Untersuchung zur Optimierung des Embryotransfers beim Pferd

Transcription

Aus dem Institut für Tierzucht und Tierhaltung
der Agrar- und Ernährungswissenschaftlichen Fakultät
der Christian-Albrechts-Universität zu Kiel
Untersuchung zur Optimierung des Embryotransfers
beim Pferd unter Feldbedingungen für die Nutzung
im Rahmen von Zuchtprogrammen
Dissertation
zur Erlangung des Doktorgrades
der Agrar- und Ernährungswissenschaftlichen Fakultät
der Christian-Albrechts-Universität zu Kiel
vorgelegt von
M.Sc. agr.
Henrik von der Ahe
aus Münster, Nordrhein-Westfalen
Dekanin: Prof. Dr. K. Schwarz
1. Berichterstatter: Prof. Dr. G. Thaller
2. Berichterstatter: Prof. Dr. E. Schallenberger
Tag der mündlichen Prüfung: 10. Mai 2012
Die Dissertation wurde dankenswerterweise finanziell durch die H. Wilhelm
Schaumann Stiftung unterstützt.
Table of contents
General Introduction
…………………………………………………………………………………………………1
Chapter One
Analysis of genetic and management factors affecting ovulation and
embryo recovery rate in German Warmblood donor mares
…………………………………………………………………………………………………7
Chapter Two
Impact of reproductive history and semen parameters on the number of
recovered embryos in German Warmblood donor mares
………………………………………………………………………………………………..29
Chapter Three
Effects on success of pregnancy rate after embryo transfer in warmblood
recipient mares and comparison of embryo transfer foals to non – embryo
transfer foals
………………………………………………………………………………………………..47
General Discussion
………………………………………………………………………………………………..71
General Summary
………………………………………………………………………………………………..87
Zusammenfassung
…………………………………………………………………………………………..……91
General Introduction
The use of equine embryo transfer has increased steadily since its first attempts in
the early 1970s. Today it is commonly used in equine practice. Causes why embryo
transfer is performed are various. It allows the production of offspring from show
mares that compete in performance tests, like dressage or jumping, without being
removed from their primary use. Increase of the annual reproduction rate in mares to
more than one foal per year is possible. Not fully grown mares (2 years old) can be
early used as donor mares for producing offspring through embryo transfer leading to
a reduction of the generation interval, where otherwise insemination and following
pregnancy of a two year old mare might not be performed. Subfertile mares,
particularly those that suffer early embryo loss, can be used for producing offspring.
The USA (15 000 transfers per year) and South America, especially Brazil (12 000
transfers per year), are leading in performing equine embryo transfer (Thibier, 2007).
The use of equine embryo transfer in Europe is actually limited but numbers of
recovered embryos and performed transfers are steadily increasing in the last
decade.
Important for the practitioner when performing ET are adapted monitoring and care of
the donor mare, synchronization of donor and recipient and conducting embryo
recovery (flushing) and following transfer itself (Hinrichs et al., 2005). Furthermore
selection and management of the recipient mare plays an important role for achieving
acceptable pregnancy rates after performing embryo transfer.
There is no difference in monitoring and inseminating the donor mare, compared with
the technique for a standard pregnancy. During estrous, follicular development on the
1
ovaries and changes in the uterus are monitored daily with ultrasonography assuring
that the number of follicles ovulated and their exact day of ovulation, are known.
Embryo recovery rate in young, fertile mares lies in a range between 55 to 80%
(Hinrichs, 1993). The efficiency of equine embryo transfer would greatly increase, if it
would be possible to superovulate donor mares. However, superovulation of donor
mares, like in cattle, inducing multiple ovulation is ineffective in horses, because of
various respond to FSH products of the donor mare. This lack of consistence
superovulatory regimen in the donor leads generally to a limit of single embyo
recovery per cycle. Although there is a breed dependent occurrence of multiple
ovulation, especially in Quarter Horse and warmblood mares, guiding to higher
embryo recovery rates and therefore a greater success of embryo transfer.
Insemination of donor mares is generally realized with fresh/cooled or frozen-thawed
semen 1 to 2 days prior to ovulation. Flushing of the uterus for recovering the
embryos is generally done on day 7 or 8, unless the embryo is used for freezing. In
this case flushing will be done on day 6, because between day 6 and 7, a acellular
capsule is formed underneath the zona pellucida which may avoid the complete
penetration of the cryoprotectant into the embryo (Seidel et al., 1986).
The earliest successful recovery of the embryo is possible 144 to 156 h after
ovulation because of the long transport, in comparison to other species, in the
fallopian tube (Battut et al., 1997; Battut et al., 2001). At day 9 the embryo has grown
to a size where danger increases to damage the embryo due to recovery, storing and
transfer (McKinnon et al., 1988; Carnevale et al., 2000).
There are several factors influencing the embryo recovery rate. Main sections are the
day of recovery (Griffin et al., 1981; Squires et al., 1995), number of ovulations
(Squires et al., 1987; Squires et al., 1987), age of the donor (Squires et al., 1982;
2
Vogelsang et Vogelsang, 1989) as well as the quality of sire’s semen (Francl et al.,
1987).
Factors that influence the pregnancy rate after transfer include the synchrony of
donor and recipient mare, management of the recipient, method of transfer as well as
the quality of the embryo. Highest pregnancy rates were obtained, if the recipient
mare ovulates 1 d before to 3 d after the donor (McKinnon et al., 1988; Squires et al.,
1995). The recipient mare should be examined with ultrasonography at day 4 and 5
after ovulation, if a corpus luteum has generated and no fluid or air was in the uterus
(Squires et al., 1999).
Method of transfer can be performed as surgical flank incision or as nonsurgical
transfer, through the vagina of the recipient mare. Surgical transfer promised a
consistent pregnancy rate of 65 to 75% (Squires et al., 1995), whereas results of
nonsurgical transfer vary among technicians, with obtained pregnancy rates of 50 to
75% (Hinrichs, 1993, McKinnon et al., 1988; Riera et al., 1993; Squires et al., 1995;
Vogelsang et al., 1985). As a result of its simplicity and in terms of animal welfare,
the nonsurgical transfer is actually the most common technique.
Size of the recipient mare in comparison to donor mare and service stallion is an
aspect which is often discussed. Transfer into smaller recipients resulted in a
retardation of fetus growth, which could not totally compensated (Allen et al., 2004).
In general, the transfer of warmblood embryos into trotter or thoroughbred mares,
which had commonly a similar size can sometimes result in a slightly reduced size at
birth, but this is fully compensated during postnatal development.
No investigations regarding differences in embryo transfer foals versus non embryo
transfer foals have done to the best of our knowledge so far. However there are
some studies about length of gestation, withers height at birth and weight at birth
(Valera et al. 2006, Cilek 2009, Satuè 2011).
3
In Chapter 1 of the present study, fixed and random effects regarding the embryo
recovery rate were investigated using a threshold mixed model and a probit link
function. Age of the donor mare at time of flushing, season of flushing, preparation of
the donor mare and the running number of flushing within season were investigated.
Estimation of heritabilities for the traits “ovulation rate” and “number of recovered
embryos” as well as phenotypic and genetic correlations between maternal and
paternal components were performed. Additional research was conducted in Chapter
2 to investigate the breeding status of the donor mare as well as the quality of the
semen. Finally in Chapter 3 the pregnancy rate of the recipient mare was
investigated with respect to the age of the recipient mare at time of transfer, season
(month of transfer), preparation of the recipient mare before transfer, synchrony
between donor and recipient mare and the age of the embryo at time of transfer. The
comparison of embryo transfer foals and non embryo transfer foals as well as colts
and fillies was done with a view to the traits length of gestation, withers height at birth
and weight at birth.
REFERENCES
Allen, W. R., Wilsher, S., Tiplady, C. and Butterfield, R. M.; The influence of maternal
size on pre- and postnatal growth in the horse: III Postnatal Growth.
Reproduction 2004; 127: 67-77
Batut, I., Colchen, S., Fieni, F., Tainturier, D., Bruyas, JF. Success rates when
attempting to nonsurgically collect equine embryos at 144, 156 or 168 hours
after ovulation. Equine Vet J (Suppl) 1997;25:60-2
Batut, I., Grandchamp des Raux, A., Nicaise, JL, Fieni, F., Tainturier, D., Bruyas, JF.
When do equine embryos enter the uterine cavity? An attempt to answer.
R&W Publications, Newmarket; 2001
Carnevale, EM, Ramirez, RJ, Squires, EL, Alvarenga, MA, Vanderwall, DK, McCue,
PM. Factors affecting pregnancy rates and early embryonic death after equine
4
embryo transfer. Theriogenology 2000; 54; 965-79.
Francl, A. T.,R. P. Amann,E. L. Squiresand B. W. Pickett. Motility and fertility of
equine spermatozoa in a milk extender after 12 hours or 24 hours at 20degrees-C. Theriogenology 1987;27:517-525.
Griffin, J. L.,R. S. Castleberryand H. S. Schneider. Influence of day of collection on
recovery rate in mature cycling mares. Theriogenology 1981;15:106-106
Hinrichs, K. Embryo-Transfer in the mare - A status-report. Animal Reproduction
Science 1993;33:227-240
Hinrichs, K.and Y.-H. Choi. Assisted Reproductive Techniques in the Horse. Clinical
Techniques in Equine Practice 2005;4:210-218
McKinnon, EL. Morphologic assessment of the equine embryo. J Amvetmed Ass
1988;192:401-6
Riera, FL., and McDonough, J.; Commercial embryo transfer in polo ponies in
Argentina. Equine Vet. J. 1993; 15(Suppl): 116-119.
Seidel, GE Jr.; Cryopreservation of equine embryos. In Squires EL (ed), Veterinary
Clinics of North America, Equine Practice: Diagnostic Techniques and
Assisted Reproductive Technology 1986;12(1):85-101.
Squires, E. L.,K. J. Imel,M. F. Iulianoand R. K. Shideler. Factors affecting
reproductive efficiency in an equine embryo transfer programme. J Reprod
Fertil Suppl 1982;32:409-414
Squires, E. L.,M. G. McClain,O. J. Gintherand A. O. McKinnon. Spontaneous multiple
ovulation in the mare and its effect on the incidence of twin embryo collections.
Theriogenology 1987;28:609-613.
Squires, E. L.,A. O. McKinnon,E. M. Carnevale,R. Morrisand T. M. Nett.
Reproductive characteristics of spontaneous single and double ovulating
mares and superovulated mares. J Reprod Fertil Suppl 1987;35:399-403.
Squires, E.and G. Seidel. Collection and transfer of equine embryos. Animal
Reproduction and Biotechnology Bulletin No 11. Fort Collins CO: Colorado
State University 1995:7-9 11-15 27-32
Squires, E. L., Mc Cue, P.M. and Vanderwall, D. The current status of equine embryo
transfer. Theriogenology 1999;51:91-104.
Thibier, M.; New records in the number of both in vivo-derived and in vitro-produced
bovine embryos around the world in 2006. IETS Newsletter 2007;25(4):15-20.
5
Valera, M., Blesa, F., Dos Santos, R. and Molina, A.; Genetic study of gestation
length in andalusian and arabian mares. Animal Reproduction Science 2006;
Vol. 95, Issue 1-2: 75-96.
Vogelsang SG, Bondioli, KR and Massey, JM.; Commercial application of equine
embryo transfer. Equine Vet. J. 1985; (3): 89-91.
