano mie˛so tryczków BF × pog (4,63 pkt.), a najniższe oceny uzyskało mie˛so jagnia˛t pog (4,30 pkt.).
Wysokie oceny punktowe za smakowitość i soczystość oraz najkorzystniejsza˛ ogólna˛ ocena˛ sensoryczna˛
40
P. Paraponiak
(wielowymiarowa analiza funkcji dyskryminacyjnej) uzyskało mie˛so tryczków mieszańców (BF ×
(BF × pog).
Zarówno skład chemiczny, cechy fizyczne, jak i wyniki oceny sensorycznej świadcza˛ o dobrej
przydatności technologicznej i wysokiej jakości mie˛sa jagnia˛t utrzymywanych w warunkach naturalnych.
QUALITY OF TABLE EGGS AS INFLUENCED BY LAYING HEN
ORIGIN*
L i d i a C z a j a 1, K r y s t i a n D z i a d e k 2, E w a G o r n o w i c z 2
1
Poultry Farm ZADROB Zakrzewo Ltd., Poznańska 11,
62-069 Pale˛dzie, Poland
2
Abstract
Eggs from commercial layers at 50 to 52 weeks of age were used in the experiment. The birds were
grown in identical management conditions and fed with the same compound feeds. The purpose of
the study was to determine differences in the physico-chemical traits of table eggs produced by
various commercial strains and being offered on the Polish market. The greatest weight of eggs
(69.26 g) and yolks (22.11 g) was noted in Rosa 1 hens. Eggs from Messa 445 demonstrated the
highest albumen height (6.72 mm), Haugh units (78.93) and the highest albumen content of the egg
(62.27%), whereas N-11 × P-11 layers produced eggs with the darkest yolk colour (10.81). The
chemical analysis of egg yolk revealed that Rosa 1 eggs had the lowest water content (48.88%),
mean lipid content (32.53%) and protein content. Eggs from Messa 443 and Astra W hens were
found to have yolks with higher lipid content (32.68% and 32.63%, respectively). The experimental
findings revealed statistically significant differences (P ≤ 0.05) in the physico-chemical traits of
table eggs from the examined commercial laying hens.
Key words: chicken, genotype, physical traits, chemical composition
Hen eggs have long been considered as a valuable food product (Pingel and
Jeroch, 1997). Numerous studies showed that the egg content is dependent not only
on the diet and environmental conditions, but also on the genetic factors (Cherian
and Sim, 1995; Rapacz et al., 1997; Roberts and Nolan, 1997; Nielsen, 1998;
Polowczyk et al., 1998; Besbes and Gibson, 1999; Rapacz et al., 2000; We˛żyk et al.,
2000; Hartmann et al., 2003). Egg quality traits demonstrate marked variability
influenced by hen genotype and age, physiological and physical conditions, and
environmental factors.
* This work was conducted as part of the research supported by the Ministry of Agriculture
and Rural Development, project no. 5101.3.
42
L. Czaja et al.
Consumers of animal food products have considerably changed their attitude to
quality over the last few years. For that reason the physical traits of table eggs such
as egg weight, albumen height, yolk colour, thickness and density of the shell as
well as the chemical composition of egg content become more and more important
(Panheleux et al., 1999; Dziadek et al., 2003; Hartmann et al., 2002; Hocking et al.,
2003). Therefore the present study was undertaken to assess the effect of bird
genotype on physico-chemical traits of table eggs.
Table eggs used in the study were taken from nine lines of commercial laying
hens: Rosa 1, Rosa 2, Messa 443, Messa 445, Astra D, Astra N, Astra W, Astra W-2
and N-11 × P-11 hybrid. Baby chicks of layers were hatched and raised on litter
under the same management conditions of a poultry house (36 boxes) at the
Chicken Testing Station in Wroniawy. Throughout the study they were kept under
the same management conditions: temperature 20°C, light programme — 16 h/day,
intensity — 3.5 W/m2 and fed ad libitum with the same diet tailored to particular
age and laying period. From 50 to 52 weeks of age, i.e. during the period when eggs
were taken for the experiment, the birds were fed with the same compound feed, the
ingredients of which are presented in Table 1.
Table 1. Composition of the compound feed (%)
Ingredients
Content (%)
Wheat
Maize
Barley
Triticale
Wheat bran
Rapeseed oil
Extracted soybean meal 48%
Meat meal 56%
Fodder chalk
Calcium-magnesium phosphate
Mineral-vitamin premix
29.00
15.00
10.00
10.00
5.00
1.00
16.00
3.20
7.40
1.40
2.00
Crude protein
Metabolizable energy (MJ/kg)
Ca
P
17.40
10.82
3.50
0.37
Physical and chemical traits were examined in 90 eggs selected at random from
each experimental group of birds. The eggs (30 eggs per group of layers) were
collected three times at weekly intervals. The analyses were conducted with eggs
laid at 50, 51 and 52 weeks of birds’ age. Egg Quality Micro Version 3.1 instrument
Quality of table eggs as influenced by hen origin
43
was used to determine the weight of whole egg, albumen, yolk and shell as well as
albumen height, yolk and shell colour. The egg content was used thereafter for the
chemical analysis of egg constituents. The content of protein was determined according to
the Kjeldahl method (PN-75/A-04048), dry matter and fat according to PN-A-86509
standard, and ash content according to the method described by Krełowska-Kułas (1993).
The results were subjected to statistical analysis. Analysis of variance was used
to confirm the differences in the egg traits among the groups of laying hens
examined. On the other hand, the simple correlation analysis was used to estimate
the relationship among the examined traits of eggs within each group of birds.
Results
The analytical data concerning table egg quality in all studied groups of layers
at the age of 50 to 52 weeks are given in Tables 2 to 7.
Eggs from Rosa 1 layers demonstrated the highest weight (69.26 g) and were
heaviest among eggs from all groups of commercial layers in this study. The highest
albumen height (6.72 mm) and Haugh units (78.93) were found in Messa 445 birds. On
the other hand, eggs laid by Rosa 2 hens had significantly (P ≤ 0.05) lowest albumen
height (6.18 mm), while Astra W eggs the lowest Haugh units (74.00). The lowest
variation of the three analysed quality traits was observed in the eggs from Rosa 1 hens.
The greatest shell weight and darkest shell colour were noted in the eggs laid by
Messa 445 hens (Table 3), whereas Rosa 2 produced eggs with the lightest shell
colour (54.29), the lowest shell weight (5.66 g) and thickness (331.02 µm) as well
as the greatest shell density (77.31 mg/cm2).
As can be seen from Table 4, which gives the percentage of egg components,
the eggs from Rosa 2 had the highest content of yolk (32.05%) accompanied by the
lowest albumen (59.44%) and shell content (8.51%). Unlike the Rosa 2 eggs, the
Messa 445 eggs demonstrated significantly lowest (P ≤ 0.05) yolk content (28.83%)
and at the same time the highest albumen content (62.27). The greatest shell weight
(9.12%) was noted in the eggs from Astra N layers.
A relatively high yolk weight was observed in the examined eggs, ranging from
19.17 g (Messa 445) to 22.11 g (Rosa 1). The eggs also demonstrated relatively
dark yellow colour within the range from 10.27 (Astra D) to 10.81 of Roche units
(N-11 × P-11 hybrids). The eggs from Messa 445 and Astra W birds exhibited
significantly (P ≤ 0.05) lowest yolk weight in comparison with eggs from the other
hen groups except the yolks of Messa 443 eggs.
The highest water content was observed in the albumen of eggs laid by N-11
× P-11 hybrids (88.60%), but the water content of egg albumen was found to be at
a relatively similar level, i.e. from 87.75% to 88.60% in the eggs from Astra W-2
and N-11 × P-11 hybrid hens, respectively. The ash content of the eggs was similar
and ranged from 2.70% (Messa 445) to 3.00% (Astra D). In protein content of the
albumen, significant differences were noted in eggs laid by Rosa 1 (9.39%), Messa
445 (9.22%) and Astra N (9.27%) birds.
Table 2. Quality traits of eggs from various groups of commercial layers at 50 to 52 weeks of age
Group
Egg weight (g)
x̄
v
Albumen height (mm)
x̄
v
Haugh units
x̄
v
Rosa 1
Rosa 2
Messa 443
Messa 445
Astra D
69.26 a
6.4
66.59 b
7.3
65.30 c
7.1
66.64 b
6.8
66.15 b
7.2
6.39 ab
15.8
6.18 b
21.0
6.30 b
22.2
6.72 a
17.6
75.95 b
10.0
74.74 b
14.0
76.12 b
13.2
78.93 a
10.5
Astra N
N-11 × P-11
Astra W
Astra W-2
65.14 c
7.0
64.42 d
6.5
65.23 c
6.7
66.89 b
6.6
6.41 ab
17.2
6.51 ab
19.1
5.98 b
21.3
6.24 b
16.7
6.69 a
20.6
77.00 ab
11.0
77.91 ab
11.3
74.00 b
13.7
76.13 ab
10.0
78.48 a
12.7
a, b, c, d — various superscripts in columns denote statistically significant difference at P ≤ 0.05.
Table 3. Shell quality of eggs from various groups of commercial layers at 50 to 52 weeks of age
Group
Shell weight (g)
x̄
v
Colour
x̄
v
Shell thickness (µm)
x̄
v
Shell density (mg/cm2)
x̄
v
Rosa 1
5.89 a
9.9
52.43 ab
11.8
Rosa 2
5.66 b
8.3
Messa 443
5.80 ab
8.6
Messa 445
5.93 a
8.7
Astra D
Astra N
Astra W
Astra W-2
5.84 ab
8.8
5.93 a
9.5
5.71 b
9.2
5.90 a
9.1
N-11 × P-11
5.78 ab
9.4
54.29 a
12.5
50.92 b
14.9
41.74 d
13.4
47.58 c
16.0
43.14 d
13.3
43.38 d
14.7
42.39 d
13.5
47.83 c
14.5
338.61 c
8.0
331.02 d
6.6
343.51 bc
7.1
348.23 ab
6.4
344.19 b
7.5
358.38 a
7.7
334.50 d
8.4
354.29 a
8.0
335.47 d
7.7
73.69 c
8.8
72.69 c
6.2
75.46 b
6.9
76.09 ab
7.0
76.09 b
7.0
77.31 a
7.7
For explanation of significance see Table 2.
77.31 bc
7.7
76.83 ab
8.3
73.94 c
7.6
Table 4. Percentage of yolk, albumen and shell in table eggs from various groups of commercial layers at 50 to 52 weeks of age
Group
% of yolk
x̄
v
% of albumen
x̄
v
% of shell
x̄
v
Astra D
Astra N
Astra W
Astra W-2
N-11 × P-11
28.83 c
6.7
30.89 b
7.2
31.18 b
6.7
30.73 b
8.1
30.60 b
6.0
31.54 b
5.3
60.58 b
3.4
62.27 a
3.3
60.27 bc
3.9
59.70 c
3.7
60.40 b
4.0
60.34 bc
305
59.82 c
3.0
8.91 b
7.1
8.91 b
7.1
8.84 bc
7.2
9.12 a
7.8
8.87 b
8.0
9.06 a
8.7
Rosa 1
Rosa 2
Messa 443
Messa 445
31.97 a
7.6
32.05 a
6.7
31.52 b
6.5
59.51 c
4.4
59.44 c
3.7
8.51 d
9.0
8.51 d
6.4
8.64 cd
7.5
Table 5. Yolk traits in table eggs from various groups of commercial layers at 50 to 52 weeks of age
Group
Yolk weight
x̄
v
Yolk colour (La Roche)
x̄
v
Rosa 1
Rosa 2
Messa 443
Messa 445
Astra D
Astra N
Astra W
Astra W-2
N-11 × P-11
22.11 a
8.4
21.30 b
7.8
19.90 de
8.0
19.17 e
7.3
20.39 d
7.8
20.26 d
6.8
19.76 e
8.6
19.93 d
7.3
21.08 c
7.6
10.61 a
7.6
10.52 a
6.4
10.43 ab
6.1
10.57 a
6.8
10.27 b
7.7
10.71 a
5.4
10.30 b
8.8
10.38 b
7.7
10.81 a
6.5
Table 6. Basic chemical composition of the albumen in table eggs from various groups of commercial layers at 50 to 52 weeks of age
Group
% of water
x̄
v
% of ash
x̄
v
% of albumen
x̄
v
Rosa 1
87.91
0.7
Rosa 2
88.18
1.3
Messa 443
88.28
0.2
Messa 445
88.07
1.2
Astra D
Astra N
Astra W
88.30
0.8
88.08
1.0
88.36
1.3
Astra W-2
87.75
0.9
N-11 × P-11
88.60
1.3
2.86 ab
14.8
2.86 ab
6.9
2.86 ab
9.4
2.70 b
5.2
3.00 a
2.9
2.84 ab
6.0
2.80 a
6.3
2.81 ab
3.5
2.82 ab
4.7
9.39 a
6.2
8.81 c
5.3
9.13 ab
3.3
9.22 a
2.8
9.00 b
5.7
9.27 a
4.2
8.76 c
3.7
9.03 ab
3.4
8.75 c
4.1
Table 7. Basic chemical composition of the yolk in table eggs from various groups of commercial layers at 50 to 52 weeks of age
Group
% of
x̄
v
% of
x̄
v
% of
x̄
v
% of
x̄
v
Rosa 1
Rosa 2
Messa 443
Messa 445
Astra D
Astra N
Astra W
Astra W-2
N-11 × P-11
water
48.88 b
1.6
49.58 a
0.7
49.10 ab
1.6
49.38 ab
1.7
49.26 ab
1.6
49.18 ab
1.1
49.18 ab
1.7
49.18 ab
0.8
49.46 ab
0.6
32.53 ab
1.5
32.46 ab
0.7
32.68 a
1.0
32.51 ab
1.1
32.57 ab
0.9
32.52 ab
0.6
32.63 a
0.8
32.28 b
1.3
32.41 ab
0.5
4.01 b
5.9
3.92 b
13.1
4.05 ab
8.5
3.94 b
3.7
4.29 a
3.0
4.00 b
3.5
4.01 b
8.6
13.37 ab
2.2
13.24 ab
2.9
13.31 ab
2.6
13.22 ab
2.8
13.34 ab
3.2
13.20 ab
2.7
13.14 b
1.7
lipids
ash
4.11 ab
3.0
4.01 b
3.9
albumen
13.50 a
2.5
13.23 ab
1.8
Table 8. Correlation among selected physical traits of table eggs from various groups of commercial layers at 50 to 52 weeks of age
Traits
Group
Rosa 1
Rosa 2
Egg weight x
% of yolk
% of shell
% of albumen
shell thickness (total)
yolk colour
albumen height
− 0.307*
− 0.244*
0.360*
0.167
0.026
0.138
− 0.377*
− 0.259*
0.426*
0.183
0.050
0.191
− 0.314*
− 0.273*
0.390*
0.134
− 0.013
0.229*
% of yolk x
shell weight
albumen height
Haugh units
shell density
− 0.144
− 0.034
0.021
− 0.016
− 0.315*
− 0.285*
− 0.238*
0.128
% of shell x
albumen weight
% of albumen
shell thickness
shell colour
shell density
− 0.348*
− 0.350*
0.506*
0.111
0.970*
% of albumen x
albumen height
Haugh units
shell density
shell thickness (total)
0.023
− 0.040
− 0.273*
− 0.171
* — correlations statistically significant (P = 0.05).
Messa 443 Messa 445
Astra W-2 N-11 × P-11
Astra D
Astra N
Astra W
Total
− 0.401*
− 0.222*
0.444*
0.121
− 0.036
− 0.052
− 0.424*
− 0.228*
0.473*
0.211*
0.068
0.020
− 0.501*
− 0.183
0.523*
0.112
0.200
0.242*
− 0.341*
− 0.210*
0.410*
0.116
0.273*
0.059
− 0.320*
− 0.352*
0.409*
− 0.053
0.146
0.120
− 0.141*
− 0.117
0.176
0.230*
0.088
0.126
− 0.351*
− 0.231*
0.404*
0.134*
0.089*
0.123*
− 0.305*
0.240*
− 0.190
− 0.173
− 0.276*
− 0.129
− 0.068
− 0.081
− 0.344*
− 0.130
− 0.056
− 0.152
− 0.314*
− 0.359*
− 0.262*
− 0.086
− 0.437*
− 0.114
− 0.048
− 0.317*
− 0.069
− 0.148
− 0.081
0.100
− 0.150
− 0.232*
− 0.231*
− 0.095
− 0.263*
0.187*
− 0.129*
− 0.106*
− 0.304*
− 0.264*
0.744*
− 0.009
− 0.924*
− 0.309*
− 0.244*
0.694*
− 0.126
0.946*
− 0.303*
− 0.355*
0.750*
− 0.101
0.948*
− 0.278*
− 0.273*
0.774*
− 0.150
0.944*
− 0.280*
− 0.364*
0.863*
− 0.241*
0.952*
− 0.181
− 0.077
0.458*
0.006
0.961*
− 0.479*
− 0.529*
0.835*
− 0.204
0.976*
− 0.217*
− 0.317*
0.502*
− 0.208*
0.953*
− 0.301*
− 0.308*
0.674*
− 0.102*
0.954*
0.283*
0.223*
− 0.099
− 0.039
0.281*
0.213*
− 0.126
− 0.032
0.116
0.052
− 0.216*
− 0.121
0.176
0.093
− 0.122
− 0.047
0.375*
0.271*
− 0.207
− 0.195
0.145*
0.067
0.041
0.005
0.134
0.048
− 0.450*
− 0.310*
0.295*
0.285*
− 0.254*
− 0.103
0.204*
0.136*
− 0.192*
− 0.114
Table 9. Correlations among selected chemical traits in the eggs from birds of various origin
Traits
Group
Rosa 1
Rosa 2
Albumen (yolk) ×
protein (albumen)
ash (albumen)
lipids (yolk)
water (yolk)
0.701*
− 0.167
− 0.755*
− 0.118
0.710*
− 0.715*
− 0.020
− 0.588
0.920*
0.162
− 0.731*
− 0.356
Lipids (yolk) ×
protein (albumen)
water (albumen)
ash (yolk)
− 0.691*
0.391
0.260
− 0.396
0.484
− 0.741*
− 0.736*
0.835*
0.094
For explanation see Table 8.
Messa 443 Messa 445
Astra W-2 N-11 × P-11
Total
score
Astra D
Astra N
Astra W
0.909*
0.244
− 0.502
− 0.317
0.898*
− 0.259
− 0.879*
− 0.537
0.615
− 0.160
− 0.951*
− 0.760*
0.789*
0.043
− 0.841*
− 0.492
0.780*
− 0.109
− 0.738*
− 0.285
0.877*
0.837*
− 0.858*
− 0.822*
0.789*
− 0.026
− 0.726*
− 0.454
− 0.616
0.530
− 0.707*
− 0.758*
0.492
0.794*
− 0.457
− 0.120
− 0.277
− 0.849*
0.448
0.141
− 0.772*
0.605
− 0.250
− 0.834*
0.396
0.533
− 0.668*
0.443*
− 0.043
49
Water content of the yolk was relatively equalized from 48.88% in Rosa 1 to
49.58% in Rosa 2 eggs. A statistically significant difference was noted between
Rosa 1 and Rosa 2 birds only.
Taking into account the yolk lipid content a statistically significant difference
was observed between Messa 443 (32.68%) and Astra W (32.63%) eggs. In the
other groups of layers, the lipid content varied from 32.28% to 32.57% in the eggs
from Astra W-2 and Astra D, respectively. Protein content of the yolk ranged from
13.14% in Astra W to 13.50% in Astra W-2 birds, and the noted difference was
statistically significant at P ≤ 0.05. In the other groups of layers that content was
equal. In the case of yolk ash content, the differences were observed among Astra
D eggs and the eggs from Rosa 1, Rosa 2, Messa 445, Astra N, Astra W and N-11
× P-11 birds.
Variation coefficient was found to range from 6.4% to 15%. Great variation was
noted in albumen height and the difference reached 6.4%. The coefficient of
variation for the water percentage in the albumen was below 2%. In the case of yolk
chemical composition, the coefficient of variation did not exceed 3.5%, except the
ash content which reached even 13.1%.
Simple correlation coefficients among certain physical traits of eggs laid by
hens of different origin are presented in Table 8. The correlation coefficients were
determined between: egg weight, shell thickness, percentage content of yolk,
albumen and shell on the one hand, and certain physical quality traits on the other.
In the total evaluation comprising all groups of birds studied, the correlation
coefficient ranged from –0.351 to 0.954 and was statistically non-significant in the
case of the albumen percentage and shell thickness only.
Correlations among certain egg physical traits of hens from various commercial
strains are given in Table 9. All correlation coefficients were found within the range
from -0.879 to 0.920. Mention should be made of the high relationship between
yolk protein content and albumen protein content in the eggs.
Discussion
The present results demonstrate differences in the physico-chemical traits of
eggs from the analysed commercial flocks of layers.
Hocking et al. (2003) examined hen eggs of various origin and observed
differences in egg properties. Eggs from a traditional strain demonstrated higher
yolk weight (19.3 g), darker yellow colour (8.6 units on the La Roche scale) and
lower Haugh units (74.2). On the other hand, eggs produced by a commercial hen
strain had significantly higher weight (65.1 g), greater albumen content (37.2 g) and
shell weight (6.16 g). However, the authors found no statistically significant
differences in the egg shell colour between the two examined hen strains. Similarly
Czekalski et al. (2000) confirmed the effect of genotype on egg quality traits.
Lenartowicz (1998) reported that heaviest eggs were produced by Messa
245 layers. In our study, heaviest eggs were laid by Rosa 1 birds, whose eggs also
50
L. Czaja et al.
had the largest yolks (22.11 g) and the lowest shell percentage. The lowest yolk
weight (13.03 g) was noted by Lenartowicz (1998) in Astra S eggs. Our study
revealed that the lightest eggs were laid by Astra W strain. Moreover, those eggs
demonstrated the lowest albumen height (5.98 mm) and Haugh units (74.00). The
latter trait can be regarded as a poor quality feature of Astra W eggs since Haugh
units are used nowadays as the main quality attribute in the international egg trade.
In this study, the greatest albumen height and Haugh units were found in Messa 445
eggs as reported by Dziadek et al. (2003) too.
As far as egg shell properties are concerned, the Astra N eggs demonstrated not
only the greatest shell weight (5.93), but also the highest coefficient of density
(77.31 mg/cm2), shell thickness (358.26 µm) and the greatest shell percentage in the
egg (9.12%). Similarly as reported by Dziadek et al. (2003), in this study Messa 445
eggs also exhibited the greatest shell weight unlike the Rosa 2 eggs which
demonstrated the lowest shell weight (5.66), density (72.69 mg/cm2) and thickness
(331.02 µm) followed by the lowest percentage of albumen (59.44%) and shell
(8.51%) in the egg. On the other hand, Rosa 2 eggs showed the lightest shell colour
and the highest yolk content in the egg (32.05%).
The experimental findings showed a relatively large variation of yolk weight.
Moreover, with the increase of yolk weight a tendency was observed to increased
yolk colour intensity.
Trziszka (2000) reported that in the freshly laid eggs the albumen contained
88.00% water and 11% protein. In this study the water content in the albumen
ranged from 87.75% (Astra W-2) to 88.60% (N-11 × P-11 strain), whereas that of
protein from 8.75% (Rosa 1, Rosa 2, Messa 445) to 9.39% in Astra D. A statistically significant difference in the ash content of egg albumen was noted only between
Astra D eggs on the one hand, and Messa 445 and Astra W eggs on the other.
The chemical composition of egg yolk was noted to be relatively equalized. The
mean content of water amounted to 49.25%, lipids 32.51%, protein 13.29% and ash
4%. In Astra hen eggs, a statistically significant difference was noted in the content
of protein, lipids and ash. In Astra W-2 eggs the protein content attained 13.50%; in
Astra D the ash content was 4.29%; whereas in Astra W eggs and in Messa
443 eggs the lipid content was 32.63% and 32.68%, respectively. A statistically
significant difference in the water content was noted between the eggs of Rosa 1
and Rosa 2 birds only.
The effect of bird origin on the magnitude and significance of correlation
coefficients was observed in this study. This was particularly noticeable in the
correlation coefficients of eggs from the N-11 × P-11 hybrid, which ranged from
–0.317 to 0.953. The effect of bird genotype on the correlation coefficient was also
observed among certain chemical components, especially between yolk protein
content and albumen protein content and between yolk lipid content and yolk ash
content.
In the recent time various commercial strains of laying hens have appeared on
the domestic market in Poland. For that reason it seemed interesting to compare the
physico-chemical traits of eggs from laying hens of various origin. This was
51
particularly interesting because there are few reports on the correlation between
performance traits of layers and quality traits of eggs aimed to determine the effect
of bird origin on those traits. Since the standard of life in many countries has
improved and the consumers are increasingly concerned with health, also desirable
sensory attributes of food become more and more important. That pertains not only
to egg freshness and yolk colour, but also to shell colour and strength. Nowadays,
the average consumer prefers fresh and large eggs of brown shell colour sold at
a relatively low price. In this study the eggs from Rosa 1 layers demonstrated the
highest egg and yolk weight. On the other hand, the highest weight, density and
thickness of the shell as well as its percentage content in the table eggs were noted
in Astra N commercial layers. These dependences in the quality traits of table eggs
were shown in birds on the same compound feed which contained 10.82%
metabolizable energy (MJ/kg) and 17.40% crude protein.
The experimental results of this study revealed significant (P ≤ 0.05) differences
in the physico-chemical traits of table eggs from layers of various commercial
strains offered on the Polish market and which best meet the requirements of the
Polish consumers (Rosa 1) for high quality table eggs.
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P o l o w c z y k K., R a p a c z J., K a p k o w s k a E., J a n i k K. (1998). Wpływ żywienia je˛czmieniem
i wiesiołkiem na poziom TC w surowicy krwi i żółtku jaj kurzych oraz TG i HDL-C w surowicy
krwi. Zesz. Nauk. Prz. Hod., 36: 321 – 326.
R a p a c z J., J a n i k A., K a p k o w s k a E., P u s t k o w i a k H. (1997). Fatty acid content in egg yolks of
commercial layers. VII European Symposium on the Quality of Eggs and Egg Products, Poland,
Poznań, pp. 59 – 67.
R a p a c z J., J a n i k A., G u m u ł k a M. (2000). Wpływ składu mieszanek na profil kwasów tłuszczowych w żółtkach jaj konsumpcyjnych u mieszańców kur nieśnych. Rocz. Nauk. Zoot., Supl., 8:
171 – 175.
R o b e r t s J.R., N o l a n J.V. (1997). Egg and eggshell quality in FIDE strains of laying hen and the
effect of calcium source and age. VII European Symposium on the Quality of Eggs and Egg
Products, Poland, Poznań, pp. 38 – 44.
T r z i s z k a T. (2000). Jajczarstwo. Wyd. AR Wrocław.
W e˛ ż y k S., C y w a - B e n k o K., G a w e˛ c k i W., K r a w c z y k J. (2000). Wpływ genotypu na jakość jaj
i mie˛sa drobiowego. Rocz. Nauk. Zoot., Supl., 5: 235 – 240.
LIDIA CZAJA, KRYSTIAN DZIADEK, EWA GORNOWICZ
Wpływ pochodzenia kur nieśnych na jakość jaj konsumpcyjnych
STRESZCZENIE
Badano jaja podchodza˛ce od komercyjnych niosek w wieku od 50. do 52. tygodnia. Ptaki
utrzymywano w identycznych warunkach i żywiono tymi samymi mieszankami. Celem badań było
określenie różnic w zakresie fizykochemicznych cech jaj konsumpcyjnych pochodza˛cych od różnych
zestawów komercyjnych doste˛pnych na polskim rynku.
Najwie˛ksza˛ mase˛ jaj (69,26 g) i żółtek (22,11 g) stwierdzono u kur Rosa 1. Jaja pochodza˛ce od kur
z rodu Messa 445 miały najwie˛ksza˛ wysokość białka (6,72 mm), jednostki Haugha (78,93) i najwyższa˛
zawartość białka w jaju (62,27%), natomiast mieszańce N-11 × P-11 produkowały jaja o najciemniejszej
barwie żółtka (10,81).
Podczas chemicznej analizy żółtka stwierdzono, że jaja pochodza˛ce od kur Rosa 1 miały najniższa˛
zawartość wody (48,88%), średnia˛ zawartość lipidów (32,53%) i białka. Żółtka jaj od kur Messa 443
i Astra W zawierały wie˛cej lipidów (odpowiednio 32,68% i 32,63%).
Na podstawie uzyskanych wyników wykazano statystycznie istotne różnice (P ≤ 0,05) w zakresie
fizykochemicznych cech jaj konsumpcyjnych pochodza˛cych od badanych komercyjnych zestawów kur
nieśnych.
COMPARING THE STRUCTURE AND CHEMICAL COMPOSITION
OF DUCK EGGS*
A d a m M a z a n o w s k i1, Z e n o n B e r n a c k i1, T o m a s z K i s i e l2
1
Department of Poultry Breeding, University of Technology and Agriculture, Mazowiecka 28,
85-084 Bydgoszcz, Poland
2
Department of Poultry Breeding, National Research Institute of Animal Production,
Dworzyska 1, 62-035 Kórnik, Poland
Abstract
The structure and chemical composition of eggs from ducks of paternal (A44, A55) and maternal
strains (P66, P77, K11) were compared at the start (week 2) and at the end of lay (week 24). Each
time, 12 eggs per strain were analysed. During lay, decreases were found in shell weight, index and
area and in shell thickness. During 24 weeks of reproduction, yolk and shell percentage in eggs
decreased, and white percentage increased. This was accompanied by lower yolk density and
higher white and shell density. During reproduction, the intensity of yolk colour decreased and pH
of yolk and white decreased substantially. Haugh units and the number of pores in eggshells
increased in ducks of all strains. The decreased value of egg structure traits could adversely affect
incubation parameters and duckling quality. The exception was the increasing number of pores in
eggshell, which could positively affect the incubation parameters of ducklings. From 2 to 24 weeks
of reproduction, also the chemical composition of eggs changed. In yolk, percentage of water and
fat were found to increase and the content of protein and ash decreased. In the white of duck eggs,
percentage of water increased and proportion of protein and ash decreased. Changes in the
structure and chemical composition of eggs require that the incubation technology be adjusted to
maintain the correct hatching results of ducklings. The best, statistically significant results in terms
of egg weight and structure were found in strains A55 and P77, and the worst in strain K11. The
best, statistically significant results of egg chemical composition were noted in strain K11, and the
worst in strain A44.
Key words: duck, reproductive traits, egg, chemical components
Over 20-year successful selection of meat-type ducks in paternal and maternal
strains to increase muscling and decrease fatness might have adversely affected the
number of eggs laid, their structure and chemical composition. Significant effects
of the direction and intensity of selection on reproductive traits of ducks and
54
A. Mazanowski et al.
structural traits of their eggs were reported by Hattenhauer et al. (1981). Egg structure
traits were also considerably influenced by the genotype of birds (Ksia˛żkiewicz,
2002), feeding (Baéza et al., 1995), day length (Baéza et al., 1995; Davis et al., 1993;
Rouvier, 1999) and the age of ducks (Mazanowski et al., 1999 a; b).
Attempts were also made to determine the reaction correlated with the level of
reproductive traits of ducks selected for meat traits (Górski et al., 1995; 1998;
Mazanowski and Adamski, 2003; Sochacka and Różycka, 1990). Studies were also
made on the morphological structure of duck eggs, especially the eggshell
(Mazanowski and Adamski, 2003; Mazanowski et al., 2004; Paganelli et al., 1974;
Testik and Karaman, 2000; Tullett and Smith, 1983; Yannakopoulos and TserveniGousi, 1987). Chemical composition of duck eggs was only studied sporadically
(Ksia˛żkiewicz et al., 1999; Niewiarowicz and Płotka, 1989).
Duck breeding in Poland includes paternal and maternal strains. The paternal
strain A44 was created in 1978 from two groups of English crossbred ducks, which
were selected on account of their high reproductive and meat traits. Despite their
great productive value, including very good muscling and low fatness, A44 ducks
were liquidated in 2003. The paternal strain A55 was created in 1983 from A44
ducks crossed with P88 Pekin ducks imported from the Danish Poultry Houses. This
strain was established from four groups of crossbreds differing in the direction of
crossing during their creation (Mazanowski et al., 1999 b). Work on the maternal
strain P66 was commenced in 1977. The strain was created from 13 groups of
between-strain crossbreds of ducks from the old strains (P11, P22, P33) and from
imported strains (P44, P55). In 1978 work was begun on the maternal strain (P77),
which was created from two groups of crossbreds (P45, P54). The strain was
founded by crossbreds with the highest reproductive and meat traits. Work on the
maternal strain K11 was started in 1974, using wild mallard (Anas platyrhynchos L.), Pekin and Orpington ducks for the mating (Ksia˛żkiewicz, 1984; Ksia˛żkiewicz and Mazanowski, 1985; Mazanowski et al., 1999 a).
The objective of this study was to compare the weight, shell and egg content
traits, and the chemical composition of eggs of ducks from two paternal and three
maternal strains during their first laying period.
The study was carried out in 2003 at the Waterfowl Breeding Farm Dworzyska,
which belongs to the National Research Institute of Animal Production (IZ Group —
HD ZADROB Zakrzewo Ltd.). Eggs from pedigree ducks of two paternal strains
(A44, A55) and three maternal strains (P66, P77, K11) were investigated. Ducks
were kept in the same environmental conditions and fed in the same way.
There were 30 males and 270 females in each strain. During reproduction, birds
were kept in a house with no access to the outside run. They were fed ad libitum
with a diet containing 19.0% protein and 2830 kcal (11.8 MJ) metabolizable energy
per 1 kg diet. The diet also contained 3.0% fat, 3.6% fibre, 10.5% ash and 13.5%
Structure and chemical composition of duck eggs
55
water. The diet was supplemented with 10 g of oat grain per bird per day.
Thoroughly mixed mineral components: chalk, mineral mixture for poultry and dry
gravel in a volumetric ratio of 1 : 1 : 4 were given separately.
In all the strains, the number of eggs laid was recorded daily for 182 days, and
weight of 20 eggs from each strain was checked individually, once a week, over the
whole production period. At the start (week 2) and at the end (week 24) of lay,
12 eggs were taken from each strain to analyse their structure and contents.
Analysis was made 24 h after the eggs were laid. Egg weight (g) was determined on
a RADWAG WPS 360 C balance. Egg shape index was determined by measuring
the width to length ratio (%). Shell colour was determined with the Quality Control
Reflectometer (QCR) from the TSS (Technical Services and Supplies) kit. Egg
surface area was calculated using the formula of Paganelli et al. (1974):
PS = 4.835 × M0.662
where:
M = egg weight.
Shell deformation was determined using a Marius instrument and shell strength
with Egg Crusher EGC 20 SW (VEIT). After removing the content of the egg, the
shell was dried for three hours at 105°C in a SUB 100 M type drier. Thereafter
Medicat 160 scales were used to evaluate weight (g) and a digital micrometer screw
to measure shell thickness (mm). Shell density was measured, after isolating about
1 – 2 g of the sample, with a kit for determining the density of solid bodies, using
the procedure of a WPS 360 C balance. When determining shell density, distilled
water (25°C) was used as the model liquid.
Shell porosity was also determined. Eggshell fragments from the large end,
small end and equator of the egg were cooked for 5 minutes in 5% NaOH solution
to remove the protein fraction from the outer and inner shell surface. After cooking
the shells were rinsed in distilled water, dried, immersed for 5 seconds in a diluted
65% nitric acid (one part acid to three parts water) and rinsed again in distilled
water. Dried fragments of the shells were coated with methylene blue on their inner
side. Shell porosity was read under a Nikon stereoscopic microscope at 4×
magnification to determine the number of pores per 0.25 cm2 of shell surface.
White and yolk were isolated from the egg content. Yolks were weighed on
a WPS 360 C balance. The weight of white was calculated as the difference
between egg weight and weights of yolk and shell. Percentages of yolk, white and
shell were related to the weight of fresh egg. Densities of yolk and white were
determined with a liquid density determination kit, using the procedure of a WPS
360 C balance. A pH-meter CP-401 (Elmetron) was used to determine pH of yolk
and white. After removing the egg’s content onto a mirrored glass table, thick white
and yolk were measured for height with a QCD instrument (Quality Control Digital
Display Unit) by TSS. Yolk diameter was determined along the chalaza using
electronic calipers. The yolk index was expressed as the yolk height to yolk
diameter ratio (%). The height of thick white (H) and egg weight (W) were used to
calculate Haugh units from the formula (Williams, 1992):
56
HU = 100 lg (H + 7.7 – 1.7 W0.37)
Yolk colour was rated on a scale of 1 to 15 (La Roche). The incidence of blood
and meat spots in the eggs was also determined.
At the start (week 2) and at the end (week 24) of lay, 12 eggs per strain were
collected. After removing and separating the egg’s contents into yolk and white,
chemical analyses were made to determine total protein according to Kjeldahl,
using 2200 Kjeltec Auto Distillation Foss Tecator according to the Polish Standard
PN-75/A-04018 (1975). The content of water (dry matter) was determined using the
drier method according to the Polish Standard PN-A-86509 (1994), and ash
following the procedure described by Krełowska-Kułas (1993). Amount of fat in
yolk was determined by Soxhlet extraction according to the Polish Standard
PN-A-86509 (1994), using Büchi 810 instrument.
The results were analysed statistically (means, variation coefficients, analysis of
variance, significant differences) with a package of statistical computer programs
developed at the National Research Institute of Animal Production.
Results
Over 182 days, the greatest number of eggs per one duck was obtained in
maternal strains P66 and P77. The lowest egg production was characteristic of ducks
from the paternal strain A44. A55 and P66 ducks had the greatest egg weight and K11
ducks the smallest (Table 1). Figure 1 shows laying intensity and egg weight in five
strains of ducks, during the entire reproductive period. In paternal strains, laying and
egg weight curves were similar in shape. Also egg production and egg weight
followed a similar pattern in the maternal strains P66 and K11, but the value of these
traits was lower than in strain K11. In the maternal strain P77, the highest egg
production and the most normal egg laying curve were found. Weight of eggs in the
maternal strain P77 was low, but increased during the reproductive period.
Table 1. Mean values (x̄) of egg laying and mean values and variation coefficients (v) of egg weight in
ducks according to strain
Group — symbol of strains
Trait
Number of eggs per 1 duck
(pcs)
x̄
Laying intensity (%)
x̄
Egg weight (g)
x̄
v
I
A44
II
A55
III
P66
IV
P77
V
K11
117 c
129 b
135 a
134 a
121 c
64.3 c
71.1 b
74.0 a
73.8 a
66.5 c
88.0 b
8.2
90.3 a
8.1
90.5 a
8.1
86.9 c
7.6
84.6 d
7.4
Means in rows followed by different letters differ significantly (P < 0.05).
57
Egg weight and shell traits differed significantly between strains, except shell
thickness and shell deformation, which did not differ at the start (week 2) and at the
end (week 24) of lay (Table 2). There were no statistically significant differences
between strains in the egg index at the start of lay, and in shell strength at the end of
lay. Comparison of strains for egg weight and shell traits at the start and at the end
of lay showed decreased egg weight, egg index, shell area and shell thickness. Shell
deformation did not change in the entire period of lay, and slight increases in shell
strength and shell colour intensity were noted.
——— laying intensity (%)
– – – egg weight (g)
Figure 1. Laying intensity and egg weight in ducks
Table 2. Mean values (x) and variation coefficients (v) of egg weight and shell traits in ducks for early (A — week 2) and late (B — week 24) laying period
Group
Trait
I
A
Egg weight (g)
x̄
v
Egg shape index (%)
x̄
v
Eggshell area (cm2)
x̄
v
Shell thickness (mm)
x̄
v
Shell deformation (µm/cm2)
x̄
v
Shell strength (N)
x̄
v
Shell colour (% of white)
x̄
v
II
III
IV
B
A
B
A
89.5 abc
9.2
85.9 a
8.2
94.7 a
7.4
87.7 a
8.3
90.9 b
6.6
79.7 ab
5.5
71.8 a
4.3
70.0 b
3.7
72.5 a
3.4
71.3 a
6.4
72.6 a
2.3
94.6 abc
6.2
92.1 a
5.4
98.3 a
4.9
93.4 a
5.5
95.7 b
4.4
0.377 a
16.7
0.374 a
11.9
29.4 a
9.8
28.6 a
12.2
28.1 a
9.5
28.4 a
10.7
29.2 a
10.7
14.9 ab
27.1
14.4 a
37.6
14.2 ab
27.4
13.2 a
37.4
50.4 a
8.6
49.4 b
11.2
50.5 a
9.9
49.3 b
11.4
0.396 a
7.9
0.376 a
7.3
B
A
I−V
B
87.0 bc
7.0
86.9 a
8.6
84.8 c
5.2
79.6 b
8.9
89.4
84.0
73.9 a
3.8
74.2 a
4.8
73.8 a
3.6
72.4 a
3.5
70.3 b
5.0
72.7
71.9
87.7 b
3.6
93.0 bc
4.7
92.8 a
5.8
91.4 c
3.4
87.6 b
5.8
94.6
90.7
0.398 a
10.1
0.385 a
11.1
0.393 a
10.4
0.373 a
10.9
26.8 a
14.5
27.3 a
15.5
28.5 a
11.1
27.2 a
16.6
28.8 a
13.9
28.2
28.2
14.1 ab
22.2
13.9 a
27.7
15.3 a
19.7
14.9 a
34.9
11.9 b
29.7
15.5 a
29.6
14.1
14.4
49.8 a
6.5
53.2 ab
9.7
51.5 a
7.2
53.6 a
8.0
49.9 a
10.1
52.5 ab
9.7
50.4
51.6
0.376 a
7.2
Means in rows, separately for A and B, followed by different letters differ significantly (P < 0.05).
A
V
A
0.393 a
7.6
B
B
0.391
0.377
Table 3. Mean values (x̄) and variation coefficients (v) for proportion and density of yolk, albumen and eggshell weight in ducks for early (A — week 2) and late
(B — week 24) laying period
Group
Trait
Proportion of egg (%):
yolk
x̄
v
white
x̄
v
shell
x̄
v
Density (g/cm3):
yolk
x̄
v
white
x̄
v
shell
x̄
v
I
II
III
IV
I−V
V
A
B
A
B
A
B
A
B
A
B
A
B
35.6 a
7.1
34.2 a
6.8
36.4 a
9.3
35.5 a
4.6
35.5 a
5.8
34.5 a
5.1
34.8 a
4.5
34.7 a
6.7
34.9 a
5.4
34.8 a
5.3
35.4
34.7
55.9 a
4.4
57.2 a
4.3
54.6 a
6.0
56.0 a
3.4
55.6 a
3.7
56.7 a
3.0
55.9 a
3.4
56.8 a
4.7
56.0 a
3.0
56.4 a
3.8
55.6
56.6
8.5 b
11.9
8.6 a
8.7
9.0 a
8.7
8.5 a
7.8
8.9 ab
5.3
8.8 a
5.3
9.3 a
6.3
8.5 a
8.9
9.1 a
8.0
8.8 a
8.4
9.0
8.6
0.381 a
5.2
0.385 a
7.1
0.392 a
4.2
0.390 a
4.7
0.390 a
3.2
0.374 a
8.6
0.390 a
4.1
0.380 a
5.0
0.396 a
3.1
0.382 a
5.7
0.390
0.382
0.336 a
7.9
0.335 ab
11.1
0.333 a
7.1
0.350 ab
11.0
0.327 a
7.6
0.330 b
10.4
0.336 a
5.9
0.357 a
11.5
0.334 a
7.4
0.339 ab
10.7
0.333
0.342
1.823 ab
20.4
1.921 a
7.9
1.932 a
5.2
1.920 a
5.2
1.690 b
29.5
1.943 a
4.1
1.899 a
6.0
1.948 a
6.3
1.949 a
2.8
1.925 a
4.5
1.859
1.931
For explanations see Table 2.
Table 4. Mean values (x̄) and variation coefficients (v) for yolk colour, yolk index, Haugh units, pH and pores of eggshell in ducks for early (A — week 2) and late
(B — week 24) laying period
Group
Trait
I
A
Yolk colour by La Roche
scale (pts)
x̄
v
Yolk index (%)
x̄
v
Haugh units
x̄
v
Mean values of pH:
yolk
x̄
v
albumen
x̄
v
Pores in egg shell
(no./0.25 cm2):
small end
x̄
v
equatorial part
x̄
v
large end
x̄
v
Blood and meat spots (%)*
x̄
II
B
A
III
B
A
IV
B
A
I−V
V
B
A
B
A
B
5.7 ab
10.8
4.5 c
25.9
5.2 b
11.8
4.6 bc
19.6
6.2 a
11.6
6.2 a
26.6
6.0 ab
12.3
5.3 b
23.1
5.8 ab
14.3
5.0 bc
19.1
5.8
5.1
38.7 b
4.4
40.6 ab
7.3
40.8 a
6.5
41.2 a
5.9
40.0 ab
7.8
40.3 abc
7.9
40.5 ab
3.2
38.7 bc
6.0
39.7 ab
5.4
38.4 c
6.4
39.9
39.8
67.5 a
14.1
80.6 a
6.8
66.5 a
17.4
76.0 a
10.4
69.0 a
15.2
80.6 a
12.0
66.4 a
15.1
73.0 a
14.1
67.0 a
20.4
78.0 a
10.9
67.3
77.6
6.06 a
1.4
5.08 b
8.5
6.01 a
1.3
4.87 bc
7.7
6.00 a
0.7
4.78 c
5.9
6.08 a
1.2
4.82 c
7.4
6.05 a
1.2
5.31 a
8.2
6.04
4.97
8.56 a
2.0
7.25 a
0.8
8.54 a
2.1
7.15 bc
0.8
8.58 a
1.9
7.13 c
0.7
8.56 a
1.2
7.24 a
0.5
8.61 a
1.5
7.23 b
1.1
8.57
7.20
13.1 b
22.9
15.8 a
13.7
16.2 a
16.2
16.2 a
12.0
13.1 b
17.1
16.1 a
12.6
14.7 ab
12.1
16.3 a
11.2
13.3 b
19.3
15.5 a
10.8
14.1
16.0
19.4 c
11.9
24.9 a
6.9
22.3 b
10.2
25.2 a
8.4
27.4 a
7.0
25.0 a
9.9
19.4 c
8.1
24.7 a
7.1
22.2 b
8.3
24.7 a
9.0
22.1
24.9
28.7 a
7.0
34.9 a
7.0
28.6 a
7.1
34.5 ab
7.2
28.0 a
10.1
34.2 ab
7.8
26.8 a
7.3
34.8 a
7.2
27.7 a
8.5
32.8 b
6.6
28.0
34.2
0
16.7
25.0
25.0
16.7
41.7
16.7
16.7
6.6
18.3
21.7
8.3
Means in rows, separately for A and B, followed by different letters differ significantly (P < 0.05);
* Significant differences were not calculated statistically.
61
Percentages of yolk and white in egg did not differ significantly between the
strains at the start and at the end of lay, and shell percentage at the end of lay only
(Table 3). Yolk density at the start and at the end of lay did not differ significantly
between the strains. White density did not differ significantly at the start of lay, and
shell density at the end of lay. Comparison of percentage egg components and their
density at the start of lay with the values at the end of lay showed that percentage of
yolk and shell in egg decreased and percentage of white increased. In the analysed
period, yolk density declined slightly, and white and shell density increased.
Table 5. Mean values (x̄) and variation coefficients (v) for chemical components of yolk for early and
late laying period in ducks
Trait
Chemical components of yolk
in early (week 2) laying period
in ducks (%):
water
x̄
v
protein
x̄
v
fat
x̄
v
ash
x̄
v
Chemical components of yolk in late
(week 24) laying period in ducks
(%):
water
x̄
v
protein
x̄
v
fat
x̄
v
ash
x̄
v
Group
I
II
III
IV
V
I−V
42.04 b
7.0
47.16 a
2.6
46.04 a
2.4
46.84 a
4.4
47.02 a
1.8
45.82
3.9
16.17 a
3.0
15.99 a
3.5
16.01 a
2.3
16.11 a
2.4
15.99 a
4.0
16.05
3.1
23.54 b
4.7
27.61 a
7.4
23.28 b
4.1
23.46 b
8.1
26.54 a
9.6
24.89
7.3
3.21 a
11.4
3.16 ab
10.5
2.97 ab
11.1
2.87 b
16.1
3.22 a
11.1
3.09
12.1
49.23 a
3.2
49.43 a
3.3
48.41 a
2.9
48.44 a
2.5
48.86 a
2.7
48.87
2.9
15.98 ab
6.1
15.23 b
6.1
16.38 a
6.3
16.20 a
6.1
15.84 ab
5.2
15.93
6.0
32.16 a
3.0
32.91 a
3.2
32.56 a
2.4
32.69 a
2.4
32.55
3.2
35.57
2.9
2.25 b
13.8
2.60 a
13.8
2.60 a
10.1
2.47 ab
9.2
2.68 a
8.1
2.52
11.1
Yolk colour and yolk index differed significantly between strains at the start
(week 2) and at the end (week 24) of lay. There were no significant differences
between strains in Haugh units. Yolk colour evaluated using a Roche colour fan
62
decreased during reproduction, and Haugh units increased (Table 4). pH of yolk and
white at the start of lay did not differ between strains. At the end of lay, differences
were statistically significant. Comparison of yolk and white pH at the start and at
the end of lay showed that they decreased at the end of production. The number of
pores at the small end and equator of the egg differed significantly between the
strains at the start of lay, and at the end they were greater in all the strains, but did
not differ significantly. The number of pores at the large end of the egg differed
significantly only towards the end of lay. When comparing the number of pores at
the start and at the end of lay, it appeared that they increased considerably during
reproduction. We also noted that the percentage of blood and meat spots increased
at the end of reproduction compared to the start of reproduction.
Between strains there were no significant differences in percentage of protein
in yolk at the start of lay, and in percentage of water and fat at the end of lay
(Table 5). Chemical components in yolk were the most abundant in strain K11 at the
start of lay, and in strains P66 and P77 at the end of lay. Comparison of the chemical
composition of yolk at the start and at the end of lay revealed that during reproduction,
percentage of water and fat increased, and percentage of protein and ash decreased.