Vogelsang, S. G.and M. M. Vogelsang. Influence of donor parity and age on the
success of commercial equine embryo transfer. Equine Veterinary Journal
1989;21:71-72.
6
Chapter One:
Analysis of genetic and management factors affecting ovulation and embryo
recovery rate in German Warmblood donor mares
H. von der Ahea, J. Tetensa, E. Stamerb, E. Kalma and G. Thallera
a
b
CAU, Institute of Animal Breeding and Husbandry, Hermann-Rodewald-Straße 6, 24098 Kiel, Germany
TiDa GmbH, Bosseer Str. 4c, 24259 Westensee/Brux, Germany
Submitted for publication in Theriogenology
7
Abstract
Embryo transfer (ET) is an important and frequently used biotechnology in animal
breeding. A main motivation to apply ET in horses is the possibility to obtain foals
from mares that are competing as sport horses. The success of ET is to a large
extent determined by the number of transferable embryos obtained from the donor
mare. Selection, management and flushing of the donor as well as paternal fertility
are factors influencing embryo recovery. The primary aims of this study were thus to
determine influencing factors including the donor mare’s age and management.
Furthermore, heritabilities as well as phenotypic and genetic correlations for the traits
“ovulation rate” (OR) and “number of recovered embryos” (RE) were estimated. Data
for these traits were obtained from 5 715 flushes of 715 donor mares from a single
stud. Threshold mixed models using a probit link function were applied. The OR was
found to be significantly affected by the age of the donor mare and the season as
well as the number of flushings within season and the preparation of the donor mare.
The fixed effects of age, season and preparation of the donor mare, but not the
number of flushings showed significant influences on the trait RE. As to the
preparation, the application of an ovulation inducing agent had the highest effect,
probably due to the optimized insemination time. The estimated maternal heritability
of OR was low. For RE, the paternal genetic component has to be considered
additionally and the estimates for both genetic components were also low. Notably,
the paternal component seems to have a stronger impact on RE. The correlation of
OR and RE was slightly positive whereas genetic correlations of maternal and
paternal components are negative indicating a genetic antagonism. In summary,
several management factors significantly affecting OR and RE were identified and
genetic components were demonstrated. Although low, these components offer a
way to genetically improve the respective fertility traits.
8
Keywords: horse, ET, German Warmblood, ovulation rate, embryo recovery,
heritability
Introduction
Today, ET is commonly used in some countries in equine reproduction practice, both
for the production of more offspring from a given mare and to obtain foals from mares
that suffer early embryo loss [1]. Furthermore, ET is used to produce offspring from
mares competing in any discipline at performance events, which naturally restricts
their ability to deliver foals. For economic reasons, the genetic improvement of
reproductive traits including the outcome of ET is of great importance for horse
breeders. On the other hand, the breeding goals for (sport-) horses are mainly
focused on jumping or dressage abilities.
Success of ET is influenced by several factors such as the ovulation rate, the day of
recovery, the age of the donor mare, and the quality of the semen.
Monitoring and inseminating the donor mare does principally not differ from the
technique of standard artificial insemination. During estrus, follicular development
and uterine status are monitored daily with ultrasonography to assess the number of
follicles and their exact time of ovulation. Donor mares are inseminated with
fresh/cooled or frozen-thawed semen 1 to 2 days prior to ovulation. After successful
fertilization, the developing embryo moves from the oviducts into the uterus at day 5
after ovulation and continues to grow rapidly in diameter. The donor mare’s uterus is
typically flushed at day 7 or 8 to, unless the embryos are to be frozen. In such cases
the recovery is performed at day 6, because at day 7 or 8 embryos have formed a
capsule which leads to a lower pregnancy rate [2] due to an incomplete permeation
of the freezant and resulting difficulties during the freezing/thawing process. Embryos
recovered at day 8 after ovulation are larger and easier to locate in the filter or Petri
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dish [1]. Thus, some embryos might be missed if the flush is performed at an earlier
stage [1]. A day 9 embryo, however, has grown to a size, where pregnancy rates
after transfer may be reduced due to potential damages to the embryo during
handling. The influence of the day of flushing on the number of recovered embryos
(RE) has been considered in several studies. Griffin et al. [3] used sixteen mature
mares and collected 24 embryos from 38 flushes five to nine days post ovulation.
They reported the highest recovery rate of 80% (8 embryos / 10 flushes) for day 6
followed by day 8 and day 7 with 67% (4/6) and 60% (9/15), respectively. Other
authors did not find significant differences between days 7-10 [4].
Horses are generally a monovular species, but there are reports that multiple
ovulations occur with frequencies ranging from 4 to 43 % [5,6]. Several aspects such
as age, breed, genetics, season, nutrition and reproductive status were investigated
in these studies and found to substantially influence findings of multiple ovulations in
the mare [5-8]. The frequency of multiple ovulations in draft horses and
Thoroughbreds ranges from 15 to 30 %, whereas light breeds and ponies showed a
lower rate of around 2 to 10 % [5,6]. Breed differences as well as individual
differences between mares indicate a genetic background for the occurrence of
multiple ovulations. The recovery of two or more embryos from a multiple ovulating
mare can substantially increase the reproductive efficiency of an embryo transfer
program [7]. The chances for obtaining a pregnancy from a given donor mare may be
doubled if both embryos are viable. For embryos collected from mares seven days
after ovulation, Squires et al. [9] reported recovery rates of 53% for single-ovulating
mares compared to 106% for double-ovulating mares. In cattle, it is common practice
to induce a superovulation in the donor in order to obtain a higher number of
embryos. In contrast, the majority of donors in the horse are spontaneous, singleovulating mares [10]. Recent studies indicate an effective increase in the number of
10
ovulations and embryos recovered in mares treated with recombinant equine FSH
and LH [11]. However, the success of superovulation schemes in the horse is still
limited and further research is needed to optimize dosage and regime [11].
Regarding the age of the donor mare, Vogelsang and Vogelsang [12] reported
significant differences between embryo recovery rates in younger compared with
older mares. Mares younger than 9 years showed a recovery rate of 60.6%, while
mares older than 17 years had a rate of only 30.1%.
The success of ET can be regarded as a complex trait influenced by a variety of
environmental and genetic factors including direct, maternal and paternal
components. The aim of the current study was to quantify potential determinants
affecting the success of embryo recovery. The analyses are focused on factors
related to the donor mare’s management including the age and treatment of the
donor mare and the season as well as the number of flushings within a season and
the day of flushing. Furthermore, heritabilities and genetic correlations were
estimated to provide parameters necessary to improve equine embryo transfer within
the context of breeding programs. Interactions between paternal and maternal
genetic components were analyzed to facilitate a differentiated insight into the ET
status of horses. To the best of our knowledge, this study is the first to examine
genetic aspects of ET in horses.
Material and Methods
The data set comprised reproduction records related to embryo transfer of 715
Warmblood donor mares obtained from a large stud farm in Northern Germany
during the years 2004 to 2008. Data on ovary and uterus control as well as on
inseminations and flushes were registered daily by veterinarians. ET donor mares
were monitored and inseminated in the same manner as mares used in the standard
11
breeding program. Mares were generally inseminated either with fresh or frozen
semen, one to two days prior to ovulation. Embryos were obtained six to nine days
after ovulation by flushing the uterus via cervix. Data were immediately recorded
manually and transferred to electronic media later on.
ET reproduction traits considered were the ovulation rate (OR) and the number of
recovered embryos (RE). The data comprised a total of 5 755 cycles of donor mares.
In 40 cases, the donor mare did not ovulate, so that flushing was not possible.
Furthermore, several incomplete records had to be removed resulting in 5 665 and 5
674 flushes for the analysis of OR and RE, respectively. Due to the comparatively
small number of ovulations with more than two ova OR was defined as a binary trait
and coded with ‘0’ for one ovum and ‘1’ for at least two ova. RE was also coded as a
binary trait with no flushed embryo (‘0’) vs. at least one embryo (‘1’). An overview is
given in Table 1.
Table 1 Overview of the analyzed reproduction traits ovulation rate (OR) and
recovered embryos per flush (RE)
Trait
Analyzed
Frequency by trait class (N)
Mean
flushes (N )
Ovulation
rate 5 665
(OR)
Embryos
recovered (RE)
5 674
1 ovum
≥ 2 ova
4 424
1 241
(78.1%)
(21.9%)
no embryo
≥ 1 embryo
2 909
2 765
(51.3%)
(48.7%)
1.22 ova/flush
0.54 RE/flush
The 2 765 positive flushes resulted in a total of 3 050 recovered embryos with one (2
506), two (252), three (15) and four (1) embryos per flush.
12
The donor age (‘Age’) at flushing was grouped into eight classes as 2, 3, 4, 5, 6 to 8,
9 to 11, 12 to 15 and ≥ 16 years old. The season of flushing (‘Season’) classes
correspond to month’s classes within years. Donors were flushed routinely between
March and September. The average number of performed flushes per month was
159. Treatment code (‘TC’) represents six different combinations of time of treatment
before insemination (last and penultimate treatment) and type of treatment (heat and
ovulation inducing agents, Table 2). The penultimate treatment was only considered
if it occurred within a time range of less than 28 days before flushing.
Table 2 Definitions and distribution of treatment codes (TC). TC 3 was excluded from
further analyses due to a small number of observations
last treatment
penultimate treatment < 28 d TC
N
%
before flushing
none
none
0
352 6.17
heat inducing agent
none or >28 d
1
169 2.96
heat inducing agent
heat inducing agent
2
106 1.86
heat inducing agent
ovulation inducing agent
3
30 0.53
none or >28 d
4
2999 52.54
heat inducing agent
5
1804 31.60
6
248 4.34
Number of flushing (‘NOF’) within breeding season was in a range between 1 and 15
and was divided into nine classes (1, 2, 3, 4, 5, 6, 7, 8, ≥9). The ‘day of flushing’
(‘Day’) was defined as the difference between the date of flushing and the date of
ovulation and ranged from day 6 to day 10. Most of the flushes were performed at
days 7 and 8 (74 %).
13
Data analysis was done applying a threshold mixed model and a probit link function
within the GLIMMIX procedure of the SAS package (SAS 2008). The following model
was used to analyse the ovulation rate (OR):
Φ ,
where is the expected probability to observe at least two ovulations and Φ
is the cumulative probability function of the standard normal distribution. ,
, and represent the fixed effects of i-th age class (i = 1,..., 8), the j-th
month of flushing (j = 1,..., 36), the k-th preparation of the donor (k = 1,..., 6) and the
l-th flushing number within season (l = 1,..., 9), respectively. The random effect of the
m-th donor mare is represented by (m = 1,..., 715).
The recovery rate was analysed using the following model:
Φ !" #$$% ,
where is the expected probability to obtain at least one embryo and Φ is
the cumulative probability function of the standard normal distribution. , ,
, and !" represent the fixed effects of i-th age class (i = 1,..., 8), the j-th
month of flushing (j = 1,..., 36), the k-th preparation of the donor (k = 1,..., 6), the l-th
flushing number within season (l = 1,..., 9), and the m-th day of flushing after
ovulation (l= 1,...,5), respectively. The random effects of the n-th donor mare and the
o-th service stallion are represented by (n = 1,..., 715) and #$$% (o = 1,...,
182), respectively.
14
To estimate the heritabilities for OR and RE as well as phenotypic and genetic
correlations between traits as well as maternal and paternal components, random
effects of mare and stallion were divided into the permanent environmental effect and
the additive genetic effect of the animal based on an additive genetic relationship
matrix. The available pedigrees contained four generations and comprised 5 092
animals in total. The number of sires was 188 with 3.8 daughters on average. For
estimation of variance components and correlations the software package ASReml
3.0 [13] was used.
Results
Within the time period from 2004 to 2008, the average number of recovered embryos
per flushing was 0.54 across 715 analyzed donor mares. The average number of
ovulations per flush was 1.22 ± 0.50, the probability of obtaining at least two
ovulations was 21.9 %.
All analyzed fixed effects (‘Age’, ‘Season’, ‘TC’, ‘NOF’) have a highly significant effect
on OR. With an increasing age of the donor mare from 2 to 15 years, the probability
of more than one ovulation increased from 0.13 to 0.30, respectively (Figure 1). Only
donor mares belonging to the age group ≥16 years showed a lower probability for
multiple ovulations (0.23). With a view to the ‘Season’ the highest probability for at
least two ovulations was in April 2004 (0.29), the lowest probability in July 2008
(0.12, Figure 2). Regarding the treatment code (‘TC’), code 5 indicates the highest
probability for multiple ovulations (0.27, Figure 3). The lowest probability for multiple
ovulations was obtained by code 0 and 1 (both 0.14). The number of flushes within
season (‘NOF’) showed that with increasing number of flushes the probability for
multiple ovulations also increased (class 1: 0.12 to class 9: 0.28, Figure 4).
15
Fixed effects of ‘Age’, ‘Season’, and ‘TC’ – but not the flushing number within
breeding season (‘NOF’) and the day of recovery (‘Day’) – had a significant influence
on the trait RE. Within the donor’s age range of 2 to 15 years, minor differences were
detected in the likelihood of recovering an embryo (0.41 to 0.46). For mares older
than 15 years, the probability of recovering an embryo decreases to 0.30 (Figure 1)
leading to a significant difference in the number of provided embryos between donors
older than 15 and younger than 16 years.
Figure 1 Probability of >1 ovulation and to recover at least 1 embryo conditional on
the age of the donor mare (F-test:0.0001; LS-Means). Different letters indicate
significant differences in least square means (p<0.05).
Season of flushing affected the probability of recovering an embryo in a range of 0.22
to 0.60. The recovery rate was higher in spring and early summer than in late
summer and autumn (Figure 2).
16
Figure 2 Probability of >1 ovulation (upper figure) and to recover at least 1 embryo
(lower figure) conditional on season (month class within breeding season). Given are
the LS-Means together with the respective standard errors (error bars).
The probability to obtain at least one embryo was significantly higher in mares whose
last treatment was an ovulation inducing agent compared to those having received a
heat inducing agent as their last treatment or no treatment at all. This seems more or
less independent from the penultimate treatment (Figure 3). The combination of heat
and ovulation inducing agent (‘TC’ = 3) was, however, not tested due to a small
number of observations. Notably, the probability in mares having received no
treatment at all is significantly higher than in mares having received an heat inducing
agent as last treatment and no ovulation inducing agent within 28 days prior to the
flushing.
17
Figure 3. Probability of >1 ovulations and to recover at least 1 embryo depending on
the treatment of the donor mare (F-test:0.0001; LS-Means). Different letters indicate
significant differences in least square means (p<0.05), error bars indicate the
respective standard error. Animals with treatment class ‘0’ (light grey bar) have
received no treatment at all. White bars represent the application of an heat inducing
agent (‘TC’ = 1 or 2) and grey bars the application of an ovulation inducing agent
(‘TC’ = 4,5 or 6) as last treatment, respectively.
The probability for a successful flushing conditional on the consecutive number of the
flushing within breeding season is in a range of 0.38 (flush number 1) to 0.44 (flush
number 6) and shows no significant influence on the embryo recovery rate (Figure 4).
With respect to the day of flushing, the probability to recover at least one embryo
ranged from 0.39 (Day 6) to 0.43 (Day 8) but shows no significant differences.
18
Figure 4 Probability of >1 ovulations and to recover at least 1 embryo conditional on
the consecutive number of flushing within breeding season (F-test:0.0001; LSMeans), error bars indicate the respective standard error. Different letters indicate
significant differences in least square means (p<0.05), error bars indicate the
respective standard error. Animals with treatment class ‘0’ light grey bar) have
received no treatment at all. White bars represent the application of an heat inducing
agent (‘TC’ = 1 or 2) and grey bars the application of an ovulation inducing agent
(‘TC’ = 4,5 or 6) as last treatment, respectively.
The heritabilities for fertility parameters for RE (h2 = 0.037 ± 0.020) and OR (h2 =
0.144 ± 0.045) within donor mares were low (Table 3). The service stallion shows a
slightly higher influence on RE than the donor mare (h2 = 0.045 ± 0.037).
Table 3 Heritabilities and proportions of the effect of the permanent environment of
mare and service stallion for RE and OR (standard errors in parentheses)
Trait
Animal
permanent environment
Heritability
RE
donor mare
0.030 ± 0.020
0.037 ± 0.020
RE
service stallion
0.040 ± 0.034
0.045 ± 0.037
OR
donor mare
0.099 ± 0.041
0.144 ± 0.045
donor
The genetic correlation between donor mare and service stallion is -0.599 ± 0.484.
The permanent environmental effect for RE is 0.030 ± 0.020 for the donor mare. The
19
service stallion exhibits a permanent environmental effect for the trait RE of 0.040 ±
0.034. For OR the permanent environmental effect of the donor mare is 0.099 ±
0.041. Correlation (and standard error) between the two traits RE and OR is 0.154 ±
0.283.
Discussion
In the period 2004 to 2008, 0.54 embryos per flush were achieved over all 715 donor
mares. This value is in agreement with Douglas [14], Squires et al. [15]. and
Vogelsang and Vogelsang [12] who found probabilities to flush an embryo ranging
from 0.53 to 0.80 in young fertile mares. The average number of ova was 1.22 per
cycle with a probability of 21.9% to observe more than one ovum. This is partly in
agreement with previous reports. The frequency of multiple ovulations in draft horses
and Thoroughbreds has been reported to range from 15 and 30 %, whereas that in
light breeds and ponies was only up to 10% [5,6]. Based on these values, a slightly
lower probability might have been expected in the German Warmblood horses
analyzed within the current study. The breeding programs of the major German
Warmblood associations have, at least in past decades, been coined by substantial
introgression
of
Thoroughbreds.
Many
Warmbloods
have
high
shares
of
Thoroughbred today of sometimes even more than 50%, which might partly explain
the high probability of multiple ovulations in the analyzed data set. Another
explanation might be an optimized preparation of the donors.
While the probability for at least two ovulations was 21.9%, the probability to obtain at
least two embryos was only 10% indicating a loss of 12% between the time of
ovulation and flushing. Vanderwall [16] reported very high (>90%) pregnancy rates in
young mares under optimal conditions (i.e., fresh, fertile semen) versus older mares
that showed a lower pregnancy rate of 85%. Although high fertilization rates were
20
achieved, embryo recovery rates on days 6 to 9 after ovulation were distinctly lower
for older mares [12,17], which indicates high embryonic losses during the first week
of gestation. In other studies, the estimated embryonic loss rate between fertilization
and day 14 was lower than 10% for young mares but up to 70% for older mares
[18,19]. Vogelsang and Vogelsang [12] reported significant differences in embryo
recovery rates in younger mares compared with older mares. While the recovery rate
in mares younger than 9 years was more than 60%, it decreased to ~30% in mares
older than 17 years. These reports are in agreement with the current study as an
increasing probability for more than one ovulation was observed in old mares, while
the embryo recovery rate decreased (Fig. 1). Thus there is an antagonism between
OR and RE regarding the age of the donor mare. Causes of reduced recovery from
older mares include uterine and oviductal pathology and an increasing early
embryonic death [18] as well as defective oocytes and an impaired maturation [2022].
Several reports based on data from commercial operations have reported a 50 %
embryo recovery rate from single-ovulating mares, some of which were subfertile
mares [23,24]. In most breeds, a low recovery rate due to the lack of multiple
ovulation increases the time required to obtain a pregnant recipient mare. This is
associated with increased costs for the whole procedure of equine embryo transfer.
Double or triple ovulations during a given cycle would result in higher embryo
recovery rates than are currently possible from single-ovulating mares [9]. Therefore,
superovulation would be desirable in mares and would highly increase the efficiency
of equine embryo transfer. Superovulation in mares is, however, still difficult and
generally embryos result only from the normally developing one (or sometimes two)
dominant follicles per cycle. The main difficulty is substantial variation in the donor
mare´s response to treatment. Although recent studies indicate the effectiveness of
21
recombinant equine FSH and LH [11], there is presently no product for
superovulation which has gained widespread acceptance for clinical use in equine
embryo transfer. Thus, most of the mares in an embryo transfer program are
currently not stimulated. This also applies to the donor mares analyzed in the current
study. However, the treatment with an ovulation inducing agent increased the
probability to recover at least one embryo by approximately 0.05 (Figure 3). If the last
treatment was an ovulation inducing agent, then ovulation rate is highest in those
animals whose penultimate treatment was heat induction. However, the recovery rate
after stimulated ovulation is high, almost irrespective of the penultimate treatment.
Based on these results, the application of a heat inducing agent might be
dispensable with respect to the number of recovered embryos. The mechanism by
which the ovulation inducing agent increases the number of recovered embryos is
most likely not related to a higher OR, but to an optimal time of insemination. The
length of the mare’s estrus cycle ranges from 15 to 26 days the duration of estrus
from 2 to12 days with a mean of 7 days [25]. Thus, the precise prediction of the time
of ovulation depends on dense ultrasonsographic monitoring. The positive effect of
stimulating the mare is the predictable time of ovarian maturation and following
ovulation. By knowing the narrow time slot in which ovulation will occur, it is possible
to inseminate the donor mare at the best time, which increases the number of
recovered embryos. Especially when using cooled, frozen or poor-quality semen,
insemination close to ovulation increases fertilization rates [26].
The number of ovulations as well as of recovered embryos is higher in spring and
early summer than in late summer and autumn. It is known that the reproduction
cycle of horses starts with an increasing day length. So the time of mating and giving
birth in the horse is spring and early summer. The results for OR as well as for RE
are in agreement with this. Hence, spring is the best time to start embryo recovery in
22
the year. Considering the consecutive number of the flushing within a season, no
significant differences were found regarding the number of recovered embryos. This
is not consistent with the result obtained from ovulation rate: the number of ovulations
increases with increasing flushing number. One would thus expect a higher number
of recovered embryos in later flushes. This discrepancy may be due to poorer quality
of the ovulated oocytes.
Generally, the flushing of donor mares took place from day 6 to day 9 after ovulation,
which is a standard procedure [27]. Flushing at day 6 is, however, preferably
performed if the embryo will be frozen after flushing. A reason for flushing the donor
mare at day 8 or later is that some embryos, especially from older donors, may be
missed if the flush is performed at an earlier stage [1]. In this study, no significant
differences could be detected between the days of flushing. This is in agreement with
a recent study by Jacob et al. [4], who reported recovery rates in a range 56 to 66%
for days 7 to 10 with the highest value achieved for day 8 (66%, 285/434). The rates
did not significantly differ between these days. The consistent performance in the
current study might be due to the experienced veterinarians who performed the
flushes.