Table 6. Mean values (x̄) and variation coefficients (v) for chemical components of white for early and
late laying period in ducks
Trait
Chemical components of white
in early (week 2) laying period
in ducks (%):
water
x̄
v
protein
x̄
v
ash
x̄
v
Chemical components of white in
late (week 24) laying period in
ducks (%):
water
x̄
v
protein
x̄
v
ash
x̄
v
Group
I
II
III
IV
V
I−V
85.87 a
1.4
85.78 a
2.2
86.09 a
0.9
85.49 a
1.0
85.94 a
1.9
85.83
1.5
11.10 a
2.7
10.90 a
3.2
10.47 b
3.7
11.08 a
3.6
11.19 a
3.9
10.95
3.4
1.16 a
7.5
1.15 a
6.2
1.17 a
6.3
1.13 a
7.2
1.13 a
10.8
1.15
7.8
90.11 bc
1.0
89.84 cd
0.8
91.13 a
0.9
90.62 ab
1.0
89.35 d
0.9
90.21
0.9
8.96 ab
9.7
9.29 a
8.1
7.99 c
9.7
8.45 bc
10.9
9.40 a
7.4
8.82
9.1
0.81 b
10.2
0.82 b
7.8
0.87 b
12.7
1.13 a
30.1
0.90
20.7
0.86 b
21.3
63
No significant differences were found between strains in percentage of water
and ash in white of duck eggs at the start of lay (Table 6). At the end of lay, there
were significant differences in percentage of water, protein and ash in egg white
between strains. In strain K11 there was the lowest content of water and the highest
content of protein and ash. High protein content in egg white was found in strain
A55. When comparing chemical composition of white at the start and at the end of
lay, it appeared that during reproduction, percentage of water increased and
percentage of protein and ash decreased.
Discussion
In the earlier studies compared to the present ones, egg production per duck in
paternal strains was greater, ranging in the first laying period from 170 to 178 eggs
(Mazanowski et al., 1999 b) during 225 days, and in the other periods from 145 to
146 eggs during 182 days (Mazanowski et al., 2004). In maternal strains in the
earlier studies, egg production was also greater in the first laying period than in the
current experiment and ranged from 152 to 159 eggs during 225 days (Mazanowski
et al., 1999 a), and from 119 to 152 eggs during 182 days in the other periods
(Mazanowski and Adamski, 2003). In paternal strains in the earlier experiment
(Mazanowski et al., 1999 b) and in other studies (Mazanowski et al., 2004), egg
weight was high and similar to that obtained in the present study. High egg weight
had a beneficial effect on the weight of young ducks.
Evaluation of egg weight and shell traits in strain A44 (Mazanowski et al.,
2004) showed a lack of significant differences in egg weight, shape index and shell
area at the start, in the middle and at the end of reproduction. Shell deformation and
shell thickness increased during reproduction. In strain A55 during reproduction,
increased egg weight, shell area and shell thickness were found, while the values of
other traits did not change significantly. Egg weight and shell traits in strains P66
and P77 did not differ (Mazanowski and Adamski, 2003). In the present study, all
strains showed decreases in egg weight, egg index (except for eggs from strain
P66), egg area and shell thickness. Shell deformation and shell strength did not
change. On the whole, most of the analysed traits, including egg weight and shell
traits, deteriorated during reproduction.
Mazanowski et al. (2004) report that percentage of yolk and thin white in eggs
of ducks from parental strains increased, while percentage of thick white and shell
decreased. Density of yolk and white decreased during reproduction, and shell
thickness increased. In maternal strains, greater percentage of yolk was found in
strain P77 than in P66. Percentage of thick white and shell was greater in strain P66.
In percentage of thin white, no differences were found between the strains
(Mazanowski and Adamski, 2003). In the present experiment, percentage of yolk
decreased and percentage of white increased, but the differences were not statistically
significant. Only the proportion of shell decreased statistically significantly. These
results point to declining egg quality during lay. Górski et al. (1998) showed that
64
A55 ducks were characterized by a significantly higher content of yolk compared to
P66 and A44, and by a highly significant yolk index compared to strain P77. Eggs
of A55 ducks were also characterized by the highest hatchability of ducklings.
In paternal strains (Mazanowski et al., 2004) density of yolk, thick white and
thin white was found to decline, and shell density increased. Maternal strains
differed in terms of yolk and shell density, but did not differ in density of thick and
thin white. In the present study, yolk density during reproduction was found to
decline, with no significant differences between the strains. Density of white and
shell differed significantly between strains and in the studied period showed an
upward tendency.
In paternal strains, yolk colour intensity evaluated using a Roche colour fan was
found to decrease. Yolk index increased in both strains, and Haugh units decreased
in strain A55. In the same experiment, pH level was constant for yolk and decreased
for thick and thin white during the entire reproduction period (Mazanowski et al.,
2004). In maternal strains, eggs of P66 ducks had a more intense yolk colour and
a higher yolk index than eggs of P77 ducks. Eggs of P77 ducks were characterized
by higher Haugh units. In terms of pH of yolk and white, no significant differences
were noted between the strains (Mazanowski and Adamski, 2003). In the present
experiment, only egg parameters expressed in Haugh units did not show significant
differences between the strains. There were no significant differences in yolk colour
and yolk index at the start and at the end of reproduction. Significant differences
were noted between strains in pH of yolk and white. A marked decrease in pH
during the reproductive period could negatively affect the hatchability of ducklings.
In eggs of ducks from paternal strains, the number of pores in shell were found
to increase during reproduction in the small and large end and in the equator of the
egg. Percentage of blood and meat spots was considerable, ranging from 25 to 33%
(Mazanowski et al., 2004). In female strains, the content of pores in the entire shell
was similar in both strains. Blood and meat spots were less numerous, from 12 to
24% (Mazanowski and Adamski, 2003). The number of pores in shell of duck eggs
differed significantly between the strains. During reproduction, the number of pores
increased, which may cause greater evaporation of water from eggs and release of
heat. Also the proportion of blood and meat spots increased from 18.3 to 21.7%.
Between eggs from duck strains, no statistically significant differences in the
content of protein were noted in egg yolk at the start of the reproductive period. The
highest content of fat was found in the eggs of A55 and K11 ducks, and of ash in
A44 and K11 ducks. At the end of the reproductive period, yolk of eggs from P66
and P77 ducks was the highest in protein, and there were large amounts of ash in all
maternal strains. On the whole, during reproduction the content of water and fat
increased in yolk, and the content of protein and ash decreased.
At the start of the reproductive period, no significant differences were observed
between strains in the content of water and ash in white of duck eggs. Only in strain
P66 was the protein content of egg whites significantly lower. At the start of the
reproductive period, the greatest percentage of water was noted in the white of eggs
from P66 ducks, and the lowest in white of eggs from K11 ducks. The greatest
65
content of protein was found in strains A55 and K11, and of ash in strain K11.
During reproduction, increased percentage of water and decreased percentage of
protein and ash were found in the white of eggs.
On the two dates of evaluation at the start (week 2) and at the end (week 24) of
the reproductive period, significantly the best results in terms of egg weight and
structure were found in strains A55 and P77, and the worst in strain K11.
Significantly the best results in terms of chemical composition of duck eggs were
found in strain K11, and the worst in strain A44.
References
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restriction, energy and protein allowances on the performances of breeder Pekin ducks. Arch.
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krajowych. Zesz. Nauk. WSRP Siedlce, Drob., 40: 7 – 16.
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PTZ, Prz. Hod., 36: 349 – 356.
H a t t e n h a u e r H., v a n T i e u H., J u n g S. (1981). Beziehungen von Körpermassen zur Fruchtbarkeit
bei Pekingenten. Arch. Tierzucht, 24, 5: 461 – 471.
K r e ł o w s k a - K u ł a s M. (1993). Badania jakości produktów spożywczych. PWE, Warszawa, p. 97.
K s i a˛ż k i e w i c z J. (1984). Cechy reprodukcyjne kaczek mieszańców o różnym udziale pekinów,
orpingtonów i dzikiej kaczki krzyżówki (Anas platyrhynchos L.). Rocz. Nauk. Zoot., 11, 2: 63 – 76.
K s i a˛ż k i e w i c z J., M a z a n o w s k i A. (1985). Zmiany masy ciała w trzech kolejnych pokoleniach
kaczek mieszańców o różnym udziale dzikiej kaczki krzyżówki (Anas platyrhynchos L.). Zesz.
Nauk. Drob., 2: 6 – 13.
K s i a˛ż k i e w i c z J., S t e˛ p i ń s k a M., K i s i e l T., R i e d e l J. (1999). Cechy fizyczne jaj i lipidy żółtek
w stadach zachowawczych kaczek typu Pekin i Cayuga. Rocz. Nauk. Zoot., 26, 3: 99 – 100.
K s i a˛ż k i e w i c z J. (2002). Reproductive and meat characteristics of Polish ducks threatened with
extinction. Czech. J. Anim. Sci., 47, 10: 401 – 410.
M a z a n o w s k i A., K s i a˛ż k i e w i c z J., S z u k a l s k i G. (1999 a). Reproductive traits of ducks from
maternal strains in two laying periods and meat traits of their offspring. Ann. Anim. Sci. — Rocz.
Nauk. Zoot., 26, 4: 191 – 203.
M a z a n o w s k i A., K s i a˛ż k i e w i c z J., K i s i e l T. (1999 b). Reproductive traits of ducks from
paternal strains in two laying periods and meat traits of their offspring. Ann. Anim. Sci. — Rocz.
Nauk. Zoot., 26, 4: 205 – 217.
M a z a n o w s k i A., A d a m s k i M. (2003). Egg content and eggshell traits in ducks from three maternal
strains raised in Poland. Ann. Anim. Sci., 3, 2: 287 – 294.
M a z a n o w s k i A., A d a m s k i M., K i s i e l T. (2004). Cechy reprodukcyjne i cechy jaj kaczek z rodów
ojcowskich. Rocz. Nauk. Zoot., 32, 1: 69 – 80.
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aminokwasowy. Zesz. Nauk. Drob., COBRD Poznań, 6: 69 – 77.
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S o c h o c k a A., R ó ż y c k a B. (1990). Próba oceny jakości jaj kaczek rasy Pekin. Prz. Nauk. Lit. Zoot.,
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hatching results and growing performance. Proceedings of the 21st World’s Poultry Congress,
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W i l l i a m s K C. (1997). Some factors affecting albumen quality with particular reference to Haugh unit
score. World’s Poultry Sci. J., 48, 1: 5 – 16.
Y a n n a k o p o u l o s A.L., T s e r v e n i - G o u s i A.S. (1987). Effect of egg and shell quality on day-old
duckling weight. Arch. Geflügelkunde, 51, 4: 157 – 159.
ADAM MAZANOWSKI, ZENON BERNACKI, TOMASZ KISIEL
Porównanie budowy i składu chemicznego jaj kaczych
STRESZCZENIE
Porównano budowe˛ i skład chemiczny jaj kaczek z rodów ojcowskich (A44, A55) i matecznych
(P66, P77, K11) na pocza˛tku (2. tydzień) i na końcu (24. tydzień) nieśności. Za każdym razem oceniono
po 12 jaj z każdego rodu. W okresie nieśności zanotowano zmniejszenie masy, indeksu i powierzchni
skorupy oraz grubości skorupy jaj kaczek rodowych. W cia˛gu 24 tygodni reprodukcji zmniejszył sie˛
w jajach kaczych procentowy udział żółtka i skorupy, a zwie˛kszył procentowy udział białka.
Równocześnie zmalała ge˛stość żółtka, a zwie˛kszyła sie˛ ge˛stość białka i skorupy jaj. W czasie
reprodukcji zmniejszyła sie˛ intensywność koloru żółtka oraz zmalał znacza˛co odczyn pH żółtka i białka,
natomiast zwie˛kszyła sie˛ wartość jednostek Haugha oraz liczba porów w skorupie jaj kaczych
z wszystkich rodów. Zmniejszenie wartości cech budowy jaj kaczek rodowych mogło mieć ujemny wpływ
na wskaźniki le˛gu i jakość piskla˛t. Wyja˛tek stanowiła zwie˛kszaja˛ca sie˛ liczba porów w skorupie jaja,
która mogła mieć korzystny wpływ na wskaźniki wyle˛gu piskla˛t.
W okresie od 2. do 24. tygodnia reprodukcji zmienił sie˛ również skład chemiczny jaj. W żółtku jaj
zanotowano zwie˛kszenie procentowego udziału wody i tłuszczu, a zmniejszenie zawartości białka
i popiołu, natomiast w białku jaj kaczych stwierdzono zwie˛kszenie procentowego udziału wody,
a zmniejszenie udziału białka i popiołu. Zmiany w budowie i składzie chemicznym jaj wymagaja˛
dostosowania do nich technologii le˛gu, w celu utrzymania prawidłowych wyników wyle˛gu piskla˛t.
Statystycznie istotnie najlepsze wyniki pod wzgle˛dem masy i budowy jaj stwierdzono w przypadku
jaj od kaczek z rodów A55 i P77, a najgorsza˛ ocene˛ uzyskały jaja od kaczek z rodu K11. Natomiast
statystycznie istotnie najlepsze pod wzgle˛dem składu chemicznego okazały sie˛ jaja od kaczek z rodu
K11, a najgorsze od kaczek z rodu A44.
EVALUATION OF SOME REGIONAL VARIETIES OF GEESE
FOR REPRODUCTIVE TRAITS, EGG STRUCTURE
AND EGG CHEMICAL COMPOSITION*
A d a m M a z a n o w s k i1, T o m a s z K i s i e l2, M a r e k A d a m s k i1
1
Department of Poultry Breeding, University of Technology and Agriculture, Mazowiecka 28,
85-084 Bydgoszcz, Poland
2
Department of Poultry Breeding, National Research Institute of Animal Production, Dworzyska 1,
62-035 Kórnik, Poland
Abstract
Regional Kielecka (Ki), Podkarpacka (Pd), Kartuska (Ka) and Suwalska (Su) geese were
investigated in their first year of reproduction. Egg production of Kielecka and Podkarpacka geese
was lower (19 eggs) compared to Kartuska and Suwalska geese, which laid more eggs (23 to 31)
that were heavier. During reproduction, body weight losses of geese ranged from 12.7 to 17.4%. In
ganders, body weight losses were only found in Kielecka and Kartuska (5.2 to 6.0%). Egg
fertilization in the regional geese declined during reproduction, while percentage of gosling
hatchability remained similar all the time. Eggs of regional geese, evaluated at the peak of egg
production, did not differ between the groups only in terms of egg index, shell percentage in egg
and shell density. No differences between eggs of particular groups were also found for Haugh
units and pH of egg white. Chemical composition of eggs at the start and at the end of lay differed
between the groups in terms of water percentage, in protein and ash percentage at the start, and in
fat percentage at the end of lay. Chemical composition of egg white differed in terms of water
percentage only at the start of lay, and of protein at the start and at the end of lay. During the
entire reproductive period, water, protein and ash in yolk decreased, and fat in yolk increased. In
egg white during the entire reproductive period, water and protein percentage decreased and ash
percentage slightly increased. Taking into account the decreased percentages of water and protein,
and the increased percentage of fat in eggs, changes in the incubation technique of eggs of regional
goose varieties should be considered. The study showed biodiversity of the geese in reproductive
traits and in structure and chemical composition of eggs.
Key words: goose, egg traits, chemical components
68
Previous studies (Mazanowski and Kiełczewski, 1999; Mazanowski and Szukalski, 2000; Mazanowski and Adamski, 2002) revealed that the highest egg
production (57 to 95 eggs) was characteristic of Astra G geese. In the same
experiments, White Kołuda geese laid from 57 to 64 eggs. Egg production of
two-way and three-way crosses with the wild graylag goose was small, ranging
from 20 to 36 eggs (Mazanowski and Chełmońska, 2000; Mazanowski et al., 2002).
The high egg production by Astra G geese, much higher than in White Kołuda
geese (Rosiński, 2000), might have negatively affected the physical traits of eggs,
leading to poorer parameters of egg incubation and gosling hatchability (Mazanowski and Bernacki, 2003).
Analyses were also made on changes in the structure and chemical composition
of eggs from two-way and three-way crosses with wild graylag geese, which laid
a small number of eggs (Mazanowski et al., 2002; Mazanowski and Bernacki,
2003). It was found that the eggs of low-producing geese are rich in nutrients. In
Poland, physical traits and chemical composition of eggs were studied, among
others, in White Kołuda and Biłgorajska geese (Puchajda, 1991; Rosiński, 2000).
Geese from Polish conservation flocks lay a small number of eggs (19 to 31). These
geese are characterized by good parameters of egg fertilization and hatchability of
goslings from fertilized eggs. The authors of this experiment believed that the
structure and chemical composition of eggs of geese that underwent no selection in
the past and today, will differ from the eggs of birds with higher egg production.
The purpose of this experiment was to evaluate reproductive traits, egg structure
and egg chemical composition in regional geese depending on their origin.
The study was carried out in 2003 at the Waterfowl Breeding Farm Dworzyska,
which belongs to the National Research Institute of Animal Production (IZ Group —
HD ZADROB Zakrzewo Ltd.). Reproductive traits, egg structure and egg chemical
composition were evaluated in four regional varieties of geese in their first year of
reproduction. The division of geese into groups is shown below:
Group
I
II
III
IV
Symbols of geese* — sex

Ki
Pd
Ka
Su
Ki
Pd
Ka
Su
No. of geese — sex

36
37
38
40

69
74
71
72
Proportion of males
to females
:
1.9
2.0
1.9
1.8
*Ki — Kielecka goose, Pd — Podkarpacka goose, Ka — Kartuska goose, Su — Suwalska goose.
The goose varieties discussed herein were described in the World Watch List
for Domestic Animal Diversity (2000).
Evaluation of geese for reproductive traits
69
During reproduction, birds were housed indoors and had access to restricted
outside runs bedded with rye straw. Throughout the study, day length was 10 hours.
Geese were vaccinated against Derzsy disease in January 2003, prior to the onset of
lay. Geese were fed ad libitum with a feed containing 90% mixture and 10% oats.
The feed contained 18.3% crude protein and 2830 kcal (11.8 MJ) metabolizable
energy in 1 kg. During reproduction, to each 100 kg of feed were added vitamin
preparations (25 g of Polfamix Z and 10 g of Polfasol B compositum). In separate
feeders, geese were also given well-mixed components: fodder chalk, mineral
mixture and gravel at the 1 : 1 : 4 ratio.
Number of eggs was recorded daily in groups according to hatching and
unhatchable eggs, following the Polish Standard PN-R-78564 (1998). Once a week,
all hatching eggs laid on one day were individually weighed to an accuracy of 0.1 g.
Prior to and during reproduction, amounts of feed given and uneaten were recorded
systematically in groups. These data were used to calculate feed intake per 1 egg
and per 1 kg of eggs. Eggs were incubated and goslings hatched in walk-in
Petersime incubators during the entire reproductive period, and the results of
successive incubations were recorded at weekly intervals. Males and females were
weighed individually to an accuracy of 5 g, prior to and at the end of lay.
At the peak of egg production (week 6 of lay), 12 eggs were taken from each
group for analysis. Eggs were analysed 24 h after laying. Egg weight (g) was
determined on a RADWAG WPS 360 C balance. Egg shape index was determined
by measuring the width to length ratio (%). Egg area (cm2) was calculated following
the formula of Paganelli et al. (1974):
PS = 4.835 × W0.662
where:
W = egg weight.
Shell colour in percentage white was determined with a QCR reflectometer
from the TSS kit, shell strength with Egg Crusher EGC 20 SW (VEIT). Shell
deformation (µm) was determined using a Marius instrument. After removing the
egg’s contents, the shell was dried for three hours at 105°C in a SUB 100 M type
drier. Thereafter Medicat 160 scales were used to evaluate weight of shell (g) and
a digital micrometer screw was used to measure shell thickness (mm). Shell density
was measured, after isolating about 1 – 2 g of the sample, with a kit for determining
the density of solid bodies, using the procedure of a WPS 360 C balance. When
determining shell density, distilled water (25°C) was used as the model liquid.
White and yolk were isolated from the egg content. Yolks were weighed on
a WPS 360 C balance. The weight of white was calculated as the difference
between egg weight and weights of yolk and shell. Percentages of yolk, white and
shell were related to the weight of fresh egg. Densities of yolk and white (g/cm3)
were determined with a liquid density determination kit, using the procedure of
a WPS 360 C balance. A pH-meter CP-401 (Elmetron) was used to determine pH of
yolk and white.
70
After removing the egg’s content onto a mirrored glass table, thick white and
yolk were measured for height (mm) with a QCD instrument (TSS). Yolk diameter
was determined along the chalaza using electronic calipers. The yolk index was
expressed as the yolk height to yolk diameter ratio (%). The height of thick white
(H) and egg weight (W) were used to calculate Haugh units from the formula
(Wiliams, 1997):
HU = 100 lg (H + 7.7 – 1.7 W0.37)
Yolk colour was rated on a scale of 1 to 15 (Roche colour fan). The incidence of
blood and meat spots in the eggs was also determined.
To determine shell porosity, eggshell fragments were taken from the large end,
small end and equator of the egg and cooked for 5 minutes in 5% NaOH solution to
remove the protein fraction from the outer and inner shell surface. After cooking the
shells were rinsed in distilled water, dried, immersed for 5 seconds in a diluted 65%
nitric acid (one part acid to three parts water) and rinsed again in distilled water.
Dried fragments of the shells were coated with methylene blue on their inner side.
Number of pores in the shell from the large end, small end and equator of the egg
was read under a Nikon stereoscopic microscope at 4× magnification, each time on
0.25 cm2 of shell surface.
At the start (week 2) and at the end (week 24) of lay, 12 eggs per group were
collected for chemical analysis. After removing and separating the egg’s contents
into yolk and white, chemical analyses were made to determine total protein
according to Kjeldahl, using 2200 Kjeltec Auto Distillation Foss Tecator according
to the Polish Standard PN-75/A-04018 (1975). The content of water (dry matter)
was determined using the drier method according to the Polish Standard PN-A86509 (1994), and ash following the procedure described by Krełowska-Kułas
(1993). Amount of fat in yolk was determined by Soxhlet extraction according to
the Polish Standard PN-A-86509 (1994), using Büchi 810 instrument.
The results were analysed statistically (variation coefficients, significant differences, standard deviation) with a package of statistical computer programs developed at
the National Research Institute of Animal Production.
Results
Laying performance of the regional geese differed significantly depending on
their origin (Table 1). Geese of groups I and II (southern varieties) exhibited lower
egg production than geese of groups III and IV (northern varieties). The greatest
number of eggs per layer was found in Suwalska geese. Northern varieties also
showed higher egg weight compared to southern varieties of geese. After 7 weeks
of production, egg production in regional varieties of geese decreased together with
increased egg weight (Figure 1). In Suwalska geese, decreased egg production and
increased egg weight were noted around 12 weeks of reproduction. The lowest feed
71
intake (Table 1) in terms of 1 egg and 1 kg of eggs was characteristic of Suwalska
geese, and the highest of Podkarpacka geese. Before and after reproduction,
Kartuska and Suwalska ganders (groups III and IV) weighed more than Kielecka
and Podkarpacka ganders (groups I and II). Kielecka and Kartuska ganders showed
a slight loss of body weight, and Podkarpacka and Suwalska ganders even a small
body weight gain. In geese of all the groups, a body weight loss of 12.7 (group IV)
to 17.4% (group III) was found.
Table 1. Mean values (x̄) and variation coefficients (v) of egg production, egg weight, feed intake, and
body weight in geese
Group — symbol of geese
Trait
Number of eggs per 1 goose
x̄
Egg weight (g)
x̄
v
Intake per 1 egg (g)*:
feed mixture
x̄
crude protein
x̄
Intake per 1 kg of eggs (g)*:
feed mixture
x̄
crude protein
x̄
Ganders — body weight (g):
before reproduction
x̄
v
after reproduction
x̄
v
difference*
x̄
%
Geese — body weight (g):
before reproduction
x̄
v
after reproduction
x̄
v
difference*
x̄
%
I
Ki
II
Pd
III
Ka
IV
Su
19 c
19 c
23 b
31 a
142.7 c
8.9
149.2 a
9.7
145.4 b
8.5
143.2 bc
10.2
1138
1162
1143
852
208
213
209
156
7945
8171
7663
5859
1454
1490
1402
1072
5071 c
12.0
5138 c
11.5
6135 a
11.1
5633 b
8.7
4766 c
12.3
5189 b
14.9
5816 a
15.4
5793 a
13.8
− 305
6.0
51
1.0
− 319
5.2
160
2.8
4546 b
10.9
4625 b
11.0
5284 a
12.4
5337 a
9.2
3806 c
15.2
3907 c
14.2
4365 b
15.2
4660 a
14.7
− 740
16.3
− 718
15.5
− 919
17.4
− 677
12.7
Ki — Kielecka goose, Pd — Podkarpacka goose, Ka — Kartuska goose, Su — Suwalska goose.
* Significant differences were not calculated statistically.
72
——— laying intensity (%)
– – – egg weight (g)
Figure 1. Laying intensity and egg weight
Suwalska geese (group IV) exhibited the lowest egg fertilization, and Kielecka
geese (group I) the highest. A significantly higher (P < 0.05) percentage of
unhatched goslings was found in southern varieties of geese, and lower in northern
varieties (Table 2). The lowest percentage of gosling hatchability from set eggs was
noted in Suwalska geese, and the highest percentage of gosling hatchability from
fertilized eggs was observed in Kartuska and Suwalska geese (groups III and IV).
Egg fertilization in groups of regional geese declined during reproduction, while
percentage hatchability of goslings from fertilized eggs remained similar during
reproduction (Figure 2). Number of goslings per goose ranged from 9 to 14.
Egg weight estimated at the peak of reproduction was the highest in Kartuska
and Suwalska geese, which were characterized by the greatest shell surface area
(Table 3). Egg shape index was similar in all the groups of geese. The thickest
eggshell was found in Suwalska geese, and the thinnest in Kielecka geese. Eggs of
Kielecka geese (group I) and Kartuska geese (group III) were characterized by the
greatest shell deformation, and those of Suwalska geese (group IV) by the lowest
shell deformation. Significantly (P < 0.05) greater egg shell strength (N) was found
in southern varieties of geese (groups I and II). The highest shell whiteness
percentage was found in Podkarpacka, and the lowest in Kielecka geese.
73
Table 2. Mean values of egg hatch and hatchability of goslings
Trait
Fertilized eggs (%)
Dead embryos (%)
Goslings (%):
unhatched
crippled and weak
healthy from set eggs
healthy from fertilized eggs
Healthy goslings per 1 goose (head)*
Group
I
II
III
IV
71.7 a
3.0 a
62.2 b
2.5 a
64.8 b
2.4 a
53.8 c
2.7 a
19.7 a
0.3 a
55.2 a
77.0 b
10
17.1 a
0.4 a
49.8 b
80.0 b
9
10.9 b
0.3 a
56.0 a
86.4 a
13
12.3 b
0.6 a
45.4 c
84.4 a
14
——— fertilized eggs (%)
– – – healthly goslings from fertilized eggs (%)
Figure 2. Fertilized eggs and healthy goslings from fertilized eggs
Yolk percentage was the greatest in Kielecka (group I) and Suwalska geese
(group IV), the eggs of which were also the lowest in white. Shell percentage and
density in eggs from all groups of geese were similar (Table 4). The greatest yolk
and white density was characteristic of the eggs of Suwalska geese, and the lowest
of Kielecka geese.
74
Table 3. Mean values (x̄) and variation coefficients (v) of egg weight and shell traits in geese
Trait
Egg weight (g)
x̄
v
Egg shape index (%)
x̄
v
Eggshell surface area (cm2)
x̄
v
Shell thickness (mm)
x̄
v
Shell deformation (µm)
x̄
v
Shell resistance (N)
x̄
v
Shell colour (% of white)
x̄
v
Group
I
II
III
IV
140.4 b
8.2
142.7 ab
7.3
149.8 a
7.3
147.2 a
5.0
67.9 a
2.6
66.9 a
5.6
68.2 a
2.6
68.3 a
5.1
127.6 b
5.4
128.9 ab
4.9
133.1 a
4.8
131.7 a
3.3
0.552 b
6.3
0.577 ab
6.4
0.588 ab
9.0
0.595 a
11.3
20.1 a
18.3
19.5 ab
14.4
20.1 a
12.1
17.4 b
15.8
21.7 a
25.9
21.5 a
38.3
16.3 b
28.2
20.6 ab
20.4
77.6 b
5.4
80.4 a
2.5
79.0 ab
4.6
79.7 ab
2.7
Table 4. Mean values (x̄) and variation coefficients (v) for proportion and density of yolk, white, and
eggshell weight in geese
Trait
Proportion of egg (%):
yolk
x̄
v
white
x̄
v
eggshell
x̄
v
Density (g/cm3):
yolk
x̄
v
white
x̄
v
eggshell
x̄
v
Group
I
II
III
IV
34.2 a
9.0
32.7 ab
8.1
32.0 b
7.1
34.7 a
5.3
53.0 bc
5.7
55.4 b
5.0
56.2 a
3.8
53.0 c
3.4
12.0 a
8.1
11.9 a
9.9
11.8 a
4.6
12.3 a
5.5
0.464 b
10.2
0.435 bc
9.4
0.419 c
10.6
0.497 a
6.1
0.356 b
8.0
0.367 ab
7.9
0.387 a
8.9
0.381 a
6.6
2.041 a
3.5
2.039 a
5.5
2.005 a
5.8
1.989 a
6.1
75
The most intense yolk colour was found in the group of Podkarpacka geese
(group II), and the least intense in Suwalska geese (group IV). Yolk index was the
highest in Suwalska geese, and lower and similar in the other groups. Haugh units
in eggs of geese from all the groups were low and similar (Table 5). pH of yolk
was similar in eggs of geese of groups I, III and IV, and lower in eggs of geese of
group II. pH of egg white did not differ significantly (P < 0.05) between the groups.
The largest number of pores at the equator and in the large end of the egg was found
in the shells of Suwalska geese, and in the small end of the eggshells of Suwalska
geese. In Podkarpacka geese, the number of pores was the lowest in shells at the
small and large end of eggs. Blood and meat spots were the most numerous in the
eggs of Suwalska and Kielecka geese.
Table 5. Mean values (x̄) and variation coefficients (v) for yolk colour, yolk index, Haugh units, pH, and
pores of eggshell in geese
Trait
Yolk colour by La Roche scale (pts)
x̄
v
Yolk index (%)
x̄
v
Haugh units
x̄
v
Mean values of pH:
yolk
x̄
v
white
x̄
v
Pores in eggshell (no./0.25 cm2)
small end
x̄
v
equator
x̄
v
large end
x̄
v
Blood and meat spots (%)*
%
Group
I
II
III
IV
4.1 bc
16.4
4.8 a
14.8
4.6 b
17.3
3.7 c
21.2
34.5 b
11.7
33.8 b
6.7
33.9 b
4.4
38.0 a
8.1
53.0 a
26.2
57.8 a
12.5
60.5 a
24.7
56.1 a
36.3
6.097 a
1.5
5.992 b
1.6
6.071 a
1.2
6.121 a
0.8
8.464 a
1.4
8.630 b
1.0
8.634 a
1.4
8.677 a
1.6
16.3 a
9.5
14.4 b
16.9
14.7 ab
20.1
15.5 ab
10.1
15.3 c
17.9
20.6 b
13.2
16.6 c
19.5
23.0 a
12.7
31.7 a
12.0
28.0 b
16.9
32.7 a
9.2
32.9 a
9.7
8.3
8.3
50.0
33.3
Table 6. Mean values (x̄) and variation coefficients (v) of chemical components in yolk and white (A — week 2, B — week 12)
Group
Trait
I
A
Chemical components of
yolk (%):
water
x̄
v
protein
x̄
v
fat
x̄
v
ash
x̄
v
Chemical components of
white (%):
water
x̄
v
protein
x̄
v
ash
x̄
v
II
B
A
III
I − IV
IV
B
A
B
A
B
A
B
41.7 c
2.4
45.4 a
4.0
47.0 b
1.8
44.4 b
3.1
48.2 a
2.3
44.2 b
2.4
48.0 a
1.8
43.8 b
2.6
46.2
2.1
44.4
3.0
17.4 a
2.2
16.7 a
3.8
17.5 a
1.3
16.8 a
3.3
16.9 b
3.7
16.6 a
2.3
17.2 a
2.0
16.7 a
2.4
17.2
2.3
16.7
2.9
32.4 a
2.0
34.5 c
2.3
32.4 a
2.6
35.4 b
1.7
31.9 a
2.4
36.0 a
2.2
31.9 a
2.6
36.5 a
1.9
32.1
2.4
35.4
2.0
2.6 a
10.2
2.6 b
11.2
2.5 a
10.9
2.9 a
6.6
2.7 a
12.6
2.8
8.7
2.6
10.4
84.0 a
0.5
84.7
1.1
84.1
0.8
2.8 ab
8.2
2.6 a
7.9
2.8 ab
9.0
82.9 b
1.6
84.3 a
1.4
83.0 b
1.5
84.0 a
0.9
86.2 a
1.0
84.3 a
0.6
86.6 a
0.7
9.5 b
2.9
9.8 a
3.3
9.4 b
3.3
9.3 b
4.0
10.3 a
3.5
9.4 b
3.3
10.1 a
2.8
9.5 ab
3.8
9.8
3.1
9.5
3.6
1.0 a
6.9
1.1 a
4.3
1.0 a
3.4
1.1 a
4.2
1.0 a
4.7
1.1 a
4.1
1.0 a
2.9
1.1 a
2.8
1.0
4.5
1.1
3.8
Means in rows, separately for A and B, followed by different letters differ significantly (P < 0.05).
77
Chemical composition of egg yolk at the start of the reproductive period
differed significantly within groups except for fat content (Table 6). Water
percentage was the highest in the eggs of Kartuska and Suwalska geese, protein in
the eggs of Kielecka and Podkarpacka geese, and ash in the eggs of Suwalska geese.
Towards the end of reproduction, the proportion of protein and ash in egg yolks did
not differ significantly between the groups. Significant differences were found in
the content of water and fat. The highest content of fat was found in the yolks of
eggs from Kielecka geese, and the highest content of fat in the eggs of Kartuska and
Suwalska geese.
From the start to the end of reproduction, water, protein and ash were found to
decline in egg yolks of geese of groups II, III and IV. Increased fat percentage was
found in all the groups. At the start of reproduction, water and protein percentage in
egg white was greater in Kartuska and Suwalska geese (groups III and IV) than in
Kielecka and Podkarpacka geese. At the end of lay, water and ash content in the
white of eggs was similar in all the groups. Only protein percentage in egg whites of
Suwalska geese was found to decrease. Mean percentages of water and protein were
lower in the white of eggs obtained at the end of lay, and mean percentage of ash
was slightly higher than the value obtained at the start of production.
Discussion
Number of eggs was 19 from one goose of southern variety and 23 to 31 from
one goose of northern variety. Number of eggs from one Astra G goose ranged from
52 to 60 (Mazanowski and Kiełczewski, 1999) and in other experiments from 77 to
103 (Mazanowski and Szukalski, 2000; Mazanowski and Kiełczewski, 2001).
Laying performance of two-way and three-way crosses with graylag ganders ranged
from 20 to 36 eggs depending on the experiment (Mazanowski and Chełmońska,
2000; Mazanowski et al., 2002; Mazanowski and Bernacki, 2003), being close to
the egg production of regional varieties of geese. This indicates that the crossbred
geese are highly similar to the regional geese. Schneider (1987), who selected geese
for egg production in 1978 – 1986, increased the number of eggs per layer from
39 to 48. Shalev et al. (1991), who carried out selection for egg production,
achieved a progress of 2.7 eggs per year. Cheng et al. (2003) reported that geese
selected for several years produced 72 eggs per bird during 156 days. Smalec
(1991) found large differences between geese from genetic reserve and conservative flocks not only in egg production, but also in egg weight, parameters of
hatchability and meat traits. In our experiment, we evaluated regional geese that
were not selected for reproductive and meat traits, i.e. two varieties of southern
geese (Ki, Pd) and two varieties of northern geese (Ka, Su). The productive traits of
these geese did not change in value compared to the findings of Smalec (1991).
Weight of eggs from Polish varieties of geese (Smalec, 1991) averaged 145.1.
Egg weight in Biłgorajska geese was much the same in the first period of lay
(144 g) and greater in the second period (173 g) (Puchajda et al., 2000). Egg weight
78
in Astra G geese in the first and second period of lay was 148.5 and 165.5 g, and in
the other studies it was 163.8 and 156.0 g, respectively (Mazanowski and
Kiełczewski, 1999, 2001; Mazanowski and Szukalski, 2000). Egg weight of Astra G
geese increased from 135.3 g at the start of lay to 148.9 g at the end of reproduction
(Mazanowski and Adamski, 2002). Egg weight in two-way crosses with graylag
geese in the second period of reproduction ranged from 180.6 to 187.9 g (Mazanowski and Chełmońska, 2000). In three-way crosses, it ranged from 145.3 to 152.1 g in
the first period of reproduction and from 182.2 to 185.3 g in the second
(Mazanowski et al., 2002; Mazanowski and Bernacki, 2003). Egg weight in Astra G
geese and in crosses with graylag geese was related to the age of geese. In our
experiment, geese were in their first period of reproduction. The weight of eggs
from these geese was small at the start of reproduction, but as in other studies it
showed an upward tendency.
Dietary and protein intake in terms of one egg of geese of southern and northern
varieties ranged from 852 to 1162 g and from 156 to 213 g in terms of one egg, and
from 5859 to 8171 g and from 1072 to 1490 g in terms of 1 kg of eggs. In Astra
G geese, feed and protein intake averaged 861 and 121 g per 1 egg and 5399 and
759 g per 1 kg of eggs (Mazanowski and Kiełczewski, 2001). In another
experiment, diet intake by Astra G geese in the first and second period of lay
averaged 779 and 657 g per 1 egg, and 5244 and 3970 g per 1 kg of eggs
(Mazanowski and Kiełczewski, 1999). Mazanowski and Szukalski (2000) calculated the intake per 1 egg and per 1 kg of eggs in Astra G geese to be 781 and
4764 g for diet and 111 and 677 g for protein. In three-way crosses with graylag
geese, intake per 1 egg and 1 kg of eggs was 1159 and 6290 g for diet, and 210 and
1137 g for protein (Mazanowski and Bernacki, 2003). The above data reveal that in
regional geese, feed intake per one egg and 1 kg of eggs was greater than in other
geese, which was associated with lower egg production and lower egg weight. In
geese of southern varieties (Ki, Pd) compared to northern varieties (Ka, Su), feed
and protein intake was greater and egg production lower, but the differences were
small.
During reproduction, a slight loss or gain in body weight is usually observed in
ganders. The loss of 5.2 to 6.0% was only found in Kielecka and Kartuska ganders,
and a slight gain in body weight of ganders was noted in the other two groups, i.e.
Podkarpacka and Suwalska (from 1.0 to 2.8%). A fairly high body weight loss was
found in all the geese, ranging from 12.7% in Suwalska geese to 17.4% in Kartuska
geese. Weight loss in Kielecka and Podkarpacka geese was 15.5 to 16.3%. In
regional ganders, a slight increase (by 4.6%) in body weight in the first year and
a decrease (by 4.7%) in the second year were noted from the start to the end of
reproduction. In Astra G geese, body weight declined by as much as 19.0 to 25.4%.
In the second experiment, body weight of White Kołuda crossbred ganders did not
decline, while in Astra G geese it decreased by as much as 17.6% (Mazanowski and
Kiełczewski, 1999; Mazanowski and Szukalski, 2000). On the whole, it is
concluded that the weight loss is greater in geese than in ganders. In ganders, even
a gain in body weight is observed during reproduction. Romanov (1999) links
79
performance of geese not only to the genotype, but also to other factors such as
feeding, environment and prophylaxis.
In high-producing geese, the loss of body weight during reproduction is usually
greater than in poor egg layers. In our study, this observation has been confirmed
for all geese except Suwalska. Therefore, decreased body weight of geese during
reproduction is now always connected with the number of eggs laid. In an
experiment with Astra G in the third laying period, body weight increased from
12.1 to 13.5% (Mazanowski and Kiełczewski, 2001). In another experiment, body
weight of ganders increased by 9.5%, and that of Astra G geese decreased by 15.2%
(Mazanowski and Adamski, 2002). In graylag, White Kołuda and Slovakian
crossbreds, a marked decrease in body weight (by 11.6 to 24.0%) occurred in both
ganders and geese. Interestingly, body weight loss in Graylag goose crossbreds is
greater than in other geese.
In the present study, the greatest fertilization of eggs was noted in Kielecka
geese (group I), and the highest hatchability of goslings from fertilized eggs in
Kartuska geese (group III). A positive relationship was found between shell
thickness and hatchability of goslings from fertilized eggs. Fertilization ranged
from 62.8 to 73.2% in Kielecka ganders crossed with Astra G geese, and from 55.8
to 75.5% in Kartuska ganders. Hatchability of goslings was 60.7 to 66.1% in
Kielecka geese, and 57.6 to 63.8% in Kartuska geese (Mazanowski and Kiełczewski, 1999). In Ka’W33 or W33’Ka ganders crossed with Astra G geese, egg
fertilization ranged from 22.7 to 72.3%, and number of goslings per one goose from
16 to 53. In Pd’W33 and W33’Pd ganders crossed with Astra G geese, egg
fertilization ranged from 34.5 to 78.0%, and the number of goslings obtained from
26 to 58.
In Ki’W33 and W33’Ki ganders crossed with Astra G geese, egg fertilization ranged
from 34.7 to 68.1%, and the number of goslings obtained from 25 to 53. In Su’W33
and W33’Su ganders, egg fertilization was high and ranged from 71.4 to 73.3%, and
the number of hatched goslings varied between 52 and 59 (Mazanowski and
Szukalski, 2000). Poorer results of egg fertilization and gosling hatchability were
obtained in similar crossbreds in other experiments (Mazanowski and Kiełczewski,
2001; Mazanowski and Adamski, 2002). In crossbreds with graylag geese, egg
fertilization ranged from 27.5 to 84.7%, and hatchability of goslings from fertilized
eggs from 64.0 to 85.4% (Mazanowski et al., 2002). In three-way crossbreds of
ganders and geese (similar ganders crossed with similar geese), very good
fertilization of eggs (83.3 to 92.7%) and good hatchability from fertilized eggs (71.7
to 79.0%) were noted (Mazanowski and Bernacki, 2003). The highest egg
fertilization was found in Kielecka geese, in which hatchability of goslings from
fertilized eggs was the lowest. Kartuska geese were characterized by medium
fertilization of eggs, but showed the greatest hatchability from fertilized eggs.
Similar patterns were noted in Kielecka and Kartuska crossbreds.
The greatest shell weight and area were characteristic of eggs of Kartuska geese
(group III), and the smallest of Kielecka geese (group I). Egg index were similar in
all the groups. Puchajda et al. (2000) reported for Biłgorajska geese lower egg
80
shape indices, both in the first and the second laying period. Shell thickness was the
smallest in Kielecka geese (group I) and the greatest in Suwalska geese (group IV).
In Biłgorajska geese, shell thickness was lower than in the present study and
amounted to 0.527 and 0.534 mm in the first and second laying periods,
respectively (Puchajda et al., 2000). The greatest shell deformation was found in the
eggs of geese of groups I and II. Shell strength (N) was greater in geese of southern
varieties than in those of northern varieties. Eggshells of Podkarpacka geese (group
II) had the greatest whiteness percentage. Egg shape indices were greater in Astra
G geese than in geese from genetic reserve flocks. Three-way crosses with the wild
graylag goose did not differ in egg weight, egg shape index, eggshell area, shell
thickness and shell deformation. Egg weight and shell parameters were greater in
the three-way crosses than in the present experiment (Mazanowski and Adamski,
2002; Mazanowski and Bernacki, 2003).
In the present study, the greatest percentage of yolk was found in the eggs of
Kielecka and Suwalska geese (groups I and IV), and the greatest percentage of
white in Kartuska geese (group III). Both percentage and density of shell in egg did
not differ significantly between the groups. Yolk and white density was the greatest
in eggs of Suwalska geese (group IV). The lowest density was characteristic of yolk
of Kartuska geese (group III) and of white of Kielecka geese (group I). In Astra G at
the peak of egg production, percentage of white was similar, yolk percentage much
the same, and shell percentage greater (Mazanowski and Adamski, 2002) compared
to our findings. In three-way crosses with graylag geese, yolk, white and shell
percentage and density (Mazanowski and Bernacki, 2003) were similar as in the
current study. In Biłgorajska geese (Puchajda et al., 2000) yolk percentage in egg
was lower than in our study, but weight of white and shell was greater in
Biłgorajska geese. This points to great differences in the structure of eggs of
regional goose varieties. These differences also depend on when the eggs were
collected for analysis.
Yolk colour in geese from Polish regional flocks, expressed in points, was the
most intense in Podkarpacka geese (group II) and the least intense in Suwalska
geese (group IV) which laid more eggs. Yolk index was the greatest in eggs of
Suwalska geese (group IV). Haugh units assumed similar and intermediate values
that were lower than in other experiments. pH of yolk was greater in eggs of the
geese analysed in the present study than in eggs of graylag crossbreds. In graylag
crossbreds, the number of pores at the small end and equator of the egg was similar
in all the experimental groups, but differed between the groups only in the large end
of egg. Geese from conservation flocks had more pores at the small and large end,
and less pores in the equator compared to graylag crossbreds (Mazanowski and
Adamski, 2002; Mazanowski and Bernacki, 2003).
At the start of lay, water in yolk was the highest in the eggs of Kartuska geese
(group III) and the lowest in the eggs of Podkarpacka geese (group II). At the end of
lay, the greatest amount of water was found in the egg yolks of Kielecka geese
(group I). During the laying period, amount of water decreased in yolks of all eggs.
At the start of lay, white percentage was the lowest in group III. During the laying
81
period, the amount of protein decreased, and that of fat increased. Increased fat
content was only found at the end of lay. The amount of fat in yolk was the greatest
in the eggs of Kartuska and Suwalska geese (groups III and IV). Changes in
percentage of ash in yolk were noted only at the start of lay, and the greatest amount
of ash was found in the eggs of Suwalska geese (group IV). Ash content decreased
during the entire laying period.
Percentage of water in eggs of Kartuska and Suwalska geese (groups III and IV)
was greater than in Kielecka and Podkarpacka geese (groups I and IV) at the start of
lay. At the end of lay, no differences were found between the groups, and
percentage of water during reproduction decreased slightly in all the groups. At the
start of lay, protein content in egg white was greater in the eggs of Kartuska and
Suwalska geese than in the eggs of Kielecka and Podkarpacka geese. At the end of
the reproductive period, eggs of Kielecka geese contained the most protein and
those of Podkarpacka and Kartuska geese the least. The amount of protein in egg
white decreased during the entire reproductive period. Percentage of ash in egg yolk
did not differ significantly between the groups at the end of lay, and in egg white at
the start and at the end of lay.
Geese of regional flocks differ in the number of eggs laid, egg weight, egg
fertilization and gosling hatchability. Egg fertilization in geese of regional varieties
(Ki, Pd, Ka, Su) decreased during reproduction, whereas percentage of gosling
hatchability was similar all the time. During reproduction, there was a body weight
loss of 12.7 to 17.4% in geese, and in ganders a slight decline in body weight in
Kielecka and Kartuska ganders (5.2 to 6.0%).
Eggs of regional geese, evaluated at the peak of egg production, did not differ
between the groups only in terms of egg index, eggshell percentage, and shell
density. Between groups no differences were noted in Haugh units and in pH of egg
white. pH of yolk was greater in the eggs of regional geese than in the eggs of other
goose varieties or crossbreds. In regional geese, more pores were found at the small
and large end of the egg, and less pores at the equator, than in other varieties of
geese.
Chemical composition of eggs at the start and at the end of lay differed between
the groups of geese in terms of percentage of water and ash at the start, and in terms
of percentage of fat at the end of lay. Composition of egg white differed in
percentage of water only at the start of lay, and in percentage of protein at the start
and at the end of lay.
During the entire period of reproduction, decreases in the percentage of water,
protein and ash, and an increase in fat were noted in yolk. In egg white, water and
protein decreased and ash slightly increased during the whole reproductive period.
Taking into account the decreased percentages of water and protein, and the
increased percentages of fat in eggs, changes in feeding and in the incubation
technique of eggs of regional goose varieties should be considered in the second
period of reproduction. The study showed biodiversity of the geese in reproductive
traits and in the structure and chemical composition of eggs.
82
References
C h e n g Y.S., R o u v i e r R., H u Y.H., T a i J.J.L., T a i C. (2003). Breeding and genetics of waterfowl.
World’s Poultry Sci. J., 59, 4: 509 – 519.
K r e ł o w s k a - K u ł a s M. (1993). Badania jakości produktów spożywczych. PWE, Warszawa, s. 97.
M a z a n o w s k i A., A d a m s k i M. (2002). Evaluation of reproductive traits and egg quality in Astra
G geese during the first reproductive period. Ann. Anim. Sci., 2, 2: 67 – 78.
M a z a n o w s k i A., B e r n a c k i Z. (2003). Porównanie wyników reprodukcji i jakości jaj potrójnych
mieszańców z ge˛sia˛ ge˛gawa˛. Rocz. Nauk. Zoot., 30, 1: 171 – 184.
M a z a n o w s k i A., C h e ł m o ń s k a B. (2000). The effects of reciprocal crossing of White Kołuda and
graylag crossbred geese with Slovakian geese. Ann. Anim. Sci. — Rocz. Nauk. Zoot., 27, 4:
85 – 103.
M a z a n o w s k i A., K i e ł c z e w s k i K. (1999). Wyniki reprodukcji ge˛si ze stad rezerwy genetycznej
i ge˛si białych kołudzkich w dwóch okresach nieśności. Rocz. Nauk. Zoot., 26, 1: 55 – 72.
M a z a n o w s k i A., K i e ł c z e w s k i K. (2001). Reproductive performance of conservative flock
× White Kołuda ganders of geese crossed with Astra G geese in the third period of laying. Ann.
Anim. Sci., 1, 1: 67 – 79.
M a z a n o w s k i A., S z u k a l s k i G. (2000). Porównanie wyników reprodukcji ge˛si mieszańców i ge˛si
białych kołudzkich w drugim okresie nieśności. Rocz. Nauk. Zoot. — Ann. Anim. Sci., 27, 1:
141 – 156.
M a z a n o w s k i A., D z i a d e k K., A d a m s k i M. (2002). Cechy reprodukcyjne i mie˛sne mieszańców
potrójnych z udziałem ge˛si ge˛gawych. Rocz. Nauk. Zoot., 29, 1: 105 – 120.
P a g a n e l l i C.V., O l s z o w k a A., A r A. (1974). The avian egg: surface area, volume, and density. The
Condor, 76, 3: 319 – 325.
P u c h a j d a H. (1991). Kształtowanie sie˛ cech użytkowych i ich parametrów genetycznych w stadzie
zachowawczym ge˛si biłgorajskich. Acta Acad. Agricult. Tech. Olst. Zoot., 34: 3 – 59.
P u c h a j d a H., P u d y s z a k K., M r ó z E., C y w a - B e n k o K. (2000). Ultrastruktura skorup i budowa
makroskopowa jaj ge˛si białych kołudzkich i biłgorajskich. Rocz. Nauk. Zoot. — Ann. Anim. Sci.,
27, 1: 65 – 78.
R o m a n o v M.N. (1999). Goose production efficiency as influenced by genotype, nutrition and
production systems. World’s Poultry Sci. J., 55, 3: 281 – 294.
R o s i ń s k i A. (2000). Analiza bezpośrednich i skorelowanych efektów selekcji w dwóch rodach ge˛si.
Rocz. AR Poznań, 309: 5 – 107.
S c h n e i d e r K.H. (1987). Erhöhung der Legeleistung bei Gänsen durch eine effektive Selektion auf
Eizahl. Tierzucht, 41, 9: 416 – 418.
S h a l e v B.A., D v o r i n A., H e r m a n R., K a t z Z., B o r n s t e i n S. (1991). Long-term goose breeding
for egg production and crammed liver weight. Brit. Poultry Sci., 34, 4: 703 – 709.
S m a l e c E. (1991). Zróżnicowanie ge˛si rezerwy genetycznej pod wzgle˛dem cech użytkowych i polimorfizmu białek surowicy krwi. Zesz. Nauk. Drob., COBRD, Poznań (rozpr. hab.), 3:
5 – 87.
W i l l i a m s K.C. (1997). Some factors affecting albumen quality with particular reference to Haugh unit
score. World’s Poultry Sci. J., 48, 1: 5 – 16.
World Watch List for Domestic Animal Diversity (2000). 3rd edition. FAO, UNDP.
83
ADAM MAZANOWSKI, TOMASZ KISIEL, MAREK ADAMSKI
Ocena cech reprodukcyjnych oraz budowy i składu chemicznego jaj wybranych odmian
ge˛si regionalnych
STRESZCZENIE
Materiał doświadczalny stanowiły ge˛si kieleckie (Ki), podkarpackie (Pd), kartuskie (Ka) i suwalskie (Su), be˛da˛ce w pierwszym roku reprodukcji. Ge˛si ze stad regionalnych różnia˛ sie˛ pod wzgle˛dem
liczby składanych jaj, masy jaj oraz zapłodnienia jaj i wyle˛gowości piskla˛t. Ge˛si kieleckie i podkarpackie zniosły mniej (19), a kartuskie i suwalskie wie˛cej (23 do 31) i cie˛ższych jaj. Masa
jaj ge˛si w okresie reprodukcji wykazywała tendencje˛ wzrostowa˛. Zużycie paszy na jedno jajo i na 1 kg
jaj było w porównaniu z innymi ge˛siami wie˛ksze, co wia˛zało sie˛ z mniejsza˛ nieśnościa˛ i mniejsza˛ masa˛
jaj. W okresie reprodukcji ubytek masy ciała ge˛si wynosił od 12,7 do 17,4%. Ubytek masy ciała
u ge˛siorów stwierdzono tylko u kieleckich i kartuskich (5,2 do 6,0%). Zapłodnienie jaj u ge˛si
regionalnych malało w czasie reprodukcji, natomiast procent wyle˛gu piskla˛t był podobny przez cały
czas.
Jaja ge˛si regionalnych oceniane w szczycie nieśności nie różniły sie˛ mie˛dzy grupami tylko pod
wzgle˛dem indeksu jaja, procentowego udziału skorupy w jaju i ge˛stości skorupy. Nie stwierdzono też
mie˛dzy grupami różnic w jednostkach Haugha i w pH białka jaja. Odczyn pH żółtka był wie˛kszy
w jajach ge˛si regionalnych niż w jajach innych odmian lub mieszańców ge˛si. Na ostrym i te˛pym końcu
jaja u ge˛si regionalnych stwierdzono wie˛cej porów, a w cze˛ści równikowej mniej porów w porównaniu
z innymi odmianami ge˛si.
Skład chemiczny jaj na pocza˛tku i na końcu nieśności różnił sie˛ mie˛dzy grupami ge˛si pod
wzgle˛dem procentowego udziału wody, natomiast w przypadku białka i popiołu na pocza˛tku, a tłuszczu
na końcu nieśności. Skład białka jaja różnił sie˛ pod wzgle˛dem procentowego udziału wody tylko na
pocza˛tku, a białka na pocza˛tku i na końcu nieśności.
W całym okresie reprodukcji zanotowano w żółtku jaja zmniejszenie udziału wody, białka
i popiołu, a zwie˛kszenie zawartości tłuszczu. W białku jaja stwierdzono w całym okresie reprodukcji
zmniejszenie udziału wody i białka, a nieznaczne zwie˛kszenie udziału popiołu. Biora˛c pod uwage˛
zmniejszenie udziału wody i białka, a zwie˛kszenie udziału tłuszczu w jajach, należy rozważyć
wprowadzenie zmian w żywieniu i technice le˛gów jaj ge˛si regionalnych w drugiej cze˛ści okresu
reprodukcji. Wykazano bioróżnorodność ge˛si w cechach reprodukcyjnych oraz w budowie i składzie
chemicznym jaj.
FACTORS AFFECTING THE OOCYTE RECOVERY RATE
FROM OVARIAN FOLLICLES OF GILTS
J a r o s ł a w W i e c z o r e k, M i r o s ł a w C e g ł a, W i e s ł a w K a r e t a
Department of Animal Reproduction Biotechnology, National Research Institute of Animal Production,
Abstract
Repeated recovery of oocytes in vivo from farm animals requires that non-invasive methods be
elaborated. In this study, equipment was selected and appropriate operational techniques were
mastered. A Circon endocamera linked to a monitor enabled the training to be carried out on
a specially made trainer (simulator). The factors analysed were vacuum pressure, needle thickness
and the size of aspirated oocytes. The results indicate that low vacuum pressure (0.2 bar)
and 0.7 and 0.8 mm thick needles are the optimum choice. The highest oocyte recovery rate was
obtained from 2 – 4 mm diameter follicles, while 5 mm follicles showed the best morphological
quality.
Key words: gilt, collection of oocytes, non-invasive technique
The growing interest in the collection and culture of mammalian oocytes results
from developments in the embryo transfer technique. The application of this
method on a larger scale is conditional on sufficient amounts of material. Maturing and then fertilized oocytes may provide an important source of oocytes
(Ka˛tska, 1995; Samiec et al., 2003; Smora˛g and Gajda, 1999). One of the
fundamental problems, which has not been completely solved, is the recovery
of oocytes. Post-slaughter methods concern the slaughter material, while from farm
animals it is necessary to repeatedly recover oocytes in vivo. Hence the need to
develop a non-invasive method that is at the same time effective.
The objective of the present study was to develop new techniques for repeated
recovery, from the same animals, of oocytes useful for culture and fertilization in
vitro. The present stage was aimed to select proper equipment and to learn how to
86
J. Wieczorek et al.
operate it using prepared organs from a training kit (trainer), which will find
application in endoscopy at the later stages of the research.
The material for analysis was taken in an abattoir from randomly chosen gilts in
good condition during their natural sexual cycle. The gilts were 8 – 12 months
of age and weighed 80 – 110 kg. Reproductive organs, collected directly after
slaughter from 60 gilts, were stored for 1.5 to 4 h at 38 – 40°C. Ovaries were
rinsed with sterile physiological salt solution at 38°C. Oocytes were aspirated
from follicular fluid by puncturing the ovarian follicles of 2 – 6 mm diameter
(Ka˛tska-Ksia˛żkiewicz, 2003). Analysis was made on 3 vacuum pressure ranges
(0.2, 0.4 and 0.6 bar), 4 needle thicknesses (0.5, 0.6, 0.7 and 0.8 mm) and 2 sizes
of aspirated follicles (2 – 4 and 5 mm). A specially made trainer (simulator) was
used for the tests. It is an encased chamber with openings for laparoscopic
equipment and contains the prepared organs. The improvements in the immobilization of ovaries and aspiration of ovarian follicles could be viewed
on a monitor thanks to the use of Circon endocamera. A modified pump used for
recovery of bovine oocytes with the ovum pick-up (OPU) procedure was also used
(Smora˛g and Gogol, 1997). During aspiration, we ensured that the distance between
the follicle and the bulk container should be minimized. Follicular fluid was
collected with 4 injection needles sized from 0.5 to 0.8 mm (25 — 21 G) and
connected to the syringe stiffly or through a tubing. One of the oocyte collection
variants was the use of our specially made equipment of 1 ml capacity and a needle
of 0.6 mm diameter (23 G) (Polish Patent Application P-361068, 2003). Prior
to aspiration of follicles, in order to prevent agglutination and sticking of cells,
the needle, ducts and the bulk container were rinsed with warm PBS solution
supplemented with heparin (50 i.u./100 ml PBS). Punctures were made between
stroma of the ovary and follicular wall to limit the loss of follicular fluid.
The aspirated fluid was introduced into the container filled with 5 ml PBS. Oocytes
were found and analysed using a stereomicroscope (25 – 40×). Then oocytes were
rinsed three times with PBS medium at 38°C and subjected to careful morphological selection. They were assayed according to the following criteria:
I (+) — oocytes morphologically normal, surrounded by a dense and cohesive layer
of the ovarian cumulus cells, having evenly granulated cytoplasm, showing no
changes; II (+/–) — oocytes with unchanged cytoplasm and 1 or 2 layers of
granulosa cells; III (–) — oocytes showing signs of cytoplasm degeneration, with
changes in the ovarian cumulus cells or with both types of changes concurrently
were classified as atresic cells (Ka˛tska-Ksia˛żkiewicz, 2003). Oocytes of groups
I (+) and II (+/–) were classified as useful for in vitro culture, and those of group III
(–) as useless.
87
Factors affecting oocyte recovery from ovarian follicles
Table 1. Aspiration of gilt ovarian follicles depending on the parameters analysed
No.
of aspirated
follicles
Parameter
total
no.
%
Oocytes collected
useful for culture
gr. I (+)
gr. II (+/–)
useless
gr. III (–)
no.
%
no.
%
no.
%
Vacuum pressure
(bar)
0.2
0.4
0.6
210
328
306
84
121
105
40.0
36.9
34.3
15
16
10
17.9
13.2
9.5
28
36
31
33.3
29.7
29.5
41
69
64
48.8
57.0
60.9
Needle
diameter (mm)
0.5
0.6
0.7
0.8
78
181
664
190
24
59
249
62
30.8
32.6
37.5
32.6
3
16
27
14
12.5
27.1
10.8
22.6
5
7
71
24
20.8
11.9
28.5
38.7
16
36
151
24
66.7
61.0
60.6
38.7
Follicle size (mm)
2–4
5
613
181
311
59
50.7
32.6
26
12
8.4
20.3
58
15
18.6
25.4
227
32
73.0
54.2
194
76
39.2
27
35.5
11
14.5
38
50.0
Oocyte recovery
equipment
Results
In the three ranges of vacuum pressure, we found improved recovery of oocytes
as the pressure decreased from 0.6 to 0.4 and 0.2 bar (34.3, 36.9 and 40.0%), as well
as increased frequency of oocytes in the group qualified for culture (39.0, 42.9 and
51.2% respectively) (Table 1). An important role was played by the needle
diameter, which was the most favourable when it exceeded 0.6 mm. Lowest
recovery rate (30.8%) was ascertained for a 0.5 mm needle and highest (37.5%) for
a 0.7 mm needle. In the morphological assessment, the number of oocytes qualified
for culture increased with increasing needle thickness, from 33.3 to 39.0, 39.3 and
61.3%. When classifying follicles according to size, the best recovery rate (50.7%)
was obtained in the group of small (2 – 4 mm) follicles, while suitability for oocyte
culture was better in the group equal to or greater than 5 mm (20.3%). The best
results were obtained when using the oocyte recovery equipment (Polish Patent
Application P-361068, 2003), both for oocyte recovery (39.2%) and the number of
oocytes qualified for culture (50%).
From a total of 120 ovaries, 1113 follicles were aspirated and 394 oocytes
(35.4% recovery) were obtained. After morphological assessment, 42.4% oocytes
were qualified for culture (60 from group I and 107 from group II), and 57.6% were
rejected (227 oocytes from group III).
Discussion
In vivo recovery of oocytes from ovarian follicles, which is widely used in human
medicine, also found application in breeding large animals (Brussow et al., 1997;
88
J. Wieczorek et al.
Graff et al., 1999; Smora˛g and Gogol, 1997). However, this method was limited by
a relatively low efficiency, which varied widely from 30 to 80%. Of the recovered
oocytes, 40 – 80% were qualified for further culture (Brussow and Ratky, 1994;
Brussow et al., 1997, 2002). The results and efficiency of this method is determined
by a number of technical factors. In our study, we tested the procedures developed
by other authors in terms of culture media, heparin and follicle aspiration method.
Treatments were magnitude of the vacuum pressure, needle thickness and size of
aspirated follicles. Laparoscopic equipment was applied to prepared reproductive
organs of gilts, which were placed in the ‘‘trainer”. The problem of ovary
stabilization method was resolved by the use of appropriate and more precise ends
of the manipulators. In all the procedures, following the first unsuccessful attempts,
we strived to minimize the distance between the follicle and the bulk container. For
the same reasons, we abandoned connecting the needle with a tubing in favour of
stiff connections that are more useful in laparoscopic technique. The results of
oocyte recovery and aspiration indicate that low vacuum pressures of 0.2 bar and
0.7 and 0.8 mm needles are optimal. Using identical parameters of vacuum pressure
and needle thickness, we achieved 35 to 79% efficiency with the OPU method, with
a much greater number of oocytes qualified for culture (Smora˛g and Gogol, 1997).
When using similar parameters of vacuum pressure and needle thicknesses in goats,
the transvaginal (OPU variant) and laparoscopic retrieval of oocytes gave similar
recovery rates of 61 and 71.5% (Graff et al., 1999). In pigs, as reported by Brussow
et al. (2002) and Torner et al. (2004), in vivo procedure assures 45 to 80% oocytes
with the laparoscopic method. Similar to our study, the above authors reported that
recovery rate and oocyte quality decreased with increasing vacuum pressure —
above all, the number of ‘‘naked” oocytes, i.e. those deprived of the ovarian
cumulus cells, increased (Brussow et al., 1997). Many authors (Brussow and Ratky,
1994; Torner et al., 2004) stress the importance of the size and maturity of aspirated
follicles. With increasing diameter of follicles, they achieved much higher efficiency and better quality of retrieved oocytes. Follicles aspirated directly before the
ovulation proved the best. In the study under discussion, a higher oocyte recovery
rate was obtained from small, non-convex follicles with low fluid pressure, while in
qualitative terms, better oocytes were obtained from mature oocytes of > 5 mm
diameter. Interesting results were obtained when specially made equipment was
used for oocyte retrieval. The recovery rate of over 39 and 50% oocytes suitable
for culture is comparable with the findings of other authors (Brussow et al.,
2002; Smora˛g and Gogol, 1997). This procedure, despite its limited efficiency,
can already now be used to aspirate follicles obtained from highly valuable
animals.
Based on the present research, it is concluded that the vacuum pressure ranges,
the needle thicknesses and the size of aspirated follicles affect the oocyte recovery
rate and oocyte quality. This means that they should be taken into account when
adapting equipment for laparoscopy.
Factors affecting oocyte recovery from ovarian follicles
89
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B r u s s o w K.P., T o r n e r H., R a t k y J., H u n t e r M.G., N u r n b e r g G. (1997). Ovum pick up in
swine: the influence of aspiration vacuum pressure on oocyte recovery from preovulatory follicles.
Acta Vet. Hung., 45 (2): 189 – 196.
B r u s s o w K.P., R a t k y J., T o r n e r H., E g e r s z e g i I., S c h n e i d e r F., S o l t i L., T u c h s c h e r e r A. (2002). Follicular and oocyte development in gilts of different age. Acta Vet. Hung., 50 (1):
101 – 110.
G r a f f K.J., M e i n t j e s M., D y e r V.W., P a u l J.B., D e n n i s t o n R.S., Z i o m e k C., G o d k e R.A.
(1999). Transvaginal ultrasound-guided oocyte retrieval following FSH stimulation of domestic
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K a˛t s k a L. (1995). Możliwości i ograniczenia pozaustrojowych metod uzyskiwania oocytów i zarodków ssaków. Biotechnologia, 3 (30): 33 – 43.
K a˛t s k a - K s i a˛ż k i e w i c z L. (2003). Dotychczasowe osia˛gnie˛cia i perspektywy hodowli przedi wczesnoantralnych pe˛cherzyków jajnikowych ssaków. Biotechnologia, 1 (60): 129 – 137.
S a m i e c M., S k r z y s z o w s k a M., S m o r a˛g Z. (2003). Effect of activation treatments on the in vitro
developmental potential of porcine nuclear transfer embryos. Czech. J. Anim. Sci., 48 (12):
499 – 507.
S m o r a˛g Z., G a j d a B. (1995). Witryfikacja oocytów i zarodków ssaków. Biotechnologia, 3 (30):
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S m o r a˛g Z., G o g o l P. (1997). Aspiracja bydle˛cych pe˛cherzyków jajnikowych pod kontrola˛ USG
u stymulowanych i nie stymulowanych jałówek i krów. Rocz. Nauk. Zoot., 24, 3: 77 – 83.
T o r n e r H., B r u s s o w K.P., A l m H., R a t k y J., P o h l a n d R., T u c h s c h e r e r A., K a n i t z W.
(2004). Mitochondrial aggregation patterns and activity in porcine oocytes and apoptosis in
surrounding cumulus cells depends on the stage of pre-ovulatory maturation. Theriogenology, 61
(9): 1675 – 1689.
JAROSŁAW WIECZOREK, MIROSŁAW CEGŁA, WIESŁAW KARETA
Czynniki wpływaja˛ce na efektywność uzyskiwania oocytów z pe˛cherzyków jajnikowych loszek
STRESZCZENIE
Przyżyciowe i wielokrotne uzyskiwanie oocytów od zwierza˛t hodowlanych wymaga opracowania
mało inwazyjnych metod. Prezentowany etap polegał na doborze sprze˛tu i aparatury oraz opanowaniu
techniki posługiwania sie˛ nimi. Endokamera Circon w poła˛czeniu z monitorem umożliwiała prowadzenie treningu na wykonanym „symulatorze — trenażerze”. Czynnikami badanymi były: wielkość
podciśnienia, grubość igły oraz rozmiar aspirowanych pe˛cherzyków. Wyniki wskazuja˛ jako optymalne
rozwia˛zania: niskie podciśnienie (0,2 bara) oraz grubość igieł 0,7 i 0,8 mm. Najwyższy odzysk oocytów
uzyskano z pe˛cherzyków o średnicy 2 – 4 mm, zaś najlepsza ich jakość morfologiczna okazała sie˛
w grupie pe˛cherzyków 5 mm.
IRON-INDUCED LUMINESCENCE OF BOAR SPERMATOZOA CELLS*
Piotr Gogol
Abstract
Induced luminescence of boar spermatozoa was assayed with a luminometer. The possibility of
increasing the ultraweak luminescence signal using luminol and the relationship of luminescence
intensity and kinetics to sperm concentration in the sample and to FeSO4 concentration in the
luminescence-inducing solution were analysed. The study showed that luminescence can be greatly
increased by adding 10 µl 5mM luminol solution to 0.7 ml sperm suspension. Sperm concentration
in the analysed sample was shown to affect the intensity and kinetics of luminescence. It was found
that for Fe(2+)-induced luminescence, the relationship between Fe(2+) ions concentration and
luminescence intensity exhibits a bell-shaped curve (characteristic of induced lipid peroxidation)
and achieves a maximum at 0.05 mM FeSO4. The results obtained prove that induced sperm
luminescence can be assayed with a luminometer and confirm that a key role in the formation of
sperm luminescence is played by free-radical processes of membrane lipid peroxidation.
Key words: boar, semen quality, ultraweak luminescence, lipid peroxidation
All living cells emit very weak electromagnetic radiation (ultraweak luminescence), which is related to the metabolic processes therein (Sławiński et al., 1992;
Godlewski et al., 1997). We distinguish spontaneous luminescence, characteristic
of undisturbed homeostasis of the cell, and induced luminescence, generated
by the influence of external physico-chemical factors (such as light, temperature
and various chemical compounds) on the cell (Sławiński, 1989). The close
association between luminescence and the state of the biological system allows
parameters of luminescence to be used for integral assessment of homeostasis
disturbances, the rate of metabolic processes and the adaptability of organisms to
a changeable environment (Sławiński, 1991; Popp, 1984). For many biological
* This work was conducted as part of the research project no. 3 P06D 040 24, financed by the State
Committee for Scientific Research.
92
P. Gogol
systems, correlations have been proved between intensity and harmfulness
of the stress factor and the parameters of ultraweak luminescence (Sławiński et al.,
1992).
Under laboratory conditions, semen is exposed to many stress factors such as
non-physiological changes in temperature, oxygen, light and other radiation,
changes in pH value, the effect of semen diluents that contain cryoprotectors,
mechanical factors connected with the centrifugation, and other factors that can
significantly alter the properties of spermatozoa. The effect of stress factors on
semen quality can be tested by determining their physiological and biochemical
parameters. Measurements of the biophysical phenomenon of ultraweak luminescence can become a new research procedure, which will give insights into sperm
biology and allow further progress in semen preservation and artificial insemination
of animals.
Research shows that central to the generation of ultraweak luminescence is the
formation and breakdown of free radicals in non-enzymatic and enzymatic
oxidative reactions of biological membrane components (Sławiński, 1989). A significant relationship between damage caused by free radicals and sperm dysfunction in animals was reported by Aurich et al. (1997). These authors demonstrated
a protective action of the antioxidant ascorbic acid towards integrity of sperm
cell membranes in stallion semen stored at reduced temperature. Other studies
with stallion semen (Baumber et al., 2000) showed a detrimental effect of free
radicals on sperm motility. Animal spermatozoa studies that employed very
sensitive equipment for measurement of ultraweak luminescence (using single
photon counting technique) demonstrated an extremely low intensity of spontaneous emission (close to background level) from physiologically normal sperm
of the bull, ram and boar. A possibility was also shown to induce luminescence of
much greater intensity than the intensity of spontaneous luminescence by exposing
spermatozoa to a variety of environmental stress factors (Laszczka et al., 1995).
Similarities were also revealed between spectral distribution of the photon emission
from sperm and spectral distribution of the photon emission induced from model
lipid systems, which supports the hypothesis about the role of free-radical processes
of membrane lipid peroxidation in decreased viability of spermatozoa (Laszczka
et al., 1993). The relationship shown between parameters of luminescence from
spermatozoa and their physiological state allowed a hypothesis that the effect
of the intensity of adverse environmental factors on sperm cell can be determined
from the parameters of induced luminescence (Gumińska et al., 1997; Sławiński et
al., 1998). The use of luminescence measurements for practical semen quality
assessment is conditional on the applicability of measuring equipment available on
the market.
This study was designed to determine the possibility of using a luminometer
for analysing ultraweak luminescence (photon emission) of boar spermatozoa and
to determine the optimal conditions of its measurement, allowing for emission
with the maximum ratio of the registered signal to the background (equipment
noise).
Iron-induced luminescence of boar spermatozoa cells
93
Preparation of semen samples
Fresh boar semen was analysed. Directly after collection, semen was diluted
with 0.9% NaCl solution and centrifuged twice (10 min/600 g) to separate sperm
from plasma. Separated sperm were diluted with 0.9% NaCl solution to the required
concentration.
Measurements of luminescence
Luminescence was measured using a Berthold AutoLumat LB953 luminometer
equipped with a cooled photomultiplier with spectral sensitivity range of
370 – 620 nm. Luminescence kinetics was recorded during 350 s and its selected
parameters were determined.
Effect of luminol concentration on an increase in the induced luminescence signal
To a 0.7 ml plasma-free sample of semen with sperm concentration of
200 million/ml was added 10 µl of the luminol solution in DMSO. Solutions with
1 mM, 5 mM, 10 mM, 15 mM and 25 mM concentrations of luminol were used.
The sample containing 10 µl DMSO without luminol served as the control.
Luminescence was induced by adding 100 µl 0.4 mM of FeSO4 solution to the
sample.
Effect of sperm concentration in the analysed sample on luminescence intensity and
kinetics
Sperm separated from the plasma was diluted to concentrations of 25, 50, 100 or
200 million/ml. To a sample of 0.7 ml was added 10 µl 5 mM of luminol solution
and placed in the luminometer. Then 100 µl 0.4 mM of FeSO4 solution was added
and luminescence kinetics was recorded.
Effect of ferrous sulfate on luminescence intensity
To a 0.7 ml plasma-free sample with sperm concentration of 200 million/ml
containing 10 µl luminol solution (5 mM) was added 100 µl ferrous sulfate (FeSO4)
solution with a concentration of 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 or 1.4 mM and
luminescence was measured.
Results
Supplementation of the semen sample with luminol increased the intensity of
luminescence. The maximum increase in the luminescence signal was obtained
when using a 5 mM luminol solution (Figure 1).
Sperm concentration of the analysed sample was shown to influence luminescence intensity and kinetics (changes in luminescence kinetics over time). As
sperm concentration of the sample increased, an increase was observed in intensity
94
P. Gogol
of luminescence (total pulses measured). With a higher concentration of sperm in
the sample, luminescence intensity increased more rapidly, and, after reaching the
maximum, underwent a decline (Figure 2). Maximum luminescence intensity was
higher and more pronounced in samples with higher sperm concentration.
Figure 1. Amplification of the induced sperm luminescence signal depending on luminol concentration
Figure 2. Effect of sperm concentration in a sample on kinetics of induced luminescence
It was found that the curve depicting the relationship between Fe(2+) ions
concentration and induced luminescence intensity exhibits a bell shape and
achieves a maximum at a final concentration of 0.05 mM FeSO4 (Figure 3).
95
Figure 3. Effect of ferrous sulfate concentration on intensity of induced luminescence of boar
spermatozoa (n = 8)
Discussion
The measurements of induced luminescence of boar spermatozoa showed that it
can be greatly increased by the use of luminol. Luminol is a chemical compound
often used to amplify chemiluminescence in reactions in which free radicals are
generated. Thanks to the introduction to the system of luminol, which after
excitation is an efficient light emitter, stronger chemiluminescence that is easier to
measure can be observed. Luminol is a non-specific luminescence amplifier and is
excited in reactions in which various reactive oxygen species (ROS) and organic
radicals are generated (Bartosz, 1995). The data obtained indicate that under the
influence of Fe ions, reactive oxygen species are generated, and their level can be
determined based on measurements of chemiluminescence dependent on luminol.
One of the major biological processes involving ROS is lipid peroxidation.
Lipids are found in all body cells and fulfil many biologically important functions.
They are primarily the building blocks of all cell membrane structures (cell
membrane, nuclear membrane, mitochondria) and determine their properties
(fluidity, permeability, enzymatic and receptor activity). In the free radical chain
process of lipid peroxidation, the emerging ROS, by initiating further peroxidation
reactions, may cause serious damage to the cells. Mammalian sperm cells are
particularly vulnerable to oxidative damage due to the high content of easily
peroxidized polyunsaturated fatty acids (Jones et al., 1979) and the low activity of
the enzymatic antioxidant system (Aitken, 1994). The attack of free radicals on
UFA-rich sperm cell membrane lipids leads to irreversible decrease of membrane
fluidity and the increase of their non-specific permeability (Ohyashiki et al., 1988),
which, in turn, leads to disturbances in the course of capacitation (Ball et al., 2001)
and decreases the ability of sperm to penetrate the egg cell (Aitken et al., 1993 b).
Because lipid peroxidation is associated with damaged structure and function of
96
P. Gogol
spermatozoa, evaluation of the intensity of this process is of great diagnostic
importance. The most common method of assessing lipid peroxidation is based on
the reaction of the final lipid oxidation product — malondialdehyde (MDA) with
thiobarbituric acid (TBA), in the acid environment and at elevated temperature. In
spermatozoa, the spontaneous process of MDA formation is very slow. However,
the process is greatly accelerated in the presence of transient metal ions, e.g. iron
(Jones et al., 1979). The mechanism of amplification (acceleration) of lipid
peroxidation by Fe ions is not completely clear. It is thought that the addition of Fe
ions to cell suspension may trigger a breakdown of lipid peroxides and hydroperoxides accumulated within the cell membrane, thus stimulating the propagation
of a chain reaction of peroxidation. An alternative concept assumes that Fe ions
initiate the peroxidation reaction by entering into reaction with reactive oxygen
species produced by the spermatozoa and by generating an exceptionally reactive
hydroxide radical (Aitken et al., 1993 a). Whatever the mechanism, it is thought that
the main source of luminescence are the lipid peroxidation processes taking place in
the cells.
If that is so, the measurement of sperm luminescence may provide information
about the intensity of one of the main biological processes leading to decreased
biological value of semen.
The association between induced, luminol-amplified luminescence of spermatozoa and the lipid peroxidation process is confirmed by the shape of a curve
depicting the relationship between luminescence intensity and concentration of Fe
ions. This curve, just as for the curve depicting the relationship between the amount
of generated peroxidation products and Fe ions concentration, is bell shaped.
The present findings prove the possibility of analysing induced luminescence of
spermatozoa with the use of a luminometer and confirm that the key role in the
generation of sperm luminescence is played by free-radical processes of lipid
peroxidation.
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PIOTR GOGOL
Luminescencja plemników knura indukowana jonami żelaza
STRESZCZENIE
Przy użyciu luminometru badano indukowana˛ luminescencje˛ plemników knura. Oceniano możliwość wzmocnienia sygnału ultrasłabej luminescencji przy zastosowaniu luminolu oraz zależność
nate˛żenia i kinetyki luminescencji od koncentracji plemników w próbce i od ste˛żenia FeSO4 w roztworze
indukuja˛cym luminescencje˛.
W wyniku przeprowadzonych prac stwierdzono możliwość znacznego wzmocnienia luminescencji
poprzez dodanie do 0,7 ml zawiesiny plemników 10 µl 5mM roztworu luminolu. Wykazano wpływ
koncentracji plemników w badanej próbce na nate˛żenie i kinetyke˛ luminescencji. Stwierdzono, że
98
P. Gogol
w przypadku luminescencji indukowanej jonami żelaza krzywa zależności pomie˛dzy ste˛żeniem jonów
żelaza a nate˛żeniem luminescencji przyjmuje kształt dzwonu (charakterystyczny dla procesu indukowanej peroksydacji lipidów), z maksimum przy ste˛żeniu 0,05 mM FeSO4.
Uzyskane wyniki dowodza˛ możliwości badania indukowanej luminescencji plemników przy użyciu
luminometru oraz potwierdzaja˛, że kluczowa˛ role˛ w powstawaniu luminescencji plemników odgrywaja˛
wolnorodnikowe procesy peroksydacji lipidów błonowych.
EFFECT OF IN VITRO CULTURE CONDITIONS
ON THE DEVELOPMENTAL POTENTIAL OF PORCINE NUCLEAR
TRANSFER EMBRYOS RECONSTRUCTED WITH CUMULUS CELLS
ANALYSED ON APOPTOSIS*
M a r i a S k r z y s z o w s k a, M a r c i n S a m i e c, Z d z i s ł a w S m o r a˛g
Abstract
In the study, effect of different culture systems on the in vitro developmental potential of porcine
nuclear transfer (NT) embryos reconstructed with cumulus cells analysed on apoptosis was
investigated. To detect the early-apoptotic changes in the cumulus cells, which had been isolated
from in vitro matured cumulus-oocyte complexes (COCs), single nuclear donor cell suspension was
subjected to dyeing with live-DNA green fluorochrome YO-PRO-1. The source of recipient cells
were in vitro matured oocytes. Maternal chromosomes were removed by chemically assisted
microsurgical technique. Reconstruction of embryos was achieved by direct microinjection of
cumulus cell karyoplasts into a cytoplasm of previously enucleated oocytes. Afterwards, reconstituted oocytes were artificially activated. The cloned embryos were cultured in 50-µL droplets of
NCSU-23/BSA followed by NCSU-23/FBS medium covered with mineral oil (Group I) or in
co-culture system either with cumulus cells (Group II) or with oviductal epithelial cells (Group III)
in 500 µL of the same medium. A total of 342 enucleated oocytes were injected with non-apoptotic
cumulus cell-derived karyoplasts. After activation, 312 reconstituted oocytes (91.23%) were in
vitro cultured for 7 to 8 days in three different physicochemical culture conditions (129, 79 and 104,
in Group I, II and III, respectively). The proportion of reconstructed embryos that cleaved was
approximate in all the experimental groups (68.9%, 89/129; 56.0%, 44/79, and 68.0%, 71/104, in
Group I, II and III, respectively). Development to morula stage was not different in three culture
conditions (29.2%, 26/89; 34.1%, 15/44; 26.8%, 19/71, in Group I, II and III, respectively).
Blastocyst development rate was similar in all groups (4.5%, 4/89; 6.8%, 3/44 and 7.0%, 5/71 in
Group I, II and III, respectively) and was not statistically different among treatment groups. In
conclusion, the used in vitro culture conditions of reconstituted porcine embryos had no significant
effect on developmental competences of porcine NT embryos and although the percentage of
morulae obtained was relatively high in all experimental groups, blastocyst formation rate decreased
considerably. However, blastocyst percentage tended to be higher in the co-culture system groups
than in the group without somatic cell feeder layer but the difference was not statistically significant.
Key words: pig, nuclear transfer, somatic cell, apoptosis, in vitro culture
* This work was conducted as a Solicited Project no. PBZ-KBN-084/P06/2002, financed from
2003 to 2005 by the State Committee for Scientific Research.
100
M. Skrzyszowska et al.
Since the first report on the successful generation of a somatic cell clone
(Wilmut et al., 1997), a large number of cloned offspring has been produced in
various livestock species, including pigs (Kato et al., 2000; Keefer et al., 2001;
Galli et al., 2003; Yin et al., 2002; Hyun et al., 2003 a). Porcine complex in vitro
production (IVP) systems, including in vitro maturation (IVM) and in vitro
fertilization (IVF) of oocytes and their subsequent in vitro culture (IVC) have been
modified so far, but are still at a low level because of a low developmental rate of
embryos to the blastocyst stage and their poor morphological qualities. Swine
embryo engineering (somatic cloning combined with transgenesis) is a particularly
important research field of assisted reproduction technologies (ARTs) due to
increasing use of the pig in studies on xenotransplantation (Galili, 2001; Thomson
et al., 2003; Lai et al., 2002) or creation of animal bioreactors of human
recombinant proteins/biopharmaceuticals (Lee et al., 2003 a). In vitro production
systems (IVM-IVF-IVC) are fundamental procedures for the acceleration of the
technologies of somatic cloning and transgenesis. Despite tremendous improvement
of somatic cell nuclear transfer (SCNT) technique in pigs, effectiveness of cloning
in this species remains at the relatively low level and in relation to the number
of the reconstructed oocytes it does not exceed an average of 10 to 20% blastocysts
and 3 to 5% piglets born (Onishi et al., 2000; Bondioli et al., 2001; Dai et al., 2002;
Ramsoondar et al., 2003).
Many studies investigating the somatic cloning of pigs confirmed that the
relatively high rate of in vitro developmental potential to the blastocyst stage can
only be attained through a preservation of appropriate requirements in all steps of
SCNT procedure. One of the most important factors which determine cloning
efficiency are nuclear-cytoplasmic maturity state of recipient oocytes and the
structuro-functional quality of nuclear donor cells (Lee et al., 2003 b; Hyun et al.,
2003 b). Moreover, right coordination between nuclear donor cell phenotype and
cell cycle stage, maternal chromosome elimination (recipient oocyte enucleation)
method, oocyte reconstruction technique and artificial activation system (Yin et al.,
2002; Hyun et al., 2003 a; Samiec et al., 2003) appear to be also significant.
Physico-chemical conditions of in vitro culture of nuclear transfer-derived embryos
affect undoubtedly their developmental competences, too (Lee et al., 2003 c; Im et
al., 2004). So far, deficit of effective methods of in vitro culture has limited
considerably morula/blastocyst formation yields in pig somatic cloning. Therefore,
it became a stimulus for a search of optimal circumstances of SCNT embryo
culture. Several media, such as modified Whitten (WM) medium (Beckmann and
Day, 1993; Onishi et al., 2000), North Carolina State University (NCSU-23)
medium (Petters and Wells, 1993; Lee et al., 2003 a; Im et al., 2004), NCSU-37
medium (Petters and Wells, 1993; Kikuchi et al., 2002; Yin et al., 2002), Charles
Rosenkrans 2 (CR2) medium (Betthauser et al., 2000), potassium simplex optimized (KSOM) medium (Machaty et al., 1998), modified Chalot, Ziomek, Bavister
(CZB) medium (Pollard et al., 1995), and Beltsville embryo culture (BECM-3)
medium (Dobrinsky et al., 1996; Onishi et al., 2000; Im et al., 2004) were used for
the successful culture of porcine embryos, which had been derived either from
Developmental potential of porcine cloned embryos
101
in vivo fertilization, in vitro production (IVP) or somatic cell nuclear transfer
(SCNT), to the blastocyst stage. In particular, NCSU-23, which had been tailored to
the nutrient and metabolic needs of pig embryos, was the most suitable, compared
to other media, for the culture of pig IVP or SCNT embryos and resulted both in the
highest blastocyst formation rate and their total cell number.
The aim of our study was to determine the effect of different culture systems on
the preimplantation developmental potential of porcine nuclear-transferred embryos
reconstructed with cumulus cells, which had been analysed on apoptosis.
Oocyte collection and in vitro maturation
Unless otherwise mentioned, all chemicals used in this study were purchased
from Sigma Chemical Co. (St. Louis, MO).
Slaughterhouse ovaries were collected from prepubertal and postpubertal gilts
as well as sows. Cumulus-oocyte complexes (COCs) were recovered by aspiration
of follicular fluid from 2- to 6-mm antral ovarian follicles using an 18-gauge needle
attached to a 10-mL disposable syringe. COCs were collected into HEPES-buffered
Tissue Culture Medium 199 (TCM 199-HEPES; Gibco BRL, Life Technologies
Inc., Grand Island, NY) supplemented with 50 IU 10 mL–1 heparin. COCs with
evenly granulated ooplasm and several uniform layers of compact cumulus cells
were washed three times in TC 199-HEPES medium with addition of 4 mg mL–1
bovine serum albumin (fraction V; BSA-V) and selected for in vitro maturation
under atmospheric conditions. Immature COCs were transferred into the wells of
four-well dishes (Nunclon Multidishes; Nalge Nunc International, Denmark) filled
with 500 µL of TC 199 maturation medium that had been overlaid with paraffin oil
and equilibrated at 39°C in atmosphere of 5% CO2 in air for 1 to 3 h. The
maturation medium comprised 25 mM L–1 HEPES and 26 mM L–1 sodium
bicarbonate-buffered Tissue Culture Medium 199 (Gibco BRL, Grand Island, NY)
supplemented with 10% porcine follicular fluid (pFF), 0.6 mM L–1 L-cysteine,
10 ng mL–1 recombinant human (rhEG7) epidermal growth factor (EGF), 1 mM L–1
dibutyryl cyclic adenosine monophosphate (db-cAMP), and 0.1 IU mL–1 human
menopausal gonadotropin (hMG). Approximately 50 to 60 COCs were cultured in
the db-cAMP- and hMG-supplemented medium for 20 h at 39°C in a 100% watersaturated atmosphere of 5% CO2 and 95% air. The oocytes were then cultured for
22 to 24 h in fresh maturation medium that did not contain db-cAMP and hMG.
After maturation, expanded cumulus cells and corona cells were completely
removed by vigorous pipetting COCs in the presence of 0.1% hyaluronidase in
500 µL of HEPES-buffered TCM 199 for 1 to 2 min. In vitro matured, Metaphase II
oocytes, selected on the basis of accepted morphological criteria, and first of all
with evenly granulated, dark cytoplasm and an intact plasma membrane, and with
distinctly expelled first polar bodies provided a source of recipient cells for
exogenous cell nuclei in the somatic cloning procedure.
102
Preparation of nuclear donor cells analysed on apoptosis
The freshly collected cumulus cells were dissociated from COCs by vortexing
in vitro matured COCs for 1 to 2 min in TCM 199-HEPES supplemented with 0.1%
hyaluronidase and washing three times in the medium with addition of 4 mg mL–1
BSA-V. To detect the early-apoptotic changes in the cumulus cells, single nuclear
donor cell suspension was subjected to dyeing with 1 µg mL–1 live-DNA green
fluorochrome YO-PRO-1 (Vybrant(T)M Apoptosis Assay Kit 4; Molecular Probes).
After 15 to 20 min of cell incubation in the diagnostic solution, assessment of
apoptosis was carried out on an epi-fluorescent microscope (Olympus IMT-2,
Tokyo, Japan) after excitation of somatic cell DNA-YO-PRO-1 dye complexes with
blue light. Cumulus cells, which had not emitted a green fluorescence, were
classified as non-apoptotic and used in the somatic cloning procedure.
Production of nuclear-transferred embryos
Enucleation procedure. Cumulus-denuded oocytes were incubated in the maturation medium supplemented with 0.4 µg mL–1 demecolcine (DMCC) and 0.05 M L–1
sucrose for 1 h at 39°C. Afterwards, the treated oocytes were transferred into a glass
micromanipulator chamber filled with TCM 199 with addition of 4 mg mL–1
BSA-V, 5 µg mL–1 cytochalasin B (CB) and 0.4 µg mL– 1 DMCC. Maternal
chromosomes (metaphase plates), which had been allocated into chemically
induced protrusion of plasma membrane, were removed microsurgically (Yin et al.,
2002). Enucleation was accomplished by gently aspirating the ooplasmic cone,
which contained condensed chromosome mass, with the aid of a bevelled
micropipette of 20- to 25-µm external diameter. Following enucleation, the
resulting cytoplasts were washed extensively in HEPES-buffered TCM 199/BSA
and held in this CB-free medium until microinjection of donor nuclei.
Reconstruction of enucleated oocytes. Reconstruction of oocytes was achieved by
intracytoplasmic microinjection of cumulus cell-derived karyoplasts. Donor cells
were selected under differential interference contrast (DIC) optics according to their
size and shape. Small cells (10 – 15 µm diameter) with a smooth, intact plasma
membrane surface were chosen as donor nuclei to be injected into enucleated oocytes.
By using an injection pipette, whose sharp, bevelled tip had an external diameter
about half smaller than the diameter of the selected cumulus cells (7 – 8 µm), the
plasma membrane was broken by gentle, repeated aspiration of the entire cells into
and out of the pipette. Then the injection pipette was introduced mechanically
through the zona pellucida (using the same slit as made during enucleation) and up to
halfway into the ooplasm of each cytoplast and the tiny karyoplast was quickly
deposited with the cytoplasm. The reconstructed oocytes were incubated in NCSU-23
medium (Petters and Wells, 1993) with 4 mg mL–1 BSA-V and 0.4 µg mL–1 at 39°C
in a 100% humidified atmosphere of 5% CO2 in air for 1 h before activation.
Activation protocol. Reconstructed oocytes were equilibrated in 500 µL of
electroporation medium for 5 min at 39°C and then transferred to electroactivation
chamber with two wire electrodes 0.5 mm apart overlaid with 10 mL of the same
medium. Activation was induced with three consecutive DC pulses of 1.0 kV cm–1
103
for 80 µs each, delivered by a BTX Electrocell Manipulator 200 (BTX, San Diego,
CA). Immediately after electroactivation, nuclear-transferred embryos were left once
more in the electroporation medium for 5 min at 39°C, before being transferred to the
NCSU-23/BSA medium supplemented with 5 µg mL–1 CB for 2 h. The electroporation medium was isotonic dielectric solution consisting of 0.3 M D-mannitol,
0.05 mM CaCl2, 0.1 mM MgSO4, and 0.2 mg mL-1 fatty acid free BSA (FAF-BSA).
In vitro culture of reconstructed embryos
After activation treatments, surviving reconstructed embryos (with intact
plasma membrane) were dissected into three experimental groups. In Group I, 20 to
30 cloned embryos were cultured in 50-µL droplets of NCSU-23 medium
supplemented with 4 mg mL–1 BSA-V, which had been overlaid with light mineral
oil. After 72 to 96 h of in vitro culture, cleaved embryos were transferred into 50-µL
drop of NCSU-23/BSA medium supplemented with 10% fetal bovine serum (FBS)
for additional 72 h. In Group II, 30 to 50 cloned embryos were co-cultured with
cumulus cells in 500 µL NCSU-23 medium supplemented with 4 mg mL–1 BSA-V
(per well of 4-well dishes). After 48 to 72 h of in vitro culture, cleavage-stage
embryos were transferred onto the cumulus cell feeder layer in the medium
supplemented with 10% FBS for additional 96 h. In Group III, 30 to 50 cloned
embryos were co-cultured with oviductal epithelial cells in 500 µL NCSU-23
medium supplemented with 4 mg mL–1 BSA-V (per well of four-well dishes). After
48 to 72 h of in vitro culture, cleaved embryos were transferred onto the oviductal
epithelial cell feeder layer in the medium supplemented with 10% FBS for
additional 96 h. In all experimental groups, reconstructed embryos were incubated
at 39°C, in a 100% water-saturated atmosphere of 5% CO2 and 95% air. At the end
of the in vitro culture period (days 6 to 7), embryos were evaluated morphologically
for morula/blastocyst formation rates.
Embryo cell number evaluation
Nuclear transfer-derived blastocysts were stained with 5 µg mL– 1 bisbenzimide
(Hoechst 33342), to analyse the number of cell nuclei using the fluorescent
microscopy. The mean number of cells was determined based on the results of
double counting.
Statistical analysis
In order to compare the number of successfully reconstructed (injected) oocytes,
the number of cleaved embryos and the number of embryos at morula and blastocyst
stages between different in vitro culture groups, Chi-square test was used.
Results
The qualitative and quantitative composition of the maturation medium prepared in our studies for in vitro culture of porcine oocytes turned out to be optimal
104
and in the established culture conditions allowed reaching nuclear and cytoplasmic
maturity for approximately 70 to 80% of recipient oocytes.
The cumulus cells, which had been isolated from in vitro matured COCs,
were diagnosed on apoptosis using YO-PRO-1 dye. After fluorescent analysis
of approximately 50 different random samples it was found that only minimal
proportion of cumulus cells revealed ultrastructural apoptotic changes. On the
basis of morphological features of the cells, it can be ascertained that these ones are
the symptoms of late destruction phase of apoptosis. The percentage of lateapoptotic cells with advanced morphological transformations did not exceed 10 to
15% of a total pool of the follicular cells isolated from in vitro matured
COCs.
Using the same medium (NCSU-23), such preimplantation development rates as
cleavage activity, morula and blastocyst formation, as well as mean total cell
number of cloned blastocysts were compared between in vitro embryo culture
groups, which were different for: 1) the total volume of culture medium compartment and number of embryos per volume unit of culture medium (20 to 30 embryos
per 50 µL of NCSU-23 in Group I vs. 30 to 50 embryos per 500 µL of NCSU-23 in
Groups II and III); 2) the presence or absence of somatic cell feeder layer (Groups II
and III vs. Group I); and 3) the type of somatic cells, which form a monolayer in the
co-culture systems (Group II vs. Group III).
Both the post-reconstruction survival rates and cleavage activity as well as
morula/blastocyst frequencies were not different among the in vitro culture groups
(P > 0.05; Table 1).
In all experimental groups, overall efficiency of in vitro morula production was
relatively high but blastocyst formation rate was decreased radically. The percentage of blastocysts tended to be slightly higher in the co-culture groups (Groups II
and III) than in the monoculture group (Group I); however, the differences were not
statistically significant (P > 0.05; Table 1).
Mean cell nuclear numbers in blastocysts derived from NT oocytes reconstructed
with cumulus cells, were 26.5 cells in Group I, and 28 cells in Groups II and III.
Table 1. Comparison of different in vitro culture systems for the developmental potential of nuclear
transfer-derived porcine embryos
Culture
system
groups
No. of injected
oocytes
Cultured
(%)
Cleaved
(%)
I
139
II
82
III
121
129
(92.8)
79
(96.3)
104
(85.9)
89
(68.9)
44
(56.0)
71
(68.0)
Developed (%)
morulae
blastocysts
26
(29.2)
15
(34.1)
19
(26.8)
4
(4.5)
3
(6.8)
5
(7.0)
105
Discussion
The results of our study showed that the used in vitro culture conditions had no
significant effect on developmental competences of porcine nuclear-transferred
embryos reconstructed with cumulus cells, which had been additionally stained
with DNA-live fluorochrome for diagnostics of apoptosis. It was found that both in
vitro cleavage activity rate and morula/blastocyst formation yields were similar in
all culture systems. But, although the percentages of NT morulae obtained were
relatively high in all experimental groups, the blastocyst frequencies decreased
considerably. However, we observed that blastocyst percentage tended to be higher
in the co-culture system groups than in the group without somatic cell feeder layer.