As to be expected for fertility parameters, the heritabilities for both analyzed traits are
low, although the maternal heritability for OR is substantially higher than for RE
(Table 3). Notably, the paternal heritability for the trait RE was higher than the
maternal component indicating that the impact of paternal fertility on embryo recovery
is higher than previously assumed. This likely applies to the pregnancy rate in
standard artificial insemination programs as well. Thus, more attention should be
given to paternal fertility traits in breeding programs. A major factor determining
paternal fertility is the semen quality, which has been analyzed in an accompanying
paper [28]. The correlation between RE and OR is slightly positive, whereas genetic
23
correlations between maternal and paternal components show a slightly negative
tendency. Despite the comparatively large data set, the estimates are afflicted with
high standard errors. Nevertheless, the results indicate a genetic antagonism, which
is consistent with the situation found in cattle. Genetic correlations between maternal
and paternal effects for most fertility traits are reported to be negative in this species
[29]. König et al. [30] emphasized the need for studies revealing the physiological
mechanisms responsible for the phenomenon of negative correlations between
maternal and paternal effects for such traits. The current study is to our knowledge
the first to estimate genetic parameters related to ET. Based on our results, we
conclude that such studies should be carried out in the future not only for cattle but
also for horses.
The low heritability of the complex fertility traits limits the possibility to breed for an
increased breeding performance. Nevertheless, the traits have a genetic component
and the application of genomic selection in the horse, which will probably be
implemented within the next years, might be a promising way to achieve genetic
improvement of fertility. However, this substantially depends on the availability of
accurate phenotypic information as used within the current study.
Conclusion
Within the current study, the use of young donor mares, the start of flushing in spring
and an induced ovulation were found to increase the number of recovered embryos
offering possibilities to optimize ET. The heritabilities for OR and RE were low as to
be expected for fertility traits. Nevertheless, the genetic component offers a way to
breed for improved fertility applying advanced breeding methodologies like genomic
selection. Especially the paternal fertility seems to affect embryo recovery. Further
24
studies presented in an accompanying paper have thus addressed the influence of
semen quality on reproductive outcome.
Acknowledgments
The authors thank the H. Wilhelm Schaumann Stiftung, Hamburg, for the financial
support of H. von der Ahe.
References
1.
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Hinrichs K. Embryo-Transfer in the mare - A status-report. Animal
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Griffin JL, Castleberry RS, Schneider HS. Influence of day of collection on
recovery rate in mature cycling mares. Theriogenology 1981;15: 106-106.
4.
Jacob JCF, Haag KT, Santos GO, Oliveira JP, Gastal MO, Gastal EL. Effect of
embryo age and recipient asynchrony on pregnancy rates in a commercial
equine embryo transfer program. Theriogenology 2012;77: 1159-1166.
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Ginther O. Reproductive Biology of the Mare. Basic and Applied Aspects. 2nd
ed Cross Plaisn, WI: Equiservice 1992.
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Bruyas JF, Battut I, Fieni F, Tainturier D. Gemellar gestation in a mare: A
major cause of abortion. Point Veterinaire 1997;28: 43-53.
7.
Squires EL, McKinnon AO, Carnevale EM, Morris R, Nett TM. Reproductive
characteristics of spontaneous single and double ovulating mares and
superovulated mares. J Reprod Fertil Suppl 1987;35: 399-403.
8.
Pascoe RR, Pascoe DR, Wilson MC. Influence of follicular status on twinning
rate in mares. J Reprod Fertil Suppl 1987;35: 183-189.
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9.
Squires EL, McClain MG, Ginther OJ, McKinnon AO. Spontaneous multiple
ovulation in the mare and its effect on the incidence of twin embryo collections.
Theriogenology 1987;28: 609-613.
10.
Squires EL. Superovulation in mares. Vet Clin North Am Equine Pract
2006;22: 819-830.
11.
Meyers-Brown G, Bidstrup LA, Famula TR, Colgin M, Roser JF. Treatment
with recombinant equine follicle stimulating hormone (reFSH) followed by
recombinant equine luteinizing hormone (reLH) increases embryo recovery in
superovulated mares. Anim Reprod Sci 2011;128: 52-59.
12.
Vogelsang SG, Vogelsang MM. Influence of donor parity and age on the
1989;21: 71-72.
13.
Gilmour AR, Gogel BJ, Cullis BR. ASReml User Guide, release 3.0. . 2009.
14.
Douglas RH. Some aspects of equine embryo transfer. J Reprod Fertil Suppl
1982;32: 405-408.
15.
Squires EL, Imel KJ, Iuliano MF, Shideler RK. Factors affecting reproductive
efficiency in an equine embryo transfer programme. J Reprod Fertil Suppl
1982;32: 409-414.
16.
Vanderwall DK. Early Embryonic Loss in the Mare. Journal of Equine
Veterinary Science 2008;28: 691-702.
17.
Woods GL, Hillman RB, Schlafer DH. Recovery And Evaluation Of Embryos
From Normal And Infertile Mares. Cornell Veterinarian 1986;76: 386-394.
18.
Ball BA, Little TV, Weber JA, Woods GL. Survival of day-4 embryos from
young, normal mares and aged, subfertile mares after transfer to normal
recipient mares. Journal of Reproduction and Fertility 1989;85: 187-194.
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19.
Ball BA, Little TV, Hillman RB, Woods GL. Pregnancy rates at Days 2 and 14
and estimated embryonic loss rates prior to day 14 in normal and subfertile
mares. Theriogenology 1986;26: 611-619.
20.
Carnevale E, Ginther O. Defective oocytes as a cause of subfertility in old
mares. Biol Reprod Monogr 1 (Equine Reproduction VI) 1995: 209-214.
21.
Brinsko SP, Ball BA, Ellington JE. In-vitro maturation of equine oocytes
obtained from different age-groups of sexually mature mares. Theriogenology
1995;44: 461-469.
22.
Bézard J. In vitro fertilization in the mare. Proceedings of the International
Scientific Conference on Biotechnics in Horse Reproduction 12 Crakow,
Poland (Abstract) 1992.
23.
Vogelsang SG, Bondioli KR, Massey JM. Commercial application of equine
embryo transfer. Equine Veterinary Journal 1985;17: 89-91.
24.
Squires E, Seidel G. Collection and transfer of equine embryos. Animal
Reproduction and Biotechnology Bulletin No 11 Fort Collins CO: Colorado
State University 1995: 7-32.
25.
Senger PL. Pathways to Pregnancy and Parturition, 2nd edition: Current
Conceptions Inc., 2011.
26.
Samper JC, Jensen S, Sergeant J, Estrada A. Timing of induction of ovulation
in mares treated with Ovuplant or Chorulon. Journal of Equine Veterinary
Science 2002;22: 320-323.
27.
Squires EL, McCue PM, Vanderwall D. The current status of equine embryo
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Von der Ahe H, Stamer E, Kalm E, Thaller G, Tetens J. Theriogenology 2013.
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Rotvieh. DGFZ Schriftenreihe 1998;11: 82-88.
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König S, Bosselmann F, von Borstel UU, Simianer H. Genetic Analysis of
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28
Chapter Two:
Impact of reproductive history and semen parameters on the number of
recovered embryos in German Warmblood donor mares
H. von der Ahea, E. Stamerb, E. Kalma, G. Thallera and J. Tetensa
a
b
Submitted for publication in Theriogenology
29
Abstract
Success of embryo recovery in horses depends on several factors such as age and
preparation of the donor mare, the day of flushing and the season. Furthermore, the
reproductive status of the donor and the quality of the sire’s semen play an important
role. The aim of the current study was to investigate effects of the donor mare’s
breeding history on the ovulation rate (OR) and the number of recovered embryos
(RE). Furthermore, the impacts on embryo recovery of semen type (fresh vs. frozen)
and quality (number and motility of spermatozoa in fresh semen) were analyzed. The
data set consisted of a total of 5 715 flushes from 715 donor mares sired by 188
service stallions. The breeding status of donors showed significant effects on the
ovulation rate, but notably not on the embryo recovery rate. The type of semen and
the motility of the spermatozoa also exhibited a significant impact on the embryo
recovery rate. Regarding the semen type, fresh semen was clearly superior to frozen
semen. In fresh semen, the proportion of motile spermatozoa substantially
determines the success of embryo recovery.
Keywords: horse, ET, German Warmblood, ovulation rate, embryo recovery, semen
quality, reproductive history
Introduction
Equine embryo transfer (ET) has gained importance in horse breeding over the last
ten years, predominantly to increase the number of foals from show mares or mares
with successful offspring. In an accompanying study based on the same large data
set, it has been shown, that the age and the preparation of the donor mare as well as
the season affect the success flushing [1]. However, embryo recovery requires
successful insemination and thus also depends on paternal fertility. The important
role of the paternal genetic component was also demonstrated in the aforementioned
30
study [1]. Furthermore, Vidament et al. [2] reported that the breeding history and the
management of the mare during the estrus period as well as the quality of the semen
are the most important factors for achieving a pregnancy, especially when using
frozen semen.
The breeding history of the mare includes the age, the reproductive performance of
previous breeding seasons as well as the breeding status, i.e. whether the mare was
pregnant or barren in the last breeding season or is a maiden mare. The reproductive
history is one of the main factors that have to be considered when selecting a mare
as a donor mare for embryo transfer [3-7]. The number of embryos decreased with a
poor reproductive history and higher ages of the donor mare. Reasons for lower
numbers of recovered embryos of older mares may be oviductal and uterine
pathology as well as early embryonic death [8]. Furthermore, the chance to obtain an
embryo from an old donor mare is low because of defective oocytes which may lead
to reduced fertility in these old mares [7].
With respect to embryo transfer, pregnancy rates around day 7 are of great interest,
because these are the days on which the donor mare is generally flushed after
insemination. When investigating groups of young maiden mares (<7 years old),
older maiden mares (>8 years old), barren mares and mares with foal at foot,
Samper [9] found that the mare’s status had a big effect on pregnancy rates. The
average first cycle pregnancy rates within the second week of gestation were 68.8%,
37.2%, 56.2% and 59.3% for each group, respectively. Samper et al. [10] reported
that the number of older maiden mares (>8 years) with a prolonged sport or show
career used for breeding is steadily increasing. Especially when bred with frozen
semen, these mares had lower pregnancy rates and were potential candidates for
failure.
31
The type and the quality of semen also affect embryo recovery. The use of fresh
semen results in a higher recovery rate as compared to frozen semen [9]. Sanchez et
al. [11] described the characteristics of frozen semen as follows: Advantages of
frozen semen are (1) continuous availability of the semen, (2) better timing of
insemination, (3) lower costs of transportation, (4) decrease in the risk of transmitting
venereal diseases, and (5) increase of the genetic pool. Disadvantages are (1) lower
pregnancy rates from some stallions, (2) increased cost to mare owner, (3) increased
costs for semen preparation and (4) risk of disease transmission.
The fertility of frozen semen is influenced by several factors including semen quality,
individual difference between stallions and the insemination dose. It is well known
that there is a variation in semen freezability among service stallions and further
between ejaculates from the same stallion. Pregnancy rates obtained with frozen
semen in commercial programs ranged from 32 to 73% per cycle [12] and from 26%
[13,14] to 66% [15] per season with most values varying from 30 to 55% for the
seasonal success rate [7,16,17].