One of the most important factors, which determine both the preimplantation
developmental potential and morphological quality of porcine cloned embryos,
seem to be physico-chemical circumstances of in vitro culture (mono- or co-culture)
such as: 1) a quantitative and qualitative composition of culture medium and its
resultant osmolarity, 2) a total volume of culture medium unit/compartment
(droplet or well of multiwelled dishes), 3) a number of embryos per volume unit of
medium, 4) gas atmosphere conditions as well as 5) incubation temperature. The
second series of in vitro culture-related factors involves: 6) the use of chemically
defined or undefined medium in the mono-culture system, 7) presence or absence of
somatic cell-derived feeder monolayer, 8) the use of somatic cell-conditioned
medium, and 9) their modifications through energy (e.g. sodium lactate, sodium
pyruvate, glucose) and/or trophic (e.g. BSA, FBS) supplementation. Several media
such as NCSU-23 (Onishi et al., 2000; Hyun et al., 2003 a; Samiec et al., 2003; Im
et al., 2004), NCSU-37 (Yin et al., 2002; Kawakami et al., 2003), BECM-3 (Onishi
et al., 2000; Im et al., 2004), CR2 (Betthauser et al., 2000) and modified WM
(Onishi et al., 2000) have been used to culture the preimplantation porcine nuclear
transfer-derived embryos so far. Of these ones, cloned embryos in NCSU-23 and
NCSU-37 medium had higher developmental competences in vitro. Therefore,
NCSU-23 was chosen as basic culture medium to test the ability to support the
development of porcine NT embryos reconstituted with cumulus cells. In the
present study, the proportions of cloned embryos that reached the blastocyst stage
(4.5 to 7.0%), were comparable to those of other reports in which nucleartransferred embryos, reconstructed also with cumulus cells, were cultured in the
same medium (Martinez Diaz et al., 2002; Lee et al., 2003 c).
The in vitro culture system of pig embryos has to be improved because there are
still problems with the low embryo survival rate and poor embryo quality (Petters
and Wells, 1993; Machaty et al., 1998). The first two days of in vitro culture appear
to have a critical effect on the developmental potential of cloned embryos, because
they are the prerequisite stage for transcriptional activation of embryonic genome
(Viuff et al., 2002), at which stage a four-cell block occurs. Our observations
showed that porcine NT embryos differed clearly in their timing of blastomere
cleavage, resulting in a wide variety of developmental embryonic stages (two- to
eight-cell stage at day 2). However, most of them were at the late four-cell stage. In
106
turn, it is probable that sequential trophic (BSA/FBS) supplementation of basic
chemically defined media such as NCSU-23 (Samiec et al., 2003; Lee et al., 2003 a)
or the use of somatic cell-derived feeder layer or somatic cell-conditioned media
(Kikuchi et al., 2002) may enhance the potential for maternal to embryonic
transition of genome activity or for overcoming the cleavage block, and subsequently for morula compaction and cavitation processes under in vitro conditions. It was
confirmed that co-culture of bovine IVP or SCNT embryos with cumulus cells
(Goto et al., 1989; Skrzyszowska et al., 2000) or oviductal epithelial cells (Eyestone
and First, 1985) has enabled a considerable increase of blastocyst formation rate and
improvement of their morphological quality. On the contrary, in our experiments the
application of porcine NT embryo co-culture either with cumulus cells or oviductal
cells did not affect significantly both the blastocyst proportion and their total cell
number. We observed only a small upward tendency in the development of cloned
embryos to blastocyst stage and in the embryo quality as compared to standard
monoculture system. These results suggest that the methods of in vitro culture,
which have been used in porcine IVP and SCNT techniques so far, seem to be
suboptimal. Moreover, the ex vivo culture conditions which are necessary for the
preimplantation development of nuclear transfer-derived embryos might differ from
those for the in vitro fertilization-derived embryos.
Further experiments are required to optimize the in vitro culture conditions of
pig cloned embryos. It is possible that either slight modification of quantitative and
qualitative compositions of the media, which are commonly used for porcine
embryo culture or a development of completely new media could contribute to both
increase of blastocysts produced in vitro and their morphological quality.
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MARIA SKRZYSZOWSKA, MARCIN SAMIEC, ZDZISŁAW SMORA˛G
Wpływ warunków hodowli in vitro na potencjał rozwojowy zarodków świni rekonstruowanych
z komórek wzgórka jajonośnego analizowanych w kierunku apoptozy
STRESZCZENIE
W pracy badano wpływ warunków hodowli in vitro na potencjał rozwojowy zarodków klonalnych
świni rekonstruowanych z ja˛der komórek wzgórka jajonośnego analizowanych w kierunku apoptozy.
W celu rozpoznania wczesnoapoptotycznych zmian w komórkach stanowia˛cych źródło dawców ja˛der
komórkowych, komórki wzgórka jajonośnego poddawane były przyżyciowemu barwieniu zielonym
fluorochromem DNA YO-PRO-1. Biorca˛ egzogennych ja˛der komórkowych w procedurze klonowania
somatycznego były dojrzałe in vitro oocyty świń. Chromosomy mateczne były usuwane przy zastosowaniu techniki mikrochirurgicznej wspomaganej chemicznie. Rekonstrukcja enukleowanych oocytów była przeprowadzana metoda˛ docytoplazmatycznej iniekcji karioplastów przygotowanych z komórek pe˛cherzykowych. Zrekonstruowane oocyty poddawane były sztucznej aktywacji metoda˛ elektroporacji. Naste˛pnie, zygoty klonalne hodowane były in vitro w 50-µL kroplach pożywki NCSU-23/BSA,
a naste˛pnie NCSU-23/FBS (grupa I) lub w 500 µL tej samej pożywki w systemie współhodowli
z komórkami wzgórka jajonośnego (grupa II), albo z komórkami nabłonka jajowodowego (grupa III).
Całkowita liczba 342 enukleowanych oocytów była poddawana iniekcji karioplastów komórek pe˛cherzykowych analizowanych w kierunku apoptozy (z użyciem YO-PRO-1). Po aktywacji, 312 rekonstytuowanych oocytów (91,23%) było hodowanych in vitro przez 7 do 8 dni w trzech różnych
fizykochemicznych warunkach hodowli (129, 79 oraz 104, odpowiednio w grupach: I, II oraz III).
Aktywność podziałowa cybrydowych zygot klonalnych utrzymywała sie˛ na podobnym poziomie
we wszystkich grupach doświadczalnych (68,9%, 89/129; 56,0%, 44/79; 68,0%, 71/104, odpowiednio
w grupach I, II i III). Odsetek zarodków w stadium moruli oraz blastocysty wynosił 29,2% i 4,5%
109
(26/89 i 4/89), vs. 34,1% i 6,8% (15/44 i 3/44) vs. 26,8% i 7,0% (19/71 i 5/71), odpowiednio w grupach:
I, II oraz III. Podsumowuja˛c należy stwierdzić, że zastosowane warunki hodowli in vitro nie miały
istotnego wpływu na kompetencje rozwojowe świńskich zarodków klonalnych, rekonstruowanych
z ja˛der komórek pe˛cherzykowych. We wszystkich wariantach hodowli in vitro odsetek uzyskanych
morul był stosunkowo wysoki, natomiast odsetek blastocyst ulegał znacznemu obniżeniu. Obserwowano
jednak tendencje˛ wzrostowa˛ w odsetku uzyskanych blastocyst w systemach współhodowli z komórkami
somatycznymi (grupa II i grupa III) w porównaniu z hodowla˛ bez udziału warstwy odżywczej (grupa I),
mimo iż różnice nie były statystycznie istotne.
EFFECT OF SOYBEAN MEAL PROTECTED WITH CA SALTS
OF FATTY ACIDS ON COWS’ YIELD, PROTEIN
AND FAT COMPONENTS IN MILK AND BLOOD*
F r a n c i s z e k B r z ó s k a
Department of Animal Nutrition and Feed Science, National Research Institute of Animal Production,
Abstract
Thirty-two Red-and-White cows were allocated to 4 groups in a randomized block design.
Pasture-fed cows received crop forage, fresh brewers’ grains and concentrate mixture. The
concentrate mixture contained 15% soybean meal (control group) and soybean meal protected
with Ca salts of fatty acids (CSFA) at rates of 15, 22 and 29% (experimental groups). This
corresponded to an intake of 1.20, 1.23, 1.87 and 2.44 kg/day, respectively. Dry matter (DM) intake
by cows averaged 22.77 kg/day, including 8.27 kg/day for the concentrate mixtures, and was not
significantly different between the groups. The cows’ milk yield averaged 30.01 kg/day and showed
an upward tendency, with no differences between the groups. The amount of milk corrected for
protein and fat content averaged 27.04 kg/day and did not differ significantly between the groups.
Giving protected (PSBM) vs. unprotected soybean meal (USBM) increased protein by 14 g/kg and
fat by 7 g/kg milk, the differences being statistically significant. There was a significant increase in
the amount of fat and protein secreted in milk, including fat by 63 g and protein by 17 g/day. No
significant differences were found in the plasma levels of glucose, total protein, urea or
triglycerides in cows in individual groups. Giving cows PSBM significantly decreased the levels of
capric, lauric and palmitoleic acids as well as of arachidonic and eicosapentaenoic acids in milk fat.
There were increases in the levels of stearic, arachidic, behenic and docosahexaenoic acids in milk
fat. Differences in the levels of these acids were not statistically significant. A significant increase in
unsaturated fatty acids (UFA) in milk, including monounsaturated fatty acids (MUFA), was found
in cows receiving 22% PSBM in the diet. PSBM resulted in an improvement in the dietetic value of
milk (DFA). It is concluded from the present research that giving PSBM to cows characterized by
an average yield of 30 kg milk/day increases milk yield non-significantly, while significantly
increasing the protein content of milk and improving the dietetic value of milk fat.
Key words: soybean meal, milk yield, protein content, milk fatty acids
* This work was conducted as part of a statutory project supported by the Ministry of Agriculture
and Rural Development, no. 2215.1.
112
F. Brzóska
Soybean meal is one of the major protein feeds used in concentrate mixtures for
high-yielding cows. Previous research has shown that rumen degradation of
soybean meal is approximately 79% after 24 h of in sacco digestion (Pieszka and
Brzóska, 2001 a). This means that almost two-thirds of soybean meal is degraded
before it flows from the rumen to the duodenum and small intestine. Considering
the high (70 – 80%) degradability of bulky feeds including pasture forage, silages
and hay, there is a scarcity of feeds with lower rumen degradability in our climatic
zone (Erdmann et al., 1984; Rossi et al., 1999). The high bacterial deamination of
protein in the rumen deteriorates the small intestine supply of cows with amino
acids, which are the substrate for the synthesis of milk protein. Several methods
have hitherto been developed to protect feed protein against degradation in the
rumen. They involve high temperature (Schroeder et al., 1995) and chemical
compounds such as formaldehyde (Subuh et al., 1996), lignosulphonates (Standford
et al., 1995) and xylose (Nakamura et al., 1992). In recent years, Ca salts of fatty
acids (CSFA) have been used to protect solvent meal protein (Kowalski et al., 1997;
Maiga and Schingoethe, 1997; Rohr et al., 1993; Rossi et al., 1999). In this method,
particles of protein feed are covered with a thin layer of CSFA. The higher melting
temperature of salts compared to rumen temperature causes the protein protected
with salts to bypass the rumen intact. It has been shown that using 200 – 300 g of
CSFA per kg soybean meal is sufficient for effective protection of protein and
enriches protein feed with energy, including unsaturated fatty acids (Klusmayer et
al., 1989; Pieszka and Brzóska, 2000; 2001 a; b). Preliminary studies on the
efficiency of feeding CSFA to cows during winter have demonstrated that they have
a beneficial effect on milk yield and on increased fat content of milk (Pieszka and
Brzóska, 2001 b). The aim of our study was to evaluate the milk yield of cows
supplemented with CSFA during the grazing period.
It was hypothesized that giving pasture-grazed cows a concentrate mixture
containing soybean meal protected with CSFA (PSBM) would increase their milk
yield, the levels of protein and fat in milk, and the amount of these secreted in milk.
It was also conjectured that the level of unsaturated fatty acids (UFA) in milk would
also increase under the influence of CSFA from linseed oil. The present study was
aimed to prove these hypotheses.
Experimental design and animals
Studies were performed at Lipowa farm of the Experimental Station Grodziec
Śla˛ski (Silesian province) with 32 Red-and-White cows in a randomized block
design. Each of the 4 groups contained 8 cows, and each of the 4 periods was
32 days long. Cows were chosen for the experiment from a herd of 135 cows
yielding an average of 6500 kg milk. Cows selected were aged 5 – 7 years, in the
third to fifth lactation, and calving between 28 February and 26 April. The
experiment commenced on 13 May and ended after 112 days. The cows’ ration
113
Soybean meal protected with Ca salts of fatty acids in cow feeding
contained pasture forage, crop forage, brewers’ grains and concentrate mixture fed
at 0.28 kg/kg milk obtained. The primary ingredients of the pasture forage were
orchard grass, perennial ryegrass and meadow fescue. A mixture of red clover and
grasses formed the crop forage. The concentrate mixture contained unprotected
soybean meal (USBM) and soybean meal protected with CSFA at rates of 15%
(PSBM-15), 22% (PSBM-22) and 29% (PSBM-29) of the mixture (Table 1).
Protected soybean meal was purchased for the experiment from INNFOSS, Poznań.
The pasture area allotted for grazing was determined each day based on its yield,
to ensure that the intake of cows during 8 hours’ grazing was approximately
8 – 10 kg DM. The grazing area was fenced off with an electric fence. Cows were
allowed constant access to water, both at pasture and indoors. Brewers’ grains and
crop forage were given to the cows indoors in the afternoon, after the cows had left
pasture. The concentrate mixtures were prepared by the author and were supplemented with Bovimix (BASF Kutno) mineral mixture at a rate of 1%. In the
barn, cows were fed individually and their intake of bulky feeds and of the
concentrate mixture was recorded. Feed intake was checked by weighing the feeds
at the start of each period and during the periods when milk yield was recorded.
Table 1. Components of feed concentrate (%)
Components
Ground wheat
Ground barley
Unprotected soybean meal
Protected soybean meal
Mineral mixture
USBM —
PSBM-15 —
PSBM-22 —
PSBM-29 —
Group
USBM
PSBM-15
PSBM-22
PSBM-29
63
20
15
63
20
56
20
49
20
15
2
22
2
29
2
2
unprotected soybean meal.
protected soybean meal, 15% of concentrate.
Sample collection and analysis
Feed samples were taken for chemical analyses in amounts of 500 g at weekly
intervals, and dried and pooled for each group and feeding period separately. Feeds
were analysed for DM, basic nutrients, acid detergent fibre (ADF) and neutral
detergent fibre (NDF). The nutritive value of each feed was calculated from the
nutrient content, using INWAR 1.3 software.
Cows were milked twice a day. Over the last three days of each experimental
period, the cows’ milk yield was recorded and milk samples taken for analysis.
Milk output was recorded with a milk meter Tru-Test, FV model. Milk was
preserved with 2-bromo-2-nitro-1,3-propanediol (GROPOL) and frozen until further analyses. On the last day of each period, milk samples were taken from the
jugular vein into heparinized tubes and centrifuged to obtain plasma. Glucose was
114
F. Brzóska
assayed in plasma from fresh blood. After thawing, the plasma was assayed for total
protein, urea and triglycerides.
The levels of basic nutrients in feeds were determined according to standard
procedures (AOAC, 1990) using nitrogen, fat (Büchi) and crude fibre analysers
(Foss-Tecator). Feed DM was determined by drying at 105°C. NDF and ADF were
determined according to Goering and van Soest’s procedure (1970). Protein, fat and
lactose in milk were determined with Milko-Scan model FT 120 (Foss Electric),
and pH, specific gravity and density of milk with the methods described in Polish
standard PN-68/A-86112. Nitrogen fractions in milk were determined by the
Kjeldahl method according to a procedure described by Gordon and Kolan (1983).
Milk fatty acids were determined as methyl esters of fatty acids using gas
chromatography (GC Varian 3400), column CP-Wax 58 (25 m × 0.53 mm × 1.0 micron)
using the method of Atwal et al. (1990). The fatty acid assay procedure is described
in Brzóska (2004).
The levels of glucose, total protein, urea and triglycerides in blood plasma were
determined enzymatically using diagnostic kits (Cormay Diagnostyka S.A., Lublin,
Poland).
Milk yield corrected for fat and protein and amount of milk were calculated
based on a formula provided by Subnel et al. (1994), where:
FPCM = Fat and Protein Corrected Milk (kg) = [0.337 + 0.116 × fat (%) +
0.06 × protein (%)] × milk (kg)
The results were analysed statistically by variance analysis and Tukey’s new
multiple range test using the Statgraphics 6.0 package.
Results
The level of USBM in the concentrate mixture of the control group was 15%,
which corresponded to an intake of 1.2 kg soybean meal/day. The level of USBM in
the concentrate mixture in respective groups was 15, 22 and 29% (Table 1), which
corresponded to an intake of 1.23, 1.87 and 2.44 kg/cow/day (Table 3). Considering
that 25% was formed by CSFA used to protect protein, the actual amount of
protected soybean consumed in the experimental groups was 0.92, 1.40 and
1.83 kg/day/cow. The nutrient content of forages and brewers’ grains concurred
with the data for this farm that had been obtained in an earlier study. Total protein
was 178.4 g/kg in the concentrate mixture of the control group and ranged from
157.3 to 184.4 g/kg in the concentrate mixtures of the experimental groups
(Table 2). DM intake by cows averaged 21.1 kg/day, including approximately
8.3 kg/day for the mixtures, and did not differ significantly between the groups
(Table 3). Intake was 1.20 kg/d for USBM, 1.23 kg/d for 15% protected
(PSBM-15), 1.87 kg/d for 22% protected (PSBM-22), and 2.44 for 29% protected
soybean meal (PSBM-29). The milk yield of cows averaged 30.01 kg/day and
115
showed an upward tendency, although it did no differ between the groups. The
amount of fat and protein corrected milk (FPCM) was on average 26.26 kg, and
26.86, 27.47 and 27.57 in the experimental groups, and did not differ significantly
between the groups either. Giving the cows PSBM significantly increased the
protein and fat content of milk. The fat content of milk increased by 7 g, and the
protein content by 14 g/kg milk, on average. There was a significant increase in the
amount of fat and protein secreted in milk in 24-h periods, including fat by 63 g and
protein by 17 g. No significant differences were found in the content or amount of
lactose secreted in milk. There were no marked tendencies regarding acidity,
renneting time or specific gravity of milk. The body weight of cows for the whole
experimental period showed a slight tendency for growth, both in the control group
and in the experimental groups.
Table 2. Nutritive value of feeds for cows (g kg–1 DM)
Nutrients
Dry matter (g kg–1)*
organic matter
crude protein
ether extract
crude fibre
N-free extractives
crude ash
NDF
ADF
Nutrients/kg in INRA-IZ
units of DM**
UFL
PDIN (g)
PDIE (g)
Concentrate
Pasture
forage
Crop
forage
Brewer’s
barley
grain
USBM
195.6
921.9
158.7
26.2
256.4
480.6
78.1
640.5
270.5
200.8
932.2
144.2
23.6
257.0
507.7
67.8
680.5
285.1
250.5
957.9
257.3
80.7
198.2
421.7
47.1
712.5
284.7
865.8
970.6
178.4
15.5
54.9
721.8
29.4
225.9
62.2
864.2
968.7
157.3
29.2
56.6
725.6
31.3
253.4
67.3
863.4
967.3
162.1
30.3
52.4
722.5
32.7
255.4
69.2
865.3
969.2
184.4
28.8
50.2
705.8
30.8
254.2
69.5
0.92
221
185
1.14
107
107
1.15
103
112
1.16
118
124
1.18
127
129
0.97
108
95
0.94
95
92
PSBM-15 PSBM-22 PSBM-29
* analytical measurement.
** calculated.
No significant differences were found in the levels of individual fractions of
nitrogen in milk except true N, the level of which was significantly lower in cows
given 15% PSBM in their diet (Table 4).
There were no significant differences in the levels of glucose, total protein,
urea, or triglycerides in plasma, although the level of total protein showed an
upward tendency as the percentage of PSBM increased in the concentrate mixture
(Table 5).
116
F. Brzóska
Table 3. Milk yield, milk composition, physical parameters and cows’ body weight change
Group
Item
Dry matter intake
(kg/day)
total
pasture forage
crop forage
brewer’s barley grain
concentrate
USBM in concentrate
PSBM in concentrate
USBM
21.98
8.00
2.60
2.20
7.98
1.20
PSBM-15
PSBM-22
PSBM-29
22.22
8.00
2.66
2.15
8.18
23.07
8.00
2.54
2.18
8.48
23.79
8.00
2.71
2.21
8.43
1.23
1.87
2.44
Milk yield (kg/day)
29.28
30.35
FPCM (kg/day)
26.26
26.86
fat (%)
3.15 Bb
3.13 Bb
protein (%)
3.15 Bb
3.08 Cc
lactose (%)
4.93
4.94
fat (g/day)
910 Bb
978 Aa
protein (g/day)
954 cC
964 bB
lactose (g/day)
1443
1554
6.30 abAB
6.50 aAB
Acidity (0SH)
Renneting time (s)
130 abAB
105 bB
Density (g/cm3)
1.0302
1.0299
BW before experiment
(kg)
574
573
BW after experiment (kg) 583
573
BW change (g/day)
+9
0
30.06
30.36
27.47
27.57
3.30 Aa
3.23 Aa
3.23 abAB
3.26 Aa
4.97
4.97
974 Aa
968 Aa
963 bB
986 aA
1490
1510
6.68 aA
6.03 bB
162 aA
148 abAB
1.0302
1.0300
546
557
+11
556
557
+21
SEM
0.23
0.00
0.08
0.05
0.06
0.51
0.47
0.04
0.02
0.01
16
15
25
0.06
6
0.0001
10
10
a, b, c — values in the same rows with different letters differ significantly (P < 0.05).
A, B, C — values in the same rows with different letters differ significantly (P < 0.01).
FPCM — fat and protein corrected milk.
Table 4. Milk nitrogen fractions (%)
Item
Total N
True protein N
NPN
Whey protein N
Casein N
N casein (% total N)
N true (% N total N)
Group
USBM
PSBM-15
PSBM-22
PSBM-29
0.494
0.460 ab
0.034
0.123
0.382
77.33
93.12
0.483
0.447 b
0.036
0.117
0.369
76.10
92.55
0.516
0.470 a
0.036
0.120
0.389
76.88
92.89
0.511
0.473 a
0.038
0.126
0.398
77.89
92.56
For explanation see Table 1 and 3.
SEM
0.004
0.004
0.001
0.002
0.004
0.33
0.21
117
Table 5. Physiological parameters of cows’ blood plasma (mg/100 ml)
Group
Item
Glucose
Total protein
Urea
Triglycerides
USBM
PSBM-15
PSBM-22
PSBM-29
36.52
8.86
38.57
8.62
35.51
8.73
36.23
8.81
35.34
8.99
35.72
9.52
36.30
9.02
37.81
8.71
SEM
0.43
0.09
1.01
0.17
All parameters in the rows were not significant.
Table 6. Fatty acid content in milk fat (g/100 g)
Item
Estimated
C8
C10
C12
C14
C16
C16:1
C18
C18:1
C18:2
C18:3
C18:3
CLA
C20
C20:4
C20:5
C22
C22:6
n-6
n-3
EPA
DHA
Calculated
SFA
UFA
MUFA
PUFA
PUFA 6
PUFA 3
DFA
OFA
UFA/SFA
DFA/OFA
PUFA/SFA
MUFA/SFA
Group
USBM
SEM
PSBM-15
PSBM-22
PSBM-29
2.10
4.09 aA
4.47 aA
12.59
30.94 abAB
2.14 aA
12.20 b
26.35 b
2.57
0.19
0.51
1.61
0.15 bB
0.10 aA
0.047 a
0.067 b
0.004
2.08
4.01 aAB
4.26 aAB
12.68
30.92 abAB
1.91 bAB
12.73 ab
25.98 b
2.62
0.19
0.51
1.69
0.19 aAB
0.09 abAB
0.045 ab
0.090 a
0.007
1.80
3.20 bB
3.43 bB
11.31
29.63 bB
1.96 abAB
13.40 ab
29.47 a
2.75
0.19
0.53
1.65
0.18 abAB
0.08 bcBC
0.038 bc
0.077 ab
0.005
2.01
3.59 abAB
3.84 abAB
12.41
31.36 aA
1.80 bB
13.72 a
26.12 b
2.75
0.19
0.54
1.74
0.20 aA
0.07 cC
0.037 c
0.091 a
0.007
0.05
0.10
0.10
0.19
0.20
0.03
0.19
0.41
0.03
0.00
0.01
0.04
0.00
0.00
0.001
0.002
0.000
66.55
33.45
28.21
4.95
2.86
0.57
45.65
54.35
0.51
0.87
0.07
0.43
66.95
33.05
27.89
5.16
2.91
0.56
45.75
54.22
0.50
0.85
0.08
0.42
64.26
36.67
31.43
5.25
3.02
0.58
50.07
49.93
0.58
1.03
0.08
0.50
66.84
33.17
28.01
5.15
2.84
0.58
46.89
53.11
0.50
0.89
0.08
0.42
0.50
0.47
0.43
0.06
0.03
0.01
0.49
0.49
0.01
0.02
0.00
0.01
b
b
bB
aA
bAB
bB
bAB
For explanation see Table 1 and 3.
b
b
bB
aA
bAB
bB
bB
a
a
aA
bB
aA
aA
aA
b
b
abAB
abAB
bAB
bAB
bB
118
F. Brzóska
Giving cows PSBM significantly decreased the levels of capric, lauric,
palmitoleic and eicosapentaenoic acids in milk fat, and increased the levels of
stearic, arachidic, behenic and docosahexaenoic acids. No significant changes were
found in the level of conjugated linoleic acid (CLA) in milk fat (Table 6).
A significant increase in UFA, including MUFA, was found in cows receiving 22%
PSBM (PSBM-22). The dietetic value of milk (DFA) was significantly improved
under the influence of PSBM.
Discussion
The synthesis of microbiological protein in the rumen does not fulfil the
requirements of high-producing cows for amino acids necessary for milk protein
synthesis (Hvelplund et al., 2001). Tables giving the nutritive value of feedingstuffs
specify that soybean meal should contain 480 to 545 g protein per 1 kg DM (Polish
nutrient requirements). Rumen degradability of USBM exceeds 60%, as confirmed
by Polish studies (Żebrowska et al., 1997; Kowalski et al., 1997; Pieszka and
Brzóska, 2001 a). The results of earlier studies indicate that the use of CSFA to
protect soybean meal reduces its rumen degradability during 24-h digestion by
approximately 27 – 49%, while the increased level of CSFA in the mixture of both
feeds increases the efficiency of its protection by 10 – 30% (Pieszka and Brzóska,
2001 a). Research has shown that the use of CSFA to protect the meal does not
reduce intestinal digestibility of protein, which is approximately 85 – 95% (Pieszka
et al., 2000). Giving young bulls protected soybean meal at 9% of the ration DM did
not negatively affect the level of volatile fatty acids or fermentation of organic
matter in the rumen, which suggests that PSBM has no effect on the synthesis of
microbiological protein in the rumen and can therefore be considered as a feed
neutral to the rumen environment (Pieszka and Brzóska, 2001 a). Good results for
the protection of soybean meal with Ca salts of palmitic and stearic acids at 10 and
25% have also been reported by Rossi et al. (1999). It is usually assumed that
MUFA and PUFA contained in vegetable oils used to protect protein show a lower
rumen stability than saturated acids, such as stearic and palmitic acid (Sukhija and
Palmquist, 1990; Gulati et al., 1997), which is probably why palmitic acid was used
to protect rapeseed meal in the MEGAPRO preparation. The dietary fat Erafet, used
to protect soybean meal in the current study, was composed of approximately 80%
waste fat and rapeseed fat and approximately 20% fish oil.
It is assumed that protected soybean meal should be used to feed cows during
the time when their requirement for amino acids is the highest, i.e. during the first
100 days of lactation. Schingoethe (1996) reports that the use of additional protein
or amino acids protected against rumen degradation is effective when the yield of
cows exceeds 5 kg milk per 100 kg body weight. In the present experiment, this
value was 28.5 kg. The results of research shown in this paper indicate that the use
of PSBM to feed cows yielding approximately 30 kg milk/day, from 2 to 4 months
postcalving, does not significantly increase the milk yield of cows, although an
119
upward tendency (within 1.0 kg milk/day) was observed. It can be assumed that the
efficiency of PSBM given to cows would be greater during the first 100 days after
calving. These findings support the results of earlier studies performed in the same
herd of cows during winter feeding and in the same period of lactation, in which the
increase in the cows’ milk yield, as influenced by soybean protected with CSFA,
was approximately 1.5 kg/day (Pieszka and Brzóska, 2001 b). It can therefore be
assumed that the investigated cows were close to the limit of milk yield at which the
supplementation of protected protein is effective and increases the milk yield of
cows. In other studies (Kraszewski et al., 2003) performed with Black-and-White
cows whose diets were supplemented during the first 100 days of lactation with 0.5
and 1.0 kg protected soybean meal (MEGAPRO), with a yield of 33 kg milk/day,
a significant increase of milk yield (1.31 and 3.02 kg/day) was obtained compared
to the group given no protected protein. In that experiment an increase in milk fat
content was also obtained, with no differences in protein content. Studies performed
in Germany with soybean meal protected against rumen degradation with xylose
(SoyPass preparation), whose rumen degradability was 40% compared to 65% for
unprotected soybean, resulted in cows’ milk yield increasing by approximately
2 – 3 kg milk/day, persisting until the fifth month of lactation (Losand et al., 1996).
Some studies obtained no increases in cows’ yield (DePeters and Cant, 1992),
although this is hard to justify. Schingoethe et al. (1996) suggest that one possible
reason is the reduced intestinal digestibility of the amino acids of protein protected
from rumen degradation, resulting from the excessive temperature used for its
protection or from the use of excessive doses of formalin to protect protein. Studies
by Pieszka et al. (2000) suggest that the method of protecting feed protein with
CSFA does not decrease the digestibility of intestinally digested protein, which is
estimated to be 95%. This way of protein protection is therefore considered
favourable from the viewpoint of protected protein quality.
The research hypothesis assumed that improved balance of amino acids as
a result of giving protein protected with CSFA and a certain amount of energy in the
form of CSFA would positively affect the synthesis of milk protein and fat. These
findings are supported by Wu et al. (1992). As a result of giving cows PSBM,
increases were obtained in the fat and protein content of milk and in the amount of
fat and protein secreted with milk in 24-h cycles. This may suggest that protection
of soybean meal with CSFA improves the conditions of amino acid supply to cows,
necessary for the synthesis of milk proteins, while fatty acids released from PSBM
stimulate the synthesis of milk fat in the mammary gland and are incorporated into
milk fat. This is also confirmed by a relationship between protein percentage and
amount of protein secreted in milk, which was estimated by Sporndly (1989) at
a positive correlation level (r = 0.37) for Holstein-Friesian cows.
An increase in the milk production of cows given protected protein or
undegradable amino acids has been found in American studies (DePeters and Cant,
1992). It is estimated that feeds with lower rumen degradability increase milk yield
by at least 5 – 10% when the level of dietary protein for cows is adequate
(Schingoethe et al., 1988). It must be remembered, however, that these studies were
120
F. Brzóska
performed with cows yielding approximately 35 kg milk/day. Relatively few results
of Polish studies on protein protected with CSFA consistently indicate that its use is
effective when feeding cows with yields above 30 kg milk/day.
Giving cows PSBM did not cause a significant increase in the level of true-N
fraction in milk, and there were no significant differences for the other N fractions.
It is thought that the level of casein-N in milk and the proportion of casein-N
in milk true-N is determined by genetic traits rather than by dietary composition.
Of great importance to the technological suitability of milk for cheese making
are the physical characteristics of milk, such as acidity, renneting time, and specific
gravity. The highest acidity was characteristic of the milk from cows receiving the
highest amount of PSBM. Distribution of this trait indicates that milk acidity is not
related to the type or amount of soybean meal given to cows. Similar relationships
were found for milk renneting. The specific gravity of milk was consistent between
the groups of cows, which indicates that it has no effect on this trait.
The levels of basic indicators of carbohydrate, protein and fat metabolism in
cow bodies, based on their blood plasma picture, showed no significant differences.
Glucose content was approximately 10% below the lower limit of physiological
norms accepted for cattle. These values may indicate a lack of available energy in
the dietary rations of cows in this experiment. On the other hand, a small but
upward tendency for cows’ body weight to increase indicates that the cows
underwent the process of synthesis and deposition of tissue fat. The total protein
content of plasma was within optimal range, and that of urea in the upper range.
This is evidence that the nutrient requirements of cows for nitrogen were
completely satisfied. The serum level of triglycerides was within the lower
reference values for cattle, which in addition to the low glucose content may be
indicative of the insufficient amount of available energy in the dietary rations. Data
on the analysed parameters of blood plasma were compared to the physiological
norms for cattle published by Kłopocki and Winnicka (1992).
Giving cows protein protected with CSFA at a rate of 22% of the diet caused
a significant increase in UFA in milk fat, which concurs with the previous research
on the use of CSFA obtained from vegetable oils and fish oil in cattle nutrition
(Brzóska et al., 1999). The decrease in saturated fatty acids in milk, accompanied
by an increase of stearic and docosahexaenoic acids, indicates an increase in the
level of fatty acids with anti-cholesterol action. These changes could result from the
fact that soybean meal was protected with CSFA containing rapeseed oil, which
slightly improved the dietetic value of milk fat.
It is concluded that giving PSBM to cows that yielded an average of 30.0 kg
milk/day increased their yield during the grazing period by 1.0 kg/day, with
a significant increase in the protein content of milk and in 24-h synthesis of protein
and fat. The yield of 30 kg milk/day seems to be a borderline value, at which giving
cows bypass protein can be expected to produce positive effects.
121
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Accepted for printing 25 XI 2004
FRANCISZEK BRZÓSKA
Wpływ śruty sojowej chronionej solami Ca-KT na wydajność krów, składniki białkowe
i tłuszczowe mleka oraz krwi
STRESZCZENIE
Doświadczenie wykonano na 32 krowach rasy czb, podzielonych na 4 grupy w układzie
analogowym. Krowy żywione na pastwisku otrzymywały ponadto zielonke˛ z upraw polowych, młóto
browarniane świeże i mieszanke˛ paszowa˛. Mieszanka paszowa zawierała śrute˛ poekstrakcyjna˛ sojowa˛
w ilości 15% (grupa kontrolna) i śrute˛ sojowa˛ chroniona˛ solami Ca-KT w ilości 15, 22 i 29% (grupy
doświadczalne). Krowy pobierały mieszanke˛ w ilości: 1,20; 1,23, 1,87 i 2,44 kg/dobe˛. Pobranie suchej
masy wynosiło średnio 20,98 kg/dzień, w tym mieszanek paszowych 8,27 kg/dzień i nie różniło sie˛
istotnie pomie˛dzy grupami. Wydajność mleczna krów kształtowała sie˛ na poziomie średnio 30,01
kg/dzień i wykazywała tendencje˛ rosna˛ca˛, jakkolwiek nie była zróżnicowana pomie˛dzy grupami. Ilość
mleka skorygowanego na zawartość białka i tłuszczu wynosiła średnio 27,04 kg/dzień i również nie
różniła sie˛ istotnie pomie˛dzy grupami.
Podawanie śruty sojowej chronionej, w porównaniu z niechroniona˛, zwie˛kszyło zawartość białka
o 14 g/kg i tłuszczu o 7 g/kg mleka, a uzyskane różnice były statystycznie istotne. Wzrosła istotnie ilość
123
tłuszczu i białka wydzielanych w mleku — tłuszczu o 63 g a białka o 17 g/dobe˛. Nie stwierdzono
istotnych różnic w zawartości glukozy, białka całkowitego, mocznika i trójglicerydów w osoczu krwi
krów z poszczególnych grup. Podawanie krowom śruty sojowej chronionej solami Ca-KT istotnie
obniżyło zawartość kwasu kaprynowego, laurynowego i palmitynooleinowego, a także kwasu arachidonowego i eikozapetnaenowego w tłuszczu mlecznym. Zwie˛kszyła sie˛ zawartość kwasu stearynowego,
arachidowego, behenowego i dokozaheksaenowego w tłuszczu mlecznym. Różnice w zawartości tych
kwasów były statystycznie nieistotne. Istotne zwie˛kszenie zawartości kwasów nienasyconych w mleku,
w tym MUFA, stwierdzono u krów otrzymuja˛cych w mieszance paszowej 22% śruty sojowej chronionej.
Pod wpływem śruty chronionej solami Ca-KT istotnej poprawie uległa wartość dietetyczna mleka
(DFA).
Na podstawie wykonanych badań można stwierdzić, że podawanie krowom, o przecie˛tnej wydajności 30 kg mleka/dzień, śruty sojowej chronionej solami CaKT nieistotnie zwie˛ksza wydajność mleczna˛,
natomiast istotnie zawartość białka w mleku oraz poprawia wartość dietetyczna˛ tłuszczu mleka.
EFFECTS OF TYPE OF LIQUID FEED AND FEEDING FREQUENCY
ON RUMEN DEVELOPMENT AND REARING PERFORMANCE
OF CALVES*
B a r b a r a N i w i ń s k a, J u l i u s z S t r z e t e l s k i
Abstract
The effects of type of liquid feed and feeding frequency on concentrate intake, rumen development
and rearing performance of calves were investigated. The experiment was performed with 36
bull-calves (37.5% Black-and-White × 62.5% Holstein-Friesian) assigned to two equal groups.
Calves received either cow’s milk (group M) or milk replacer (group P). In both groups, calves
were given liquid feeds once daily (subgroups-1) or three times daily (subgroups-3). In each
subgroup, three calves were slaughtered at the age of 36, 60 and 90 days. Calves were fed
according to the IZ-INRA (2001) standards feeding. Nutrient intake and conversion, weight of the
ruminal wall and ruminal contents, the content of volatile fatty acids (VFA) in ruminal fluid, and
the size of ruminal papillae were analysed. Calves from group M compared to those from
group P had a higher intake of liquid feed (P ≤ 0.03), achieving higher weight gains (P ≤ 0.02) and
showing better nutrient conversion per kg of weight gain (P ≤ 0.04). In calves of group M, ruminal
fluid contained more butyric acid (P ≤ 0.03) in total VFA. Calves fed three times daily compared to
those fed once daily until 36 days of age had a higher intake of liquid feed and concentrate
(P ≤ 0.02) and were characterized by higher daily gains (P ≤ 0.04) and higher percentage of butyric
acid in total VFA of ruminal fluid (P ≤ 0.03). Weight of ruminal walls and size of ruminal papillae
were not related to the type of liquid feed and frequency of its feeding (P > 0.05). Calves in the
subgroup given milk three times daily to 36 and to 60 days of age were characterized by the highest
daily weight gain (P ≤ 0.02), the highest content of butyric acid (P ≤ 0.04) and the lowest content of
propionic acid (P ≤ 0.02) in total VFA of rumen. The results suggest that the type of liquid feed has
no effect on the size of ruminal papillae, but may stimulate the direction of rumen fermentation.
Key words: calve, liquid feed, frequency, rumen, VFA, ruminal papillae
Research on the development of ruminal digestive function indicates a major role
of solid feed intake and digestion (Warner et al., 1956; Baldwin, 2000). The effects
of chemical composition, physical structure and amount of concentrate given on
digestive processes in the developing rumen have been determined (Greenwood et
al., 1977; Beharka et al., 1998). However, although during their first weeks of life
* This work was conducted as part of NRIAP statutory activity, project no. 23217.1/2003.
126
B. Niwińska and J. Strzetelski
calves ingest no solid feeds, volatile fatty acids appear in digesta of the developing
rumen as products of microbiological fermentation of liquid feed, which has entered the
rumen due to insufficient closure of the reticular groove (Lane and Jesse, 1997). It
appears that the type of liquid feed affects the direction of microbiological fermentation
during the first weeks of calves’ life, which may be reflected in the functional
development of the rumen. Research findings conclusively show that products of
microbiological digestion stimulate the rumen development, both by stabilizing ruminal
wall metabolism and by enlarging the surface of ruminal mucosa (Sander et al., 1959;
Baldwin, 1998; Lane et al., 2000). However, there are no current studies analysing the
effect of type and form of liquid feed on functional development of calf rumen.
The objective of this study was to find out if the type of liquid feed and feeding
frequency affect microbiological fermentation in the rumen, the size of ruminal
papillae, the intake of concentrates, and rearing performance of the calves.
Animals, feeding and management
The studies were performed with 36 Black-and-White bulls aged 7 – 9 days and
assigned to two experimental groups of equal size. Calves received either milk
(group M) or milk replacer (group P). In both groups, calves were given liquid feeds
to 56 days of age once daily (subgroups-1) or three times daily (subgroups-3). In each
subgroup, three calves were slaughtered at the age of 36, 60 and 90 days. Calves were
fed in keeping with the IZ-INRA (2001) standards feeding. Crude protein in the milk
replacer was provided by soy protein concentrate (60%) and dried whey (40%).
Calves were given limited amounts of hay (0.1 kg/day during milk feeding and 0.2 kg
after weaning), and ad libitum concentrate containing (%): crushed barley grain, 50;
wheat bran, 6; soybean meal, 17; crushed wheat grain, 25; and mineral mixture, 2.
The mineral mixture contained (g/kg): Ca, 165; P, 90; Mg, 36.2; Na, 72.5; Fe, 2.24;
Se, 0.011; Zn, 2.95; Cu, 1.31; Co, 0.08; Mn, 3.0; I, 0.09; biotin, 0.0015; vitamin E,
0.20; and vitamin A, 11250 IU. Feed intake was recorded daily by weighing feed
refusals. Animals were kept in individual cages with automatic drinkers and plastic,
perforated floors. Liquid feed was given in nipple buckets. Body weights were
checked at the start of the trial and at slaughter. In the calculations we used mean
body weights of calves from two consecutive days. The calves were always weighed
at the same hour (in the morning, prior to feed intake).
Slaughter of animals
Two to four hours after the morning feeding, calves were sacrificed at a commercial abattoir. After preparing the digestive tract, clamps were inserted in the rumen
atrium and in the reticulorumen to prevent outflow of digesta. Weight of rumen with
digesta was determined, rumen digesta samples were taken, and after removing the
digesta, the weight of rumen without the digesta was determined. From the front part
of the dorsal sac, parallel to the rostral groove, fragments of ruminal wall (0.5 × 7.0
Effects of liquid feed on rearing performance of calves
127
cm) were taken and placed in 10% formalin solution (Greenwood et al., 1997).
Weight of calf carcass was determined following the regulations of the Ministry of
Agriculture and Rural Economy (Law Gazette, No. 161, item 1566).
Preparation and assessment of histological preparations
Histological preparations of the ruminal wall were made and assessed at the
Department of Pathomorphology of the Jagiellonian University Medical College.
A tissue processor Shandon Inc. (Great Britain) was used. Paraffin blocks were made,
the sections of which were stained with the hematoxylin-eosin technique. Acquisition
of images was performed using a Zeiss Axioscop microscope (Zeiss GmbH, Germany)
with Plan-NEOFLUAR 2.5x lens and CCD ZVS-47DE camera (Optronics Inc., USA),
connected via RGB line with GraBIT PCI card (Soft Imaging System GmbH,
Germany). From each fragment of ruminal wall, long and short axis of 20 ruminal
papillae was measured using the Arbitrary Distance function of the image analysis
system. General appearance of the papillae, the presence of inflammatory processes,
and intensity of the keratotic processes were analysed. Keratosis with parakeratotic
layer was found in the presence of dark, pycnotic nuclei in the eosinophilic,
homogeneous horny layer. For the assessment, a Zeiss Axioscop microscope (Zeiss
GmbH, Germany) with a set of Plan-NEOFLUAR 2.5, 10, 20 and 40x lens was used.
Chemical analyses
The chemical composition of the feeds was determined according to AOAC
(1990). Samples of ruminal contents were assayed for pH using an ELMETRON
CP-411 pH-meter, and volatile fatty acids (VFA) after the ruminal contents filtrate
was centrifuged with 24% metaphosphoric acid using VARIAN 3400 gas chromatograph (column CP-Wax 58, 25 m, 0.53 mm, 1 micron; detector 260°C, operating
temperature 90 – 200°C).
Calculations
The weight of ruminal contents and ruminal wall was expressed as % of
slaughter weight of calf carcass. The elongation index (We) was calculated as the
width to length ratio of the ruminal papillae. Daily concentrate intake was
calculated by dividing the amount of concentrate (after accounting for feed refusals)
by the number of experimental days (age of calves at slaughter — age at start of
experiment). The results were analysed statistically with two-way analysis of
variance (type of liquid feed and feeding frequency), using Statistica ver. 5.0
(1997). Differences were assumed to be not significant at P > 0.05.
Results
The chemical composition and nutritive value of the feeds are given in Table 1.
Both liquid feeds contained about 32.2 g digestible protein per 1 kg.
128
Table 1. The chemical composition and nutritive value of the feeds
In 1 kg DM:
Feed
Concentrate
Meadow hay
Cow’s milk
Milk replacer 2
1
2
Dry
matter
crude
protein
(g)
ether
extract
(g)
fibre
(g)
875.3
821.5
125.6
947.0
189.1
85.7
266.7
215.8
15.7
18.1
319.3
154.4
47.5 61.4
331.7 108.1
59.7
10.1 77.1
ash
(g)
UFL PDIN
(g)
1.17
0.58
1.90
1.53
digestible
PDIE
protein1
(g)
(g)
130
57.2
131
74.2
253.3
204.9
Digestibility coefficients of crude protein acc. to INRA (1989).
Liquid feed contained 166 g powder per 1 kg solution.
Calves from group M had a higher daily intake of liquid feed (P ≤ 0.03), and to
60 days also a higher intake of concentrate compared to calves from group
P (P ≤ 0.01; Table 2). Calves fed once daily had a lower intake of liquid feed
(P ≤ 0.07), and to 36 days of age a higher intake of concentrate (P ≤ 0.02) compared
to calves fed three times daily.
Table 2. Daily intake of feeds by the calves (kg)
Item
Feeding period
(from — to days of age3):
7 – 36
liquid feed
concentrate
7 – 60
liquid feed
concentrate
7 – 90
liquid feed
concentrate
1
2
3
Liquid feed
M
P
6.61
0.07
5.49
0.08
5.76
0.24
3.97
1.19
P=
1
Frequency
Interaction2
P=
Mean SEM
6.39
0.04
0.02
0.02
6.08
0.08
0.3
0.02
*
N
5.31
0.18
5.61
0.15
0.07
0.21
5.46
0.17
0.22
0.02
*
*
3.46
1.24
3.74
1.29
0.07
0.60
3.60
1.26
0.13
0.07
*
N
1
3
0.01
0.72
5.73
0.11
5.14
0.11
0.02
0.01
3.23
1.33
0.03
0.20
Level of significance for differences between means.
Significance of interactions: N — P > 0.05; * — P ≤ 0.05.
Actual age of calves at slaughter (days): 36.75 ± 2.2; 59.8 ± 2.5 and 89.5 ± 4.3.
Regardless of the type and frequency of liquid feed, the weight of ruminal
contents was similar (P > 0.05) (Table 3). In the ruminal fluid of calves from group
M, a higher content of butyric acid in total VFA (P ≤ 0.05) was found compared
with group P. At 36 days of age, a higher proportion of butyric acid in total VFA
was characteristic of the ruminal contents of calves fed three times a day compared
to those fed once a day (P ≤ 0.03).
Weight of ruminal walls of the calves was not dependent on the type of liquid
feed or feeding frequency (P > 0.05; Table 4). Ruminal papillae of calves receiving
liquid feed once daily compared to three times daily were characterized by a higher
coefficient of We at 36 (P ≤ 0.01) and 60 days of age (P ≤ 0.03).
129
Table 3. Weight, pH and concentration of VFA in ruminal fluid
Item
Liquid feed
M
P
P=
Frequency
1
3
P=
Mean SEM
Interaction1
36 days of age:
2.99 2.62 0.05
2.81 0.51
weight of ruminal contents2 2.74 2.89 0.51
133.75 177.06 0.22 120.01 190.81 0.07 155.41 22.9
total VFA3
56.43 61.62 0.06 57.28 60.76 0.10 59.02 1.80
% mol4 C2
26.58 28.46 0.06 29.96 25.07 0.10 27.52 1.92
C3
12.45 6.2 0.01
8.58 10.07 0.03
9.33 1.08
C4
2.12 2.17 0.60
1.91 2.40 0.11
2.15 0.2
ratio
C2/C3
2.13 4.59 0.01
3.49 2.49 0.06
2.98 0.5
C3/C4
pH
min
5.73 5.6 0.49
5.84 5.48 0.12
5.66 0.12
max
5.9
5.9 0.91
5.99 5.79 0.39
5.90 0.12
N
*
*
*
*
*
*
N
N
60 days of age:
weight of ruminal contents 9.62 8.56
total VFA
136.6 118.2
% mol C2
53.3 53.14
30.2 35.9
C3
10.75 6.53
C4
1.76 1.48
ratio
C2/C3
2.81 5.5
C3/C4
pH
min
6.24 5.93
max
6.38 6.01
0.62 11.37 6.79 0.08
9.09 1.21
0.53 134.37 120.56 0.61 127.43 18.7
0.91 55.5 50.94 0.32 53.22 2.2
0.03 32.18 33.92 0.41 33.05 1.87
0.03
7.3
9.98 0.06
8.64 1.67
0.30
1.73 1.50 0.60
1.62 0.15
0.06
4.41 3.40 0.08
4.03 0.69
0.31
6.24 5.94 0.21
6.09 0.18
0.19
6.23 6.16 0.42
6.20 0.19
N
*
N
*
*
*
N
*
*
90 days of age:
weight of ruminal contents 9.03 11.1
total VFA
129 177.9
% mol C2
54.23 56.01
32.64 34.62
C3
6.72 5.62
C4
1.66 1.62
ratio
C2/C3
4.86 6.16
C3/C4
pH
min
6.07 5.83
max
6.35 6.03
0.29 10.11
0.30 160.9
0.62 55.47
0.07 33.15
0.02
6.15
0.81
1.67
0.07
5.39
0.60
5.93
0.51
6.13
N
N
N
N
N
N
N
N
N
1
2
3
4
10.10
146
54.78
34.11
6.19
1.60
5.51
5.97
6.27
0.91 10.08 1.12
0.72 153.45 20.5
0.80 55.12 1.35
0.81 33.63 1.25
0.92
6.17 0.28
0.60
1.64 0.11
0.91
5.48 0.27
0.91
5.95 0.21
0.79
6.20 0.18
Significance of interaction: N — P > 0.05; * — P ≤ 0.05.
Weight of ruminal contents was expressed as % of calf carcass weight postmortem.
Total VFA = C2 + C3 + iso-C4 + C4 + iso-C5 + C5.
mmol/100 mmol total VFA.
Type of liquid feed did not affect the intake of concentrate per kg of weight gain
(P > 0.05; Table 5). Calves from group M grew faster (P ≤ 0.02) and converted
liquid feed and nutrients per kg weight gain to greater advantage (P ≤ 0.01) than
calves from group P. To 36 days of age, calves receiving liquid feed three times
daily were characterized by higher daily gains than those fed once daily (P ≤ 0.04).
Both to 36 and to 60 days of age, calves from the subgroup receiving cow’s
milk three times daily were characterized by the highest daily weight gain
(P ≤ 0.02), the highest content of butyric acid (P ≤ 0.04) and the lowest content
of propionic acid (P ≤ 0.02) in total VFA of ruminal fluid compared to the other
subgroups (Table 6).
130
Table 4. Weight of ruminal wall, size of ruminal papillae, and advancement of inflammatory and
keratinization processes
Item
36 days of age:
Weight of ruminal wall2
Ruminal papillae:
length (µm)
width (µm)
We
keratinization
inflammatory infiltration
of the mucosa
Liquid feed
P
1.78
1.69 0.60
3
P=
1
3
1.91
1.56 0.08
Mean SEM
933 814
0.41 766 982
0.20 852
75
341 286
0.32 334 294
0.41 303
24
0.39 0.40 0.89
0.45 0.34 0.01
0.39 0.02
+
+
+
+
N
N
*
++
++
0.09
++
++
3.03
2.62 0.11
1.72
Interaction1
N
90 days of age:
Weight of ruminal wall
5.38 5.50
Ruminal papillae:
length (µm)
2650 2334
width (µm)
436 474
0.18 0.25
We
keratinization
++
++
of the mucosa
+
+
2
Frequency
0.17
60 days of age:
Weight of ruminal wall
3.02 2.63
Ruminal papillae:
length (µm)
1113 1075
width (µm)
404 375
0.40 0.38
We
keratinization
+
+
of the mucosa
+
++
1
P=
M
0.13
N
0.92 1063 1125
0.81 1099
98
0.53 437 342
0.07 392
28
0.81
0.43 0.35 0.03 0.391 0.03
+
+
N
*
N
0.08
+
+
5.66
5.21 0.08
2.82
0.24
N
0.41 2594 2390
0.21 2658
179
0.40 453 459
0.92 445
19
0.05
0.19 0.24 0.10
0.19 0.02
++
++
N
N
N
+
5.44
+
Significance of interactions: N — P > 0.05; * — P ≤ 0.05.
Weight of ruminal wall expressed as % of hot carcass weight.
Intensity of processes: + observable, ++ advanced.
Table 5. Daily weight gains and feed conversion per kg of weight gain
Item
1
Liquid feed
M
P
2
3
From 7 to 36 days of age:
701 410
Daily gains (g day–1)
Conversion per kg of weight gain:
liquid feed (kg)
9.29 13.14
concentrate (kg)
0.10 0.20
dry matter (kg)
1.32 2.93
crude protein (g)
339 627
PDI (g)
296 502
UFL
2.20 3.76
P=
4
Frequency
1
3
5
6
0.01 486
620
0.01 11.99 10.43
0.42
0.23 0.06
0.01
2.15 2.09
0.01 489 477
0.01 424 372
0.04
3.15 2.76
P=
7
Mean SEM
Interaction1
8
9
10
0.04 554
62
*
0.11 11.2
1.6
0.08
0.15 0.03
0.81
2.12 0.28
0.89 483
19.4
0.09 398 184
0.80
2.97 0.43
*
N
*
*
*
*
131
Table 5 — contd.
1
2
3
714 466
Daily gains (g day–1)
liquid feed (kg)
7.93 10.82
concentrate (kg)
0.33 0.24
dry matter (kg)
1.32 1.97
crude protein (g)
323 413
PDI (g)
292 381
UFL
2.26 2.87
939 731
Weight gains (g day –1)
concentrate (kg)
1.25 1.80
dry matter (kg)
1.84 2.42
crude protein (g)
354 453
PDI (g)
281 351
UFL
2.32 2.94
4
5
6
0.01 586
594
7
8
9
10
0.91 590
46
*
0.01
9.14 9.61
0.11
0.31 0.26
0.01
1.67 1.62
0.01 372 365
0.01 339 334
0.01
2.59 2.54
0.89
9.38 0.51
0.31
0.29 0.03
0.52
1.64 0.10
0.70 369
16.0
0.80 337
15.5
0.83
2.57 0.11
*
N
*
*
*
*
0.02 835
0.89 835
*
834
0.06
1.5
1.55
0.01
2.08 2.19
0.02 393 413
0.02 307 324
0.02
2.57 2.69
41
0.61
1.53
0.80
2.13
0.82 403
0.90 316
0.89
2.63
0.06
0.06
8.7
6.0
0.05
N
*
*
*
*
Significance of interaction: N — P > 0.05; * — P ≤ 0.05.
Table 6. Weight gains, DM intake, and %mol propionic acid and butyric acid in total VFA of calf rumen
liquid in experimental subgroups in periods characterized by a significant interaction of experimental factors
Liquid feed
Frequency
Daily weight gain (g)
DM of liquid feed (kg)
DM of the diet (kg)
At 36 days of age:
%mol1 propionic acid
butyric acid
Daily weight gain (g)
DM of liquid feed (kg)
DM of the diet (kg)
At 60 days of age:
%mol1 propionic acid
butyric acid
M
1
589 ABb
1.15 Bb
1.32 b
33.3 a
11.2 ABa
698 a
P
3
811 Aa
1.17 Bb
1.33 b
19.89 b
13.7 Aa
729 a
1
392 Bb
2.09 Aa
2.99 a
26.6 ab
5.9 Bb
473 b
3
427 Bb
P=
0.01
2.04 ABa 0.01
2.86 a
0.01
30.3 a
6.4 Bb
458 b
0.02
0.01
0.02
0.95 B
1.35 B
1.04 B
1.29 B
1.75 A
2.00 A
1.67 A
1.94 A
0.01
0.01
33.9 ABb
8.0 ab
26.6 Bb
13.5 a
30.3 Bb
6.6 b
41.3 Aa
6.4 b
0.01
0.04
1
mmol/100 mmol total VFA.
a, b — values in rows with different letters differ significantly (P ≤ 0.05).
A, B — values in rows with different letters differ highly significantly (P ≤ 0.01).
132
Discussion
The concentration of volatile fatty acids in the ruminal fluid was not different
from the mean values reported for calves aged 2 to 10 weeks by Beharka et al.
(1998), but clearly showed differences between calves receiving milk or milk
replacer. The rumen content of milk fed calves was characterized by a higher
proportion of butyric acid in total VFA. Particularly noticeable differences shown in
the youngest calves to 36 days of age (P ≤ 0.01) suggest that they were induced by
the type of liquid feed, because the intake of concentrate was similar in both groups
(average of 0.08 kg day–1; P ≤ 0.72). The higher content of butyric acid as a product
of developing microbiological fermentation in the rumen is favourable. As the
ruminal wall metabolism stabilizes, butyric acid becomes the main source of
energy, and its use is directly proportional to the concentration in the ruminal fluid
(Baldwin and Jesse, 1992; Lane et al., 2000). Favourable development of the
digestive tract in milk-fed calves is also supported by the higher daily gains and
better nutrient conversion to 90 days of age (P ≤ 0.02). At the present stage of
research, however, it is difficult to indicate which constituents of milk increased the
content of butyric acid. Recent studies indicate that milk is better than milk replacer
in satisfying the needs of a young organism not only because of its chemical
composition, but also due to the presence of bioactive components (Neeser et al.,
1991; Molkentin, 2000; Zabielski, 2001).
Greenwood et al. (1997) and Baldwin (2000) demonstrated that the increase in
the weight of ruminal wall is dependent on the increase in the DM intake of solid
feed. In our experiment, calves on the milk diet from 7 do 60 days of age had
a higher intake of concentrate compared to calves on the milk replacer (P ≤ 0.01),
yet this had no effect on differences in the weight of ruminal wall (P ≤ 0.09). It
appears that this phenomenon was due to the insufficient intake of concentrate,
which in this period averaged 0.17 kg day–1 in both groups of calves. In their
studies, the reported authors used animals that were weaned earlier (28 or 49 days
of age) and had a higher daily intake of solid feeds (about 0.50 kg day–1).
The differences in VFA content of the calf ruminal fluid, found in our study, did
not affect the size of ruminal papillae. Also Zitman et al. (2003) and Lesmeister et
al. (2004) did not find a relationship between the type of feed and the size of
papillae from the anterior part of the dorsal sac. The lack of differences in the size
of ruminal papillae could result from great variation of these structures, as indicated
by McGavin and Morrill (1976). Hugi et al. (1997) and Lane and Jesse (1997)
report that mainly insulin appears to be responsible for the increase in the
absorptive surface and metabolism of the ruminal wall. Possibly, an analysis of
insulin level in the blood of calves could help to clarify the effect of type of liquid
feed on rumen development.
The inflammatory infiltrations observed in the ruminal wall and the keratinization processes could be due to excessive grinding of the concentrate mixture,
despite the fact that it contained grain components in the crushed form. Greenwood
et al. (1997) demonstrated that the finer particles a feed contains, the greater the
133
extent of keratinization of rumen mucosa. On the other hand, Cozzi et al. (2002)
report digestive disorders as the main cause of advanced keratinization processes. In
our studies, we did not find any effect of the type of liquid feed or feeding frequency on the
advancement of inflammatory states and keratinization. Although the intake of
concentrate by calves aged 36 days was small, the inflammatory and keratinization
processes were most noticeable in calves at this age. In young calves receiving solid
feed, the immature ruminal wall is unable to metabolize the entirety of fermentation
products in the rumen, and as a result, their concentration increases and pH value
decreases (Beharka et al., 1998). The low intake of solid feeds is also associated with
low saliva secretion, which also leads to decreased pH value, and additionally the
buffering role of saliva is stronger in older calves. Our findings support the suggestion
of Beharka et al. (1998) that there is a relationship between pH of ruminal content and
health status of ruminal wall, because the most advanced inflammatory changes of the
mucosa were found in calves with the lowest pH values of ruminal content.
The results obtained indicate that the type of liquid feed may stimulate the
direction of fermentation in the developing rumen. Calves in the subgroup given milk
three times daily were characterized by the highest daily weight gain and the greatest
content of butyric acid in total VFA of the ruminal fluid. Both the type of liquid feed
and feeding frequency as well as differences in the concentration of VFA in ruminal
contents, did not affect the weight of the ruminal wall and size of the ruminal papillae.
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BARBARA NIWIŃSKA, JULIUSZ STRZETELSKI
Wpływ rodzaju i cze˛stotliwości skarmiania paszy płynnej na rozwój żwacza oraz wyniki
odchowu ciela˛t
STRESZCZENIE
Badano wpływ rodzaju paszy płynnej i cze˛stotliwości jej skarmiania na pobranie mieszanki
treściwej, rozwój żwacza i wyniki odchowu ciela˛t. Doświadczenie przeprowadzono na 36 ciele˛tachbuhajkach, mieszańcach rasy czarno-białej z holsztyńsko-fryzyjska˛ (62,5% hf), przydzielonych do
dwóch równoliczebnych grup. W grupie M — ciele˛ta otrzymywały mleko krowie a w grupie
P — preparat mlekozaste˛pczy. W obu grupach pasze płynne podawano ciele˛tom jeden raz dziennie
(podgrupy-1) lub trzy razy dziennie (podgrupy-3). W każdej podgrupie ubijano po trzy ciele˛ta w wieku
36, 60 oraz 90 dni. Ciele˛ta żywiono wg zaleceń systemu IZ-INRA (2001). Oceniano pobranie
i wykorzystanie składników pokarmowych, mase˛ ściany żwacza i treści żwacza, zawartość lotnych
kwasów tłuszczowych (LKT) w płynie żwacza oraz wymiary kosmków żwaczowych. Ciele˛ta z grupy
M w porównaniu do ciela˛t z grupy P pobrały dziennie wie˛cej paszy płynnej (P ≤ 0,03), osia˛gaja˛c wyższe
przyrosty masy ciała (P ≤ 0,02) i lepiej wykorzystuja˛c składniki pokarmowe paszy na 1 kg przyrostu
masy ciała (P ≤ 0,04). Płyn żwacza ciela˛t grupy M zawierał wie˛cej kwasu masłowego (P ≤ 0,03) w sumie
LKT. Ciele˛ta pojone trzykrotnie w cia˛gu dnia w porównaniu z ciele˛tami pojonymi jednokrotnie do
36. dnia życia pobierały dziennie wie˛cej paszy płynnej oraz mieszanki treściwej (P ≤ 0,02), charakteryzowały sie˛ również wyższymi dziennymi przyrostami masy ciała (P ≤ 0,04) oraz wyższym udziałem
kwasu masłowego w sumie LKT płynu żwacza (P ≤ 0,03). Masa ścian żwacza ciela˛t oraz wymiary
kosmków żwaczowych nie zależały od rodzaju paszy płynnej oraz od cze˛stotliwości jej skarmiania
(P > 0,05). Ciele˛ta w podgrupie karmionej trzykrotnie mlekiem do 36. oraz do 60. dnia życia
charakteryzowały sie˛ najwyższym dziennym przyrostem masy ciała (P ≤ 0,02), również najwyższa˛
zawartościa˛ kwasu masłowego (P ≤ 0,04) oraz najniższa˛ kwasu propionowego (P ≤ 0,02) w sumie LKT
żwacza. Wyniki badań sugeruja˛, że rodzaj paszy płynnej nie wpływa na wymiary kosmków żwaczowych, jednak może stymulować kierunek fermentacji żwaczowej.
EFFECTS OF CONJUGATED LINOLEIC ACID (CLA)
ON PIG PERFORMANCE, CARCASS QUALITY, CHEMICAL
COMPOSITION OF MEAT AND SERUM LIPID PROFILE*
Beata Szymczyk
Department of Animal Nutrition and Feed Sciences, National Research Institute of Animal Production,
Abstract
Effects of feeding different levels (0.0, 0.1, 0.2, 0.4 and 0.6%) of conjugated linoleic acid (CLA) on
pig performance, carcass quality and serum lipids were determined. The results of fattening in the
61 to 108 kg weight range depended on the dietary level of CLA. The addition of 0.4% CLA
significantly increased weight gain (P < 0.05) and improved feed conversion (P < 0.01) in relation to
the control group. The 0.6% CLA supplement worsened the fattening results but they were better
than in the group given no CLA supplement. Pigs receiving CLA were characterized by lower
fatness than the control animals. Statistically highly significant decreases in backfat thickness of
neck and loin and in mean backfat thickness from 5 measurements were obtained. Backfat
thickness at the C-1 site proved lower (P < 0.01) in the CLA groups and ranged from 1.07 (0.1%
CLA) to 0.91 cm (0.4% CLA), compared to 1.17 cm in the control group. Increasing the dietary
level of CLA caused a linear increase in the fat-to-meat ratio, from 6.81 in the control group to 8.40
in the group fed with 0.6% CLA. A greater loin eye area and meatiness (P < 0.01) and a higher
proportion of meat in ham (P < 0.05) were also found in groups with CLA. Chemical analysis of
m. longissimus showed highly significant differences between the groups in the content of crude fat
(reduction from 1.23% in the control group to 0.95 and 0.97% at 0.4 and 0.6% CLA, respectively)
and in cholesterol, which decreased in the LD muscle of pigs from 63.99 in the group given no CLA
to 49.53 mg/100 g (0.6% CLA). The supplementation of CLA had a negative influence on the
content of total cholesterol (TC), LDL-cholesterol and triacylglycerols (TAG) in serum. In the
control group the level of these lipid fractions was lower, while for TC and TAG the differences
were even highly significant.
Key words: pig, conjugated linoleic acid, performance, carcass quality, serum lipids
136
B. Szymczyk
Conjugated linoleic acid (CLA) is a collective name for the mixture of
positional and geometric isomers of octadecadienoic acid (C18:2) in which the
double bonds are separated by a single C-C bond. CLA isomers are present mainly
in ruminant meat and dairy products, while meats from monogastric animals are
relatively low in CLA (Lawson et al., 2001). Recent interest in CLA results mainly
from its beneficial health effects. CLA was found to be antiatherogenic and
hypocholesterolemic (Nicolosi et al., 1997). In a number of studies, pure CLA was
shown to modify lipid metabolism and to reduce body fat. CLA has been reported to
have strong fat-to-lean repartitioning effect in mice (Chardigny et al., 2003),
broilers (Du and Ahn, 2003) and pigs (Ostrowska et al., 2003). In addition, Dugan
(2001) and Barowicz et al. (2002) found trends for reduced feed conversion ratio
and increased weight gains in pigs fed CLA during the finisher phase of fattening
period.
In this context, feeding CLA to pigs may be a means of providing pork
meat with several beneficial functional properties and with desirable economic
value. Therefore, the main objective of the present study was to determine the
effects of dietary CLA isomers on pig performance, carcass quality and serum lipid
profile.
Animals and diet
Eighty crossbred fatteners [ ( Polish Landrace × White Polish Large)
× Pietrain] with an average initial body weight of 30 kg were randomly assigned
to five groups with 16 animals (8 barrows and 8 gilts) each. Animals were housed
individually in pens with free access to water. Pigs were weighed before the
beginning of experiment and then every 2 weeks throughout the fattening period.
Feed intake was recorded throughout the experiment in order to calculate feed
conversion ratio. Fatteners were fed with a grower diet from 30 to 60 kg of body
weight and a CLA-enriched finisher diet from 61 kg to slaughter body weight. The
five dietary treatments consisted of five graded levels (0.0, 0.15, 0.30, 0.61, 0.92%)
of the commercial CLA product (Natural Lipids Ltd., Norway) containing 65%
CLA isomers (27.6% of cis-9, trans-11 and 27.5% of trans-10, cis-12 isomers).
Thus the resulting dietary CLA concentrations were 0.0, 0.1, 0.2, 0.4 and 0.6%. The
CLA was substituted for sunflower oil. Pigs were fed twice daily on a restricted
scale according to nutrient requirements of pigs. Composition of diets and their
nutrient content are given in Table 1. Pigs were slaughtered when their average
weight reached about 108 kg. All right sides of carcasses were dissected and carcass
measurements were collected at 24 h postmortem. Meatiness was calculated using
regression equation according to Borzuta (1998).
137
Dietary CLA, carcass quality and serum lipids
Table 1. The ingredient composition and nutrient content of experimental diets (g/kg)
Item
Ground wheat
Ground barley
Soybean meal
Limestone
Dicalcium phosphate
NaCl
Premix Lutamix
L-lys (96 %)
Vitamin E (10 %)
Sunflower oil
Calculated nutrient content (g/kg)
crude protein (g)
crude fat (g)
crude fibre (g)
ME (MJ)
Lys (g)
Met + Cys (g)
Thr (g)
Ca (g)
P (g)
Diet
grower
finisher
250
501
200
10
10
3
5 (PT1)
1
140
650
160
10
10
3
5 (PT2)
1
1
20*
20
173.30
35.60
45.70
13.08
9.30
5.67
5.80
7.06
6.10
157.50
32.30
49.50
12.92
8.27
5.30
5.83
7.03
5.90
* The CLA source was substituted for sunflower oil at 1.5, 3.0, 6.1 and 9.2 g/kg in groups II-V, respectively.
Sample collection and chemical analysis
The samples of m. longissimus were obtained from the area of the last thoracic and
first lumbar vertebra 24 h post-mortem to determine chemical composition and
cholesterol content. The total tissue lipids were extracted according to the method
of Folch et al. (1957). Total cholesterol content of pork lipid extracts was analysed
using the Rhee et al. (1982) method. Chemical composition of meat was determined
by standard methods (AOAC, 1995).
Blood samples for estimation of lipid level were taken from the jugular vein.
Serum total cholesterol (TC) content was analysed enzymatically according to
Allain et al. (1974) and its high-density lipoprotein (HDL) fraction according to
Warrick et al. (1982). The low-density lipoprotein (LDL) fraction was calculated
according to Friedewald et al. (1972): LDL = TC — HDL — TAG/5. Triacylglycerol (TAG) content was estimated according to Mc Gowan et al. (1983). Total
lipids were analysed enzymatically using Beckman DU640 spectrophotometer and
Cormay diagnostic kits.
The effects of dietary CLA on pig performance, carcass quality and serum lipid
profile were analysed using two-way (dietary level of CLA × sex) ANOVA
generated by the STATISTICA v. 5.1 package. Duncan’s multiple range test was
138
B. Szymczyk
used to determine the significance of differences between treatment means at the
P < 0.05 and P < 0.01 levels of significance.
Results
Production indicators obtained in the first period of rearing (30 – 60 kg) were
similar in all the groups, which was due to using the identical feed mixture for all
experimental fatteners. Daily gains ranged from 743 to 757 g. Feed conversion per
1 kg gain ranged from 2.79 to 2.87 kg. The results of the experimental fattening in
the 61 – 108 kg weight range depended on the level of conjugated linoleic acid
(CLA) in the mixtures (Table 2). CLA increased in the feed up to 0.4% caused
gradual, statistically significant increases in weight gains (P < 0.05) in relation to
the control group as well as improvements in feed conversion (P < 0.01). The 0.6%
CLA supplement markedly worsened the fattening results, but in this case too,
production indicators were better than in the group given no CLA supplement. No
statistically significant differences were observed in production indicators between
gilts and barrows, regardless of the group. The dietary CLA supplement had
a significant effect on carcass analysis (Table 2). Fatteners receiving CLA were
characterized by substantially lower fatness than the control animals. Statistically
highly significant decreases in backfat thickness of neck and loin and in mean
backfat thickness from 5 measurements were obtained, from 2.23 cm in the control
group to 1.96 cm in the group with the highest CLA level. Backfat thickness at the
C-1 site proved lower (P < 0.01) in the CLA groups and ranged from 1.07 (0.1%
CLA) to 0.91 cm (0.4% CLA), compared to 1.17 cm in the control group.
In fatteners given CLA-supplemented feed, significantly higher (P < 0.05) slaughter yield (79.57 – 80.11%) was obtained than in the control group (78.76%). A greater
loin eye area and meatiness (P < 0.01) and a higher proportion of meat in ham
(P < 0.05) were also found. The above parameters achieved the highest values in the
group receiving 0.4% CLA, whereas in the 0.6% CLA group slightly worse results of
carcass analysis were observed. The high CLA level in feed had no negative effect on
the proportion of meat and fat in carcasses. Increasing the concentration of CLA in
feed in the successive experimental groups caused a linear increase in the fat-to-meat
ratio, from 6.81 in the control group to 8.40 in the group receiving the highest CLA
supplement (P < 0.01). Carcass analysis showed that some parameters were related to
the sex of animals. For gilts, a greater loin eye area and lower backfat thickness at the
C-1 site were found than for barrows (P < 0.01). A more favourable fat-to-meat ratio
and higher meatiness were also noted (P < 0.05).
Analysis of the chemical composition of meat showed highly significant
differences between the groups in the content of crude fat, which decreased from
1.23% in the control group to 0.95 and 0.97% (at 0.4 and 0.6% CLA respectively)
and in total cholesterol, which decreased in the m. longissimus of pigs from 63.99 in the
group given no CLA to 49.53 mg/100 g in the meat of animals receiving 0.6%
dietary CLA (Table 3). Sex of animals influenced only the content of crude fat
(P < 0.01), which was markedly lower in the meat of gilts than in barrows.
Table 2. Effect of conjugated linoleic acid (CLA) on fattening and slaughter results
Source of variation
dietary level of CLA (%)
Item
0.0
Average daily weight gain (g)
Feed conversion ratio (FCR)
Cold dressing yield (%)
Ham weight (kg)
Backfat of ham with skin (%)
Meat of ham (%)
Weight of neck (kg)
Backfat of neck with skin (%)
Loin with skin weight (kg)
Backfat of loin with skin (%)
Loin eye area (cm2)
Average backfat thickness from
5 measurements (cm)
Backfat thickness measurement
in C-1 (cm)
Lean-fat ratio
Meatiness (%)
760 a
3.89 Aa
78.76 a
10.13
15.30 a
77.29 a
7.40
14.86 A
8.52
24.88 A
53.82 Aa
2.23 a
1.17 Aa
6.81 A
57.99 A
0.1
0.2
779 ab
798 b
3.79 ABab
3.77 ABbc
79.64 b
79.57 b
10.28
10.25
14.78 a
14.30 ab
77.92 ab
78.82 ab
7.18
7.42
13.78 AB
12.78 BC
8.66
8.46
23.09 AB
22.34 B
58.31 Abb
56.82 Bb
2.17 a
1.07 ABb
7.09 A
59.32 AB
2.02 b
1.04 ABb
7.58 AB
59.98 ABC
sex
0.4
0.6
800 b
3.73 Bb
79.67 b
10.26
13.95 b
79.5 b
7.51
12.39 C
8.55
21.28 BC
58.76 Bb
779 ab
3.88 ABac
80.11 b
10.46
13.47 ab
78.88 ab
7.39
12.05 C
8.48
19.81 C
58.75 Bb

SEM

Significance
of effects
%
CLA
sex
interaction
779
3.82
79.36
10.35
13.72
78.89
7.34
12.89
8.50
21.41
58.39
786
3.81
79.74
10.19
14.42
78.10
7.43
13.45
8.57
23.10
56.20
4.042
0.018
0.133
0.068
0.124
0.293
0.046
0.120
0.061
0.042
0.490
x
xx
x
NS
x
x
NS
xx
NS
xx
xx
NS
NS
NS
NS
NS
NS
NS
NS
NS
x
xx
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
2.00 b
1.96 b
2.05
2.11
0.038
x
NS
NS
0.91 Bb
8.13 B
61.70 C
0.97 Bb
8.40 B
61.17 BC
0.97
7.91
60.87
1.09
7.30
59.19
0.026
0.150
0.353
xx
xx
xx
xx
x
x
NS
NS
NS
x — P < 0.05; xx — P < 0.01; NS — P ≥ 0.05.
Table 3. Composition (%) and cholesterol level (mg/100 g) of longissimus muscle of pigs
Source of variation
Item
Dry matter (%)
Crude protein (%)
Ether extract (%)
Total cholesterol (mg/100 g)
sex
0.0
0.1
0.2
0.4
0.6
24.91
22.55
1.23 A
63.99 A
24.96
22.46
1.13 AB
62.14 AB
24.81
22.01
1.02 BC
60.28 BC
25.38
22.37
0.95 C
58.00 C
25.02
22.32
0.97 BC
49.53 D
SEM

24.97
22.41
0.95
58.28
25.06
22.26
1.17
59.31
0.100
0.101
0.040
0.775
Significance
of effects
%
CLA
sex
interaction
NS
NS
xx
xx
NS
NS
xx
NS
NS
NS
NS
NS
A, B, C, D — values in the same rows with different letters differ significantly (P < 0.01).
xx — P < 0.01; NS — P ≥ 0.05.
Table 4. Effect of dietary level of CLA (%) on serum lipid profile in pigs
Source of variation
Lipid ftactions
Total cholesterol-TC (mg/dl)
HDL-C fraction (mg/dl)
LDL-C fraction (mg/dl)
HDL-C : TC ratio
Triacylglycerols (mg/dl)
Total lipids (mg/dl)
sex
0.0
0.1
0.2
0.4
0.6
82.59 A
34.27 A
39.24 a
0.415 a
44.22 A
363.05
89.18 BC
36.07 AC
41.07 ab
0.405 a
57.12 BC
379.23
98.37 D
41.13 B
44.58 b
0.418 a
51.41 AB
399.77
92.59 BCD
40.89 B
41.99 ab
0.441 b
50.67 AB
392.88
86.65 AC
38.93 BC
37.83 a
0.450 b
63.10 C
379.73
a, b — values in the same rows with different letters differ significantly (P < 0.05).
x — P < 0.05; xx — P < 0.01; NS — P ≥ 0.05.

89.79
38.50
40.59
0.430
56.04
387.92
SEM

89.96
38.01
41.29
0.422
50.57
377.94
1.099
0.627
0.742
0.007
1.481
4.960
Significance
of effects
%
CLA
sex
interaction
xx
xx
x
x
xx
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
141
Table 4 gives average values of selected lipid parameters of fatteners’ blood
serum. Contrary to expectations, the supplementation of CLA had a negative
influence on the content of total cholesterol (TC), LDL-cholesterol and triacylglycerols (TAG) in serum. In the control group the level of these lipid fractions was
lower, while for TC and TAG the differences were even highly significant. There
was a tendency for the level of total lipids to increase in the CLA groups, but the
differences were not confirmed statistically. At the same time, in all the CLA
supplemented groups there were increases in the level of the beneficial HDLcholesterol (P<0.01) and its proportion in total cholesterol (P < 0.05).
Discussion
Effect of CLA on growth indicators
The results obtained in the present experiment indicate that CLA exerts a strong
effect on the body weight of pigs, probably by modifying the whole metabolism. It
was shown that a 0.1 – 0.4% supplement of dietary CLA had a beneficial effect on
the production parameters of the pigs. This is in agreement with Dugan et al.
(2001). In the 75 – 110 kg weight range, when using 0.25 and 0.5% CLA
supplement, these authors observed a gradual improvement in fattening results
compared to the control, unsupplemented group. A decrease in weight gains and
poorer feed conversion in relation to the groups given lower CLA levels, similar to
the present study at a 0.6% CLA level, were observed by Eggert et al. (2001) who
used 1% CLA, while Thiel et al. (2001) at the same level of CLA achieved an
improvement in fattening performance. However, most of the recent studies with pigs
failed to observe a significant effect of CLA on the production results (Ostrowska et
al., 2003; Dugan et al., 2004; Migdał et al., 2004). Differences in the results obtained
are hard to explain. It is believed that they are related to differences in the
concentration of isomers, and thus in the biological activity of the CLA preparations,
in the nutrient content of feed mixtures, sometimes in the duration of experiment, and
in the potential effect of animal genotype. These issues need further study.
In the present experiment with pigs, sex of animals was found to exert no effect on
fattening results regardless of the CLA level. This conforms with the studies of Dugan
et al. (2001). The latter suggest that weight gains and feed conversion in pigs of
different sex are similar when isocaloric diets in the rationed feeding system are used.
Effect of CLA on carcass fatness and carcass analysis
The present results of carcass analysis have fully confirmed the potential ability
of CLA to reduce fatness, with an accompanying gain in weight of muscles.
Repartition of protein and fat in the body was reflected, among others, in the
reduced backfat thickness, increased loin eye area, a significant increase in
meatiness and in the fat-to-meat ratio. These results were confirmed by the majority
of studies with pigs fed on a CLA supplement (Dugan et al., 2001; Wiegand et al.,
2002; Ostrowska et al., 2003; Migdał et al., 2004). It is worth noting that the
142
B. Szymczyk
significant reduction in carcass fatness with preserved high production parameters
was already observed for relatively low (up to 0.5%) levels of CLA in feed
(Wiegand et al., 2002). In the present work, optimal effects were obtained with
a 0.4% supplement of CLA. The higher CLA levels tended to worsen the results of
carcass analysis, which was also confirmed in the above studies. This phenomenon
needs further study.
It is worth noting the special role of the C18:2 trans 10, cis-12 isomer in
repartition of protein and fat in the organism. Most authors agree that this isomer
shows a considerably higher activity in reducing adipose tissue than other CLA
isomers. Attempts at clarifying this phenomenon indicate the inhibiting effect of the
C18:2 trans-10, cis-12 isomer on proliferation of fat cells in adipocytes (Evans et al.,
2002). In mice, this isomer increased lypolysis and decreased the activity of
lipoprotein lipase in adipocytes, inhibited lipogenesis (Brown et al., 2001), and
reduced the activity of stearoyl-CoA desaturase (an enzyme from the enzymatic
system of ∆9-desaturase), which takes part in the synthesis of fatty acids in adipocytes
(Choi et al., 2000). Thus, reduction of fatty tissue in model animals could be due not
so much to the decreased number of adipocytes as to their decreased size.
The hypocholesterolemic action of CLA in the above studies was manifested by
the reduction of total cholesterol (TC) level in the m. longissimus, despite the fact that this
indicator is considered to be relatively stable and independent even of the sex of
pigs. This is confirmed by the study of Migdał et al. (2004), in which a 2%
supplement of CLA did not cause significant changes in the level of TC in the
m. longissimus. Regrettably, the available literature reveals no information on why
CLA changes the level of cholesterol in pork, but it is suggested that the decrease of
this lipid in muscle tissue may be directly related to the reduction in the amount of
intramuscular fat (Chizzolini et al., 1999).
The effect of CLA on serum lipid profile
The effect of CLA on serum lipid profile and atherosclerosis is not consistent.
Unlike in experiments with mice, in pig studies (Stangl, 2000) a dietary CLA
supplement caused a significant increase in the level of TC and triacylglycerols
(TAG). Unfavourable changes in serum lipid profile of pigs, both in the current
study and in the previously mentioned experiment of Stangl (2000) could result
from the considerably lower activity of lipoprotein lipase in the liver of pigs than in
mice. Whereas Tischendorf et al. (2002) found no significant effect of dietary CLA
supplement for pigs on serum lipid profile, although CLA caused a marked increase
in the proportion of saturated fatty acids at the cost of monounsaturated fatty acids
in the cholesterol-associated fraction of very low density lipoproteins (VLDL).
In conclusion, dietary CLA supplements increased lean tissue, decreased fat
deposition in pig carcasses and reduced cholesterol level in meat. It was shown that
a 0.1 – 0.4% supplement of dietary CLA had a beneficial effect on the production
parameters of the pigs. CLA did not reduce the content of TC, LDL-cholesterol and
TAG in serum but in all the CLA supplemented groups there were increases in the
level of the beneficial HDL-cholesterol and its proportion in TC.
143
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Accepted for printing 25 II 2005
BEATA SZYMCZYK
Wpływ sprze˛żonego kwasu linolowego (CLA) na wskaźniki produkcyjne, skład chemiczny
i jakość tuszy oraz profil lipidów w surowicy krwi tuczników
STRESZCZENIE
Badano wpływ różnych poziomów CLA (0,0; 0,1; 0,2; 0,4 i 0,6%) w paszy dla świń na wskaźniki
produkcyjne, jakość tuszy i profil lipidów w surowicy krwi. Wyniki tuczu świń w przedziale od 61 do
108 kg zależały od poziomu sprze˛żonego kwasu linolowego (CLA) w paszy. Dodatek 0.4% CLA istotnie
podwyższył przyrosty masy ciała (P < 0,05) i poprawił wykorzystanie paszy (P < 0,01) w stosunku do
grupy kontrolnej. Dodatek 0,6% CLA pogorszył wyniki tuczu, jednak były one lepsze niż w grupie
karmionej pasza˛ bez dodatku CLA.
Świnie otrzymuja˛ce CLA charakteryzowały sie˛ mniejszym otłuszczeniem niż zwierze˛ta w grupie
kontrolnej. Stwierdzono statystycznie wysoko istotna˛ redukcje˛ grubości słoniny z pie˛ciu pomiarów oraz
na karku i w pole˛dwicy. Grubość słoniny w punkcie C-1 w grupach karmionych z dodatkiem z CLA
mieściła sie˛ w granicach od 1,07 (0,1% CLA) do 0,91 cm (0,4% CLA), podczas gdy w grupie kontrolnej
uzyskano wartość 1,17 cm. Wzrost poziomu CLA w paszy powodował korzystny liniowy wzrost
stosunku tłuszczowo-mie˛snego z 6,81 w grupie kontrolnej do 8,40 w grupie żywionej z dodatkiem 0,6%
CLA. U świń otrzymuja˛cych CLA stwierdzono też wie˛ksza˛ powierzchnie˛ oka pole˛dwicy i mie˛sność
(P < 0,01) oraz wie˛kszy udział mie˛sa w szynce (P<0,05). Analiza chemiczna m. longissimus wykazała
wysoko istotne statystycznie różnice mie˛dzy grupami w zawartości tłuszczu (spadek z 1,23% w grupie
kontrolnej do 0,95 i 0,97 odpowiednio w grupach z dodatkiem 0,4 i 0,6% CLA) oraz cholesterolu,
którego zawartość obniżyła sie˛ z 63,99 w grupie bez dodatku CLA do 49,53 mg/100 g (0,6% CLA).
Dodatek CLA do diety dla świń negatywnie wpłyna˛ł na zawartość cholesterolu ogólnego (TC),
frakcji LDL i triacylgliceroli (TAG) w surowicy krwi. W grupie kontrolnej poziom tych frakcji lipidów
był niższy, a w przypadku TC i TAG różnice te były nawet wysoko istotne statystycznie.
EFFECTS OF CONJUGATED LINOLEIC ACID (CLA) ON FATTY ACID
COMPOSITION, LIPID OXIDATION AND QUALITY OF PORK MEAT*
Beata Szymczyk
Department of Animal Nutrition and Feed Sciences, National Research Institute of Animal Production,
Abstract
Effects of feeding different levels (0.0, 0.1, 0.2, 0.4 and 0.6%) of conjugated linoleic acid (CLA) in
diet on the fatty acid composition, lipid oxidation and quality of pork were determined. No
significant differences and only a tendency towards increased colour lightness (L), redness (a) and
yellowness (b) were found in the meat of fatteners in the groups fed with 0.1, 0.2 and 0.4% CLA in
relation to the control group. In the group receiving 0.6% CLA, a considerably lighter meat colour
was observed (P < 0.05). Technologically the most beneficial water holding capacity (WHC) was
characteristic of the meat of pigs given 0.1 and 0.2% CLA (P < 0.05). In the other groups
supplemented with CLA, WHC was better than in the control group (P≥0.05). There were no
significant differences in the pH of meat 45 minutes post-slaughter and after 24-h cooling of
carcasses. The increased CLA level in feed was accompanied by linearly decreased thiobarbituric
acid-reactive substances (TBARS) in meat after 6-month storage at –20°C. The CLA supplement
to the pig diets exerted a strong influence on the composition of fatty acids (FA) both in the lipids of
m. longissimus muscle and in backfat. For the m. longissimus a significant (P < 0.05) increase in the
level of saturated fatty acids (mainly C16:0 and C18:0) accompanied by a decrease in monounsaturated fatty acids (MUFA) were noted. There were no statistical differences between the groups
in the level of polyunsaturated fatty acids (PUFA) in meat. The growing CLA level in the diets
caused a linear increase of CLA isomers in total FA (P < 0.01). In the case of backfat the
incorporation of CLA isomers was more effective than in meat. The C18:2 cis-9, trans-11 isomer
was incorporated into the fatteners’ tissues much more efficiently than the C18:2 trans-10, cis-12
isomer. No CLA isomers were present in the meat of the control fatteners.
Key words: pig, conjugated linoleic acid, fatty acids, meat quality
A new class of functional (i.e. health-promoting) components of animal origin
comprises conjugated linoleic acid (CLA) isomers. CLA are the positional and
geometric (cis, trans) conjugated isomers of linoleic acid (18:2n-6), present mainly
146
B. Szymczyk
in ruminant milk and meat. Chicken meat and pork are poor sources (0.1 – 0.2% of
total fatty acids) of CLA isomers (Terpstra, 2004). In a number of reliable studies,
pure CLA were shown to decrease the risk of cancer and atherosclerosis, to enhance
immune responses, reduce body fat and decrease oxidative stress in experimental
animals (Riserus et al., 2004; Terpstra, 2004). Consequently, animal nutrition
strategies have been developed to enrich monogastric animal meat with CLA
isomers (Wiegand et al., 2002; Ostrowska et al., 2003; Dugan et al., 2004). On the
other hand, repartition of carcass fat to lean and modification of tissue lipid
composition using high dietary levels of CLA in some instances negatively affected
pork quality (Dugan et al., 2001). Wiegand et al. (2002) and Ostrowska et al. (2003)
noted a linear increase in the content of saturated fatty acids with a concurrent drop
in the amount of monounsaturated fatty acids in animals given diets with growing
CLA levels. In addition, whereas strong antioxidative properties were shown by the
C18:2 trans-10, cis-12 isomer regardless of its concentration, the C18:2 cis-9,
trans-11 isomer, at concentrations above 200 µM, intensified the oxidation
processes (Leung and Liu, 2000).
The objective of this study was to evaluate the effects of supplementing the
growing pig diets with different levels of CLA on fatty acid composition, lipid
oxidation and quality of meat.
Animals and treatments
Forty barrows and forty gilts [ ( Polish Landrace × White Polish Large)
× Pietrain] with an average initial body weight of 60 kg were randomly assigned
to five dietary treatment groups (0.0, 0.1, 0.2, 0.4 and 0.6% CLA) with 16 animals
(8 barrows and 8 gilts) each. Animals were housed in individual pens and had free
access to water. Pigs were weighed before initiation of treatments and then every
2 weeks until they reached an average slaughter weight of about 108 kg. Feed
consumption was recorded throughout the experiment. The basal feed mixture
composition and nutrient content were given in a previous paper (Szymczyk, 2005).
The CLA source (Natural Lipids Ltd, Norway) contained 65% CLA (Table 1) and
was added to the diets at concentrations of 0.15, 0.30, 0.61 and 0.92% to provide the
desired level of CLA. The CLA was substituted for sunflower oil.
Pork quality measurements
The samples of m. longissimus were obtained from the carcass (from the area of
the last thoracic and first lumbar vertebra) 24 h postmortem to determine colour,
water holding capacity (WHC), chemical and fatty acid composition, total cholesterol and thiobarbituric acid-reactive substances (TBARS) content. In addition
a samples of backfat were taken to determine fatty acid composition. The pH of m.
longissimus was determined at 45 min and at 24 h postmortem using a portable
CP-215 pH-meter with glass ERH-12-6 electrode (HYDROMET). Meat colour was
147
Dietary CLA and pork quality
measured in five locations of loin chop and mean values were calculated for colour
lightness (L*), redness (a*) and yellowness (b*) with Minolta Chromometer
CR-310, using D65 lighting, a 2° standard observer and a measuring aperture of
8 mm, standardized to a white tile. Water holding capacity was measured using the
Grau and Hamm method (1953). Lipid oxidation of meat (TBARS) was determined
at 24 h postmortem and after 6-month storage at –20°C using the Salih method as
described by Pikul et al. (1993).
Table 1. Fatty acid composition (% of total fatty acids) of sunflower oil and conjugated linoleic acid
(CLA) source fed to pigs
Fatty acids
12:0
14:0
16:0
16:1
18:0
cis-9-18:1
cis-11-18:1
18:2
trans-18:2
CLA isomers:
total
cis-9, trans-11-18:2
trans-8, cis-10-18:2
cis-11, trans-13-18:2
trans-10, cis-12-18:2
other CLA
18:3
20:0
20:1
22:0
24:0
Total SFA1
Total MUFA2
Total PUFA3
Sunflower oil
CLA-65%*
0.1
6.3
0.1
5.4
0.1
4.0
32.5
3.9
25.5
0.8
61.1
0.2
0.6
57.1
27.6
27.5
2.0
0.8
0.3
0.3
0.6
0.2
11.3
26.6
62.1
0.2
9.7
32.6
57.7
* Natural Lipids Ltd. — Hövdebygda Norway (1999).
1
SFA — saturated fatty acids, 2MUFA — monounsaturated fatty acids, 3PUFA — polyunsaturated fatty acids.
Total cholesterol content of loin lipid extracts was analysed using the Rhee et al.
(1982) method. Chemical composition of meat was determined by standard
methods (AOAC, 1995).
Fatty acid analysis
The total tissue lipids (backfat and m. longissimus) were extracted according to
the method of Folch et al. (1957). They were saponified (10 min, 75°C) in 0.5M
KOH/Me-OH and then methylated (10 min, 75°C) in 14% BF3/Me-OH. Finally,
148
B. Szymczyk
fatty acid methyl esters were extracted with hexane and analysed on a HewlettPackard (model 5890) gas chromatograph, equipped with a BPX 70 fused silica
capillary column (100 m × 0.22 mm i.d. × 0.25 m; SGE International, Australia), and
a flame ionization detector. Helium was the carrier gas used at a split ratio of 50:1.
The operating conditions were as follows: the temperature of injector was 210°C, and
that of detector was 240°C. The initial oven temperature was 160°C for 35 min,
increasing progressively by 3°C/min to 210°C, and held constant at 210°C for 10 min.
The fatty acid percentage was integrated and calculated using the HP ChemStation
Computer Program. Fatty acid methyl esters were identified by comparison of their
retention times with authentic standards purchased from Sigma-Aldrich (Poland) and
the CLA reference standards (cis-9, trans-11; trans-10, cis-12 isomers) were obtained
from Larodan Fine Chemicals AB (Malmö, Sweden).
The effects of dietary CLA on pork quality were analysed by using two-way
ANOVA generated by the STATISTICA v. 5.1 package. Duncan’s multiple range
test was used to determine the significance of differences between treatment means
at the P < 0.05 and P < 0.01 levels of significance.
Results
No significant differences and only a tendency towards increased colour
lightness (L), redness (a) and yellowness (b) were found in the meat of fatteners in
the groups supplemented with 0.1, 0.2 and 0.4% CLA in relation to the control
group (Table 2). In the group receiving the highest CLA level, a considerably
lighter meat colour was observed, as confirmed by the high values of lightness (L)
and yellowness (P < 0.05).
Technologically the most beneficial water holding capacity (WHC) was
characteristic of the meat of pigs given 0.1 and 0.2% CLA (P < 0.05). In the other
groups supplemented with CLA, WHC was better than in the control group, but the
differences were not significant.
There were no significant differences in the pH of meat 45 min postmortem
and after 24-h cooling of carcasses. It should be noted, however, that in all CLAsupplemented groups, pH values in the m. longissimus directly after slaughter were
slightly lower, and after 24 h they were higher and more favourable than in the
control group.
Mention must be made of the content of malonaldehyde (TBARS) in the
m. longissimus of pigs. It was shown that after 6-month storage of meat at –20°C
TBARS level was the highest in the control group (P < 0.01). It was also found that
the increased CLA level in feed was accompanied by decreased TBARS in meat
(Figure 1). In the first four groups, this tendency was confirmed after 12 months of
storage. Only in the group receiving 0.6% CLA was there an unfavourable,
statistically highly significant increase in TBARS, up to the control group level.
Table 2. Physicochemical traits of longissimus muscle
Source of variation
Item
0.0
45-min pH
24-h pH
TBARS1 after 6 months’ storage
(mg/kg)
TBARS after 12 months’ storage
(mg/kg)
Colour of meat:
lightness (L)
redness (a)
yellowness (b)
Water holding capacity (%)
0.1
0.2
sex
0.4
0.6
SEM

Significance
of effects
%
CLA
sex
interaction
6.08
5.48
5.96
5.51
5.98
5.55
6.06
5.65
6.02
5.58
5.99
5.56
6.05
5.55
0.026
0.027
NS
NS
NS
NS
NS
NS
0.486 A
0.474 A
0.422 B
0.369 C
0.330 C
0.425
0.408
0.009
xx
NS
NS
0.835 A
0.622 B
0.598 B
0.476 C
0.835 A
0.705
0.649
0.028
xx
NS
NS
53.19 ab
15.85
5.41 a
24.56 b
53.26 ab
16.07
5.55 ab
25.29 ab
55.06 b
15.92
6.13 b
25.28 ab
0.355
0.083
0.101
0.268
x
NS
x
x
NS
NS
NS
x
NS
NS
NS
NS
52.12 a
15.61
5.30 a
26.20 a
52.10 a
15.88
5.47 a
24.54 b
1
TBARS — thiobarbituric acid-reactive substances.
a, b — values in the same rows with different letters differ significantly (P < 0.05).
x — P < 0.05; xx — P < 0.01; NS — P ≥ 0.05.
53.27
15.77
5.51
25.55
53.01
15.96
5.63
24.79
150
B. Szymczyk
Figure 1. Effect of dietary level of CLA (%) on TBARS content (mg/kg) in M. longissimus after storage
at – 20°C for 6 and 12 months
The CLA supplement to the pig diets exerted a strong influence on the
composition of fatty acids (FA) both in the m. longissimus (Table 3) and in backfat
(Table 4). For the m. longissimus a significant (P < 0.05) increase in the level
of saturated fatty acids (mainly C16:0 and C18:0) accompanied by a decrease
in monounsaturated fatty acids (MUFA) were observed. There were no statistical
differences between the groups in the level of polyunsaturated fatty acids
(PUFA). The growing CLA level in the diets caused a linear increase of CLA
isomers in total FA (P < 0.01). The C18:2 cis-9,trans-11 isomer was incorporated
into the m. longissimus much more efficiently than the C18:2 trans-10, cis-12
isomer (Figure 2). No CLA isomers were present in the meat of the control
fatteners.
The content of fatty acids in backfat generally showed a similar tendency as in
the m. longissimus, but differences between particular experimental groups were
clearer (Table 4). In the CLA-supplemented groups, the amount of saturated fatty
acids increased highly significantly to 52.75% at 0.6% CLA supplement compared
to 40.79% in the control group. The sum of MUFA also decreased considerably,
from 40.2% in the control group to 29.05% in the group supplemented with 0.6%
CLA. For the sum of PUFA, a slight decrease was found in the groups supplemented with CLA (P < 0.05). Similar to the m. longissimus, an increase in the level
of isomers, due to the growing CLA level in feed mixtures, was also observed
in backfat. In this case the incorporation of CLA isomers was more effective than
in meat and concerned not only the above two isomers, but also the other
(undetermined) CLA isomers present in the supplemented preparation (Figures 2
and 3).