The aim of the current study was to determine the influence of the mare’s
reproductive status on the ovulation rate and the number of recovered embryos in
German Warmblood horses. Furthermore, the effect of type and quality of semen on
the number of recovered embryos was analyzed.
Material and Methods
The available data consisted of information about preparation, insemination and
flushing of 715 Warmblood donor mares on a large stud farm located in Northern
Germany. Data of 5 715 flushes were collected in a five year period ranging from
2004 to 2008. Donor mares were selected by the head of the stud, based on genetic
32
merit and not on reproductive history. Reproduction data (e.g. ovary and uterus
control, inseminations and flushes) were registered daily by veterinarians. Donor
mares were checked and artificially inseminated using the same procedures as for a
standard artificial insemination and generally inseminated with fresh or frozen semen,
one to two days prior to ovulation. Embryos were obtained six to nine days after
ovulation by flushing the uterus via cervix.
Analyzed reproduction traits were the ovulation rate (OR) and the number of
recovered embryos (RE). From the total data set comprising 5 715 flushes, all
incomplete records were removed including 40 cases with no ovulation at all. This
resulted in 5 665 and 5 674 available flushes for OR and RE, respectively. Due to the
small number of ovulations with more than two ova (1 241 flushes) OR was defined
as binary trait with one ovum (‘0’) vs. at least two ova (‘1’). The 2 765 positive flushes
(3 050 recovered embryos) resulted in the recovery of 1 (N= 2 506), 2 (N = 252), 3 (N
= 15) or 4 (N = 1) embryos. The fraction of flushings with more than one embryo was
hence only 9.66% and RE was also coded as binary trait with no flushed embryo (‘0’)
vs. at least one embryo (‘1’). The data set has previously been described in detail by
von der Ahe et al. [18].
Factors possibly influencing OR and RE considered with specific attention within the
current study were the reproductive status of donor in the end of the previous
breeding season (‘Status’), the use of fresh vs. frozen semen (‘Semen’) and the
semen quality reflected by the parameters ‘number of spermatozoa’ (‘Mil’) and the
‘motility of spermatozoa’ (‘Mot’). The latter two parameters were only available for
fresh semen.
Starting the data acquisition in 2004 as the first flushing season of donor mares, the
breeding status was set to missing, because there was no information on prior use of
the donor. ‘Status’ was divided into three classes (Table 1): mares that were
33
pregnant at the end of the preceding breeding season, mares that were barren and
mares that were used as donor mares in the previous season. For mares entering the
procedure during time of data acquisition, the reproductive history was usually
unknown. These mares were thus excluded from further analyses.
Table 1 Definitions and number of observations for different classes of the
reproductive status of the donor mare for the analyzed traits OR and RE
Number of flushes per trait (N)
Reproductive status
OR
RE
1 932
1 932
(53.19%)
(53.11%)
338
338
season
(9.31%)
(9.29%)
3: Mare was already used as donor in
1 362
1 368
(37.50%)
(37.60%)
3 632
3 638
1: Mare was pregnant in last breeding
season
2: Mare was barren in last breeding
last breeding season
Total
For the classification of the effect ‘Semen’, fresh semen was contrasted to frozen
semen and semen collected from one ejaculation to semen collected from different
ejaculations. Due to a small number of observations (N = 10) for fresh semen
collected from different ejaculations, this class was pooled with fresh semen from a
single ejaculation resulting in three different classes as summarized in Table 2.
Table 2 Summary of the three classes of semen status
Semen status
Observations (N)
1: Fresh semen
1 955
(60.75%)
2: Frozen semen from one ejaculation
1 012
(31.45 %)
3: Frozen semen from different ejaculations
251
(7.80%)
Total
3 218
34
The semen quality parameters ‘Mil’ (= number of spermatozoa) and ‘Mot’ (= motility
of spermatozoa) were divided into 3 classes according to the number of spermatozoa
per insemination and the proportion of motile spermatozoa (Table 3).
Table 3 Definitions and number of observations for the semen quality parameters
Number
of
spermatozoa
per Proportion
insemination (‘Mil’)
Class
Definition1
1
≤100 mio
Inseminations (N)
718
Definition1
≤45 %
120-200 mio
449
Total
1
≥ 250 mio
332
Inseminations (N)
486
(32.42%)
50-60%
(29.95%)
3
motile
spermatozoa (‘Mot’)
(47.90%)
2
of
574
(38.29%)
≥65 %
439
(22.15%)
(29.29%)
1 499
1 499
Number and motility of spermatozoa were not recorded on a continuous scale
Further fixed effects included in the models were the age of the donor mare (‘Age’)
with classes 2, 3, 4, 5, 6 to 8, 9 to 11, 12 to 15, ≥16 years old, the season of flushing
(‘Season’) with 36 classes representing different months and the treatment of the
donor mare. Seven different treatment classes (‘TC’) are defined based on the last
and penultimate treatment prior to flushing. Mares having received no treatment at all
belong to class ‘0’. The remaining mares are grouped into animals whose last
treatment was a heat inducing agent (classes ‘1’ to ‘3’) or an ovulation inducing agent
(classes ‘4’ to ‘6’). The further classification is done based on the penultimate
treatment within 28 days before flushing, which can be none (classes ‘1’ and ‘4’), a
heat inducing agent (classes ‘2’ and ‘5’) or an ovulation inducing agent (classes ‘3’
and ‘6’). TC 3 (last treatment = induction of heat, penultimate treatment = induction of
ovulation) was discarded due to a small number of observations (N = 30). The
consecutive number of flushing within years (‘NOF’) was divided into 9 classes (1, 2,
35
3, 4, 5, 6, 7, 8, ≥9). The impact of these effects on OR and RE has been analyzed
previously [18]; a more detailed description can be found there.
A threshold mixed model with a probit link function as implemented in the GLIMMIX
procedure of the SAS package [19] was applied to data analysis. For OR, the
following model was used:
Φ ##& ,
where is the expected probability to observe at least two ovulations and Φ
is the cumulative probability function of the standard normal distribution. ,
, and represent the fixed effects of i-th age class (i = 1,...,7), the j-th
month of flushing (j = 1,...,28), the k-th preparation of the donor (k = 1,...,4) and the lth flushing number within season (l = 1,...,9), respectively. ##& is the m-th
reproductive status of the donor mare (m = 1,…,3). The random effect of the n-th
donor mare is represented by (n = 1,...,506). Due to the reduced number of
mares with complete information on reproductive history, the number of factor levels
is reduced for some effects.
The embryo recovery rate was analysed using the following model:
Φ ' #$$% ,
where is the expected probability to obtain at least one embryo and Φ is
the cumulative probability function of the standard normal distribution. ' stands for
36
the m-th investigated effect (m = 1,…,3), which can be the reproductive status of the
donor mare (##& ), the sort of used semen (( ), the number of spermatozoa
()%$ ) or the motility ()# ). , , and represent the fixed effects
of i-th age class (i = 1,...,7 for ‘Status’ and 1,…,8 for the other investigated effects),
the j-th month of flushing (j = 1,...,28 for ‘Status’, 1,…,19 for ‘Semen’ and 1,…,15 for
‘Mil’ and ‘Mot’), the k-th preparation of the donor (k = 1,...4), the l-th flushing number
within season (l = 1,...,9), respectively. The random effects of the n-th donor mare
and the o-th service stallion are represented by (n = 1,...,506 for ‘Status’,
1,…,535 for ‘Semen’ and 1,…,409 for ‘Mil’ and ‘Mot’) and #$$% (o = 11,...,142 for
‘Status’, 1,…,93 for ‘Semen’ and 1,…,50 for ‘Mil’ and ‘Mot’), respectively. Due to a
reduced number of complete records, the number of factor levels is reduced for some
effects.
The random effects of the donor mare and the stallion were analyzed previously [18]
and only included here to correctly set up the model. The results were not further
regarded in the current study.
Results
Different impacts of the donor mare’s reproductive status on the two traits OR and
RE were observed. No effect was found for RE, while OR is significantly influenced
(Figure 1). The probability for obtaining at least two ovulations is significantly lower in
mares that have been pregnant in the previous season (0.17) as compared to mares
that have been barren or donors (0.27).
There are no significant differences between the groups regarding the probability of
obtaining an embryo. However, there is a slight tendency that mares previously used
as donors or pregnant in the last season have a higher probability to deliver an
37
embryo of 0.48 and 0.47, respectively, as compared to barren mares with a
probability of 0.44.
0.35
0.6
b
b
Probability of recovering at least one embryo
Probability of >1 ovulation
0.30
0.25
0.20
a
0.15
0.10
0.05
0.00
0.5
0.4
0.3
0.2
0.1
0.0
Pregnant
Barren
Donor
Pregnant
Reproductive status in last season
Barren
Donor
Reproductive status in last season
Figure 1 Probability of more than one ovulation and recovering at least one embryo,
respectively, conditional on mare’s reproductive status in the previous breeding
season (F-test: 0.0001; LS-Means).
The type of semen has a strong impact on RE (Figure 2). The probability for
recovering at least one embryo when using fresh semen (0.52) differs significantly
from the probability when inseminating with frozen semen from one (0.43) or different
ejaculations (0.36). No significant difference can be found between the two types of
frozen semen.
38
0.7
Probability of recovering at least 1 embryo
0.6
a
0.5
b
b
0.4
0.3
0.2
0.1
0.0
Fresh
Frozen from
single ejaculation
Frozen from
different ejaculations
Type of semen
Figure 2 Probability of recovering at least one embryo conditional on the type of
semen (F-test: 0.0001; LS-Means)
The number of spermatozoa per insemination showed no significant effect on RE.
The use of 120 to 200 million spermatozoa per insemination resulted in a probability
of 0.57 to recover an embryo, while the other classes (≤100 mio and ≥250 mio) had
an equal probability of 0.51. The motility of the spermatozoa on the other hand
exhibited a significance influence on the recovery rate (Figure 3). Semen with a
motility greater than 65% resulted in a significantly higher probability to obtain an
embryo (0.59) than semen with a motility of 50 to 60 % (0.52) and semen with a
motility less than 45% (0.47).
39
0.7
b
a
Probability of recovering at least 1 embryo
0.6
a
0.5
0.4
0.3
0.2
0.1
0.0
≤ 45%
50−60%
≥ 65%
Proportion of motile spermatozoa
Figure 3 Probability of recovering at least 1 embryo depending on the proportion of
motile spermatozoa in fresh semen (F-test: 0.0001; LS-Means)
The fixed effects of the donor mare’s age, the season, the treatment of the donor and
the consecutive number of the flushing have previously been analyzed [18]. It was
shown that all effects significantly influence OR. All effects except for the number of
flushings exhibited a significant influence on RE. These results were confirmed within
the current study with two exceptions, which are probably due to the reduced sample
size: the effect of flushing number was not significant for OR and the treatment was
not significant for RE.
Discussion
A successful flushing requires a successful insemination. Vidament et al. [2] reported
that the breeding status and the management of the mare during the estrus period as
40
well as the quality of the semen are the most important factors for achieving a
pregnancy, especially when using frozen semen. Furthermore, numerous other
studies have shown that the reproductive history is one of the main factors that have
to be considered when using a mare as a donor for embryo transfer [3-7]. The
number of embryos decreased with a poor reproductive history and higher ages of
the donor mare.