Table 3. Fatty acid composition (% of total identified fatty acids) of longissimus muscle
Source of variation
Selected fatty acids
14:0
16:0
16:1
18:0
18:1
18:2
18:3
20:1
20:4
22:4
22:5
22:6
CLA isomers
Total SFA1
Total MUFA2
Total PUFA3
sex
0.0
0.1
0.2
0.4
0.6
1.09
22.67 a
3.08
11.98 A
41.54 a
14.49 a
0.30
0.70
3.24
0.49
0.35
0.11 ab
0.00 A
35.74 a
45.32 A
18.94
1.14
23.25 ab
3.01
11.89 A
41.09 a
14.10 b
0.30
0.68
2.96
0.48
0.34
0.09 b
0.13 B
36.28 a
44.96 AB
18.76
1.14
24.25 ab
3.10
12.06 AB
39.95 ab
13.43 c
0.30
0.64
3.16
0.51
0.36
0.10 ab
0.18 B
37.45 ab
44.11 AB
18.68
1.17
24.41 b
3.08
12.73 B
39.40 b
14.04 b
0.30
0.66
2.66
0.45
0.34
0.12 a
0.34 C
38.31 bc
43.18 BC
18.51
1.18
24.96 b
3.00
13.26 C
38.89 b
14.15 b
0.30
0.60
2.43
0.48
0.40
0.12 a
0.44 D
39.08 c
42.29 C
18.63
x — P < 0.05; xx — P < 0.01; NS — P ≥ 0.05.
1

1.13
24.07
3.04
12.29
39.27
14.20
0.30
0.63
3.21
0.49
0.37
0.12
0.22
37.43
43.66
18.91
SEM

1.16
23.62
3.08
12.50
40.27
13.88
0.30
0.68
2.93
0.45
0.34
0.10
0.21
37.28
44.17
18.55
0.019
0.202
0.043
0.126
0.401
0.197
0.000
0.092
0.096
0.015
0.011
0.006
0.213
0.383
0.338
0.018
Significance
of effects
%
CLA
sex
interaction
NS
x
NS
xx
x
x
NS
NS
NS
NS
NS
x
xx
x
xx
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
x
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
xx
NS
xx
NS
Table 4. Fatty acid composition (% of total identified fatty acids) of backfat
Source of variation
Selected fatty acids
14:0
16:0
16:1
17:0
18:0
18:1
18:2
18:3
20:1
20:2
22:5
CLA isomers
Total SFA1
Total MUFA2
Total PUFA3
sex
0.0
0.1
0.2
0.4
0.6
1.21 A
23.10 Aa
1.84 A
0.41 AB
15.11 A
37.64 Aa
17.09 Aa
0.77 a
0.81 Aa
0.81 A
0.08 a
0.17 A
40.79 Aa
40.29 A
18.92 a
1.33 B
23.11 Aa
1.97 A
0.38 A
15.31 A
37.79 Aa
16.47 Aab
0.74 ab
0.84 Ab
0.76 A
0.07 b
0.31 B
41.09 Aa
40.56 A
18.35 b
1.47 C
23.69 Aa
1.88 A
0.48 AB
17.11 B
35.67 Ab
16.03 Ab
0.72 ab
0.77 Aa
0.69 B
0.07 b
0.56 C
43.46 Ab
39.05 A
18.07 b
1.57 C
24.26 Ab
1.81 A
0.46 B
19.76 B
32.97 Bc
16.63 Aab
0.76 a
0.74 Aa
0.68 B
0.06 c
0.98 D
46.05 Bc
35.60 B
18.35 b
1.91 D
27.53 Bc
1.46 B
0.44 AB
22.87 C
27.12 Cd
15.32 Bc
0.67 b
0.47 Bc
0.42 C
0.06 c
1.53 E
52.75 Cd
29.05 C
18.20 b
A, B, C, D, E — values in the same rows with different letters differ significantly (P < 0.01).
x — P < 0.05; xx — P < 0.01; NS — P ≥ 0.05.
1

1.47
24.21
1.80
0.43
17.33
34.33
16.67
0.77
0.69
0.68
0.07
0.72
44.24
36.98
18.91
SEM

1.53
25.06
1.79
0.44
17.93
34.30
16.05
0.69
0.75
0.67
0.07
0.70
44.96
36.87
18.17
0.032
0.321
0.035
0.009
0.358
0.550
0.181
0.013
0.019
0.017
0.001
0.057
0.705
0.588
0.227
Significance
of effects
%
CLA
sex
interaction
xx
xx
xx
xx
xx
xx
xx
x
xx
xx
x
xx
xx
xx
x
NS
xx
NS
NS
xx
NS
xx
xx
x
NS
NS
NS
x
NS
xx
NS
NS
NS
NS
NS
NS
x
x
x
NS
NS
NS
x
NS
x
153
Figure 2. Effect of dietary level of CLA (%) on CLA isomers concentration in m. longissimus
(% of total fatty acids)
Figure 3. Effect of dietary level of CLA (%) on CLA isomers concentration in backfat
(% of total fatty acids)
Discussion
It was shown in the current study that CLA supplement improves the
physico-chemical characteristics of pork by positively influencing its dietetic value.
The meat of animals on the CLA diet was characterized by slightly greater redness
(a) and yellowness (b) and a higher coefficient of lightness (L) regardless of the sex
of animals. These observations correspond with the findings of Wiegand et al.
(2002), although Joo et al. (2002) obtained a drop in yellowness under the influence
of growing CLA levels in feed. Special mention must be made of the higher L and
154
B. Szymczyk
b values found here in the group supplemented with the highest CLA level, both in
relation to the control group and the other CLA-supplemented groups. It is thought
that yellowness reflects the intensity of the meat lipid autooxidation processes
(Wiegand et al., 2002). Du and Ahn (2003) observed lower values of both lightness
(L) and yellowness (b), with higher redness (a), in the 7-day cold-stored meat of
chickens fed a 5% CLA supplement compared to the 0% CLA group. This may
support the hypothesis about the higher oxidative stability of meat enriched with
CLA.
The results obtained point to a relationship between the presence of CLA in
carcass and better water holding capacity. This is confirmed by Hayek et al. (1999),
who suggest that the incorporation of CLA isomers into the fraction of phospholipids and the effect they exert on the composition of fatty acids, may reduce the
permeability of cell membranes, thus limiting the drip loss. This is probably due to
decreased susceptibility of phospholipids to oxidation by reduction of their content
of linoleic (C18:2) and arachidonic acids (C20:4) and the concurrent increase in the
level of saturated acids (Belury et al., 2002). Water holding capacity of meat is
highly correlated to its pH value. It was shown that WHC rapidly decreased when
pH was approximately 5 (isoelectric point), because then the electric load of protein
approached neutral (Toldra, 2003). Optimal pH of pork meat ranged from 5.6 to 5.8.
In the present study, in the CLA-supplemented groups there were slightly higher pH
values than in the control group, which was reflected in better WHC. Importantly,
in all CLA-supplemented groups the meat obtained was PSE (pale, soft, exudative)
and DFD-free (dark, firm, dry) when using pH level as a criterion for assessment
(Hartwig et al., 1990). This supports the observations of Dugan et al. (2004), Joo et
al. (2002), Wiegand et al. (2002) and Ostrowska et al. (2003).
The gradual decrease in meat, after 6-month storage, of malonaldehyde (TBARS)
together with an increased level of CLA in feed is a highly desirable change and
attests to increased oxidative stability of pork enriched with CLA. This is supported
by the studies of Joo et al. (2002), in which a growing (from 1 – 5%) level of CLA
in the mixtures significantly reduced, in relation to the 0.0% CLA group, the level
of TBARS in the meat stored at 4°C for 7 days. Similar results were obtained by
Wiegand et al. (2002) for pigs and Du and Ahn (2003) for broiler chickens. Du and
Ahn (2003) suggest that the decreased susceptibility of CLA-enriched meat to
oxidation results from the higher content in fat of saturated fatty acids and from
greater oxidative stability of CLA isomers in relation to other polyunsaturated fatty
acids. Further studies are needed to explain the dramatic increase of TBARS (to
0.835 mg/kg meat) observed in the present study in the group given the highest CLA
level after 12 months of storage at –20°C. The studies of Leung and Liu (2000) to
some degree can justify the reasons for this phenomenon. Strong antioxidative
properties were shown by the C18:2 trans-10, cis-12 isomer, regardless of its
concentration. Whereas the C18:2 cis-9, trans-11 isomer, at concentrations above
200 µM, began to exhibit the opposite action and intensified the oxidation processes.
The increased level of CLA in feed caused a linear increase in the concentration
of CLA isomers in both backfat and intramuscular fat (Tischendorf et al., 2002;
155
Ostrowska et al., 2003). Literature data also confirm that the C18:2 cis-9, trans-11
and C18:2 cis-11, trans-13 isomers are more efficiently incorporated into pig
tissues than trans-8, cis-10, trans-10, cis-12 and the other CLA isomers present in
the preparation. Tischendorf et al. (2002) showed an advantage in the tissue
incorporation process of C18:2 cis-9, trans-11 and other cis, cis isomers in relation
to C18:2 trans-8, cis-10 and other trans, trans isomers. Badinga et al. (2003), when
using CLA supplement in the feeding of broilers noted a significantly higher
relative proportion of the cis-9, trans-11 isomer in liver fat in relation to C18:2
trans-10, cis-12 and C18:2 trans-9, cis-11 isomers. By contrast, no differences were
observed in the relative proportion of CLA isomers in the experimental preparation
in relation to the analogous proportions of isomers in backfat, liver lipids and
triacylglycerols of the cardiac muscle of fatteners (Kramer et al., 1998).
An effect was shown of the CLA supplement to pig diets on the composition of
fatty acids in depot and intramuscular fats. Wiegand et al. (2002) and Ostrowska et
al. (2003) noted a linear increase in the content of saturated fatty acids, mainly
myristic (C14:0), palmitic (C16:0) and stearic acids (C18:0), with a concurrent drop
in the amount of monounsaturated fatty acids, mainly oleic (C18:1) and arachidonic
acids (C20:4), in animals given diets with growing CLA levels. This phenomenon
was observed in both the m. longissimus and backfat, but was more conspicuous for
backfat. Sebedio et al. (1997) suggest that C18:2 trans-10, cis-12 rather than the
cis-9, trans-11 isomer has the greatest effect on this phenomenon. Probably, the
action of this isomer is related to its effect on the activity of the ∆9-desaturase
enzymatic system, which is responsible for the conversion of saturated stearic acid
(C18:0) into monounsaturated oleic acid (C18:1) in the liver. Also Chuang et al.
(2001) showed that the C18:2 trans-10, cis-11 isomer inhibits this enzyme and
decreases the content of oleic acid in liver, bone tissue and muscles. In studies with
pigs, Ostrowska et al. (2003) also suggest that the increased proportion of SFA and
the reduction of arachidonic acid (C20:4) in depot fat are directly related to
a change in the activity of ∆5, ∆6 and ∆9-desaturase systems in liver under the
influence of CLA. Changes in the content of arachidonic acid found in the above
experiments, and confirmed in the present study, are of special importance. They
support the hypothesis that CLA alters the metabolism of linoleates and arachidonates, while reducing the synthesis of eicosanoids (especially prostaglandin E2), the
main precursor of which is arachidonic acid. This is of no small importance
considering the association of eicosanoids with neoplastic processes, especially
with the proliferation of cancerous cells by induction of ornithine decarboxylase
and DNA synthesis (Belury, 2002).
In conclusion, our studies show that feeding CLA to pigs is an effective way to
obtain CLA-enriched pork meat and thus the potential health-related benefits of
CLA consumption in humans. Further studies are certainly required to determine
the optimum dietary concentration of CLA isomers needed to obtain functional
pork and to avoid the adverse effect of CLA supplementation such as increased level
of saturated fatty acids and a considerably lighter meat colour and the increased
TBARS level in meat after 12-month storage in the group fed with 0.6% CLA.
156
B. Szymczyk
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BEATA SZYMCZYK
Wpływ sprze˛żonego kwasu linolowego (CLA) na skład kwasów tłuszczowych,
oksydacje˛ lipidów oraz jakość mie˛sa wieprzowego
STRESZCZENIE
Określano wpływ różnych poziomów (0,0, 0,1, 0,2, 0,4 i 0,6%) sprze˛żonego kwasu linolowego
w paszy na skład kwasów tłuszczowych i oksydacje˛ lipidów oraz jakość mie˛sa wieprzowego. Nie
stwierdzono istotnych różnic a jedynie tendencje˛ do wzrostu wskaźnika jasności (L), wysycenia
w kierunku czerwieni (a) i żółci (b) w grupach z dodatkiem 0,1, 0,2 i 0,4% CLA. W grupie z dodatkiem
0.6% CLA mie˛so miało istotnie jaśniejsza˛ barwe˛ (P < 0,05). Najkorzystniejsza˛ z technologicznego
punktu widzenia wodochłonnościa˛ (WHC) charakteryzowało sie˛ mie˛so świń żywionych z dodatkiem 0,1
i 0,2% CLA (P < 0,05). W pozostałych grupach z CLA uzyskano również lepsza˛ wodochłonność mie˛sa
niż w grupie kontrolnej (P ≥ 0,05). Nie stwierdzono istotnych statystycznie różnic w pH mie˛sa
mierzonego 45 minut po uboju oraz po 24 h schładzania tuszy. Wzrost poziomu CLA w paszy
powodował liniowe obniżanie zawartości TBARS w mie˛sie po 6 miesia˛cach przechowywania w warunkach chłodniczych (–20°C). Dodatek CLA w paszy spowodował istotne zmiany w składzie kwasów
tłuszczowych (FA) lipidów, zarówno w m. longissimus, jak i w słoninie grzbietowej. W m. longissimus
zanotowano istotny (P < 0,05) wzrost poziomu nasyconych kwasów tłuszczowych — SFA (głównie
C16:0 i C18:0), przy równoczesnym spadku poziomu jednonienasyconych kwasów tłuszczowych
(MUFA). Nie stwierdzono istotnych zmian w poziomie wielonienasyconych kwasów tłuszczowych
(PUFA) w mie˛sie. Wzrost poziomu CLA w paszy spowodował liniowy wzrost udziału izomerów CLA
w sumie FA (P < 0,01). W przypadku słoniny grzbietowej wbudowywanie izomerów CLA było bardziej
efektywne niż w przypadku m. longissimus. Izomer C18:2 cis-9, trans-11 był wbudowywany do tkanek
tuczników efektywniej niż izomer C18:2 trans-10, cis-12. Nie stwierdzono obecności izomerów CLA
w tkankach tuczników w grupie kontrolnej.
EFFECT OF THE FORM OF RAPESEED AND LINSEED IN LAMB DIETS
ON SOME HEALTH QUALITY PARAMETERS OF MEAT*
B r o n i s ł a w B o r y s 1, A n d r z e j B o r y s 2
1
National Research Institute of Animal Production in Kraków,
Experimental Station Kołuda Wielka, 88-160 Janikowo, Poland
2
Meat and Fat Research Institute, Jubilerska 4, 04-190 Warszawa, Poland
Abstract
Twenty-four ram lambs fattened intensively to 30 – 35 kg body weight were investigated in
4 groups which were fed complete isoproteic and isoenergetic diets. A standard diet was used in
group C (control). The experimental diets contained 10% double-low rapeseed and linseed at a 2 : 1
ratio, whole seeds in group E1, 50% whole and 50% ground seeds in group E2, and 100% ground
seeds in group E3. Nutritional factors were found to exert no effect on growth rate or utilization of
dietary nutrients. Feeding oilseed diets increased slaughter yield, the proportion of muscle tissue in
the leg and loin eye area, with greater external fatness of half-carcasses. The use of ground oilseeds
increased the level of intramuscular fat. There was a favourable tendency for lower cholesterol
concentration in the muscles of lambs receiving oilseeds. The parameters of meat health quality,
based on the fatty acid profile of intramuscular fat, were generally less favourable in lambs
fattened with oilseed diets, mainly because of the lower proportion of polyunsaturated fatty acids
(PUFA). Feeding oilseeds also increased the concentration of conjugated linoleic acid (CLA), the
grinding of seeds having a distinctly positive effect on the CLA content of the muscles.
Key words: fattening of lambs, oilseeds, meat quality
Plant oils are important in the nutrition of slaughter animals as a source of
nutrients, mainly energy nutrients, and as a factor modifying raw meat quality. The
effects of using oilseeds on these two aspects can depend on both the quantity and
composition of dietary nutrients and on the form of feeding. In ruminants, the form
of the oilseeds contained in diets determines the degree of their ‘‘protection” against
digestive processes in the forestomachs and thus their nutritive utilization and
effects in modifying meat quality (Bas and Morand-Fehr, 2000; Oprza˛dek and
Oprza˛dek, 2003).
* This work was conducted as part of the research project no. 6 P06Z 059 21 financed by the
Ministry of Science and Information Society Technologies.
160
B. Borys and A. Borys
In adult ruminants, the nutritive value of unsaturated fatty acids (UFA) from
plant fats ‘‘protected” against ruminal biohydrogenation by feeding them in the
form of whole seeds, has been established (Bas and Morand-Fehr, 2000; Raes et al.,
2004; Wood et al., 2003). However, in young lambs the forestomachs of which have
not fully developed their functions, the effect of plant fats on growth and body
composition can also be determined by the form of oilseed administration. The lack
of relevant studies in the available literature has stimulated us to explore this issue.
This study was performed to determine the effect of using oilseeds (double-low
rapeseed and linseed) and of their form in complete diets on the fattening
performance of lambs and on some health quality parameters of lamb meat.
Intensively fattened ram lambs from a population of prolific sheep (Polish
Merino, Finn and Romanov sheep) and their F1 crosses with the Texel meat breed
(50% each) were investigated. Lambs were fed ad libitum a complete diet
supplemented with 100 g hay per 1 kg of concentrate diet. Lambs were fattened
from weaning at 7 – 8 weeks of age to 30 – 35 kg body weight. There were 4 groups
of lambs receiving isoproteic and isoenergetic diets. The ingredients, nutritive value
and chemical composition of the diets are given in Table 1. Diet composition and
the form of oilseed were the experimental factors. In the control group (C) standard
diet without oilseeds was used, whereas the experimental diets of groups E1, E2 and
E3 had their components partly replaced with double-low rapeseed and linseed at
a 2 : 1 ratio, at 10% of diet weight. Whole oilseeds were used in group E1,
50% whole and 50% ground oilseeds in group E2, and 100% ground oilseeds in
group E3. The animals were investigated in groups of 10 lambs per feeding group.
Carcass analysis was performed and meat composition and quality determined in
6 animals per feeding group (a total of 40 animals). Twenty-four lambs were tested
postmortem. Lambs were slaughtered and their right half-carcasses partially
dissected in accordance with the procedures used at the National Research Institute
of Animal Production in Balice, Poland (Nawara et al., 1963).
The raw longissimus dorsi muscle was analysed for water, protein and fat
content (AOAC, 1990), and colour lightness using a Specol 11 spectrophotometer
(Rd-O attachment, 730 µm wavelength). Cooked muscle was measured for
tenderness (Warner-Bratzler shear machine) and tested organoleptically for palatability, aroma, tenderness and juiciness on a 5-point scale by a panel of 5 evaluators.
Cholesterol level was determined in the musculus semitendinosus using an Agilent
6890 Plus gas chromatograph equipped with a flame-ionization detector on an HP-1
column (25 m × 0.20 mm × 0.11 µm). The fatty acid profile of the diets, intramuscular fat and CLA level in intramuscular fat (m. semitendinosus) were analysed
according to the procedures of Kramer et al. (1997) as modified by Borys et al. (1999).
These determinations were made on an Agilent 6890 N gas chromatograph with
a flame-ionization detector on a BPX 70 column (60 m × 0.25 mm × 25 µm).
Health quality of lamb meat as related to oilseed form
161
Statistical analysis of the results was performed using the ANOVA procedure of
the Statistica 6.0 PL packet and Duncan’s multiple range test.
Results
In keeping with the methodological assumptions, the control and experimental
diets were of similar nutritive value expressed as feed unit for meat production (UFV)
and protein digestible in the intestine (PDI) (Table 1). However, both the chemical
composition and fatty acid profile of diets E were differentiated by the 10%
supplement of full-fat rapeseed and linseed compared to the control diet (C). E diets
contained more crude protein (by 27.2% on average), almost 2.5 times more fat,
52.3% more fibre and 25.4% more ash. The principal changes in the composition of
fatty acids were an increased level of oleic acid (C18:1) dominant in rapeseed oil (by
27% on average), of linolenic acid (C18:3) dominant in linseed oil (by 49.0%) and of
stearic acid C18:0 (by 25.9%) in the fat of E diets, which was accompanied by
decreases in palmitic (C16:0) and linoleic acids (C18:2), by 42.8 and 35.0%
respectively (Table 1). In general, compared to C diets, the fats in E diets contained
more UFA and less saturated fatty acids (SFA) and consequently showed a higher
UFA:SFA ratio (by 57.5% on average). Of UFA acids, monounsaturated fatty acids
(MUFA) increased by 24.2% and polyunsaturated fatty acids (PUFA) decreased by
9.7%, but the PUFA:SFA ratio was 34.2% higher in E diets than in C diets.
No great differences were observed between the groups in terms of weight gains
or daily intake of the diets (Table 2). The intake of diets and nutrients per unit of
weight gain did not show great differences between the experimental and control
groups or among the experimental groups. Both the rate of lamb growth (daily gains
higher than 265 g in all groups) and the unit intake of diets and nutrients are
indicative of the normal course of fattening in all the lamb groups.
Feeding oilseed diets was found to affect some parameters of slaughter value
depending on the physical form of the oilseeds in the diet (Table 3). Lambs from all
E groups surpassed C lambs in slaughter yield (by an average of 3.8 percentage
units, NS), in percentage muscle tissue in the leg (by 2.1 percentage units, NS) and
in the loin eye area (by 2.1 cm2, i.e. 17.1%, significant for groups E1 and E2 at
P ≤ 0.05), with greater external fatness measured over the ribs (by 60.0% on
average, significant for group E2 at P ≤ 0.01). It is worth noting that E2 lambs, fed
with 50% whole and 50% ground seed diets, had the highest slaughter yield, being
related to the highest external carcass fatness in this group.
The analysed nutritive factors were found to have some effect on the quality of
lamb meat, but the effect was not significant (Table 3). The muscles of lambs
fattened with ground oilseed diets (groups E2 and E3) contained slightly more
intramuscular fat than those of the control lambs (by 4.4% on average, NS), while
the muscles of E1 lambs fed whole oilseed diets contained 6.6% less intramuscular
fat (NS). Feeding oilseed, in any form, had a beneficial effect on lowering
cholesterol concentration in lamb muscles (on average 7.4% lower in E groups than
in C groups), but the differences were not significant.
162
Table 1. Characteristics of the diets
Item
Components (%):
dried forage
barley grain
ground wheat
rapeseed meal
dried sugar beet pulp
double-low rapeseed
linseed
MM
Polfamix ‘‘O-K”
Nutritive value per kg:
UFV
PDIE (g)
Diet — Group
C
E1
10.0
25.0
25.5
20.0
18.0
E2
0.5
1.0
10.0
20.0
13.0
25.5
20.0
6.72
3.32
0.5
1.0
0.88
100
0.87
97
6.71
3.31
E3
6.73
3.33
Chemical components (g/100 g):
dry matter
protein
fat
fibre
ash
88.6
13.6
1.8
6.5
4.6
89.8
17.9
5.5
8.7
5.5
89.6
17.0
4.9
9.7
5.6
89.3
17.0
5.2
11.3
6.2
Fatty acid profile
Major fatty acids (g/100 g fat):
C16:0
C16:1
C18:0
C18:1
C18:2
C18:3
C20:0
C20:1
SFA
UFA
MUFA
PUFA
UFA:SFA
PUFA:SFA
DFA:OFA
Ω6:Ω3 PUFA
11.6
0.5
1.8
37.1
31.7
13.8
0.4
0.8
15.1
84.3
38.8
45.5
5.583
3.013
6.47
2.297
7.0
0.3
2.3
47.3
21.2
19.3
0.5
0.9
10.5
89.3
48.9
40.4
8.469
3.829
11.17
1.099
6.8
0.4
2.2
45.6
20.9
21.0
0.4
0.9
10.3
89.3
47.4
41.9
8.670
4.068
11.30
0.995
6.1
0.3
2.3
47.0
19.7
21.4
0.4
0.9
9.7
89.7
48.6
41.1
9.247
4.237
12.43
0.921
1
whole seeds,
2
50% whole seeds and 50% ground seeds, 3 100% ground seeds.
There were no statistically significant differences in measurements of colour
lightness of raw muscles or muscle tenderness after cooking. No greater effects of
feeding rapeseed and linseed on the organoleptic score of cooked muscles were
found, with a tendency towards higher scores for all parameters in group E2, in
which 50% ground oilseeds were used.
163
Table 2. Fattening results
Diet — Group
Item
Body weight (kg):
start of fattening
end of fattening
Days of fattening
Daily gains (g)
Intake of concentrate (kg/animal/day)
Intake per kg gain:
complete diet
UFV
PDIE
C
E1
E2
E3
19.1
34.1
56
273
0.99
18.9
34.1
57
266
1.04
19.3
35.6
58
281
0.98
19.0
34.2
58
267
0.96
3.55
3.39
390
3.89
3.69
416
3.50
3.32
374
3.67
3.47
391
SEM
0.53
0.42
1.41
5.88
Table 3. Slaughter value and meat quality
Item
Slaughter yield (%)
% valuable cuts
Tissue composition of leg (%):
muscles
fat
bones
Loin eye area (cm2)
Fat layer over ribs (mm)
Content per 100 g muscle tissue:
water (g)
protein (g)
fat (g)
cholesterol (mg)
WB tenderness (kG)
SP colour (%)
Sensory score (max 5 pts.)
palatability
aroma
tenderness
juiciness
Diet — Group
C
E1
E2
E3
SEM
43.5 A
43.5
45.9
43.4
49.9 A
43.9
46.2
43.8
0.82
0.29
72.1
13.0
14.9
12.3 ab
3.5 A
74.0
11.9
14.0
14.6 b
4.6 a
73.3
13.2
13.4
14.9 a
6.9 Aa
75.4
11.2
13.4
13.8
5.2
0.49
0.43
0.32
0.35
0.43
76.63
21.02
1.35
68.0
5.37
46.3
76.88
20.85
1.26
62.6
5.42
43.1
76.75
20.83
1.42
62.7
5.12
43.9
76.67
20.94
1.39
63.6
5.73
47.2
0.12
0.04
0.10
0.87
0.19
0.92
4.27
4.20
4.18
4.43
4.22 b
4.20
4.25
4.43
4.45 ab
4.35
4.37
4.53
4.18 a
4.20
4.17
4.38
0.04
0.05
0.04
0.05
aa, bb — P ≤ 0.05, AA — P ≤ 0.01.
For the most part, the major fatty acids in the intramuscular fat of the lambs
were not significantly differentiated by feeding oilseeds (Table 4). More distinct
differences, some statistically significant, occurred for long-chain PUFA acids
C18:2, C20:4, C20:5 and C22:5, which were less abundant in the fats of
experimental lambs, by 28.9, 19.9 and 26.5% on average. By far the lowest
proportion of these acids was found in E2 lambs, and the differences of 45.2 and
39.8% in relation to the control group C were significant at P ≤ 0.01. Changes in the
164
proportion of PUFA in the muscle fat of E lambs resulted in lower health quality
parameters of intramuscular fat based on this group of acids (PUFA:MUFA by
18.9%, PUFA:SFA by 20.6% on average), although the differences were not
significant (Table 4). By far the greatest decline of these parameters appeared in
group E2. There were also decreases in both Ω3 PUFA (C20:5 and C22:5) and Ω6
PUFA (C18:2 and C20:4), which caused the ratio of these acid groups to become
similar in all the groups.
Table 4. Fatty acid profile (g/100 g fat)
Item
Major fatty acids:
C14:0
C15:0
C16:0
C16:1
C17:0
C17:1
C18:0
C18:1
C18:2
C18:3
C20:4
C20:5
C22:5
CLA:
g/100 g fat
mg/100 g muscle
SFA
UFA
MUFA
PUFA
UFA : SFA
PUFA : MUFA
PUFA : SFA
Ω3 PUFA
Ω6 PUFA
Ω6:Ω3 PUFA
Diet — Group
C
E1
E2
E3
SEM
2.28
0.85
21.02
2.10
1.70
0.78
12.98
40.38
8.27
1.17
3.85 A
0.57
1.08 A
2.48
0.78
21.00
2.22
1.80
0.87
13.30
41.90
7.30
1.13
2.97
0.52
0.85
2.88
0.72
22.02
2.45
1.72
0.77
13.25
42.65
6.50
1.12
2.11 A
0.38
0.65 A
2.41
0.75
21.23
2.15
1.68
0.68
14.05
40.35
8.05
1.12
3.13
0.47
0.88
0.14
0.02
0.22
0.06
0.03
0.03
0.24
0.47
0.33
0.04
0.20
0.03
0.06
0.29
3.88
39.47
59.83
43.67
16.17
1.52
0.37
0.42
3.08
13.08 a
4.23
0.33
4.10
40.08
59.18
45.32
13.87
1.48
0.31
0.35
2.73
11.21
4.17
0.36
4.92
41.22
57.33
46.30
11.63
1.42
0.25
0.29
2.37
9.42 ab
4.00
0.46
6.68
40.75
58.38
43.58
14.80
1.43
0.34
0.36
2.72
12.28 b
4.63
0.04
0.54
0.39
0.41
0.50
0.63
0.02
0.02
0.02
0.12
0.51
0.13
aa, bb — P ≤ 0.05, AA — P ≤ 0.01.
In the muscles of lambs of all experimental groups, CLA was higher than in the
control group. This concerned CLA concentration in both intramuscular fat and
muscle tissue (Table 4). CLA concentration in the muscles of E1 lambs, where
whole seeds were used, was 5.7% higher than in group C, while the respective
differences for groups E2 (50% ground seeds) and E3 (100% ground seeds) were
26.8 and 72.2% (all not significant).
165
Discussion
Major changes in the chemical composition of E diets that contained rapeseeds
and linseeds, compared to the control group C (above all a higher content of fat and
changes in the fatty acid profile) were proportionate to changes in the chemical
composition and fatty acid profile of the components of the standard and
experimental diets. Similar findings were obtained by Bas and Morand-Fehr (2000)
and Raes et al. (2004).
The intake of feeds and nutrients and the growth rate of the lambs, which were
generally good and similar across the groups, indicates that neither the 10% dietary
supplement of oilseeds nor the proportion or form of oilseeds adversely affected the
palatability of feeds or influenced feed utilization. The lack of negative effects from
using 10 – 22% raw oilseeds in diets for fattened lambs confirms the findings of
Borys et al. (2004), Piechnik et al. (1999) and Pakulski and Osikowski (1993). Our
results did not corroborate those of some authors (Mandiki et al., 1999; Matthäus,
1997), who showed that oilseeds are of limited use in the feeding of young lambs
because of the presence of antinutritive factors which adversely affect the
physiological state of animals, and diet palatability (feeds consumed less eagerly).
Differences in parameters of slaughter value were quite characteristic, indicating that the use of rapeseeds and linseeds in lamb fattening favoured the
development of muscle tissue (proportion in leg, loin eye area), while the
unfavourable increase in the fatness concerned only external fat, with no effects on
the content of fatty tissue in the leg or on intramuscular fat. Such trends in the
parameters of the E groups, with a change in the source of energy and an increase in
the concentration of diet components (mainly fat), can be attributed to the beneficial
effect of the increased supply of UFA in oilseed diets. The observed trend for
compromised fattening performance and parameters of slaughter value and meat
quality in E1 lambs may indicate a certain decrease in the digestibility of dietary
nutrients when only whole oilseeds are used in the diet.
Under conditions of intensive fattening to high weight standards, the fat content
of the analysed lamb muscles (1.35 g/100 g on average) is considered low, and is
lower than the optimal value due to the organoleptic attributes of the meat (1.5%).
This might be associated with the high percentage (50%) of Texel genes in the
analysed lambs. The tendency towards a higher muscle organoleptic score for lambs
from group E2 can be attributed to the highest content of intramuscular fat, close to
the above reference value. Other studies are inconclusive as to the effect of using
plant oils in lamb fattening on carcass fatness and over-fatness of muscle tissue.
Yamamoto et al. (2003) reported a significant increase in the fatness of lambs given
rapeseeds and linseeds, unlike Borys et al. (2004) and Piechnik et al. (1999), who found
this influence to be very small. The effect of many naturally interrelated genetic and
environmental factors on parameters of fatness and their high individual variation
makes it difficult to compare the effects of single factors on this group of traits.
A characteristic although non-significant tendency for lower cholesterol concentration in the muscles of lambs fed on oilseeds was most likely due to the
166
DFA:OFA (hypocholesterolemic:hypercholesterolemic) ratio, which was almost
twice as high for E than for C diets (Table 1). Decreased cholesterol concentration
in the muscles of lambs fattened with rapeseed and linseed oil diets was also
reported by Borys et al. (2004), Piechnik et al. (1999), and Yamamoto et al. (2003).
Overall, the analysed indicators of meat health quality based on the fatty acid
profile of intramuscular fat were less favourable in the groups of lambs receiving
oilseed diets than in the control group. This was mainly due to the lower proportion
of PUFA in the fat of lambs from E groups, which resulted from the lower
proportion of PUFA acids in the fat of oilseed diets than in the control diet. The
least beneficial parameters were those in group E2, possibly due to the highest
fatness of carcasses and over-fatness of muscle tissue in this group, which is
normally accompanied by an increase in the degree of saturation of tissue fats.
These findings are not supported by our earlier studies (Borys et al., 2004) or the
results of other authors (Demirel et al., 2004; Macedo et al., 2003; Piechnik et al.,
1999; Potkański et al., 2001; Raes et al., 2004; Wachira et al., 2002; Wood et al.,
2003), who reported generally beneficial effects of using rapeseeds or linseeds, or
rapeseed or linseed oils, on the fatty acid profile of intramuscular fat in lambs. Our
results could be related to the combined use of rapeseeds and linseeds and their
form, which could affect the intensity and pattern of fat metabolism in the
forestomachs.
Despite the lack of differences in the CLA content of the muscles of E and
C lambs, there was a tendency towards a favourable effect of feeding rapeseeds and
linseeds on the increased content of this desirable component in lamb meat. At the
same time, a considerable increase in performance was found when ground oilseeds
were fed (in groups E2 and E3). The beneficial effect of oilseeds on CLA
proportion in muscles was probably due to the absolute content of C18:2 linoleic
acid, considered by many authors to be the main precursor of CLA in ruminant
meat and milk, which was almost twice as high (by 90.5% on average) in E than
in C diets (Mir et al., 2000; Pisulewski et al., 1999). The beneficial effect of
grinding oilseeds on the CLA level in the meat of fattened lambs may be ascribed to
more intense ruminal synthesis of CLA as a result of easier access of forestomach
microorganisms to fatty acids when rapeseeds and linseeds are ground. We also
found that using a combination of rapeseeds and linseeds had a beneficial effect on
the CLA content of the meat of fattened lambs in our earlier study (Borys et al.,
2004). In addition, one study on the use of rapeseed and linseed in lamb fattening
(Macedo et al., 2003) and a number of experiments using linseeds or linseed oil
(Cooper et al., 2004; Czauderna et al., 2004; Demirel et al., 2004; Raes et al., 2004;
Szumacher-Strabel et al., 2004) have revealed a moderate effect of rapeseed oil and
a distinctly beneficial effect of linseed oil on the increase of CLA in lamb meat.
It is concluded that:
1. The growth rate of lambs and the intake and consumption of feeds and
nutrients per unit of weight gain were not differentiated by the use of full-fat
rapeseeds and linseeds in complete diets for fattened lambs (at a rate of 10% and at
a ratio of 2 : 1), regardless of their form (whole; 50 or 100% ground).
167
2. Under intensive fattening of lambs to 30-35 kg body weight, the application
of oilseeds had a beneficial effect on some parameters of slaughter value and meat
quality, on increased slaughter yield, and on the proportion and development of
muscles, with carcass fatness that is higher but within optimal values.
3. The use of oilseeds had a beneficial effect on reducing cholesterol in the
muscle tissue of the lambs (regardless of the degree of grinding) and on increasing
CLA content, in particular when ground oilseeds were used.
4. No significant effect of using rapeseeds and linseeds on the fatty acid profile
of intramuscular fat was found, with a tendency for slightly poorer results in terms
of health quality, resulting from the lower proportion of long-chain PUFA C20:4,
C20:5 and C22:5.
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M e x i a A.A., M a c e d o R.M.G., S a k a g u t i E.S. (2003). Proximate composition and fatty acid
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Congress of Meat Science and Technology, Campinas (Brazil), pp. 11 – 12.
BRONISŁAW BORYS, ANDRZEJ BORYS
Wpływ formy nasion rzepaku i lnu w mieszankach dla tuczonych jagnia˛t na wybrane parametry
jakości zdrowotnej mie˛sa
STRESZCZENIE
Badania zrealizowano na 24 jagnie˛tach tryczkach tuczonych intensywnie do masy ciała 30 – 35 kg.
Utworzono 4 grupy jagnia˛t żywionych pełnoporcjowymi mieszankami izobiałkowymi i izoenergetycznymi. W grupie C (kontrolna) stosowano mieszanke˛ standardowa˛, a w skład mieszanek doświadczalnych
wchodziło 10% nasion rzepaku 00 i lnu w proporcji 2 : 1 — w grupie E1 całe nasiona, w E2 po 50% całe
i śrutowane, w E3 w 100% śrutowane.
169
Nie stwierdzono wpływu badanych czynników żywieniowych na tempo wzrostu i wykorzystanie
składników pokarmowych pasz. Żywienie z udziałem nasion oleistych wpływało na wzrost wydajności
rzeźnej, udziału tkanki mie˛śniowej w udźcu i powierzchni oka pole˛dwicy, przy wie˛kszym otłuszczeniu
zewne˛trznym półtusz. Stosowanie rozdrobnionych nasion oleistych wpływało na wzrost zawartości
tłuszczu śródmie˛śniowego. Stwierdzono korzystna˛ tendencje˛ do obniżonej zawartosci cholesterolu
w mie˛śniach jagnia˛t żywionych nasionami oleistych. Wyróżniki jakości zdrowotnej mie˛sa, oparte na
profilu kwasów tłuszczowych tłuszczu śródmie˛śniowego, kształtowały sie˛ ogólnie mniej korzystnie
u jagnia˛t tuczonych mieszankami z nasionami oleistych, głównie z powodu niższego udziału wielonienasyconych kwasów tłuszczowych (PUFA). Równocześnie żywienie oleistymi wpływało na wzrost
zawartości SKL, przy wyraźnie korzystnym efekcie rozdrabniania nasion na zawartość tego składnika
w mie˛śniach.
IODINE CONTENT OF GREEN FODDER AND CEREAL CROPS
IN POLAND
P i o t r S t r z e t e l s k i 1, F r a n c i s z e k B r z ó s k a 2, W i t o l d B r z e z i ń s k i 3
1
Department of Plant Physiology, Faculty of Horticulture Agricultural University, Al. 29 Listopada 54,
31-425 Kraków, Poland
2
3
Research Institute for Cultivar Testing, 63-022 Słupia Wielka, Poland
Abstract
The iodine content of feeds from plants grown in Poland was investigated. A total of 257 samples
were taken from 40 Cultivar Testing Stations located all over Poland, under varying soil,
environmental and climatic conditions. Iodine content was analysed in 5 species of fodder plants
(meadow fescue, orchard grass, red clover and white clover, silage maize) and in 4 cereal grains
(winter wheat, spring barley, winter rye, corn cobs). The iodine content of fodder crops and cereals
was determined colourimetrically after alkaline mineralization of the samples. Iodine content was
moderate and averaged 129±0.7 in grasses, 106±0.1 in clovers, and 94±0.8 µg I/kg of dry matter
(DM) in maize. In cereal grains it was 55±0.4 (winter wheat), 38±0.4 (spring barley), 53±0.4 (winter
rye) and 48±0.3 µg I/kg of DM (maize). In two successive years, the iodine content of the same
species and cultivars of fodder plants differed by more than 100% for fodder plants and by
approximately 20% for cereal grains. In samples originating from different locations and soils, the
difference was approximately 30%. In terms of the iodine content of fodder plants, soils can be
classified as follows: alluvial > brown > muck > brown leached > rendzina > podzolic. The
relationship between species of fodder plant and year of cultivation indicates that absorption by
plants of iodine from the soil is largely dependent on the distribution of temperatures and amount
of precipitation in particular years and growing seasons. Grasses accumulated greater amounts of
iodine than legumes or maize, while cereal grains were low in iodine.
Key words: iodine, green fodder, cereal
172
P. Strzetelski et al.
Iodine is one of the major elements essential for the bodies of humans and
animals to function properly. However, this element is omitted from plant
physiology and biochemistry textbooks because it is considered unnecessary for the
growth and vital functions of plants. However, small amounts of iodine — higher in
the regions of silt and clay soils and river deltas, lower in the regions of mountain
soils — are found in plants overlying calcareous rocks, sandstones or granite (Fuge,
1987).
The iodine content of plants depends on the soil iodine concentration, plant
species and fertilization used (Kapil and Singh, 2003; Moiseyev et al., 1984; Voight
et al., 1988). In most soils, the amount of iodine assimilable by plants (i.e.
water-soluble iodine) is not greater than 25% of its total content (Fuge and Johnson,
1986). Soluble forms of iodine are readily absorbed by plants, but iodine
availability is low for plants because it is strongly bound to soil organic matter.
Letunova et al. (1987) report that iodine is accumulated by soil microorganisms
and, as it degrades, is gradually released into the soil to become available to plants.
Chinese studies point to relationships between increased iodine concentration in
plants and increased iodine content in soil. Huan-xin et al. (2003) hold that iodine
concentration in plants depends on the type of plant and physiological stage of its
development, as well as on the form of iodine found in the soil (Burte et al., 1991).
Iodine levels have been found to be higher in vegetative, and lower in reproductive,
parts of plants (Ban-nai and Muramatsu, 2003).
A search of world and Polish literature reveals little data on the iodine content
of fodder plants and cereal grains.
This study was designed to determine the level of iodine in grasses, legumes,
cereals and silage maize from different regions of Poland, during two consecutive
growing seasons.
Plant samples were taken from 40 Cultivar Testing Stations belonging to the
Research Institute for Cultivar Testing (COBORU) in Słupia Wielka. The Cultivar
Testing Stations from which the plants were sampled are located in different areas
of Poland (Figure 1).
Plants were cultivated in different climatic, soil and fertilizer conditions
(Tables 1 and 2). The conditions were determined from assessment bulletins on
pasture grasses, maize, small-seeded legumes, and cereals (issue 1094 from 1996
and issues 1092, 1113, 1114, 1120 and 1133 from 1997). The Cultivar Testing
Stations from which the plants were sampled, are situated at different elevations
(from 7 to 590 m above sea level) and are characterized by a broad soil complex
starting from a good wheat complex, to a very poor rye complex, to a mountain
oat-pasture complex.
The type of soil ranged from podzolic to loose sand to silty sand and covered
podzolic, podzolic-brown, brown, brown leached, muck, rendzina and alluvial soils.
173
Iodine content of green fodder and cereal crops in Poland
Soil acidity (pH) ranged from 5.0 to 7.4 and the amount of precipitation in the plant
cultivation area varied between 323 and 751 mm/year. In total, during 1996 and
1997, iodine content was analysed in 59 grass samples, 32 clover samples, 24 green
maize forage samples, 118 grain samples, and 24 corn cob samples.
Figure 1. Regions of Poland in which plant samples for iodine content analysis were taken
Table 1. Characteristics of climatic, soil and fertilizing conditions in 1996 and 1997 during sampling
of fodder plants
Item
Meadow
fescue
Orchard
grass
White
clover
Red
clover
Silage
maize
Altitude (m)
Soil pH (KCl)
Precipitation (mm)
7 – 530
5.1 – 6.9
516 – 774
7 – 590
5.0 – 6.9
478 – 751
40 – 568
5.3 – 6.8
323 – 677
290 – 590
4.8 – 6.1
527 – 677
230 – 440
5.6 – 7.4
360 – 680
80
60 – 80
40 – 50
20 – 30
60 – 100
100 – 160
30 – 60
80 – 100
100 – 160
92 – 160
45 – 120
91 – 180
Fertilization (kg/ha pure element):
nitrogen (N)
80
60 – 80
phosphorus (P2O5)
40 – 50
potassium (K2O)
Over the two consecutive years of the trial (1996 and 1997), the following
species and cultivars of plants and numbers of samples from different areas of
Poland were analysed:
— meadow fescue (Festuca pratensis Huds.), cv. Skrzeszowicka, 21 samples from
11 and 10 locations, respectively,
— orchard grass (Dactylis glomerata L.), cv. Bara, 38 samples from 25 and
13 locations, respectively,
174
Table 2. Characteristics of climatic, soil and fertilizing conditions in 1996 and 1997 during sampling
of cereal grains
Item
Winter
wheat
Winter
rye
Fertilization (kg pure element per ha):
nitrogen (N)
phosphorus (P2O5)
potassium (K2O)
Corn
cob
year
1996 1997 1996
Soil value on a 100-point scale of ISSPC*
Soil pH in KCl (% analyses):
pH > 6.5
pH from 6.5 to 5.6
pH < 6.5
Spring
barley
1997 1996 1997 1996 1997
76
77
56
55
73
73
74
74
32
55
13
23
66
11
25
53
22
20
53
27
21
64
15
12
61
27
7
7
8
6
7
8
92
74
102
94
75
100
75
77
100
80
79
99
93
74
103
81
76
100
77
90
110
130
95
128
* According to the Polish Institute of Soil Science and Plant Cultivation.
— red (meadow) clover (Trifolium pratense L.), cv. Dajana (4 samples) and
Jubilatka (12 samples), each year from 8 locations,
— white clover (Trifolium repens L.), cv. Romena (14 samples) and Rema
(2 samples), from 7 and 9 locations, respectively,
— green fodder from silage maize (Zea mays L.) and corn cob, cv. Rassant
(8 samples), Greta (8 samples) and Antiope (8 samples), each year from 12
locations,
— spring barley grain (Hordeum sativum L.), cv. Rodos (38 samples) and Start
(2 samples), each year from 20 locations,
— winter wheat grain (Triticum vulgare L.), cv. Almari (35 samples) and Kobra
(3 samples), each year from 19 locations,
— winter rye grain (Secale cereale L.), cv. Motto (30 samples) and Warko
(10 samples), each year from 20 locations.
A total of 257 plant samples were analysed and only varieties included, or
pending inclusion, in the list of cultivated plants were considered. Legumes, winter
and spring rape and legume-cereal mixes were the forecrop to winter wheat, winter
rye and spring barley. Root crops, rape and peas were the forecrop to corn cob.
Grasses and clovers were mown and there were three crops.
Sampling and chemical analysis of plants
Samples for analysis originated from the first crop, which was collected during
the first and second year of growth. Grasses were cut at the heading stage and clover
at the budding stage. Silage maize was collected at the wax stage when dry matter
in whole crops reached approximately 30%. Forage samples were taken on the day
of cutting and divided into two portions. Immediately after the sample was taken
and ground, the first portion was analysed for dry matter content at 105°C and the
175
second, after drying with a forced-air drier at 60°C to a solid mass, was analysed for
iodine content. Cereal seeds were sampled on the day of harvesting when they
reached the stage of technological maturity and transported to a laboratory, where
they were analysed for dry matter, as described above, and for iodine content. The
iodine content of the fodder crops and cereal was assayed colourimetrically after
alkaline mineralization of the samples (Bobek and Kołczak, 1960).
Statistical calculations
The results of the plant iodine-content analysis were interpreted based on
two-way analysis of variance using the effects of species and year of cultivation,
mean values for fodder crops or cereals in different years of growth, standard error
(SE), standard deviation (SD) and a range of values for the whole set of values.
Results
The iodine content of meadow grasses, clovers and silage maize varied widely
from 48.2 to 170.4 µg/kg DM (Table 3). The highest iodine content (148.4 µg/kg DM)
was found in orchard grass (P ≤ 0.05 or P ≤ 0.01) and the lowest (94.0 µg/kg DM) in
silage maize (P ≤ 0.01). A significant difference in the iodine content of fodder crops
between successive years of cultivation was found, as well as a significant
relationship between species and year of cultivation (P ≤ 0.01). Mean iodine content
was 128.7 ± 7.4 µg/kg DM in meadow grasses and 106.3 ± 10.3 µg/kg DM in clovers.
Table 3. Iodine content of green fodder crops (g/kg DM)
Species/Year
No.
of samples
Mean iodine
level
Meadow hay
Orchard grass
White clover
Red clover
Silage maize
21
38
16
16
24
108.9
148.4
111.7
101.0
94.0
1996
1997
Interaction
67
48
152.7 bB
72.9 aA
P ≤ 0.01
Overall mean
Range
115
bB
Cc
bBC
bB
aA
112.8
48.2 – 170.4
SE
SD
8.3
6.5
9.6
11.0
7.8
9.2
98.5
a, b, c — values in rows marked by different letters differ significantly (P ≤ 0.05).
A, B, C — values in rows marked by different letters differ highly significantly (P ≤ 0.01).
The effect of soil type on the iodine content of grasses can be classified as
follows: alluvial > brown > muck > podzolic > brown leached, and that on the
iodine content of clovers as: brown leached > rendzina > podzolic.
176
Table 4. Iodine content of cereal grains and corn cobs (g/kg DM)
Species/Year
No.
of samples
Mean iodine
level
Winter wheat
Spring barley
Winter rye
Corn cob
38
40
40
24
1996
1997
Interaction
71
71
56.9 bB
40.1 aA
not significant
3.2
3.2
142
48.5
24.5 – 103.1
3.1
Overall mean
Range
55.5
37.6
52.6
48.3
cC
aA
cC
bB
SE
SD
4.0
3.9
3.9
2.8
24.8
a, b, c — values in rows marked by different letters differ significantly (P ≤ 0.05).
A, B, C — values in rows marked by different letters differ highly significantly (P ≤ 0.01).
The iodine content of cereal grains and corn cobs varied between 24.5 and
103.1 µg/kg DM (Table 4). The highest iodine content was found in winter cereals:
wheat — 55.5 µg/kg DM and rye — 52.6 µg/kg DM, between which no statistically
significant difference was found (P > 0.05). The lowest iodine content (37.6 µg/kg DM)
was characteristic of spring barley (P ≤ 0.01), in which the concentration of this
element was even lower than in corn cobs (48.3 µg/kg DM). A significant
difference was also found in the iodine content of cereal grains and corn cobs
between successive years of cultivation (P ≤ 0.01). However, there was no significant relationship between species and year of cultivation (P > 0.05). Similarly to
green fodder crops, higher iodine content was observed in cereal grains and corn
cobs when they were grown in cohesive soils of the alluvial type and in brown soils,
and lower iodine content in plants grown in muck, rendzina and podzolic soils.
Discussion
The fodder plants analysed in this experiment contained much more iodine than
has been reported by some authors. Anke et al. (1993) showed that the iodine
content of grasses and green silage maize forage in Germany averaged 59 and
66 ± 25 µg/kg DM, respectively. Studies by Heinrich and Wenk (1989) revealed
that the iodine content of grass forage and legume forage ranged from 49 to
66 µg/kg DM, whereas Georgiewskii et al. (1981) reported that the iodine content
of pasture grass, red clover, lucerne and vetch-oat mixture ranged from 40 to
70 µg/kg DM. Studies in India (Bedi, 1999) demonstrated that iodine content varies
according to stage of growth, from 19 to 46 µg/kg DM in maize, and from 20 to
30 µg/kg DM in oat forage. Drebickas (1993) reports that in Lithuania the iodine
content of green fodder crops grown as cattle feed ranges from 35 to 173 µg/kg DM.
These values are very close to the iodine concentrations obtained in our study for
grasses, clovers, and green silage maize forage (48 – 170 µg/kg DM).
177
The higher iodine content of plants grown in alluvial-type soils than in rendzina
or podzolic soils and the significant relationship found between plant species and
year of cultivation indicate that the iodine content of the analysed plants depends on
the type of soil and climatic conditions during successive growing seasons, and thus
on the site of cultivation, which is also related to the distance from the sea and
altitude above sea level. Underwood (1971), when summing up the results of
research from the 1950s, reports that the iodine content of plants varies considerably according to their species and variety, the type of soil in which they grow, the
type of fertilizers used, and the climatic and seasonal conditions. The same author
also reports that the iodine content of fodder plant leaves can be hereditary. This
seems to be confirmed by the findings of Anke et al. (1993) in Germany, which
indicate that plants from sealing and clay soils found in lowland areas, catchment
areas and seaside areas contain more iodine than plants from transported soils
overlying granite or limestone beds. The significant relationship between year of
cultivation and species allows a conclusion that the accumulation of iodine in
herbaceous plants depends on the environmental conditions prevailing in a particular year.
Yuita (1994) reports that the mean iodine content of the world’s population of
terrestrial plants is 0.42 mg/kg. In special cases, the iodine content of beetroot
parenchyma is 0.6 – 2.6 mg/kg, 0.1 – 2.4 mg/kg in cabbage (Sheppard et al., 1993),
and 0.08 µg/g fresh matter in hay (Voight et al., 1988).
The iodine content of cereal grains and corn cobs was almost twice as low as
that of fodder crops. Mean values for wheat (55 µg/kg DM) and barley
(38 µg/kg DM) were slightly lower than those specified by the NRC (2000) in
tables of the mineral composition of feeds (0.10 and 0.05 mg/kg DM, respectively).
Anke et al. (1993) hold that the iodine content of cereals is usually very low.
Presumably, the movement of iodine from vegetative to reproductive organs
requires active transport through the phloem. During plant growth, the absorbed
iodine is stored mainly in the green parts of the plant. In the final period of growth,
when metabolic processes slowly cease in leaves and conductive tissues and seeds
are formed, the storage of iodine in cereal grains can be limited. This is supported
by Maćkowiak and Grossl (1999), who showed that iodine absorbed by rice plants
is accumulated primarily in roots, stalks and leaves, while the amount of iodine
transported to grain is exceptionally low. This indicates that iodine transport
through the phloem is very low. Ban-nai and Muramatsu (2003), who investigated
the transport of radioactive iodine from the soil to different parts of some plant
species (lettuce, radish, onion, turnip, aubergine, wheat, water dropwort), observed
a tendency for higher iodine accumulation in the leaves than in the stalks, fruits or
grains of these species. It was found that iodine from the soil is transported through
the root system and accumulated mainly in the leaves of plants. The authors suggest
that iodine behaves similarly to chlorine during transport within the plant.
The observed differences in iodine content related to the site of cultivation
indicate that the decisive factor for the iodine content of cereal grains and corn cobs
is the type and geological origin of the soil in which the cereals are grown.
178
Cultivation conditions applied during plant experiments at the COBORU were
similar. It can therefore be supposed that a high level of iodine in soil is a factor that
could favourably affect the intake and deposition of iodine in cereal seeds.
The higher (by approximately 30%) iodine content found in cereal seeds and corn
cobs in the first than in the second year of cultivation suggests that humidity as well
as soil and air temperature conditions could have influenced iodine uptake by plants
and its accumulation in seeds. The lack of a significant relationship between
species and year of cultivation leads us to believe that particular species of cereals
show similar capacity to store iodine in seeds during successive years of cultivation.
To conclude, the iodine content of fodder crops and of the seeds of cereals
grown in Poland depends on the species, environmental conditions in a given year,
and the type of soil in which these plants are cultivated. The mean iodine content of
green fodder crop forages in Poland is 113 µg/kg DM (range 48 – 170 µg/kg DM),
whereas the iodine content of cereal grains averages 49 µg/kg DM (range
25 – 103 µg/kg DM).
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Accepted for printing 20 XII 2004
PIOTR STRZETELSKI, FRANCISZEK BRZÓSKA, WITOLD BRZEZIŃSKI
Zawartość jodu w zielonych roślinach pastewnych i zbożach w Polsce
STRESZCZENIE
Badano zawartość jodu w paszach z roślin uprawianych w Polsce. Próbki w ilości 257 pochodziły
z 40 Stacji Oceny Odmian zlokalizowanych na obszarze całego kraju. Rośliny uprawiane były
w zmiennych warunkach glebowych i środowiskowych oraz klimatycznych. Zawartość jodu badano
w roślinach pastewnych 5 gatunków (kostrzewa ła˛kowa, kupkówka pospolita, koniczyna czerwona,
koniczyna biała, kukurydza na kiszonke˛) oraz w ziarnie 4 gatunków zbóż (pszenica ozima, je˛czmień
jary, żyto ozime, kolby kukurydzy). Zawartość jodu w roślinach pastewnych i zbożach oznaczono
metoda˛ kolorymetryczna˛ po alkalicznej mineralizacji próbek.
Zawartość jodu była umiarkowana i wynosiła średnio: w trawach 129 ± 0,7, w koniczynach
106 ± 0,1, a w kukurydzy 94 ± 0,8 µg J/kg suchej masy. Zawartość w ziarnie zbóż kształtowała sie˛
naste˛puja˛co: w pszenicy ozimej 55 ± 0,4; je˛czmieniu jarym 38 ± 0,4; życie ozimym 53 ± 0,4 i w kukurydzy
48 ± 0,3 µg J/kg suchej masy. Różnica w zawartości jodu w dwóch kolejnych latach, w roślinach
pastewnych tych samych gatunków i odmian, wynosiła ponad 100% w przypadku roślin pastewnych
i około 20% w ziarnie zbóż. W próbkach roślin pochodza˛cych z różnych miejscowości i gleb różnica
wynosiła około 30%. Pod wzgle˛dem zawartości jodu w roślinach pastewnych gleby można uszeregować
180
naste˛puja˛co: mady > gleby brunatne > gleby murszowe > gleby bielicowe > gleby brunatne wyługowane
> re˛dziny > gleby bielicowe.
Stwierdzone zależności pomie˛dzy gatunkiem rośliny pastewnej a rokiem jej uprawy wskazuja˛, że
pobieranie jodu przez rośliny z gleby w dużym stopniu zależy od rozkładu temperatur i ilości opadów
atmosferycznych w poszczególnych latach i sezonach wegetacyjnych. Trawy kumulowały wyższe ilości
jodu niż rośliny motylkowate i kukurydza, zaś ziarna zbóż były bardzo ubogie w jod.
VALIDATION OF A METHOD FOR DETERMINING AMINO ACIDS
IN ACID HYDROLYSATES OF FEEDS*
R o b e r t G a˛s i o r, K r y s t y n a Ś l u s a r c z y k, M a r t a S z c z y p u ł a
National Research Institute of Animal Production, Central Laboratory,
Abstract
A description is provided of a method for determining aspartic acid, threonine, serine, glutamic
acid, proline, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine
and arginine. A total of 12 feeds were analysed: two poultry diets (starter and grower), a pig diet,
meat-and-bone meal, rapeseed, barley, two oat cultivars, maize, wheat, soybean, and field bean.
Analyses of blank samples showed they were not required to adjust the amino acid content of the
analysed feed samples. Amino acid stability was found in five standard solutions over at least
110 days (variation coefficients from 0.55% to 3.06%) and five feed hydrolysates were stable for at
least 30 days (variation coefficients from 0.31% to 5.72%). Repeatability of the amino acid
determination method was 5%, reproducibility 6.4%, and uncertainty 7.1%. Recovery ranged
from 85.6% (tyrosine) to 100.7% (isoleucine). The linearity of the calibration curve was 4 – 140 mg/L
for aspartic acid, threonine, serine, glutamic acid, proline, valine, leucine, tyrosine, phenylalanine
and histidine; 140 mg/L for glycine, alanine and isoleucine; 7.5 – 240 mg/L for lysine and arginine.
Residual coefficients of variation ranged from 0.9% to 8.5%, with correlation coefficients
r2 ≥ 0.999. The limit for quantitative determination was 4 mg/L for aspartic acid, threonine, serine,
glutamic acid, proline, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine and
histidine, and 7.5 mg/L for lysine and arginine. The limit of repeatability, assumed to be double the
value of repeatability, was 10%. It was established that the repeatability of sample analyses and of
the amino acid level in our own reference samples would be checked during routine analyses.
Key words: amino acids, feeds, determination methods
Because amino acids are important in the nutrition of humans and animals,
various techniques are being developed for their determination in raw materials and
animal and plant products. The basic technique for determining amino acids in acid
hydrolysates prepared for analysis of samples is chromatography. Two basic
chromatographic techniques are used to determine amino acids.
182
R. Ga˛sior et al.
The first is Reversed-Phase High-Performance Liquid Chromatography (RP
HPLC). Chromatographic analyses based on RP HPLC methods have been
described by Sarwar and Botting (1993), Jen-Kun Lin and Chiu-Hwa Wang (1980),
Beale et al. (1990), Beaver et al. (1987), Deyl (1986), and Lam et al. (1984).
The second type is Ion-Exchange Chromatography (IEC). This technique has
been described by Bütikofer et al. (1991), Gehrke et al. (1985) and Rayner (1985).
Both techniques were discussed by Elkin (1984), who compared RP HPLC based on
o-phthaldialdehyde (OPA) derivatization with IEC based on ninhydrin derivatization. He found that both procedures are consistent for determination of most amino
acids except aspartic acid, glutamic acid and alanine. Until recently, it was
impossible to determine cysteine and proline with RP HPLC (with OPA), but recent
studies indicate that this is possible. These amino acids can be determined with the
above method after prior oxidation (Czauderna and Kowalczyk, 1998; Czauderna et
al., 2003). While the RP HPLC technique is used mainly, though not exclusively,
for blood tests, the widest application of the IEC technique is in analyses of feed
and food hydrolysates (Zumwalt et al., 1987; Rayner, 1985). It is thought that RP
HPLC procedures should be applied mainly for analysis of materials that are low in
amino acids and physiological fluids that contain amino acids absent from feed and
food hydrolysates (e.g. hydroxylysine, ornitine, cystathionine).
The IEC technique is recommended for determination of amino acids in feeds as
it allows nutritionally important amino acids to be determined.
The most reliable results possible are obtained by performing analyses after
previous validation of the analytical method (PN-EN ISO/IEC 17025:2001). This
can be done by characterizing the method through determination of its parameters
such as repeatability, reproducibility, recovery, linearity, and the limit for quantitative determination (Dobecki, 1998).
Repeatability is the accuracy of a method under assumed conditions of
repeatability, i.e. an error expressed for example as a standard deviation or variation
coefficient obtained in tests of the same subject, using the same method, in the same
laboratory, by the same performer, using the same equipment, and within a short
time period.
Reproducibility is the accuracy of a method under assumed conditions of
reproducibility, i.e. an error expressed for example as a standard deviation or
variation coefficient obtained in tests of the same subject, using different methods or
the same method, in the same laboratory, by different performers, using different or
the same equipment, and within a longer time period.
Uncertainty is a parameter connected with the result of a measurement and it
describes the scatter of values that can reasonably be ascribed to the measured value
(Dobecki, 1998; Arendarski, 2003). According to the PN-ISO 10012-1:1998
standard, uncertainty of measurement is the ‘‘result of a procedure aimed at
estimating the range in which the actual value of the measurand is contained,
usually with a given likelihood”. Uncertainty can be expressed using a standard
deviation or variation coefficient. Uncertainty can also be a multiple of these
parameters.
Validation of a method for determination of amino acids
183
Recovery is a parameter that accounts for losses during the analysis. It can be
calculated by comparing the level of a component in the standard solution prepared
in the same way as the sample (standard subjected to the sample preparation
procedure) with the level of this component in the standard solution that did not
undergo the sample preparation procedure (external standard solution).
Linearity expresses the directly proportional relationship between apparatus
readout values and concentrations of the substance being determined. Linearity can
be described using parameters such as the square of the correlation coefficient r2
and the residual coefficient of variation CVres, which describes the % mean square
deviation of points from the calibration curve.
The limit for quantitative determination (Dobecki, 1998) is the lowest level of
a substance that can be determined quantitatively with sufficient reliability. This
can be the lower limit of the linearity range, determined by obtaining the required
accuracy of the result.
Other characteristics of the analytical method can include checking the stability
of standard solutions and feed hydrolysates and determining the effect of blank
sample on the analysis results. The elements of the characteristics described above
are part of validation in its widest meaning, which involves adjusting the method to
the conditions and capabilities of a laboratory and to the customer’s requirements.
Validation tests can be used as a basis for determining the ways of controlling the
quality of test results during routine analyses. Existing published material on the
determination of amino acids in feeds does not include some of the above validation
parameters and the present study makes an attempt to fill this gap.
Reagents
The following reagents were used: citric acid monohydrate, analytically pure
(Chempur, Piekary Śla˛skie, Polska), sodium hydroxide, pure (POCH, Gliwice,
Poland), caprylic acid 98%, pure (Chempur, Piekary Śla˛skie, Poland), concentrated
(36%) hydrochloric acid, analytically pure (POCH, Gliwice, Poland), ninhydrin
solution (Beckman, cat. no. 338069); eluents: Na-E buffer, pH = 3.3 (Beckman, cat.
no. 338057), Na-F buffer, pH = 4.3 (Beckman, cat. no. 338058), buffer Na-D,
pH = 6.3 (Beckman, cat. no. 338056), Na-R buffer, pH = 13 (Beckman, cat.
no. 727411), and redistilled water (MilliQ-Plus apparatus). For hydrolysis, 6 N hydrochloric acid solution was prepared by dissolving concentrated hydrochloric acid
in water at a 1:1 ratio (v/v). Post-hydrolysis samples were dissolved using a buffer
(pH 2.2) obtained after dissolving 42 g citric acid monohydrate, 16.8 g sodium
hydroxide, 32 ml concentrated hydrochloric acid and 0.2 ml caprylic acid in 1.8 l
redistilled water, determining the pH of the buffer with a hydrochloric acid solution,
and completing with redistilled water to 2 l volume.
The amino acid standards used (Sigma-Aldrich-Fluka, St. Louis, USA) were:
aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine,
184
R. Ga˛sior et al.
isoleucine, leucine, tyrosine, phenylalanine, histidine hydrochloride hydrate
(His*HCl*H2O), lysine hydrochloride (Lys*HCl), and arginine hydrochloride
(Arg*HCl).
Equipment
We used a Beckman System Gold amino acid analyser equipped with 126 AA
solvent module, 502E autosampler, UV-VIS 166 detector, Spherogel IEX High
Performance Sodium Column, and 232 post-column reactor. Data were integrated
using Gold Nouveau software (Beckman). The calculations were performed using
Microsoft Excel. Samples were prepared using sealed ampoules (50 ml), a forcedair drier, an evaporator, evaporating flasks (250 ml), Filtrak 389 filter papers, 0.22
µm syringe filters, and a feed mill with a 1 mm sieve (without cooling).
HPLC chromatographic analysis
The following amino acids were determined: lysine (Lys), arginine (Arg),
aspartic acid (Asp), threonine (Thr), serine (Ser), glutamic acid (Glu), proline (Pro),
valine (Val), leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), histidine (His),
glycine (Gly), alanine (Ala), isoleucine (Ile). The conditions for chromatographic
analysis were: UV-VIS detection, λ wavelength: 570 nm, 20 µl injection, column
temperature (1) 50oC for 10 min, column temperature (2) 75oC for 30 min, reactor
temperature 130oC. Initial parameters of eluent flow (0.67 ml/min): Na-E buffer
(67%), ninhydrin (33%), temperature 50oC. Further eluents were subjected to the
following programme: Na-E, Na-F od, Na-D (sample programme: up to 10 min —
NaE, 10 to 31 min — NaF, 31 to 67 min — NaD, over 67 min — column
stabilization). Time of analysis with column stabilization and regeneration was
approximately 80 min.
Standard solutions for analysis
The basic standard solution was prepared by dissolving the following approximate amounts of amino acids (GM — molar mass of standard substance) in
500 ml of buffer pH 2.2 in measuring flask (500 ml): 0.045 g lysine hydrochloride
(Lys*HCl; GM = 182.6); 0.052 g arginine hydrochloride (Arg*HCl; GM = 210.7);
0.033 g aspartic acid (GM = 133.1); 0.030 g threonine (GM = 119.1); 0.026 g serine
(GM = 105.1); 0.037 g glutamic acid (GM = 147.1); 0.029 g proline (GM = 115.1);
0.029 g valine (GM = 117.1); 0.033 g leucine (GM = 131.2); 0.023 g tyrosine
(GM = 181.2); 0.021 g phenylalanine (GM = 165.2); 0.052 g histidine hydrochloride
hydrate (His*HCl*H2O; GM = 209.6); 0.019 g glycine (GM = 75.07); 0.022 g alanine (GM = 89.09); 0.017 g isoleucine (GM = 131.2); 0.015 g cystine (GM = 240.3);
0.0105 g cysteic acid hydrate (GM = 187.2) or cysteic acid (GM = 169.2); 0.0093 g
methionine (GM = 149.2). Portions were weighed with a reading accuracy of
0.0001 g. Cystine, cysteic acid and methionine were not determined according to
this method but added to the standard to facilitate the interpretation of chromatograms. To make the amino acids dissolve more easily, several drops of 6 N HCl were
added to the standard solution. A second standard solution was prepared by 4-fold
185
dilution of the basic standard solution using buffer pH 2.2. Both solutions, undiluted
and diluted 4-fold, were used for analysis together with the samples. The amino
acid content of the samples was calculated based on a calibration curve plotted in
Gold Nouveau software.
Preparation of samples
The following feeds were analysed: grower diets for poultry, a pig feed,
soybean, wheat and horse bean (analysis of stability of feed hydrolysates, analysis
of reproducibility), starter diets for poultry, meat-and-bone meal, rapeseed, barley,
oats, naked oats, maize, wheat, soybean (analysis of repeatability). Each feed was
mixed and ground in a mill with a 1 mm sieve. Prior to sampling for analysis, the
representative sample, taken from several places, was again mixed and weighed.
The sample was weighed into an ampoule (50 ml) so as to contain 40 – 80 mg crude
protein determined by Kjeldahl, noting the weighed portions with a reading
accuracy of 0.0001 g, and 40 ml 6 N HCl (1 – 2 mg crude protein per 1 ml 6 N HCl)
was added. The ampoule was sealed and the sample was hydrolyzed at 110 ± 3oC
for approximately 22 h. Following hydrolysis, the ampoule and its contents were
cooled, the hydrolysate was filtered through a Filtrak 389 medium filter paper into
an evaporating flask (250 ml) and the sediment was flushed three times with
redistilled water. The sample was evaporated dry on an evaporator at 65 ± 3oC,
rinsing the system twice with redistilled water. The sediment was dissolved in 15 ml
of buffer pH 2.2, mixed from time to time for 0.5 h, and the solution was transferred
quantitatively into a measuring flask (100 ml), rinsed with buffer pH 2.2 and filled
to the marker line. Approximately 2 ml of the solution was then transferred to
a chromatography vial, filtering it through a 0.22 µm syringe filter.
20 µl of this sample solution was inserted into a chromatographic column using an
autosampler.
Calculation of results
The amino acid content of samples was calculated using Microsoft Excel
according to the following formula:
P = 100 ×
Cp × V × GM
ng × R × 1000
(g/kg)
where:
P — amino acid content of the sample (g/kg),
Cp — amino acid concentration in sample solution as calculated in Gold
Nouveau chromatography software (µmol/ml),
V — volume of buffer pH 2.2 (100 ml), to which the sample is diluted after
evaporation in the evaporator,
GM — molar mass of amino acid,
ng — weighed portion of the sample in (g).
186
R. Ga˛sior et al.
R — recovery determined by comparing the peak area of amino acid
undergoing the sample preparation procedure with the peak area from an external
standard. Recovery was calculated according to the following formula:
R = 100 ×
(Aproc × Cs)
(As × Cproc)
where:
Aproc — peak area of amino acid in the standard subjected to the procedure,
Cproc — concentration of amino acid in the standard subjected to the procedure
(µmol/ml),
Cs — concentration of amino acid in external standard (µmol/ml).
Preparation of blank samples
Four blank samples were analysed to test the purity of the reagents, the
efficiency of the chromatography system and their effect on the results of analyses.
They were prepared as described in the Preparation of samples procedure but
without adding a feed sample. 20 µl of the blank sample solution was placed into
the chromatographic column using an autosampler.
Testing the stability of standard solutions
On 5 successive dates spanning a period of 110 days, basic standard solutions of
amino acids were prepared according to Standard solutions for analysis. Each
solution was diluted at a 1:1 ratio (v/v) with buffer pH 2.2, and then placed in
a refrigerator at 2 – 8oC. In these solutions, the concentrations were approximately
0.25 mmol/L for most of the amino acids and approximately 0.12 mmol/L for Ile,
Tyr and Phe. The exact concentrations were calculated by weighing each standard
substance to a reading accuracy of 0.0001 g. At the same time, all solutions were
analysed in replication after 110 days and a slope coefficients of line taken from the
equation y = ax were compared, which were defined as the relation between the
peak area of amino acid and its concentration in the standard solution. The mean
value (from 2 determinations) of the a coefficient for a given amino acid, calculated
for standard solution stored for 1 day, was treated as 100%. The a coefficients from
the other storage dates (days 8, 21, 49 and 110) were expressed in relation to this
value. Variations in the mean values of the a coefficients after standardization,
between dates of standard solution storage, were expressed using between-group
coefficients of variation CVm. They were calculated using the formula:
CVm =
100 . SD
xsr
where:
SD — standard deviation of the a coefficients between standard solution storage
dates.
187
Testing the stability of feed hydrolysates
In replication (n = 2), following the Preparation of samples procedure, 5 hydrolysates of the following feed samples were prepared: a grower diet for poultry, a pig
feed, soybean, wheat, and field bean. Each hydrolysate was poured into 5 chromatography vials and placed in a cooler at 2 – 8oC. Analyses were performed using an
amino acid analyser at 1, 9, 17, 23 and 30 days after preparation of feed sample
hydrolysates (5 storage dates). Between-group coefficients of variation (CVm) were
determined between average levels (n = 2) of amino acids in hydrolysates from
5 storage dates. When calculating CVm, the formula presented in the Testing the
stability of standard solutions was used.
Testing the calibration linearity and the limit of quantitative determination
The initial, concentrated standard solution of the amino acid mixture was
prepared by dissolving approximately 0.12 g of the standard substances Lys*HCl
and Arg*HCl and approximately 0.06 – 0.07 g of the other standard substances in
500 ml of buffer pH 2.2 in a measuring flask (500 ml). In this solution, the
concentrations of amino acids were approximately 1.64 and 1.36 mmol/L (Lys,
Arg) and 0.78 – 1.34 mmol/L (other amino acids). The concentrations expressed in
mg/L were: approx. 240 mg/L (Lys, Arg), approx. 120 mg/L (Ile, Ala, Gly) and 140
mg/L (other amino acids). Accurate concentrations were calculated by weighing
each standard substance on scales to an accuracy of 0.0001 g. The concentrated
initial standard solution of amino acids was diluted 2-, 4-, 8-, 16- and 32-fold. All
solutions were analysed in replicate. Calibration curves were generated for each
amino acid and residual coefficients of variation were calculated as well as the
correlation coefficients r2 for the regression equation y = ax + b.
The lowest point of the calibration curve plotted for a given amino acid
determined the limit for its quantitative determination.
Repeatability and reproducibility tests
In order to determine repeatability, 9 (k = 9) feed samples (starter diet for
poultry, meat-and-bone meal, rapeseed, barley, oats, naked oats, maize, wheat,
soybean) were analysed in replicate (n = 2) for the concentration of amino acids
after acid hydrolysis. The pooled coefficient of variation (CVkn) was calculated
for each amino acid using Microsoft Excel according to the formula:
CVkn =
√
∑ CVn22
k
k
The coefficient of variation (CVn2) for feed sample determination in replicate
(n = 2) was calculated using the formula:
188
R. Ga˛sior et al.
CVn2 = 100 ×
SDn2
Xsr
where:
k
— number of samples,
SDn2 — standard deviation from two measurements of a sample,
Xsr — mean from two measurements of a sample.
To determine reproducibility, two people (n = 2) performed independent analyses of the same 5 feed samples (grower diet for poultry, pig feed, soybean, wheat
and field bean). Analysis of each sample was carried out in replicate
(k = 5 × 2 = 10). Calculations were performed in Microsoft Excel, using analogous
formulae as for calculation of pooled coefficients of variation accompanying
repeatability.
Recovery tests
External standards
Two standard solutions of the amino acid mixture were made by dissolving
approx. 0.06 – 0.07 g of standard substances Ile, Tyr and Phe, and approx. 0.12 g of
the other standards in 500 ml of buffer pH 2.2 in a measuring flask. In these
solutions, the concentrations of amino acids were: approx. 120 – 140 mg/L (Ile, Tyr,
Phe) and approx. 240 mg/L (other amino acids). Accurate concentrations were
calculated by weighing each standard substance on a scales with an accuracy of
0.0001 g.
Standards subjected to the sample preparation procedure
Another two initial standard solutions of the amino acid mixture were prepared
by dissolving approx. 0.06 – 0.07 g of standard substances Ile, Tyr and Phe and
approx. 0.12 g of the other standards in 500 ml of 6 N HCl in a measuring flask
(500 ml). In this solution the concentrations of amino acids were: approx.
120 – 140 mg/L (Ile, Tyr, Phe) and approx. 240 mg/L (other amino acids). Accurate
concentrations were calculated by weighing each standard substance on a scales
with an accuracy of 0.0001 g. The initial standard solutions of the amino acid
mixture were subjected to the Preparation of samples procedure but without
weighed portions of the sample, replacing 40 ml of 6 N HCl with 40 ml of the
solution of a standard and diluting the sediment after evaporation of hydrolysate in
50 ml (instead of 100 ml) of buffer pH 2.2. The concentrations calculated for these
standard solutions are given in Table 5 (expected concentrations).
Method of recovery determination
The recovery determined accounted for losses during sample preparation. For
this purpose, two samples of the external standard solutions and two samples of the
standard solutions subjected to the sample preparation procedure were analysed as
described in HPLC chromatographic analysis. Recovery was determined by
comparing the peak area of an amino acid derived from the standard subjected to
189
the sample preparation procedure with the peak area derived from the external
standard, and calculated according to the formula provided in the Calculation of
results.
Results
Determining the effect of blank sample on analysis results
Five amino acids found on chromatograms of blank samples were at the
detector noise level, therefore it was not necessary to adjust the level of amino acids
in the analysed feed samples.
Stability of standard solutions and feed hydrolysates
Between-group coefficients of variation CVm (standard solutions) ranged from
0.55% (Gly) to 3.06% (Arg). The standardized (for standard solution from day 1,
a = 100) values of the coefficients a from 5 storage dates (days 1, 8, 21, 49 and 110)
ranged from 95.29 to 107.36. The mean values of standardized coefficients a and
between-group coefficients of variation CVm obtained during the stability tests of
standard solutions, are shown in Table 1.
Table 1. Stability of standard solution during 110 days expressed as the coefficient of variation CVm for
standardized coefficients of a slope for y = ax line (for solution stored for 1 day a = 100)
Standardized coefficients of a slope for y = ax line
after standardization; days
Amino acid
Asp
Thr
Ser
Glu
Pro
Gly
Ala
Val
Ile
Leu
Tyr
Phe
His
Lys
Arg
CVm
(%)
110
49
21
8
1
99.68
101.61
99.59
98.60
95.29
100.52
101.39
99.28
101.52
100.73
100.78
99.59
100.72
102.08
106.10
100.72
101.30
99.43
101.09
97.92
101.42
101.53
101.19
100.75
101.97
103.95
101.84
101.69
103.02
107.36
101.80
101.17
101.96
100.80
98.98
100.95
101.48
101.61
96.33
103.12
103.03
96.78
107.29
103.11
106.98
101.26
102.59
100.89
101.42
100.31
100.30
101.57
101.27
101.82
102.70
102.80
101.25
101.90
103.40
102.51
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
0.87
0.92
1.05
1.12
2.05
0.55
0.66
0.98
2.21
1.29
1.62
1.97
2.82
1.36
3.06
Between-group coefficients of variation CVm between mean levels (n = 2) of
amino acids in feed hydrolysates from 5 storage dates (days 1, 9, 17, 23 and 30)
ranged from 0.27% (Asp, poultry diet) to 5.72% (Pro, pig feed). The results of feed
hydrolysate stability tests are given in Table 2.
190
R. Ga˛sior et al.
Table 2. Stability of feed hydrolysates during 30 days expressed as the coefficient of variation CVm
for standardized coefficients of a slope for y = ax line (for solution stored for 1 day a = 100)
Amino
acid
Asp
Thr
Ser
Glu
Pro
Gly
Ala
Val
Ile
Leu
Tyr
Phe
His
Lys
Arg
CVm between dates of storage: 1, 9, 17, 23, 30 days (%)
field bean
wheat
soybean
pig feed
poultry diet
0.27
0.92
0.36
0.46
6.12
0.33
1.11
1.38
1.22
0.46
0.65
0.43
0.67
0.31
0.64
1.09
1.24
0.40
0.52
4.72
0.41
1.17
1.66
2.03
0.51
1.73
1.16
1.21
0.49
0.40
0.49
0.69
0.35
0.45
4.11
0.38
0.92
1.15
2.56
0.63
1.16
0.72
0.75
0.47
0.23
0.75
1.11
1.27
0.63
5.72
0.42
1.14
1.82
1.55
0.49
0.87
0.65
0.77
0.52
0.63
0.31
1.95
1.57
0.47
4.41
0.52
1.08
0.89
1.66
1.50
1.64
1.27
0.58
0.30
0.69
Linearity of calibration and the limit of quantitative determination
Calibration performed based on the amino acid standards showed a linearity of
4 – 140 mg/L for Asp, Thr, Ser, Glu, Pro, Val, Leu, Tyr, Phe and His; 4 – 140 mg/L
for Gly, Ala and Ile; and 7.5 – 240 mg/L for Lys and Arg. The correlation
coefficients r2 for all amino acids were at least 0.999. Residual variation coefficients of the regression equation for solutions of amino acids in the above range of
concentrations varied between 0.9% and 8.5%. They were lower in a narrower
range of concentrations and varied between 0.3% and 4.2% (Asp, Thr, Glu, Pro,
Val, Leu, Tyr, Phe, His in a range of concentrations from 4 to 70 mg/L), between
1.3% and 1.8% (Gly, Ala, Ile in a range of concentrations from 4 to 60 mg/L), 2.8%
(Ser in a range of concentrations from 8 to 140 mg/L), 0.2% (Lys in a range of
concentrations from 7.5 to 120 mg/L) and 1.9% (Arg in a range of concentrations
from 7.4 to 30 mg/L). The limit of quantitative determination was 4 mg/L for Asp,
Thr, Ser, Glu, Pro, Gly, Ala, Val, Ile, Leu, Tyr, Phe and His, and 7.4 mg/L for Lys
and Arg.
Repeatability and reproducibility
Cumulative coefficients of variation (CVkn) did not exceed 5% for repeatability
and 6.4% for reproducibility. The values of the pooled coefficients of variation for
repeatability and reproducibility are shown in Table 3.
The concentrations of amino acids in selected feed samples (grower diet for
poultry, pig diet, soybean, wheat and field bean) are given in Table 4.
191
Table 3. Repeatability and reproducibility expressed as pooled coefficients of variation CVkn
Amino acid
CVkn (%) for repeatability
CVkn (%) for reproducibility
Asp
Thr
Ser
Glu
Pro
Gly
Ala
Val
Ile
Leu
Tyr
Phe
His
Lys
Arg
2.7
3.1
3.3
2.5
2.5
2.3
2.0
2.2
2.9
2.0
4.4
2.5
2.2
2.7
2.6
4.0
1.8
3.0
1.6
6.3
1.4
1.8
2.0
1.7
1.1
4.2
0.9
1.5
1.6
3.5
Table 4. Level of amino acids in sample feeds (g/kg)
Amino acid
Grower diet
for poultry
Pig feed
soybean
wheat
field bean
Asp
Thr
Ser
Glu
Pro
Gly
Ala
Val
Ile
Leu
Tyr
Phe
His
Lys
Arg
20.04
8.02
10.75
41.63
14.21
8.73
9.55
9.41
7.69
17.34
6.77
10.12
5.35
10.97
12.74
9.28
4.59
6.09
29.43
12.06
5.74
5.31
6.08
4.32
9.13
3.00
6.07
3.07
5.77
6.88
54.71
19.25
23.71
83.72
25.19
20.30
20.58
22.20
20.13
37.16
17.54
23.44
12.73
30.60
32.86
6.99
3.29
4.88
31.31
11.56
4.69
4.19
4.88
3.51
7.73
2.12
5.01
2.58
3.31
4.83
30.32
9.54
12.42
43.99
11.33
11.51
10.68
12.13
10.20
20.46
7.50
11.01
6.88
16.80
24.49
Recovery
Recovery rates of amino acids ranged from 85.6% (Tyr) to 100.7% (Ile),
averaging 94.6%. Recovery of Asp, Thr, Ser, Glu, Pro, Gly, Ala, Val, Ile, Leu, Tyr,
Phe, His, Lys and Arg exceeded 93% and recovery was 87.5% and 85.6% for Ser
and Tyr, respectively (Table 5).
Uncertainty
The present authors determined the uncertainty of amino acid determination in
acid hydrolysates assuming a significance level of P ≤ 0.05. The uncertainty
determined based on variation encompassing all stages of sample preparation and
chromatographic analysis of amino acid solutions was 7.1%.
192
R. Ga˛sior et al.
Table 5. Predicted and actual concentrations of amino acids in standard solutions subjected to the sample
preparation and the calculated mean recovery of amino acids
Amino
acid
Asp
Thr
Ser
Glu
Pro
Gly
Ala
Val
Ile
Leu
Tyr
Phe
His
Lys
Arg
Mean
1st standard
2nd standard
1st standard
2nd standard
solution subjected solution subjected solution subjected solution subjected
Mean
to the procedure to the procedure to the procedure to the procedure recovery
— predicted
— predicted
— actual
— actual
(%)
concentrations
concentrations
concentrations
concentrations
(mg/L)
(mg/L)
(mg/L)
(mg/L)
0.191
0.180
0.165
0.200
0.206
0.197
0.202
0.198
0.104
0.198
0.112
0.106
0.209
0.196
0.169
0.193
0.182
0.167
0.200
0.197
0.197
0.207
0.199
0.103
0.195
0.106
0.111
0.201
0.196
0.181
0.187
0.168
0.142
0.193
0.191
0.184
0.203
0.191
0.105
0.191
0.096
0.100
0.199
0.188
0.162
0.189
0.172
0.148
0.187
0.186
0.187
0.197
0.186
0.104
0.188
0.091
0.107
0.193
0.189
0.171
97.9
94.0
87.5
94.9
93.5
94.1
97.7
94.9
100.7
96.5
85.6
95.3
95.7
96.2
95.0
94.6
Discussion
Determining the effect of blank sample on analysis results
The practical lack of amino acids in blank samples indicated that the purity of the
reagents, flasks and other analytical instruments was sufficient. It must be stressed,
however, that following analyses laboratory glass was thoroughly cleaned with
specialized washing agents, rinsed with normal and then distilled water, and dried.
Stability of standard solutions and feed hydrolysates
Based on the low variability of the results of amino acid determinations between
standard solutions stored for different times at 2 – 8oC, it was found that these
solutions were stable for at least 110 days after they were made. Based on similar
observations, it was stated that the solutions of feed hydrolysates stored at 2 – 8oC
were stable for at least 30 days after preparation.
Linearity of calibration and the limit of quantitative determination
Amino acid solutions were characterized by good linearity, with high coefficients of correlation and slight deviations of the calibration points towards the
plotted regression curve y = ax + b. The limit of quantitative determination, described as the lower limit of linearity and amounting to 4 mg/L for most amino acids
and 7.5 mg/L for Lys and Arg, is sufficient for analysing feed sample hydrolysates
193
for the concentration of amino acids. For example, if analysis were performed with
a weighed portion of 1 g of the analysed sample, the limit of quantitative
determination would be 0.4 and 0.75 g/kg, respectively.
Repeatability, reproducibility
The coefficient of variation (CV), sometimes called the relative standard
deviation (RSD), is the basic parameter to express determination error. The
coefficients of variation of the analysed substances depend, among others, on the
errors they contain. These can include errors of determination by the apparatus
itself (0.2 – 5.52%; Schuster, 1988; Qureshi and Qureshi, 1989; Bütikofer et al.,
1991), but can also include other errors, e.g. errors at all stages of sample
preparation (0.9 – 4.5%; Sarwar et al., 1983; 1988) and errors of determination at
different laboratories that use different methods (Dobecki, 1998), different laboratory assistants or different time of determinations (3.6 – 16.4%; Sarwar et al., 1983).
The low coefficients of variation obtained in our study for amino acids (2.0 – 4.4%
for repeatability conditions and 0.9 – 4.0% for reproducibility conditions) are
similar to the coefficients of variation obtained in the previously mentioned studies
and are indicative of good repeatability (analyses performed by one and the same
person) and reproducibility (analyses performed by two people independently) of
the entire sample preparation process and chromatographic analysis. The repeatability and reproducibility of the method were determined based on the coefficients of
variation calculated. It was assumed that the repeatability was not less than the
pooled coefficient of variation for determinations performed with the same
method, using identical material, in the same laboratory, by the same laboratory
assistant, and during the same time period. Reproducibility was defined as being not
less than the pooled coefficient of variation for determinations performed with the same
method, using identical material, in the same laboratory, by different laboratory
assistants at different times.
Recovery
The recovery rate of ten amino acids after acid hydrolysis (Asp, Glu, Gly, Ala,
Leu, Tyr, Phe, Lys, His, Arg) ‘‘in general” (Gehrke et al., 1985) exceeds 95%. In
our study, recovery rates for Asp, Ala, Leu, Phe, Lys, His and Arg also exceeded
this value, those of Glu (94.9%) and Gly (94.1%) were similar, and that of Tyr was
significantly lower (85.6%). A similarly low recovery rate was also characteristic of
Ser (87.5%). The low recovery rates of Tyr and Ser observed in the current study
are consistent with information given in the above publication stating that these
amino acids can degrade during acid hydrolysis of samples. Although Gehrke et al.
(1985) report that Thr can also be degraded, the recovery rate determined in our
study was fairly high (94%).
Uncertainty
The greatest effect on the uncertainty of measurement is exerted by the
repeatability of the method and related errors of analysis such as: weighing error,
194
R. Ga˛sior et al.
hydrolysis, pipetting, transfer, errors due to inaccuracy of the measuring equipment
used during sample preparation, and error in chromatographic determination. It is
worth noting at this point that repeatability expressed as the pooled coefficient of
variation CVkn comprises the above errors only when the results calculated from two
replicates concern chromatographic determinations of solutions derived from two
parallel weighed samples. If a sample were weighed without replicates, and the
solution obtained after its treatment were analysed twice on the analyser, the
CVkn would cover only the error of chromatographic determination. In more general
terms, if the sample were separated into two parts at a certain stage of analysis (i.e.
the procedure of sample preparation together with its chromatographic determination), then the calculated CVkn would encompass all errors occurring from the
moment of separating this sample. If routine analysis of the sample is performed in
replicate (n = 2), the error of the mean value of these replicates is not an error
described by the pooled coefficient of variation, but is lower. Thus the error of
mean result (n = 2) is the coefficient of variation of the arithmetic mean CVn2sr,
which can be described by the following equation:
CVn2sr =
CVkn
√n
=
CVkn
√2
= 0.707 × CVkn
This variation coefficient for the arithmetic mean corresponds to the standard
uncertainty ux (Dobecki 1998; Arendarski, 2003) that determines the error of mean
result at a confidence level of approx. 68% (P ≤ 0.32). It is generally acknowledged
(Dobecki, 1998; Arendarski, 2003) that the uncertainty of the calculated result is an
uncertainty extended to a confidence level of 95% (P ≤ 0.05). Thus the uncertainty
designated as U0.05 is described by the formula:
U0.05 = 2 × CVn2sr ≈1.4 × CVkn
In summary, if routine analyses are performed in replicate, i.e. when n = 2, then
the uncertainty of measurement that encompasses errors during sample preparation
and chromatographic analysis can be defined as double the value of the variation
coefficient of the arithmetic mean (P ≤ 0.05) or as repeatability (expressed through
the pooled coefficient of variation CVkn) multiplied by 1.4. In this way, the
determined uncertainty is approximately the uncertainty of the whole analytic
method, under the assumption that its contribution to the total uncertainty is
dominant, while other components of uncertainty can, in keeping with the
conclusions resulting from Gauss’s law of propagation of errors, be omitted. The
uncertainty, together with the result treated as a mean from measurements, is of
practical importance during its interpretation and determines the range of tolerance
in which the actual value of the determination result should have a probability of
95%. The uncertainty determined in the present study is relatively low and includes
195
the error of analyses performed in one laboratory. It should be controlled during each
analysis of the samples by checking repeatability and may be higher in the case of
analyses which fail to meet the criterion of repeatability despite repeated tests.
Controlling the quality of analysis results
It was decided that during the analyses, the repeatability of sample analysis and
the level of amino acids in our own reference sample would be verified.
Checking the repeatability of sample analyses — the criterion of repeatability
To determine the criterion of when to reanalyse a sample, the limit of
repeatability was determined (Dobecki, 1998). It is assumed that this is double the
value of repeatability and equates to 10%. More precisely, this means that during
conversion of results, a condition must be satisfied stating that the coefficient of
variation for measurements of a sample performed using the same method, in the
same laboratory, by the same laboratory assistant, and at the same time, must not
exceed double the value of repeatability (10%) in more than 5% of cases. If the
condition cannot be fulfilled, the analysis should be repeated. Once the limit of
repeatability is exceeded again, the limit of repeatability and uncertainty of the
analysis of a sample or series of samples needs to be extended.
Checking the level of amino acids in our own reference sample
Soybean, like cereals, is characterized by a stable amino acid composition, and
for this reason was chosen as our reference material. From the original soybean
sample (200 g), 6 samples were separated, which were assayed for the level of
amino acids. The original sample, stored in a tightly sealed container, is used to
control the quality of analysis results (PN-EN ISO/IEC 17025:2001, p. 5.9) during
routine determinations of amino acids.
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579 – 587.
B e a v e r R.W., W i l s o n D a v i d M., J o n e s H e l e n M., H a y d o n K e i t h D. (1987). Amino
acids analysis in feeds and feedstuffs using precolumn phenylisothiocyanate derivatization and
liquid chromatography — preliminary study. J. Assoc. Off. Anal. Chem., 70, 3: 425 – 428.
B ü t i k o f e r U., F u c h s D., B o s s e t J.O., G m ü r W. (1991). Automated HPLC-amino acid determination of protein hydrolysates by precolumn derivatization with OPA and FMOC and comparison with classical ion exchange chromatography. Chromatographia, 31, 9/10: 441 – 447.
C z a u d e r n a M., K o w a l c z y k J. (1998). Determination of free amino acids in blood plasma by
high-performance liquid chromatography with fluorescence detection. J. Anim. Feed Sci. (7):
453 – 463.
196
R. Ga˛sior et al.
C z a u d e r n a M., K o w a l c z y k J., N i e d ź w i e d z k a K.M., W a˛s o w s k a I. (2003). High efficient
method for determination of some acids and glutathione by liquid chromatography. J. Anim. Feed
Sci. (12): 199 – 215.
D e y l Z. (1986). Review. Profiling of amino acids in body fluids and tissues by means of liquid
chromatography. J. Chrom., 379: 177 – 250.
D o b e c k i M. (1998). Zapewnienie jakości analiz chemicznych. Assurance of chemical analyses quality
(in Polish). Instytut Medycyny Pracy im. prof. dra med. J. Nofera Press, Łódź.
E l k i n R. (1984). Quantitative amino acid analysis of feedstuff hydrolysates by reverse phase liquid
chromatography and conventional ion-exchange chromatography. J. Assoc. Off. Anal. Chem., 67,
5: 1024 – 1026.
G e h r k e C.W., W a l l L a r r y L.Sr, A b s h e e r J.S., K a i s e r F l o y d E., Z u m w a l t R.W. (1985).
Sample preparation for chromatography of amino acids : acid hydrolysis of proteins. J. Assoc. Off.
Anal. Chem., 68, 5: 811 – 821.
J e n - K u n L i n, C h i u - H w a W a n g (1980). Determination of urinary amino acids by liquid
chromatography with ‘‘dabsyl chloride”. Clin. Chem., 26/5: 579 – 583.
L a m S., A z u m a y a H., K a r m e n A. (1984). High-performance liquid chromatography of amino acids
in urine and cerebrospinal fluid. J. Chrom., 302: 21 – 29.
Q u r e s h i A., Q u r e s h i R. (1989). Determination of free amino acids in biological samples —
problems of quantitation. J. Chrom., 491: 281 – 289.
R a y n e r C a r l J. (1985). Protein hydrolysis of animal feeds for amino acid content. J. Agric. Food
Chem., 33, 4: 722 – 725.
S a r w a r G., B o t t i n g H.G., P e a c e R.W. (1988). Complete amino acid analysis in hydrolysates of
foods and feces by liquid chromatography of precolumn phenylisothiocyanate derivatives. J. Assoc.
Off. Anal. Chem., 71, 6: 1172 – 1175.
S a r w a r G., B o t t i n g H. G. (1993). Evaluation of liquid chromatographic analysis of nutritionally
important amino acids in food and physiological samples. J. Chrom., 615: 1 – 22.
S a r w a r G., C h r i s t e n s e n D.A., F i n l a y s o n A.J., F r i e d m a n M., H a c k l e r I.R., M a c k e n z i e
S.L., P e l l e t t P.L., T k a c h u k R. (1983). Inter- and intra-laboratory variation in amino acid
analysis of food proteins. J. Food Sci., 48: 526 – 531.
S c h u s t e r R.(1988). Determination of amino acids in biological, pharmaceutical, plant and food
samples by automated precolumn derivatization and high-performance liquid chromatography.
J. Chrom., 431: 271 – 284.
Z u m w a l t R.W., A b s h e e r J.S., K a i s e r F.E., G e h r k e C.W. (1987). Acid hydrolysis of proteins for
chromatographic analysis of amino acids. J. Assoc. Off. Anal. Chem., 70, 1: 147 – 151.
ROBERT GA˛SIOR, KRYSTYNA ŚLUSARCZYK, MARTA SZCZYPUŁA
Walidacja metody oznaczania aminokwasów w kwaśnych hydrolizatach pasz
STRESZCZENIE
Scharakteryzowano metode˛ oznaczania kwasu asparaginowego, treoniny, seryny, kwasu glutaminowego, proliny, glicyny, alaniny, waliny, izoleucyny, leucyny, tyrozyny, fenyloalaniny, histydyny, lizyny
i argininy. Wykonano analizy ła˛cznie dwunastu pasz: dwóch mieszanek dla drobiu (starter i grower),
mieszanki dla trzody chlewnej, ma˛czki mie˛sno-kostnej, rzepaku, je˛czmienia, dwóch odmian owsa,
kukurydzy, pszenicy, soi i bobiku.
197
Przeprowadzone analizy prób ślepych nie wskazywały na konieczność korygowania o ich wyniki
zawartości aminokwasów w analizowanych próbkach pasz. Stwierdzono trwałość aminokwasów
w pie˛ciu roztworach wzorcowych w cia˛gu co najmniej 110 dni (współczynniki zmienności od 0,55% do
3,06%) i trwałość pie˛ciu hydrolizatów pasz w cia˛gu co najmniej 30 dni (współczynniki zmienności od
0,31% do 5,72%). Powtarzalność metody oznaczania aminokwasów wynosiła 5%, odtwarzalność 6,4%,
a niepewność 7,1%. Odzysk wahał sie˛ od 85,6% (tyrozyna) do 100,7% (izoleucyna). Sporza˛dzona
krzywa kalibracji charakteryzowała sie˛ liniowościa˛ w zakresie 4 – 140 mg/L dla kwasu asparaginowego,
treoniny, seryny, kwasu glutaminowego, proliny, waliny, leucyny, tyrozyny, fenyloalaniny, histydyny;
140 mg/L dla glicyny, alaniny, izoleucyny; 7,5 – 240 mg/L dla lizyny i argininy. Resztowe współczynniki zmienności wynosiły od 0,9% do 8,5%, a współczynniki korelacji r2 co najmniej 0,999. Granica
oznaczenia ilościowego kwasu asparaginowego, treoniny, seryny, kwasu glutaminowego, proliny,
glicyny, alaniny, waliny, izoleucyny, leucyny, tyrozyny, fenyloalaniny, histydyny wynosiła 4 mg/L,
a lizyny i argininy 7,5 mg/L. Granica powtarzalności, przyje˛ta jako podwojona wartość powtarzalności,
wynosiła 10%. Ustalono, że podczas wykonywania rutynowych analiz be˛dzie sprawdzana powtarzalność
analizy próbek oraz zawartości aminokwasów we własnej próbce referencyjnej.