Within the current study, a significant influence of the mare’s breeding status on OR
was found, while RE did not significantly differ between groups. The OR was
significantly lower if the donor mare was pregnant in the previous breeding season.
Stress resulting from the foal at foot and lactation possibly reduces OR, which could
be expected to reduce RE in an equivalent way. This was, however, not observed. A
reduced OR combined with an unaltered RE means that the fertilization rate and/or
the survival rate of the early embryo is higher in those mares. A possible explanation
might be a randomly increased use of fresh semen in the mares that were pregnant
in the previous season. This would lead to an increased embryo recovery as
discussed below. In fact, ~65% of those mares were inseminated with fresh semen
compared to ~58% in the other groups, which might partly explain this observation.
The type and the quality of semen also affected embryo recovery. The use of fresh
semen resulted in a probability to recover an embryo of 0.52 was thus clearly
superior to the use of frozen semen, which resulted in probabilities of 0.43 (1
ejaculation) and 0.36 (>1 ejaculation). One explanation for the reduced pregnancy
rates after insemination with frozen semen was suggested by Dobrinski et al. [20]
and Ellington et al. [21]. They demonstrated an impaired adhesion of previously
frozen spermatozoa to the oviductal epithelium. This reduces the ability of the mare
to create reserves of spermatozoa within the reproductive tract. Since insemination
usually takes place prior to ovulation, the adhesion ensures the presence of viable
41
spermatozoa at the time of ovulation. As a consequence, the predictability of
ovulation is crucial when using frozen semen requiring dense ultrasonographic
monitoring and/or the use of an ovulation inducing agent. Alternatively, several
consecutive inseminations can be performed, which increases the costs and reduces
the number of potential offspring from a given stallion. It is known that there is a
variation in semen freezability between different stallions as well as between different
ejaculations from the same stallion. Thus, pregnancy rates obtained with frozen
semen in commercial programs were reported to vary in wide range from 32 to 73%
per cycle [12]. When there are large differences between different ejaculations from
the same stallion, the probability of failure for a given dose of frozen semen can be
reduced by pooling semen from different ejaculations. Within this study, no significant
differences were observed between single ejaculation and pooled semen. There is,
however, a tendency that doses from a single ejaculation resulted in a higher embryo
recovery rate. This might be due to the different handling of pooled semen or the fact
that pooling is preferably done in stallions with poor or rather varying freezability.
For those inseminations performed with fresh semen, data on the number and
motility of spermatozoa were available. Motility showed a significant influence on the
embryo recovery rate, while the number of spermatozoa did not exhibit a significant
effect. Even for doses with less than 100 mio spermatozoa the probability to recover
an embryo was 0.51. The usage of a much smaller dose might result in inferior
recovery rates, but this was naturally not attempted. For doses with a satisfying
quality there is no improvement above a certain number of spermatozoa. For frozen
semen, the impact of dose size might be more pronounced.
The use of highly motile spermatozoa (≥65%) resulted in at least one embryo in 59%
of cases, whereas the use of spermatozoa with a lower motility (≤45%) resulted in a
success rate of 47%. Hence, the important fact for recovering at least one embryo is
42
the use of high motile spermatozoa (≥65 %) for insemination. This is in agreement
with the results of Varner and Blanchard (unpublished) reported by Brinsko [22]. They
found a pregnancy rate per cycle of only 48% for fresh semen, when the progressive
sperm motility was <60%. In the case of using >60 % motile sperm, the pregnancy
rate per cycle was 67%. Notably, we did not find the previously reported influence of
the donor mare’s treatment when analyzing the fresh semen characteristics. This is
probably due to the fact that the fresh semen can survive for a longer time in the
female oviduct making the time span between insemination and ovulation less
important.
Conclusion
Although the breeding history of the donor mare influences the ovulation rate, no
differences in embryo recovery were found between groups with different histories
within the current study. The use of fresh semen leads to higher embryo recovery
rates than the insemination with frozen semen. Moreover, the motility of spermatozoa
significantly influenced the number of recovered embryos. When using frozen semen,
the treatment of the donor mare is of great importance to optimally determine the
time of ovulation.
Acknowledgments
The authors thank the H. Wilhelm Schaumann Stiftung, Hamburg, for the financial
support of H. von der Ahe.
43
References
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Von der Ahe H, Stamer E, Kalm E, Thaller G, Tetens J. Analysis of genetic
and management factors affecting ovulation and embryo recovery rate in
German Warmblood donor mares. Theriogenology 2013.
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Vidament M, Dupere AM, Julienne P, Evain A, Noue P, Palmer E. Equine
frozen semen: Freezability and fertility field results. Theriogenology 1997;48:
907-917.
3.
Squires EL, Imel KJ, Iuliano MF, Shideler RK. Factors affecting reproductive
efficiency in an equine embryo transfer programme. J Reprod Fertil Suppl
1982;32: 409-414.
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McKinnon AO, Squires EL, Voss JL, Cook VM. Equine embryo transfer.
Compendium on Continuing Education for the Practicing Veterinarian 1988;10:
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5.
Vogelsang SG, Vogelsang MM. Influence of donor parity and age on the
1989;21: 71-72.
6.
Squires E, Seidel G. Collection and transfer of equine embryos. Animal
Reproduction and Biotechnology Bulletin No 11 Fort Collins CO: Colorado
State University 1995: 7-9; 11-15; 27-32.
7.
Carnevale E, Ginther O. Defective oocytes as a cause of subfertility in old
mares. Biol Reprod Monogr 1 (Equine Reproduction VI) 1995: 209-214.
8.
Ball BA, Little TV, Weber JA, Woods GL. Survival of day-4 embryos from
young, normal mares and aged, subfertile mares after transfer to normal
recipient mares. Journal of Reproduction and Fertility 1989;85: 187-194.
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Samper JC. Management and fertility of mares bred with frozen semen.
Animal Reproduction Science 2001;68: 219-228.
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10.
Samper JC, Hearn PH, Ganheim A. Pregnancy rates and effect of extender
and motility and acrosome status of frozen-thawed stallion spermatozoa.
Proceedings of the 40th Ann. Conv. Amer. Assoc. Equine Pract., 1994;41-43.
11.
Sanchez R, Gomez I, Samper JC. Artificial insemination with frozen semen. In:
Samper JC (ed), Equine Breeding Management and Artificial Insemination 2nd
Revision edition edition: Saunders, 2008;175-185.
12.
Loomis PR. The equine frozen semen industry. Animal Reproduction Science
2001;68: 191-200.
13.
Klug E, Treu H, Hillmann H, Heinze H. Results Of Insemination Of Mares With
Fresh And Frozen Stallion Semen. Journal of Reproduction and Fertility 1975:
107-110.
14.
Tischner M. Results Of Artificial-Insemination Of Horses In Poland In Post-War
Period. Journal of Reproduction and Fertility 1975: 111-114.
15.
Salazar-Valencia F. Embryo recovery rates in mares of the pasofino
Colombiano breed and deep freezing stallion semen in the tropics.
Theriogenology 1983;19: 146-146.
16.
Palmer E, Magistrini M. Automated analysis of stallion semen post-thaw
motility. Acta veterinaria Scandinavica Supplementum 1992;88: 137-152.
17.
Samper JC, Hellander JC, Crabo BG. Relationship between the fertility of
fresh and frozen stallion semen and semen quality. Journal of reproduction
and fertility Supplement 1991;44: 107-114.
18.
Von der Ahe H, Tetens J, Stamer E, Kalm E, Thaller G. Analysis of genetic
and management factors affecting ovulation and embryo recovery rate in
German Warmblood Donor Mares. Theriogenology 2013.
19.
Inc. SI. SAS, SAS 9.2 TS Level 1 M0 edition. Cary, NC, USA, 2008.
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20.
Dobrinski I, Thomas PGA, Ball BA. Cryopreservation reduces the ability of
equine spermatozoa to attach to oviductal epithelial cells and zonae pellucidae
in vitro. Journal of Andrology 1995;16: 536-542.
21.
Ellington JE, Samper JC, Wright WR. Stallion sperm interactions with oviduct
cells in vitro as an indicator of field fertility. Proceedings the 14th International
Congress Animal Reproduction, Stockholm, Sweden, 2000;276.
22.
Brinsko SP. Insemination doses: How low can we go? Theriogenology
2006;66: 543-550.
46
Chapter Three:
Effects on success of pregnancy rate after embryo transfer in warmblood
recipient mares and comparison of embryo transfer foals to non – embryo
transfer foals
H. von der Ahe*, E. Stamer†, J. Tetens*, E. Kalm* and G. Thaller*
*
†
47
ABSTRACT
Considering the procedure “equine embryo transfer” there are several points where a
decrease of the success rate, due to different influencing factors, is possible. The
result of the first pregnancy determination after embryo transfer in the recipient mare
is influenced by the season, the preparation of the recipient mare and the age of the
embryo at time of transfer. Comparing embryo transfer foals to non – embryo transfer
foals, there are only differences in the length of gestation. However, the gender of the
foal, the age of the mare at time of birth and the season influences analyzed traits,
like length of gestation, withers height at birth and weight at birth.
Keywords: horse, ET, German Warmblood, foal, recipient
INTRODUCTION
The management of recipients is the main factor affecting the success of equine as
well as bovine embryo transfer for example. Generally, recipients should show
normal cyclic activity and abnormalities or pathological findings of the genital tract
should be absent (Squires et al., 1999). Furthermore, the ultrasonographic monitoring
of ovulation and uterine changes is of great importance.
Estrus synchrony of donor and recipient mares substantially affects the success of
embryo transfer, i.e. the pregnancy rate (Squires et al., 1982; Squires et al., 1985;
Squires et al., 1985; McKinnon et al., 1988; Squires et al., 1995). Due to individual
variation of the heat length, the exact timing of ovulation in donor and recipient is,
however, difficult. An approach to handle this is the employment of induced ovulation
in the recipient once an ovulation has been detected in the donor mare. The highest
initial pregnancy rates of >75% at day 12 and around 65% at day 50 will be achieved
when the recipient mare’s ovulation occurs within a time range of two days before to
48
three days after the donor mare’s ovulation (McKinnon et al., 1988; Carney et al.,
1991). The decrease in pregnancy rates of more than ten percent between day 12
and 50 is similar to the amount of early embryonic loss of 13.4% found in mares
inseminated with fresh semen (Carney, et al., 1991) and also, comparable results
have been reported by Villahoz et al., (1985).
Considering the method of transfer, there is a difference between surgical and
nonsurgical transfer. Some investigators found lower pregnancy rates after
performing nonsurgical transfer (27% vs. 53%, Imel et al., 1981; 45% vs. 72%,
Iuliano et al., 1985). Other authors reported acceptable pregnancy rates after
nonsurgical transfer like 75 to 77% (Hinrichs and Kenney, 1987; Wilson et al., 1987).
As a result of its simplicity and in terms of animal welfare, the nonsurgical transfer is
actually the most common technique.
Additionally, the quality of the transferred embryo will affect the pregnancy rate.
Equine embryos can be graded applying an approach similar to the procedure used
in bovine embryos. Using fresh high quality embryos will then result in significantly
higher pregnancy rates. A day 12 pregnancy rate of 70 to 75% can be achieved by
using high quality embryos, followed by a day 50 pregnancy rate of 65% (Squires et
al., 2002). The transfer of large embryos will on the other hand reduce the pregnancy
rate (Squires et al., 1985 [No. 11]; Iuliano et al., 1985).