PREVALENCE AND CONTROL OF GASTROINTESTINAL
AND PULMONARY NEMATODES IN DEER IN A CLOSED BREEDING
POPULATION
A g n i e s z k a C i s e k, A l e k s a n d r a B a l i c k a - R a m i s z, B o g u m i ł a P i l a r c z y k
Department of Animal Hygiene and Prophylaxis, Agricultural University, Doktora Judyma 6,
71-466 Szczecin, Poland
Abstract
Parasitic infections of animals living in the wild, especially the gastrointestinal and pulmonary
nematodoses can generate great losses among domestic and free-living ruminants. The aim of this
study was to estimate the prevalence and usefulness of Panacur anthelmintic for the control of
gastrointestinal and pulmonary nematode invasions in deer. The anthelmintic treatment was
administered in the spring of 2000 in an enclosed breeding station located in Krajanka
(Złotów forest district, Poland). Panacur was given twice at 10-day intervals, at a dosage of 7.8 mg
per kg of body weight. The efficiency of the anthelmintic was established after 7 and 14 days of
administration. The preparation proved effective for both gastrointestinal and pulmonary
nematodes. Treatment efficiency was 87.75 – 99.40% for gastrointestinal nematodes and
74.26 – 82.13% for lungworms.
Key words: Panacur, treatment effectiveness, deer, gastrointestinal nematodes, pulmonary nematodes
The problem of parasitoses is still present. During their lifetime game animals
live in a human-modified environment the negative factors of which result in
metabolic and immune disorders. These disorders are conducive to an increase in
infectious and invasive diseases. Invasive diseases may cause losses manifesting
themselves as poorer weight gains, reproductive disorders, and increased incidence
of other diseases. In recent years, constant monitoring of diseases in game animals
has become an essential part of effective hunting management. These activities
should also include systematic control of the parasitological situation and implementation of prophylaxis programmes developed specifically for hunting
grounds or breeding centres of game animals.
Of the several invasive disease control methods for hunting grounds, one of the
most effective is the administration of antiparasitic drugs, oral remedies being the
200
A. Cisek et al.
easiest to use. Animals kept in an enclosed area of a pen are particularly exposed to
constant contact with invasive forms of parasites, which is why regular and effective
deworming is an essential veterinary procedure. The aim of this study was to evaluate
the prevalence of gastrointestinal nematodes and to test the usefulness of Panacur
anthelmintic for controlling gastrointestinal and pulmonary nematodes in deer.
The additional winter and spring feeding period was selected as a good time to
administer a dewormer to the animals. This procedure was carried out at a breeding
centre in Krajanka (Złotów forest district) in March 2000. The centre covers an area
of 800 ha. Animals were given Panacur anthelmintic twice at 10-day intervals. The
dosage of active substance, i.e. fenbendazole, was 7.8 mg per kg of body weight.
Each time the anthelmintic was given for 3 days. Panacur was added to a compound
feed for forest animals, which had been prepared by the Krajna Grain Works.
The effectiveness of the anthelmintic was determined by a coproscopic
examination of the animal faeces, which were collected from the ground near
feeding racks.
Treatment efficacy was evaluated using the following formula:
S(%) =
L – L1
L
× 100
where:
S(%) — effectiveness of treatment,
L
— mean egg/larval count before deworming,
L1 — mean egg/larval count after deworming.
The test for gastrointestinal nematodes was performed using the Willis-Schlaff
flotation method. Parasite egg count was determined using the McMaster method
[eggs per g (epg) of faeces]. The presence and amount of pulmonary nematode
larvae were determined according to the Baermann method (larval count per 5 g of
faeces). Parasites were identified based on their structure, using the normal
procedure.
The deworming results were analysed statistically using STATISTICA software. The T-Student’s test was used for statistical calculations.
Results
Deworming is an effective method of parasite control in free-living animals. An
appropriate antiparasitic preparation must be chosen that shows little toxicity, is
highly effective and has a broad spectrum of activity.
Table 1. The results of anthelmintic treatment of deer at the breeding station Krajanka — gastrointestinal nematodes
Before treatment
Species
Red deer
Roe deer
Fallow deer
7 days after treatment
O
Z
E.I
(%)
L
(I.i.)
O
Z
E.I
(%)
L
(I.i.)
S
(%)
O
Z
E.I
(%)
L
(I.i.)
S
(%)
20
72
54
15
57
39
75.00
79.20
72.20
102
163
98
15
59
35
7
19
11
46.66
32.20
31.43
6
7
12
94.20
96.32
87.75
16
62
42
2
5
5
12.5
8.06
11.90
2
1
2
98.03
99.40
97.96
O — total number of faecal samples analysed.
Z — number of faecal samples in which gastrointestinal nematode eggs were found.
E.I — prevalence of gastrointestinal nematode invasion.
I.i. — intensity of invasion (number of gastrointetinal eggs per 1 g of faeces).
L — average number of gastrointestinal nematode eggs.
S — effectiveness of treatment.
Table 2. The results of anthelmintic treatment of deer at the breeding station Krajanka — pulmonary nematodes
Before treatment
Species
Red deer
Roe deer
Fallow deer
O
Z
E.I
(%)
L
(I.i.)
O
Z
E.I
(%)
L
(I.i.)
S
(%)
O
Z
E.I
(%)
L
(I.i.)
S
(%)
15
40
28
14
17
16
93.33
42.50
57.14
450
403
338
18
42
25
10
12
6
55.5
28.57
24.0
100
95
87
77.7
76.43
74.26
15
42
20
2
1
1
13.5
2.30
5.00
86
72
64
80.84
92.13
81.10
202
A. Cisek et al.
Data on the prevalence of deer infestation with gastrointestinal and pulmonary
nematodes in the analysed breeding station are given in Tables 1 and 2. Gastric
nematodes were found in 75% of red deer, 79.20% of roe deer and 72.20% of fallow
deer (Table 1). Invasion intensity was highest in roe deer [163 egg per g (epg) of
faeces], followed by red deer (102) and fallow deer (98). Pulmonary nematodes
appeared in 93.33% of red deer, 42.50% of roe deer and 57.14% of fallow deer
(Table 2). Invasion intensity was 450, 403 and 338 larvae per 5 g faeces,
respectively.
Panacur was found to be highly effective in controlling gastrointestinal
nematodes in all the animal species analysed (Table 1). After 7 days of administration, the treatment was most efficient in roe deer (96.32%) followed by red deer
(94.20%) and fallow deer (87.75%). After 14 days of administration, treatment
efficiency was 99.40% in roe deer, 98.03% in red deer and 97.96% in fallow deer.
Intensity of infection was found to decrease considerably. Seven days after the
preparation was administered the maximum intensity of invasion was found in
fallow deer (12 epg) compared to 6 and 7 epg in red deer and roe deer, respectively.
On day 14 the decrease in the intensity of infection with gastric nematodes was
even greater: 1 epg in roe deer and 2 epg in red deer and fallow deer were found.
The preparation also proved highly effective for pulmonary nematodes (Table 2).
After 7 days of treatment, efficacy was greatest in red deer (77.7%), followed by roe
deer (76.43%) and fallow deer (74.26%). After 14 days, the treatment efficiency of
Panacur was 82.13% in roe deer, 80.84% in red deer and 81.10% in fallow deer. On
day 7 after administration of the drug infection intensity was 100 larvae per 5 g of
faeces in red deer, 95 in roe deer and 87 in fallow deer. After 14 days the intensity
dropped even further to 86 larvae per 5 g of faeces in red deer, 72 in roe deer and 64
in fallow deer.
For both gastrointestinal and pulmonary nematodes, treatment was most
effective in roe deer, and was 17.27% higher for gastrointestinal than for pulmonary
nematodes.
The treatment results were analysed statistically. Statistically significant differences were found between the infection of animals prior to deworming (group A),
7 days after deworming (group B) and 14 days after deworming (group C). For the
infection with gastrointestinal nematodes, highly significant differences were found
in red deer between the group of animals before deworming and the group 7 days
after deworming. A highly significant difference also appeared in red deer between
group A before deworming and group C 14 days after deworming. There were
significant differences between the groups of red deer 7 and 14 day after
deworming. Similar patterns were found in fallow deer. In roe deer, highly
significant differences were found between groups A, B and C.
For infection of animals with pulmonary nematodes, highly significant differences were found in red deer and roe deer between animals from group B (day 7 after
deworming) and group C (day 14 after deworming), and significant differences
appeared between group A (before deworming) and animals 7 days after deworming. In fallow deer, highly significant differences were noted between the group
Prevalence and control of gastrointestinal and pulmonary nematodes in deer
203
of animals prior to deworming and the group of animals 7 days after deworming.
A highly significant difference was also evident between fallow deer before
deworming (group A) and 14 days after deworming. There was a significant
difference between the fallow deer of group B and those of group C, 14 days after
deworming.
Discussion
Both in Poland and abroad, most antiparasitic preparations have so far been
administered in feed. Demiaszkiewicz et al. (2000 a) used Eprinex Pour-On
(eprinomectin) and showed it to be 100% effective for pulmonary nematodes. The
pour-on method of application involves restraining animals, results in stress and
increases the costs of treatment. Among the other drugs currently used in Poland are
Ivomec premix (ivermectin) (Malczewski et al., 1998; Demiaszkiewicz et al.,
2000 b) and Ceremix (Kryński et al., 2000). The use of Ivomec premix on a farm in
Kosewo (Malczewski et al., 1998) proved 90.3 – 99.6% effective for pulmonary
nematodes. The effectiveness of this preparation was confirmed by Demiaszkiewicz
et al. (2000 b) through multiple administration of Ivomec. These authors reported
the preparation to be 98.4 – 100% effective. The effectiveness of Cermix (ivermectin) applied in the Rypin hunting ground was much lower. In Cermix-dewormed roe
deer, the invasion intensity dropped by 20% (Kryński et al., 2000). In Wielkopolska, benzoimidazole preparations were used to control the Capreocaulus capreoli
pulmonary nematode and an effectiveness of 52.8 – 78% was obtained (Kozakiewicz et al., 1986). Special resistance to antiparasitic agents among pulmonary
nematodes is characteristic of the Elaphostrongylus cervi. Some authors suggest
that antiparasitic drugs do not completely eliminate this nematode but only inhibit
excretion of the larvae (Demiaszkiewicz et al., 2001; Kutzer, 1990). Recently,
Demiaszkiewicz (2001) achieved very good results in controlling elaphostrongylosis in domestic ruminants using Eprinex Pour-On. Swiss authors were successful in using ivermectin, fenbendazole and flunixin to treat elaphostrongylosis in
goats (after Demiaszkiewicz, 2001 and Kutzer, 1990).
Among foreign papers mention should be made of the administration of Panacur
in the form of 1.5% granules and 4% pellets. In Germany, Düwel et al. (1979)
administered 5 mg fenbendazole per kg body weight to red deer, roe deer and
fallow deer for 5 successive days and achieved high effectiveness against gastrointestinal nematodes. Panacur was also used in red deer in Austria (Kutzer and Prosl,
1979) with 100% success. Miško et al. (1982) used Panacur (Panacur 1.5% pellets)
in red deer in Czechoslovakia, reaching 100% treatment efficiency against gastric
nematodes and 61.6% against pulmonary nematodes. In the Soviet Union Nazarova
and Sigacheva (1981) supplemented the feed of red deer with thiabendazol
(0.075 g/kg b.w.), cambendazole (20 mg/ kg b.w.) and Nilverm (10 – 15 mg/kg
b.w.). These drugs proved effective against Spiculopteragia spp., Capillaria spp.
and Nematodirus spp. Rhodes (1993) successfully used Albendazole in liquid form
204
A. Cisek et al.
(Valbazen 10 mg/kg b.w.) and in capsule form (Proftril 3.8 g). Both forms of the
drug were effective against gastrointestinal and pulmonary nematodes, but the
capsule form proved more effective. Forstner and Weber (1992) treated farmed
fallow deer with levamizole (7.5 mg/kg b.w) in drinking water over a period of one
year and achieved a considerable reduction in the faecal egg count of gastrointestinal nematodes. In Great Britain, Connan (1997) administered fenbendazole and
ivermectin to farmed red deer to successfully control invasions of gastric nematodes, taking into account the effects of these drugs on the individual developmental
stages of nematodes. The study showed 100% effectiveness for ivermectin in
mature and developing nematodes and 95% effectiveness for the dormant stage of
the larvae. Fenbendazole reduced the dormant stage of larvae by 75%. A year later,
the same author injected farmed red deer with avermectin (ivermectin and
doramectin) at a rate of 0.4 mg per kg body weight. This treatment proved effective
both for Trichostrongylidae nematodes and Dictyocaulus pulmonary nematodes.
The high effectiveness of Ivomec was also shown by Rhebein et al. (1993) who
treated fallow deer in Austria with ivermectin at a rate of 0.5 mg per kg body
weight. Treatment efficacy (Capillaria bovis and Dictyocaulus control) was
greatest at the 6th week of the treatment. After 12 weeks, the authors found some
Capillaria bovis eggs in the faeces of the investigated animals. Another pour-on
medicine applied to the skin (Waldrup et al., 1998) was moxidectin (0.5 mg/kg
b.w). The authors showed this drug to be 100% effective for Dictylocaulus
viviparus and 99.9% effective for larval stages and mature gastrointestinal nematodes of the subfamily Ostertagiinae.
The results obtained show that Panacur is effective and can be recommended to
control parasites in free-living animals. It is worth noting the high efficiency of this
drug compared to other preparations from the benzoimidazole group. In addition,
when added to the feed mixture the preparation is eagerly eaten by animals. It is
safe and shows low toxicity. In addition to the administration of dewormers to
animals, the incidence of parasites in the natural environment is also influenced by
factors such as the observation of hygiene in and around feeding racks, changing
feeding rack location, culling emaciated animals, and eliminating intermediate
hosts of pulmonary nematodes.
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AGNIESZKA CISEK, ALEKSANDRA BALICKA-RAMISZ, BOGUMIŁA PILARCZYK
Prewalencja i zwalczanie inwazji nicieni żoła˛dkowo-jelitowych i płucnych u zwierzyny płowej
w hodowli zamknie˛tej
STRESZCZENIE
Inwazje pasożytnicze zwierza˛t wolno żyja˛cych, a zwłaszcza nematodozy przewodu pokarmowego
i płuc, moga˛ być przyczyna˛ wielu strat u przeżuwaczy domowych i zwierza˛t wolno żyja˛cych. Celem
badań była ocena prewalencji oraz przydatności preparatu Panacur w zwalczaniu inwazji nicieni
żoła˛dkowo-jelitowych i płucnych u zwierzyny płowej.
Badania przeprowadzono na terenie ośrodka hodowlanego znajduja˛cego sie˛ w Krajance na terenie
nadleśnictwa Złotów, w miesia˛cach wiosennych 2000 r. Zwierzynie podano dwukrotnie w odste˛pach
206
A. Cisek et al.
dziesie˛ciodniowych preparat Panacur w dawce 7,8 mg na 1 kg masy ciała. Skuteczność preparatu
oceniano po 7 i 14 dniach po jego podaniu.
Preparat okazał sie˛ skuteczny zarówno w przypadku nicieni żoła˛dkowo-jelitowych jak i płucnych.
U nicieni żoła˛dkowo-jelitowych skuteczność wynosiła 87,75 – 99,40%. W odniesieniu do nicieni
płucnych preparat okazał sie˛ nieco mniej skuteczny (74,26 – 82,13%).
OPTIMUM SCALE OF PIG FATTENING ON POLISH FAMILY FARMS
IN LIGHT OF ECONOMIC STUDIES*
Stanisława Okularczyk
Department of Technology, Ecology and Economics of Animal Production,
National Research Institute of Animal Production, 32-083 Balice n. Kraków, Poland
Abstract
Total production costs and net agricultural income in a closed cycle of pig fattening were
investigated in 49 family farms in Poland. Standard calculations comparing average annual wages
outside agriculture with those obtained on the analysed farms were performed. The analysis
accounted for two variants: pig fattening based on farm-produced feed and on 50% purchased
feed. The calculations accounted for differences in the numbers of pigs sold per sow per year. The
calculations showed that 21 sows are enough to obtain income parity for fattening with
farm-produced feed, where 25 fatteners are sold per sow, and 34 sows when only 16 fatteners are
sold. On farms that buy 50% of their feeds, the number of sows necessary to achieve income parity
in the closed cycle should range from 33 to 51.
Key words: fatteners, scale of production, scale criteria, optimization
The concentration of herds of all livestock species in Poland is favourable for
both farmers, from the viewpoint of more efficient and more profitable production
on a larger scale, and for the processing industry, because of the simplified purchase
system and better quality of raw materials (Woś, 2004; Okularczyk, 2004 a; b). The
differentiation of pig herds in Poland has taken place over the last decade
(Małkowski, 2002; Woś, 2001; Zegar, 2002). For many years, the volume of
purchase from one producer in Poland has been the lowest in Europe (Woś, 2001;
Zegar, 2002). Large differences in the profitability of pig fattening between regions
have increased the concentration of pig production in northern Poland to the cost of
southern and eastern Poland, particularly over the last 14 years. For instance, the
supply in Wielkopolska already accounts for 26% of live pigs purchased in Poland,
compared to less than 4% of national supply in the Subcarpathian and Małopolska
* This work was conducted as part of the NRIAP statutory activity, project no. 4502.1.
208
S. Okularczyk
regions (Okularczyk, 2001; 2004 b; Woś, 2004). Pig fattening, compared to other
lines of specialization, is one of the simplest and least connected with land
resources in organic terms. For these reasons, among others, since the profitability
of pig fattening improved many farms have been characterized by an extremely
high scale and concentration of pig farming that deviates considerably from the
optimum, especially in terms of ecological safety (Flis and Lewicki, 1994; Woś,
2001; 2004; Okularczyk, 2004 b). This unchecked growth in the scale of pig
fattening in some areas of the Lake District and Pomerania disturbs the sustained
development of these regions and poses a threat to the economic safety of
established pig producers who fatten pigs on a lower scale using their own feeds
(Okularczyk, 2004 a; Kisiel, 1999). This also leads to market turbulence as a result
of uncontrolled and unpredictable fluctuations in the prices of live pigs. Thus, it
contributes to the instability of prices and, secondarily, to fluctuations in the
population of pigs all over Poland (Małkowski, 2002; Woś, 2004). Authors studying
the organizational systems of pig production agree that to maintain the ecological
safety of the surroundings and the biological equilibrium of the soils, at least
600 – 800 sq m of feed area, which is at the same time an absorption area for
excrements, should be provided per fattening pig (Okularczyk, 2004 b; Woś, 2004).
The stability of the pig population in EU countries results in part from the
monitoring of current supply and demand and early government regulations, but
mainly from the popularity of the closed cycle of production. This system
eliminates piglet market turbulence like that found in Poland, which leads to
unpredictable fluctuations in the population of pigs. Economists are guided by the
economic and social rule to give preference to the scale of production that brings
agricultural income at least equal to average wages outside agriculture, i.e. parity
income. However, we speak of a certain minimum resulting only from the Polish
agrarian structure, in which the volume of production does not exceed the stocking
density of 35 structural sows (Woś, 2004; Zegar, 2002). This scale is comparable to
that used by farmers in most EU countries. However, it is too small for full
technological development and profitable use of the latest production techniques on
these pig farms. It could be treated as a starting volume for gradual development
were it not for the fact that when making investment decisions, farmers must plan
the target size of their pig farms (Woś, 2004). Another principal criterion guiding
Polish farmers, which has in the past been used by German, French and English
farmers, is the amount of land resources owned (Woś, 2004; Zegar, 2002). Efficient
production processes and profitable pig fattening are possible only when the scale is
based on scientifically determined economic criteria. The present results, calculations and analyses serve to determine these criteria.
The results of studies performed on the farms and standard calculations were
used. The studies were performed in 49 purposely selected family farms in the
Optimum scale of pig fattening on Polish farms
209
Małopolska, Wielkopolska, Subcarpathian and Lower Silesia regions. Specialization
in the closed cycle of pig production was the criterion for selection. The factors
analysed were: the scale of production measured by the number of sows and annual
sales of fatteners; the proportion of farm-produced and purchased feeds in pig
nutrition; the direct and indirect costs of managing the foundation herd; the direct
and indirect costs of pig rearing and fattening; the monetary value of pigs sold, and
of selected and culled pigs. Net agricultural income was calculated per whole pig
farm, as was unit income. To compare incomes and to determine income parity,
average annual wages outside agriculture were calculated for the study period. Net
agricultural income was calculated for each farm and on average per 1 structural
sow on these farms. The analysed farms were divided into 2 groups: those which
feed pigs with their own feeds supplemented only with concentrates, and those
which purchase at least 50% of their feeds.
Multi-variant calculation was performed for the scale of production that allows
parity agricultural income to be obtained depending on the number of fatteners sold
per sow in pig farms purchasing 50% feeds, and another calculation of parity scale
in which the farms were completely independent of the feed market. For these
calculations, average net agricultural income per 1 structural sow and per 1 fattener
were computed for both fattening variants (Okularczyk, 2004 a; b).
Results
The scale of production on the studied farms, expressed as the number
of structural sows, ranged from 19 to 53. The number of fatteners sold per year
per sow ranged from 16 to 25, with most (34, i.e. 69%) of the farms selling
18 – 20 fatteners per sow per year. Net agricultural income from fattening 1 animal
varied considerably between the pig farms which used only farm-produced feeds
supplemented with concentrates and those which purchased over 50% of the feed
mixtures. The difference in income was 34.7% on average. Mean agricultural
income per 1 fattener given only farm-produced feeds was 98 zloty (Figure 2)
compared to only 64 zloty on the farms which used 50% commercial feeds.
Agricultural income per 1 structural sow depended mostly on the origin of feeds
(farm-produced or commercial mixtures) and on the number of fatteners sold. Sows
from which 25 fatteners were sold (this was the case on two pig farms only), yielded
an income of 1600 zloty in farms buying commercial feeds to 2450 zloty using only
farm-produced feeds (Figures 1 and 2). During the study period, the average annual
wages outside agriculture for two people in Poland were 52 800 zloty. Thus,
agricultural income of 52 800 zloty in the closed cycle of pig fattening for farms on
which two people are employed full-time is the parity income. Figure 3 illustrates
the scale of pig production expressed as the number of structural sows that allows
this parity income to be obtained on the condition that 16 to 25 fatteners are sold per
sow. With an average income of 64 zloty per fattener on farms selling 25 pigs per
sow, income parity was obtained when there were 33 sows, compared to 51 sows
210
S. Okularczyk
when only 16 pigs were sold. Figure 4 gives the scale of production that allows
parity agricultural income to be obtained on farms in which fattening is based 100%
on farm-produced feeds. On these farms, average income per fattener was 98 zloty
and when 25 fatteners per sow were sold, only 21 sows were needed to obtain
income parity, compared to a minimum of 34 sows when only 16 fatteners were
sold.
Figure 1. Net agricultural income per sow in the closed cycle of production based
on 50% purchased feeds
Figure 2. Net agricultural income per sow in the closed cycle of pigs independent
of the feed market
211
Figure 3. Scale of production allowing for parity agricultural income. Fattening with 50% purchased
feeds
Figure 4. Scale of production allowing for parity agricultural income. Fattening with 100%
farm-produced feeds
Discussion
Pork production is not subject to the quota system and its supply is regulated by
economic and market factors. The scale of Polish live pig production on family
farms is limited by land resources and the financial scope for investment. When the
fodder area is too small, many farms are permanently dependent on the feed market,
which makes fattening less profitable. Here, the value added from production is, in
212
S. Okularczyk
fact, intercepted by the feed industry. This is confirmed by the results of the present
study. Farms operating independently of the feed market and fattening pigs entirely
with their own feeds (supplemented with concentrates) generate an income higher
by an average of 34 zloty per fattener, which is as much as 35% more than farms
which base their production on commercial mixtures. To obtain a parity agricultural
income, farmers who have an incomplete fodder base should produce on a higher
scale. When selling the same number of 25 fatteners per sow per year, a farmer
must keep a herd 30 sows larger to obtain the same parity income of 52 800 zloty.
Despite this parity, the wages of a farmer attending to 51 sows are relatively
lower than the wages of a farmer attending to 21 sows, in terms of different
technical and economic indicators, i.e. per animal, hour, month, stall, kg live
animal, etc.
The present findings also point to a very important economic factor, namely the
number of fatteners sold per sow per year. This is the second factor (after the origin
of feed) which determines the profitability of pig production. The difference in the
income per structural sow, from which 25 vs. 16 pigs were sold, is 576 zloty, i.e.
36%.
The scale of production that allows parity agricultural income to be obtained on
farms dependent on the feed market (Figure 3), which ranged from 33 to 51 structural sows, is considered relatively high for the Polish private sector. Assuming that
in the future these farms will enlarge their feed areas to the size of standard areas
and abandon the purchase of feeds, their incomes will rise 33.4% above parity.
Another economic aspect of the scale of pig production, along with incomplete
production of farm-produced feeds for the generation of the same income, is the
cost of a pig farm with 30 additional stalls for sows and their offspring (Figures 3
and 4). This shows that the ownership of a standard feed area (1.5 to 1.7 ha per
structural sow) that guarantees higher profitability from fattening, also yields many
indirect benefits, i.e. allows for production in a smaller and cheaper pig farm, limits
labour requirements when the foundation herd is smaller, and reduces several other
direct and indirect costs of pig farm operation.
Figure 4 gives the scales of production that allow for parity agricultural income.
It accounts for farms independent of the feed market, where average income per
fattener is 98 zloty. Here, the production scales depend on the percentage of
fatteners sold per sow per year. The parity scale decreases with increasing
percentage of fatteners sold per sow per year and ranges from 21 to 34 sows in the
closed cycle. The number of family farms in Poland that produce fatteners on this
scale is not large, although it has grown in recent years.
The increase in the number of fattener herds with more than 50 pigs per year
during 1977 – 2004, is shown in Table 1. From 1977 to 2004, the number of such
suppliers of pigs has increased several-fold. However, parity income on farms producing live pigs can only be achieved when the scale of production is 21 – 34 sows
and feeds are produced on the farm, or when the scale exceeds 50 sows and the
feeds are purchased.
213
Table 1. Population of pigs (1,000) and structure of production
Years
1977
1978
1979
1980
1981
1982
1983
1984
1985
1986
1987
1988
1989
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
End of 4th quarter
20
21
20
18
19
17
15
17
19
19
19
20
18
19
20
21
17
19
20
17
18
19
18
16
17
18
18
16
591
108
983
734
081
564
900
207
170
619
373
169
686
739
725
074
422
138
343
697
497
275
224
988
494
998
439
800
% of farms selling over 50 pigs
16
16
17
17
18
18
18
19
19
19
19
21
24
27
30
31
33
41
44
46
49
51
53
54
57
58
60
62
Source: author’s analysis based on Main Statistical Office data.
References
F l i s M., L e w i c k i C. (1994). Strawność składników pokarmowych i energii nasion bobiku u świń
o różnej masie ciała. Acta Acad. Agric. Olst. Zoot., 39: 3 – 10.
K i s i e l M. (1999). Rynek zbóż i pasz. Wyd. IERiGŻ, Warszawa, ss. 99.
M a ł k o w s k i J. (2002). Żywiec rzeźny. Wyd. IERiGŻ, Warszawa, ss. 109.
O k u l a r c z y k S. (2001). Możliwości obniżania kosztów produkcji żywca wieprzowego. Raport
z PB 1H0621610, KBN.
O k u l a r c z y k S. (2004 a). Opłacalność produkcji zwierze˛cej w latach 1991 – 2003. Raport badań
statut., 43102.1, IZ Balice.
O k u l a r c z y k S. (2004 b). Profitability trends in pig production in 1991 – 2003. Ann. Anim. Sci., 4, 1:
197 – 203.
W o ś A. (2001). Koniunktura rolnicza. Wyd. IERiGŻ, Warszawa, ss. 22.
W o ś A. (2004). W poszukiwaniu modelu rozwoju polskiego rolnictwa. Wyd. IERiGŻ, Warszawa.
Z e g a r J. (2002). Dochody rolnictwa i gospodarstw indywidualnych według rachunków narodowych.
Wyd. IERiGŻ, Warszawa, ss. 43.
214
S. Okularczyk
STANISŁAWA OKULARCZYK
Optymalna skala tuczu świń w polskich gospodarstwach rodzinnych w świetle badań
ekonomicznych
STRESZCZENIE
Badano koszty całkowite produkcji oraz dochody rolnicze netto z tuczu świń w cyklu zamknie˛tym
w 49 gospodarstwach rodzinnych. Przeprowadzono rachunek normatywny porównawczy średniej płacy
rocznej uzyskiwanej poza rolnictwem z osia˛ganymi w badanych gospodarstwach. W analizie uwzgle˛dniono naste˛puja˛ce warianty: tucz świń oparty na paszach własnych oraz w 50% na paszach zakupionych.
W obliczeniach uwzgle˛dniono różnice w liczbach sprzedanych tuczników rocznie od maciory.
Na podstawie rachunku stwierdzono, że aby uzyskać parytet dochodowy w tuczu świń, opartym na
paszach własnych, w przypadku sprzedaży od 1 lochy 25 tuczników, wystarczy obsada 21 macior,
natomiast przy sprzedaży tylko 16 szt. obsada ta musi wynosić 34 lochy. W gospodarstwach kupuja˛cych
50% pasz obsada ta, w celu uzyskania parytetu dochodowego, powinna wynosić od 33 do 51 macior
odchowywanych w cyklu zamknie˛tym.
ECONOMIC EFFICIENCY OF LENGTHENING THE PRODUCTIVE LIFE
OF LAYING HENS THROUGH MOULTING*
Z o f i a S o k o ł o w i c z1, J ó z e f a K r a w c z y k2
1
Department of Animal Production Technology, University of Rzeszów, M. Ćwiklińskiej 2,
35-601 Rzeszów, Poland
2
Abstract
The aim of this study was to evaluate the economic efficiency of lengthening the productive life of
laying hens through moulting, on the Polish market in 2002 – 2004. The study involved 14 000
Hyline layers, used from July 2002 to June 2003, and 6200 layers moulted at 64 weeks of age and
used until week 111 (May 2004). Forced moulting produced the expected economic effects despite
lower production parameters in the second period of egg production. This pattern of hen moulting
profitability resulted from the fact that during the period under discussion, the increase in egg
prices was higher than the increase in feed prices, while eggs obtained from moulted hens were
characterized by great weight and thus were sold at a higher price. The increased mortality of hens
after moulting, in both our studies and those of other authors, suggests that the practice of
moulting is detrimental to hens and should be limited.
Key words: laying hens, moulting, economic efficiency
The increasingly high costs of rearing laying hens, resulting from the rising
prices of chicks, feeds and energy carriers, make it desirable to extend the
productive life of layers. This is possible through the practice of forced moulting.
McDaniel and Aske (2000), who performed studies in 5 US states, noted
a statistically significant relationship between feed and egg prices and the number
of moulting flocks. Bell (2003) estimated that in some US states, as many as 75% of
commercial flocks are subjected to periodical moulting, which allows not only egg
producers, but also pedigree farms and hatcheries, to reduce expenditure. Forced
moulting allows the productive life of hens to be extended and pullet rearing costs
* This work was conducted as part of the NRIAP statutory activity, project no. 4421.1.
216
Z. Sokołowicz and J. Krawczyk
to be spread over a greater number of eggs laid during two periods of egg
production — before and after moulting. Furthermore, moulted hens lay heavier
eggs that are sold for higher prices (Pietras, 1997). In many countries, the rate of
egg production increase is greater than the scope for expanding the hatching base.
This leads to periodic shortages of breeding stock on the market, which can be
offset by lengthening the productive life of hens through moulting (Noles et al.,
1969; Zeelen, 1975).
The aim of this study was to evaluate the economic efficiency of lengthening
the productive life of laying hens through moulting, on the Polish market in
2002 – 2004.
In the study, 14 000 Hyline laying hens were used from July 2002 to June 2003
(18 – 64 weeks of age). At 64 weeks of age, when egg production was 69%, 6878
used layers were sold for 3.50 zloty/hen and 6200 layers were subjected to forced
moulting by starvation. The second period of egg production was 37 weeks (until
May 2004), after which 111-week-old hens were sold for 2.50 zloty/bird. Premoulting hens were designated as flock A1 and moulted hens as flock A2. In both
periods of egg production, layers were kept in batteries of cages and fed on an ad
libitum basis under environmental conditions recommended for Hyline flocks.
The practice of forced moulting covered four periods: I — preparation of flock
for moulting (7 days), day length shortened from 16 to 8 hours; II — forced
moulting (8 days), illumination with red light for 8 hours per day, deprivation of
feed, 8-hour access to water per day; III — resting (2 weeks), day length 8 hours,
feed given every second day, water provided for 8 hours a day; IV — bringing hens
into egg production (4 weeks), gradual lengthening of the day by approximately
3 hours daily to reach 16 hours, increasing diets to 100 g per day, water provided for
8 hours a day.
Productive results such as mortality, culling rate, egg production, egg weight,
and feed intake per hen and per egg laid were monitored throughout the trial.
Production costs and income from the sale of eggs and culled hens were also
recorded, taking into account 2002 – 2004 prices on the farm on which the
experimental flock was kept.
Results
During the first year of productive life, flock A1 achieved peak of lay (91.68%)
at week 11 of the test (Table 1, Figure 1). During the second period of production,
point of lay started at 72 weeks of age and peak of lay in flock A2 (76.07%)
occurred at week 7 of this period (Figure 1). Pre-moulting hens were characterized
by higher egg production and greater persistency of lay than after moulting. For 33
217
Profitability of moulting in laying hens
weeks, egg production in flock A1 exceeded 80%, while flock A2 failed to reach this level,
and the value of this trait was observed to decrease considerably from 20 weeks of
production. When the flock was closed down, egg production was 69.42% in flock A1
and only 40.78% in flock A2 (Table 1). In addition in flock A2, which showed a much
lower level of egg production during the entire productive period, a difference of 35%
was noted between peak of lay and final week of lay compared to 17.43% in flock A1.
Table 1. Performance of hens in the first and second period of egg production
Period of egg production — flock
Item
I — flock A1
Highest egg production at peak of lay (%)
Age at point of lay >80% (weeks)
Number of weeks with egg production >80%
Mean egg production in terms of SP (%)
Egg production at flock liquidation (%)
Decline in egg production between peak of lay and last week
of production (%)
Number of eggs per SP layer (pcs)
Number of eggs per SS layer (pcs)
Mortality (%)
Mean feed intake (g/egg)
Mean feed intake (g/hen/day)
II — flock A2
91.68
29
34
79.02
69.42
76.07
58.72
40.78
17.43
248
263
5.7
147.5
119
35.29
152
166
8.3
196.8
125.3
Note: SP — early term, SS — mid term.
%
Figure 1. Egg production (%)
This pattern of egg production and the shorter period of hen utilization after
moulting were the reasons for the number of eggs per early-term and mid-term layer
being 248 and 263 in flock A1, compared to 152 and 166 respectively in flock A2
(Table 1).
Table 2. Economic results in the first and second period of production
1st period of production (flock A1)
Item
total
zloty
Feed
Hens
Other costs, including:
energy
veterinary care and prophylaxis
egg filler flats
others
costs of moulting
Total costs
2nd period of production (flock A2)
costs
397
120
114
20
7
50
36
%
costs
zloty/layer
zloty/egg
799.70
000.00
899.30
111.30
980.00
388.00
420.00
62.87
18.96
18.14
3.17
1.26
7.96
5.75
28.41
8.57
8.20
1.43
0.57
3.59
2.60
0.11
0.03
0.03
0.005
0.002
0.01
0.009
632 699.00
100.00
45.18
0.17
total
zloty
145
21
51
4
2
9
18
15
219
%
760.90
700.00
882.39
845.22
805.73
945.33
887.51
398.60
343.29
66.45
9.90
23.65
2.20
1.28
4.53
8.60
7.04
100.00
Income
Item
total
zloty
Sale of eggs
Sale of hens
Total sale value
Gross income
708
45
4 754
121
505.90
773.00
278.90
579.90
%
93.93
6.07
100.00
zloty/layer
zloty/egg
23.51
3.50
8.37
0.78
0.46
1.60
3.05
2.48
35.38
0.15
0.02
0.055
0.005
0.003
0.01
0.02
0.017
0.225
zloty/layer
zloty/egg
41.53
2.24
43.77
8.39
0.27
0.015
0.28
0.055
Income
zloty/layer
zloty/egg
50.60
3.28
53.88
8.69
0.190
0.014
0.204
0.034
total
zloty
257
13
271
90
485.25
917.50
402.75
604.10
%
94.87
5.12
100.00
219
In the analysed flock, mortality and culling rate during the first period of egg
production was 5.7%. Hen mortality was 4.7% during moulting and 8.3% on
average in the second year of productive life.
One layer of flock A2 consumed 6.3 g more feed daily than in flock A1, which
with the lower egg production of these hens after moulting, led to 49.3 g greater
intake of feed/egg in this group (Table 1).
zł
Figure 2. Egg prices (zloty/egg)
zł
Figure 3. Feed prices (zloty/egg)
220
The price of the eggs laid by layers averaged 0.19 zloty in flock A1 and 0.27
zloty in flock A2 (Table 2). The increase in egg prices in the second period of hen
utilization was high (42%), with the feed price increase averaging 8.2%. As is
evident from Figure 2, the price of the eggs obtained from moulted hens was initially
high but started to decline distinctly from 16 weeks of production, despite an
upward trend in feed prices during this period (Figure 3). The price of the eggs
obtained from flock A1 to 7 weeks of production was low (mainly because of their
low weight), but showed a steady upward trend with a stable price for feeds during
this period (Figures 2 and 3). It was calculated that 16% of the eggs obtained from
flock A1 were sold for less than 0.20 zloty/egg, mainly because of their low weight
during early egg production, while all the eggs laid in flock A2 were sold for over
0.20 zloty/egg.
Between the analysed flocks of hens, there was a clear change in the structure of
production costs in the two different time periods (Table 2). The greatest outlays in
egg production were for feeds, which cost 28.41 zloty/layer and 0.11 zloty/egg in
flock A1, and 23.51 zloty/layer and 0.15 zloty/egg in flock A2, accounting for 62.87
and 66.45% of total expenditure, respectively. During the first period of production, layer rearing costs to 18 weeks of age were 8.57 zloty and accounted
for almost 19% of total outlays. During the second period, hen value dropped to
3.50 zloty/bird, accounting for only 9.9% of total costs. The other costs of
production were only 18.14% in flock A1 and as much as 23.65% in flock A2.
Production costs per layer were higher by 9.8 zloty in flock A1 than in flock A2, the
difference resulting mainly from the greater value of young hens (Table 2).
However, thanks to the better laying performance of hens during the first period of
productive life, the production costs per egg were 0.17 zloty in flock A1 compared
to 0.225 zloty in flock A2.
Table 3. Correlation coefficients between egg and feed prices in the 1st and 2nd period of hen egg
production
Item
Mean egg price (zloty/egg)
Mean feed price (zloty/kg)
Correlation coefficients: egg price/feed price
Flock
A1
0.19
0.73
0.0828*
A2
0.27
0.79
0.1796
Change (%)
42
8.2
* — correlation coefficient significant at P > 0.05.
With this pattern of production costs and egg prices during 2002 – 2004, hen
moulting was profitable and despite the costs involved, it brought the producer
a profit of 8.39 zloty/hen and 0.055 zloty/egg (Table 2). At the same time,
a significant relationship was found between feed prices and egg prices during the
first period of egg production (years 2002/2003), while in 2003/2004 the rate of
feed price increase was high, with a marked decrease in egg prices noted from the
221
13th week of evaluation. Accordingly, the correlation between these major factors
of income was low and not significant (Table 3, Figures 2 and 3).
Discussion
The age of moulting was dictated by the studies of other authors who consider
the period between 54 and 67 weeks of hens’ age as economically optimal for hen
moulting (Bar et al., 2001; Hanzan and Yalcin, 1992).
Resumption of lay in the second period of production took place within
approximately 6 weeks after normal feeding restarted, which is in line with the
study of Pietras (1997). Egg production increased strongly until 8 weeks then
declined slightly, with a dramatic decline noted at 36 weeks of production.
Ultimately, the average egg production of moulted hens as well as the number of
eggs laid per layer were lower than before moulting, which is supported by the
findings of Bernacki and Mazanowski (1997) and Górski et al. (2002). Average egg
production in flock A2 was 58.72%, whereas in studies by other authors in moulted
flocks this indicator ranged from 71.2 to 79% depending on the moulting method
(Yilmaz and Sahan, 2002; Bell, 2003). Shippee et al. (1979) reported that lower egg
production in the second period of hen production results from the excessive loss of
body weight during moulting. Therefore, many authors indicate that moulting
efficiency is greater when feed is supplemented with zinc, which reduces feed
intake more gently without causing excessive decreases in hen body weight
(Pietras, 1997; Yilmaz and Sahan, 2002). In addition, Park et al. (2004) concluded
that dietary zinc supplemented during moulting increases the weight of eggs, and
thus, indirectly, agricultural income. Bar et al. (2001) demonstrated a significant
effect of age of layers on their post-moulting productivity. Hens moulting at 431
days of age laid more eggs in the second production cycle than those moulted at 501
or 571 days, but the weight of the eggs of younger hens was slightly lower. It is
appropriate to use the flock as long as egg production does not drop below 45%
(Sokołowicz et al., 2004); otherwise, egg production is effectively unprofitable. In
the analysed flock, egg production dropped below 45% at week 37 of the second
year of production and during this time the flock was closed down since further
utilization of the hens would not have compensated for the costs incurred.
When comparing the health of moulted hens with the results obtained during the
first year, a 26% higher level of mortality was found in the second period of
production, as corroborated by Górski et al. (2002).
In our study, we found moulting and thus lengthening of the productive cycle to
be profitable despite the slightly lower flock productivity compared to the findings
of other authors (Rolon et al., 1993; Górski et al., 2002; Yilmaz and Sahan, 2002;
Bar et al., 2001). The main reason for this pattern of egg production profitability in
the second period of lay was the high egg price obtained by the farm’s owner.
Economic analysis showed that if egg prices in the second period of production had
been the same as in the first period, it would have been unprofitable to subject this
222
flock to moulting considering the low productivity. The high prices obtained for
eggs during the second period of hen utilization were the consequence of the
producer’s strong marketing efforts, as poultry products were difficult to market
during this period. The high feed prices prevalent between September 2003 and
June 2004 throughout Europe due to droughts and poor cereal crops were not
always compensated by the increase in egg prices expected by the producers, since
during the last years before Poland’s accession to the European Union, there was an
oversupply of eggs on the Polish market that caused egg prices to decline. It was
impossible to rectify this situation through exports which were effectively limited
by EU quotas and WTO agreements. Based on the observations of the Polish egg
market after accession to the European Union, it can be assumed that currently hen
moulting should be more profitable due to favourable relations between egg and
feed prices, but under the new legal conditions moulting must not be achieved
through starvation. Bell (1996) points out that the forced moulting of hens is
detrimental to their bodies, and extending the production cycle using this method
can lead to the incidence of Salmonella enteritidis in flocks. Because McDaniel and
Aske (2000) confirmed a statistically significant relationship between feed and egg
prices and the number of moulting flocks, it can be assumed that among Polish egg
producers also there will be a trend towards lengthening the productive cycle of
hens through their moulting.
In summary, the forced moulting of hens produced the expected economic
results despite the lower productive parameters of layers obtained during the second
period of lay. This pattern of hen moulting profitability resulted from the fact that in
the discussed years there was a higher increase in egg than feed prices, and as
follows from the studies of Banaś (2004) a significantly higher effect of meat and
egg sale prices than of feed prices on agricultural income has been observed on the
poultry market. In addition, moulted hens yield eggs that are heavier and of
a sufficiently higher price. The increased mortality of hens after moulting, both in
our studies and those of other authors, may suggest that this procedure is
detrimental to birds and should be limited.
References
B a n a ś K. (2004). Wpływ poziomu cen na opłacalność produkcji kurcza˛t brojlerów. Rocz. Naul. Zoot.,
31, 2: 299 – 307.
B a r A., R a z a p h k o v s k y V., W a x E., M a l k a Y. (2001). Effect of age molting on postmolting
performance. Poultry Sci., 80 (7): 874 – 878.
B e l l D. (1996). An egg economics update. Cooperative Extension, University of California, 185: 1 – 4.
B e l l D. (2003). Historical and current molting practices in the U.S. table egg industry. Poultry Sci., 82
(6): 965 – 970.
B e r n a c k i Z., M a z a n o w s k i A. (1997). Ocena cech kur towarowych pochodza˛cych po kogutach
New Hampshire (N11) i Plymuth Rock (P02) w dwóch okresach nieśności. Zesz. Nauk. ATR.
Bydg., Zoot., 29, 210: 17 – 25.
G ó r s k i J., W i t a k B., B i e s i a d a - D r z a z g a B. (2002). Analiza wyników produkcyjno-ekonomicznych u kur w dwóch okresach nieśności w chowie na ściółce i w klatkach. Rocz. Nauk. Zoot., Supl.,
16; 103 – 108.
223
H a n z a n A., Y a l c i n S. (1992). Effect of molting age on the second cycle performance of broiler
breeders. Brit. Poultry Sci., 33: 189 – 193.
M c D a n i e l B.A., A s k e D.R. (2000). Egg prices, feed costs and the decision to molt. Poultry Sci., 79:
1242 – 1245.
N o l e s R.K., L o n g L.H., F o r s t o n J.C. (1969). Determining the optimum replacement policy for
commercial layers. Poultry Sci., 48: 636 – 645.
P a r k S.Y., B i r k h o l d S.G., K u b e n a L.F., N i s b e t D.J., R i c k e S.C. (2004). Effects of high zinc
diets using zinc propionate on molt induction, organs and postmolt egg production and quality in
laying hens. Poltry Sci., 83 (1): 24 – 33.
P i e t r a s M. (1997). Wybrane aspekty wymuszonego przepierzania drobiu. Rozpr. hab., IZ, Kraków.
R o l o n A., B u h r R., C u n n i n g h a m D.L. (1993). Twenty four hour feed withdrawal and limited
feeding as alternative methods for induction of moult in laying hens. Poultry Sci., 72: 776 – 785.
S h i p p e e R.L., S t a k e P.R., K o h e n U., L a m b e r t J.L., S i m m o n s R.W. (1979). High dietary zinc
or magnesium as forced-resting agents for laying hens. Poultry Sci., 58: 949 – 954.
S o k o ł o w i c z Z., K r a w c z y k J., D y k i e l M. (2004). Ekonomiczna analiza nieśności w stadzie kur
mie˛snych. Zesz. Nauk. PTZ, 72 (4): 137 – 143.
Y i l m a z B., S a h a n U. (2002). The effects of different forced molting methods on egg production and
egg quality in egg layers. Arch. Geflügelkunde, 66: 133.
Z e e l e n H.H.M. (1975). Technical and economic results from forced molting of laying hens. World’s
J. Poultry Sci., 31: 57 – 67.
ZOFIA SOKOŁOWICZ, JÓZEFA KRAWCZYK
Efektywność ekonomiczna wydłużania okresu użytkowania kur nieśnych poprzez przepierzanie
STRESZCZENIE
Celem pracy była ocena efektywności ekonomicznej wydłużania okresu użytkowania kur nieśnych
poprzez ich przepierzanie w latach 2002 – 2004. Materiał badawczy stanowiło 14 000 kur nieśnych
Hyline, użytkowanych od lipca 2002 r. do czerwca 2003 r. oraz 6200 niosek przepierzonych w 64.
tygodniu ich życia i użytkowanych do 111. tygodnia (maj, 2004 r.).
Stwierdzono, że sztuczne przepierzanie kur przyniosło spodziewane efekty ekonomiczne, mimo
niższych parametrów produkcyjnych w II okresie nieśności. Takie kształtowanie sie˛ opłacalności
przepierzania kur wynikało z faktu, że w omawianych latach nasta˛pił wyższy wzrost cen jaj niż wzrost
cen pasz, a poza tym jaja uzyskiwane od kur przepierzonych charakteryzowały sie˛ duża˛ masa˛, z czym
wia˛zała sie˛ ich wyższa cena zbytu. Wzrost liczby padnie˛ć kur po przepierzeniu, zarówno w naszych
badaniach jak i innych autorów, może sugerować, że zabieg ten nie jest oboje˛tny dla ptaków i winien być
ograniczany.
INSTRUCTIONS TO AUTHORS OF RESEARCH PAPERS
PUBLISHED IN THE ‘‘ANNALS OF ANIMAL SCIENCE”
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226
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Jan Kowalski1, Maria Anna Rokicka2, Adam Nowacki3
Department of Zoology, Jagiellonian University, św. Anny 12, 30-017 Kraków, Poland
2
Department of Immuno- and Cytogenetics, National Research Institute of Animal Production,
3
Experimental Station of the National Research Institute of Animal Production,
39-331 Chorzelów, Poland
1
abbreviated title (5-6 words as in the paper title or synonyms)
source of research financing, e.g. work financed from:
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227
3.
4.
5.
6.
7.
Papers published in periodicals: Jenkins K.J., Hidiroglou M. (1991). Tolerance of the preruminant
calf for excess manganese or zinc in milk replacer. J. Dairy Sci., 74: 1047-1053.
Papers published in multi-author monographs, occasional publications, symposium or congress proceedings: Miller E.L. (1982). Forage protein in ruminant animal nutrition. The nitrogen
needs of ruminants. In: D.J. Thomas (Editor), Proceedings of an International Symposium on Protein
Requirements for Cattle. Kansas State University, Kansas City, KN, pp. 254 – 269.
Manuals and multi-author books: Bock H.D., Eggum B.O., Low A.G., Simon O., Żebrowska T.
(1989). Editors. Protein metabolism in farm animals: evaluation, digestion, absorption and
metabolism. Oxford, UK, Oxford University Press (1989), 452 pp.
Manuals and books: Cuhna T.J. (1991). Horse feeding and nutrition. San Diego, USA, Academic
Press, Inc. (1991). Second edition, 445 pp.
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volume) with full names of the author(s) and title of the paper.
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