Size of the recipient mare in comparison to donor mare and service stallion is an
aspect which is often discussed. Studies in Newmarket (Great Britain) where
Thoroughbred embryos were transferred to pony recipient mares and vice versa
showed an imbalance between the genetically determined size of the embryo and
recipient mare’s influence on intrauterine and postnatal development. Transfer into
smaller recipients resulted in a retardation of fetus growth, which could not totally
compensated (Allen et al., 2004). Thoroughbred foals that were transferred to a pony
49
recipient mare, showed signs of immaturity after birth (Ousey et al., 2004). In general,
the transfer of warmblood embryos into trotter or thoroughbred mares, which had
commonly a similar size can sometimes lead to a slightly reduced size at birth, but
this is fully compensated during postnatal development.
Satuè et al., (2011) reported a mean gestational length of 332.4 ± 12.1 days in
Carthusian broodmares, which was significantly prolonged (5.7 days) when mares
give birth to colts (336.8 ± 11.0 days) rather than fillies (331.1 ± 12.8 days). They also
found a 5.3 days shorter pregnancy in mares 8 to 12 years (330.8 ± 11.0 days) old in
contrast to those of 13 to 17 years (336.1 ± 8.9 days). Valera et al., (2006) found
mean length of gestation of 336.8 ± 0.48 days in Andalusians or Spanish Purebreds
and 340.3 ± 0.63 days in Arabian mares. They found that two of the main factors
significantly affecting gestational length are the age of the mother and the sex of the
foal. Cilek (2009) investigated Purebred Arabian foals and found a significant
influence of sex of the foal on the birth weight. The weight at birth was 46.27 ± 0.40
kg for colts, whereas fillies weighted 45.22 ± 0.40 kg at birth. Allen et al., (2002)
found a mean duration of gestation of 339.0 ± 3.0 days in Thoroughbred mares. The
mean foal birth weight was 53.1 ± 2.6 kg.
MATERIAL AND METHODS
The data set comprised 3 130 embryo transfers performed between 2004 and 2008
on a large stud farm in Northern Germany. 910 recipient mares were used for embryo
transfer in this period. The data set was reduced due to missing information (e.g.
birth date of recipient mares), resulting in 2 927 evaluable transfers. Frozen embryos
were also excluded, because only 59 of these embryos were transferred in the period
50
from 2004 to 2008. Table 1 gives an overview of the number of embryo transfers per
year.
Table 1 Number of embryo transfers per year (2004 to 2008)
year
performed embryo transfers [n]
2004
552
2005
549
2006
536
2007
605
2008
685
total
2 927
The number of transfers per recipient lay in a range of 1 to 11 with an average of 3.4
transfers per recipient mare over the five year period. The number of transfers within
years and recipient mares was between one and four, with an average of 1.4
transfers per recipient and year.
Reproduction data of recipient mares were registered daily by the veterinarians and
promptly stored on record sheets. The description of the results of pregnancy
determination (PD) is shown in Table 2.
51
Table 2 Description of pregnancy determination (PD) at different times after
ovulation (2 927 transfers)
positive (n)
positive (%)
PD I (16 d after ovulation)
1955
66.8
PD II (45 d after ovulation)
1706
58.3
PD III (150 d after ovulation)
1543
52.7
d = days
The analyzed reproduction trait is PD I of the recipient mare around 9 days after
transfer (Table 3). PD I was defined as a binary trait: in the case of a negative PD I
result the binary code is 0, and in the case of a positive PD I result, the code is 1.
Table 3 Description of analyzed trait first pregnancy determination (PD I), 16 d after
ovulation
binary code
0
trait
pregnancy determination (PD I)
1
n transfers
n
%
2 927
972
33.2
n
%
1955 66.8
Influencing factors:
Age classes of recipient mare’s age at time of transfer were defined as 2, 3, 4, 5, 6,
7, 8, 9, 10+11, 12+13, ≥ 14 years old. The average age of recipient mares at time of
transfer was 7.5 years within the five year period.
Generally, the transfer season ranged from March to September. Number of average
transfers per month was 88.8 over the five year period.
52
The treatment code (TC) represented six different combinations between point of
treatment before ovulation (last and second last treatment) and type of heat and
ovulation inducing agents (Table 4). TC 3 was discarded due to small number of
observations (n = 50).
Table 4 Definition of treatment code (TC)
last treatment
second last treatment
TC
none
none
0
heat inducing agent
none
1
heat inducing agent
heat inducing agent < 28 d
2
heat inducing agent
heat inducing agent > 28 d
1
heat inducing agent
ovulation inducing agent < 28 d
3
heat inducing agent
ovulation inducing agent > 28 d
1
none
4
heat inducing agent < 28 d
5
heat inducing agent > 28 d
4
ovulation inducing agent < 28 d
6
ovulation inducing agent > 28 d
4
*d=day
Embryos were flushed and transferred at an age lying in the range of 6 to 9 days.
The synchrony between the donor and recipient mare regarding the day of ovulation
was divided into six classes. The difference of time of ovulation ranged from 0 days
up to 5 days.
Significance of fixed effects was analyzed using the GLIMMIX procedure of the SAS
package (SAS 2008) with a threshold mixed model and a probit link function:
Pregnancy determination [model]:
E [πijklmnop] = Φ (Agei + Seasonj + TCk + AgeEmbl + Synm + recipn + donoro + stallionp)
53
where
E [πijklmnop] = Expected probability to obtain a positive PD
Φ
= Cumulative probability function of standard normal distribution
Agei
= Fixed effect of i-th age (i = 1,...,11)
Seasonj
= Fixed effect of the j-th month of transfer within year (j = 1,..., 33)
TCk
= Fixed effect of the k-th treatment of the donor (k = 1,..., 6)
AgeEmbl = Fixed effect of the l-th age of the embryo (l = 1,..., 4)
Synm
= Fixed effect of the m-th synchrony of day of ovulation (m = 1,..., 6)
recipn
= Random effect of the n-th recipient mare (n = 1,..., 861)
donoro
= Random effect of the o-th donor mare (o = 1,..., 586)
stallionp
= Random effect of the p-th service stallion (p = 1,...,152)
The data set of the ET and Non-ET foals contained 4 288 records. The foals
descended from 192 sires. Table 5 gives an overview of foals born from 2005 to
2009.
Table 5 Distribution of number of foals born between 2005 and 2009
year
born foals [n]
2005
735
2006
916
2007
886
2008
922
2009
829
total
4 288
54
Due to missing data of the pedigree 4 268 foals were analyzable. The number of
analyzable ET foals was 1 611 (37.8 %), whereas the number of Non-ET foals were 2
657 (62.2 %).
Analyzed traits are the gestational length (GL), withers height at birth (HB) and the
weight at birth (WB). The data sets contained 3 436 records, 2 283 records and 1 322
records for gestational length, withers height at birth and weight at birth, respectively.
The fixed effect ‘ET’ was divided into two classes, embryo transfer foals and non
embryo transfer foals. The fixed effect ‘Sex’ represented two classes, colts and fillies.
The fixed effect ‘Season’ represented the month of birth of the foal and was only
used for analyzing gestational length.
The fixed effect ‘Age_moth’ was the age of the recipient mares as well as of the
mares, who were pregnant with their own foals.
The models were analyzed using the SAS procedure MIXED and the REML method
(SAS Institute, 2008). The resulting model for the analysis of the data sets was
yijklmn = µ + ETi + Sexj + Seasonk +Age_mothl + stallionm + eijklmn
where
yijklmn
= n-th observation of the analyzed trait (GL/HB/WB)
µ
= overall mean
ETi
= Fixed effect of the i-th type of foal (ET/Non-ET) (i = 1, 2)
Sexj
= Fixed effect of the j-th gender of the foal (j = 1, 2),
Seasonk
= Fixed effect of the k-th month of birth of the foal (k = 1,…, 27 for GL),
Age_mothl
= Fixed effect of the l-th Age of the mother at time of birth
(l = 1,…,12 for GL, l = 1,…, 11 for HB, l = 1,…, 6 for WB)
55
stallionm
= Random effect of the m-th service stallion
(m = 1,…, 159 for GL, m = 1,…, 146 for HB, m = 1,…, 118 for WB)
eijklmn
= Random residual effect.
RESULTS
The 3 130 performed transfers resulted in 1 611 living born foals, which is a success
rate of 51.5 %. After PD I, 66.7 % of the pregnancy determinations were positive. The
failure of pregnancy after PD I was quite low, with 94.5 % and 97.7 % positive results
at PD II and PD III, respectively.
The following fixed effects were investigated in terms of affecting PD I around nine
days after transfer to the recipient mare. Month of transfer (Season), preparation of
the recipient mare (TC) and the age of embryo at time of transfer (AgeEmb) showed
a significant influence on PD I. The age of the recipient mare at time of birth
(Age_moth) and the synchrony of donor and recipient mare regarding the day of
ovulation (Syn) showed no significant influence on the result of PD I.
Regarding the season, number of positive pregnancy results (PD I) were high in
spring and early summer, but generally decreased in midsummer. The probability
(and standard error) to obtain a positive PD I ranged from 0.46 ± 0.05 (June 2008) to
0.86 ± 0.03 (May 2004). Pregnancy rates were generally lower in 2008 than in other
years. The years 2005, 2007 and 2008 showed also higher pregnancy rates in late
summer and autumn.
The probability (and standard error) of obtaining a positive PD I with regard to the
preparation of the recipient mare (Figure 1) was between 0.61 ± 0.02 (for TC = 0)
and 0.75 ± 0.04 for (TC = 6). Only TC = 0, representing recipient mares, that got no
treatment at all, showed a significant lower probability for a successful PD I. Between
56
classes 1 and 2 (last application was a heat inducing agent) and classes 4, 5 and 6
(last application was an ovulation inducing agent) no significant differences were
detected.
b
b
b
b
b
a
a+b
different superscripts indicate significant differences in least square means (P<0.05), according to Bonferroni correction
Figure 1 Probability of obtaining a positive pregnancy determination (PD I)
depending on the treatment of the recipient mare (TC) before transfer
(F-test: 0.0001; LS-Means)
The age of the embryo at time of transfer played an important role. The probabilities
(and standard error) of obtaining a positive PD I were between 0.62 ± 0.03 (Day 9 old
embryos) and 0.74 ± 0.02 (Day 7 old embryos). Whereas there is no significant
difference regarding PD I between Day 7 and Day 8 embryos, 9 day old embryos
showed a significant lower probability for a positive PD I (Figure 2). Day 6 embryos
did not differ significantly from seven, eight or nine day old embryos with respect to
the PD I.
57
a,b
a+b
a
a
b
Figure 2 Probability of obtaining a positive pregnancy determination (PD I)
depending on the age of embryo (d=days) at time of transfer
The age of the recipient mare at time of transfer showed no significant influence on
PD I, although there was a slight tendency for better pregnancy results in younger
mares (two to seven years old). The probability (and standard error) of obtaining a
positive PD I ranged from 0.63 ± 0.03 (recipient mares ≥ 14 years old) to 0.72 ± 0.03
(recipient mares that were 2 and 6 years old), respectively.
The synchrony, defined as the difference of days of ovulation of donor mare and
recipient mare, had no significant influence on PD I. The probability (and standard
error) for a positive PD I were in a range of 0.67 ± 0.03 (one day difference) to 0.72 ±
0.04 (five days difference).
Within the years 2005 to 2009 the number of living born foals was 4 288, divided into
1 611 ET foals and 2 657 Non-ET foals. Table 6 show the results of gestation length
58
(GL), height at birth (HB) and weight at birth (WB) compared as ET and Non-ET foals
(= type of foals) as well as male and female foals.
Table 6 Values and standard errors [se] of the traits gestation length, height at birth
and weight at birth regarding to ET and Non-ET foals as well as to male and female
foals
Gestation length
Height at birth
Weight at birth
(days) [se]
(centimeter) [se]
(kilogram) [se]
ET
333,1a [0.35]
104,2 a [0.15]
57,3 a [0.31]
Non-ET
333,8b [0.32]
104,4 a [0.14]
57,7 a [0.26]
male
334,5a [0.33]
104,6a [0.15]
58,2a [0.28]
female
332,5b [0.33]
103,9b [0.15]
56,8b [0.28]
a+b
The length of gestation after ET was significantly shorter than those in mares who
were pregnant with their own foals. Withers height at birth and weight at birth showed
no significant differences between ET and Non – ET foals. All three traits were
significant influenced by the gender of the foal. Length of gestation in colts was two
days longer than fillies. Colts were 0.7 centimeters taller than fillies. Weight at birth in
colts was 1.4 kilogram higher than in fillies. There was no interaction between the
type of the foal (ET/Non-ET) and the gender of the foal.
Regarding the age of the mare at time of birth, there was a significant influence on
gestational length (Figure 3), on withers height at birth (Figure 4) and weight at birth
(Figure 5). Values for gestational length were between 331.9 d (age class = 5) and
336.5 d (age class = 17). Standard errors lay in a range of 0.41 (age class = 3) and
59
0.70 (age class = 11). Mares at an age of ≥17 years showed a higher length of
gestation than younger mares. Furthermore, mares at an age of three to seven years
old were significantly shorter in foal than mares at an age of 11 to 16 years old.
338
c
Length of gestation (d)
337
336
b
335
334
333
a
a
a
3
4
a
a,b
a,b
b
b
a,b
a
332
331
330
a-c
5
6
7
8
9
10
Age of (recipient) mare
11
12
14
17
Figure 3 Influence of the age of the mare on the trait gestational length
Withers height at birth was also significantly influenced by the age of the mare at time
of birth. Values for withers height at birth were between 101.2 cm (age class = 3) and
105.6 cm (age classes = 9 and 10). Standard errors lay in a range of 0.20 (age
classes = 3 and 4) and 0.34 (age class = 10). Younger mares (≤7 years old) and
mares older than 13 years gave birth to smaller foals.
60
Withers height at birth (cm)
e
106
105
c,g
104
c,f
g
a
101
100
3
a-g
d,f
e
e,f
b
103
102
f,g
e
4
5
6
7
8
9
10
11
13
16
Figure 4 Influence of the age of the mare on the trait height at birth
The age of the mare at time of birth showed also a significant influence on weight at
birth. Values for weight at birth were between 53.0 kg (age class = 3) and 60.2 kg
(age class = 9). Standard errors lay in a range of 0.40 (age class = 3) and 0.46 (age
class = 6). Mares, younger than six years old, gave birth to foals with lighter weights.
Although there was no significant difference between the age classes 9 and 12;
mares, older than eleven years showed a tendency to bear foals with slighter weights
at birth.
61
62
d
d
c,d
60
Weight at birth (kg)
c
58
b
56
54
a
52
50
3
a-d
4
6
8
9
12
Figure 5 Influence of the age of the mare on the trait weight at birth
For analyzing gestational length the model contained additionally the fixed effect
season, which was the month of birth of the foal (Figure 6). The season showed a
significant effect on gestational length. Values for gestational length regarding the
season were between 326.3 d (May 2007) and 340.1 d (February 2009). Standard
errors lay in a range of 0.69 (May 2009) and 1.2 (February 2006 and January 2008).
62
342
340
Length of gestation (d)
338
336
334
332
330
328
326
324
322
320
2005
2006
2007
2008
2009
Month of birth
Figure 6 Influence of the season on the trait length of gestation (F-test: 0.0001; LSMeans)
DISCUSSION
There is a large variation in pregnancy rates after nonsurgical transfer, ranging from
50 to 75 % (Squires et al., 1995; Mc Kinnon et al., 1988; Riera et al., 1993;
Vogelsang et al., 1985). This is in agreement with our results showing a positive
result of PD I of 66.7 % and a success rate of 51.5% regarding the living born foals.
Considering the method of transfer, there is a difference between surgical and
nonsurgical transfer. But there were also lower pregnancy rates found after
performing nonsurgical transfer (27% vs. 53%, Imel et al., 1981; 45% vs. 72%,
Iuliano et al., 1985) as well as other authors reported acceptable pregnancy rates
after nonsurgical transfer like 75 to 77% (Hinrichs and Kenney, 1987; Wilson et al.,
1987).
63
No significant differences in PD I were detected depending on the age of the
recipient mare at time of transfer. This is in contrast to the influence of the age on
embryo recovery rate in donor mares founded in previous studies, where mares older
than 16 years donate fewer numbers of embryos. Hence, the ability to become
pregnant is independent of the age of the recipient mare in contrast to the production
of an embryo, which depends on the age of the donor mare.
With respect to the season, there is a significant influence on PD I. Higher numbers
of positive PD I were achieved in spring and early summer, but generally decreased
in midsummer. In spite of the fact, there is no clear trend between the years. It is
generally known that the reproduction cycle of horses starts naturally when daylight is
extending. Hence, spring and early summer are the best time to achieve acceptable
pregnancy rates after embryo transfer. For the years 2005, 2006 and 2008 we found
a rise of positive pregnancy determinations in late summer and autumn. This
increase may be depending on the weaning of the foals, because mares with no foal
at foot are often more likely to become pregnant.
The treatment of the recipient mare differs only between recipient mares that got no
treatment at all and processed mares independent of the treatment were heat
inducing agents or ovulation inducing agents. Treated mares showed higher numbers
of positive PD I than untreated mares. The management of the recipient mare plays
an important role and therefore the timing and detection of the ovulation is of great
importance for the success of embryo transfer. In treated mares this is more
predictable than in untreated mares.
With a view to the age of the embryo at time of transfer, best pregnancy rates were
obtained when transferring Day 7 embryos or Day 8 embryos. Day 9 embryos
resulted in significant lower pregnancy rates due to potential damage during the
flushing and handling before transfer. This result is in agreement with Squires et al.
64
[11], (1985) and Iuliano et al., (1985), who reported reduced pregnancy rates after
the transfer of large embryos. Day 6 embryos showed no significant differences
regarding the pregnancy rate compared to day 7, day 8 and day 9 embryos, which is
an important result with view to frozen embryos (flushing at day 6 after ovulation).
Especially for frozen embryos the viability and therefore acceptable pregnancy rates
plays an important role, because of the forces of the freezing and thawing process.
Estrus synchrony of donor and recipient mares substantially affects the success of
embryo transfer, i.e. the pregnancy rate (Squires et al., 1982; Squires et al., 1985;
Squires et al., 1985; McKinnon et al., 1988; Squires et al., 1995). Due to individual
variation of the heat length, the exact timing of ovulation in donor and recipient is,
however, difficult. An approach to handle this is the employment of induced ovulation
in the recipient once an ovulation has been detected in the donor mare. The highest
initial pregnancy rates of >75% at day 12 and around 65% at day 50 will be achieved
when the recipient mare’s ovulation occurs within a time range of two days before to
three days after the donor mare’s ovulation (McKinnon et al., 1988; Carney et al.,
1991). Although this results have been found previously, in our study no significant
influence of synchrony of donor and recipient could be detected. Recipient mares
ovulated in a range of zero days up to five days after the donor mare, followed by
comparable pregnancy rates after embryo transfer.
The decrease in pregnancy rates of more than ten percent between day 12 and 50 is
similar to the amount of early embryonic loss of 13.4% found in mares inseminated
with fresh semen (Carney, et al., 1991) and also, comparable results have been
reported by Villahoz et al., (1985). No value for the covariance parameter estimates
could be found for the recipient mare in consideration of the first pregnancy
determination. This indicates that there is no variation between different recipient
mares.
65
Foals
The comparison of embryo transfer and non embryo transfer foals showed only a
significant influence with respect to the length of gestation. Comparing colts and fillies
revealed in significant differences in all analyzed traits. No interaction was found
between the type of the foal (ET foal/Non – ET foal) and the gender.
Valera et al., (2006) found mean length of gestation of 336.8 ± 0.48 days in
Andalusians or Spanish Purebreds and 340.3 ± 0.63 days in Arabian mares. They
found that two of the main factors significantly affecting gestational length are the age
of the mother and the sex of the foal. Allen et al., (2002) found a mean duration of
gestation of 339.0 ± 3.0 days in Thoroughbred mares. Satuè et al., (2011) reported a
mean gestational length of 332.4 ± 12.1 days in Carthusian broodmares, which was
significantly prolonged (5.7 days) when mares give birth to colts (336.8 ± 11.0 days)
rather than fillies (331.1 ± 12.8 days). In our study we found a longer length of
gestation for colts of 2 days. The range is not that big as in the study above, but
showed a significant longer length of gestation for colts rather than fillies as well.
Satuè et al., (2011) also found a 5.3 days shorter pregnancy in mares 8 to 12 years
(330.8 ± 11.0 days) old in contrast to those of 13 to 17 years (336.1 ± 8.9 days). This
is also in agreement with our results, that mares (>10 years old) are significantly
longer in foal than younger mares. Younger mares (≤7 years old) exhibit a
significantly shorter gestational length (- 4 days) than mares older than 17 years.
The comparison of ET and Non – ET foals resulted in a difference of gestational
length. ET foals showed a shorter length of gestation than Non – ET foals. Although
there are high standard errors in the present study, it is obvious, that the season (=
month of birth) shows a significant influence on length of gestation, which decreases,
when the foals are born in late summer or autumn. But there is no clear trend
66
between the years. Maybe there is a hormonal influence on the foal due to
decreasing daylight, triggering the birth.
Concerning withers height at birth no significant difference was detected between ET
and Non – ET foals. However, there was a significant difference regarding the gender
of the foal. Colts were 0.7 cm taller than fillies, maybe due to a significant longer
length of gestation. Satuè et al., (2011) found also a significant influence of the
mare’s age on withers height at birth, agreeing with our results, showing that younger
mares (≤ 6 years old) and mares, older than 16 years, give birth to smaller foals.
There is a significant influence of the mare’s age with an increasing height until the
age of mare of 9, 10, 11, but decreasing in mares older than 11 years old.
No significant influence could be detected between ET and Non-ET foals regarding
withers height at birth as well as the weight at birth.
With respect to the weight at birth Satuè et al., (2011) found younger mares (≤ 6
years) foaling offspring with little weights compared with mares older than eight years
foaling heavier offspring. Cilek (2009) investigated Purebred Arabian foals and found
a significant influence of gender of the foal on the birth weight as well. The weight at
birth was 46.27 ± 0.40 kg for colts, whereas fillies weighted 45.22 ± 0.40 kg at birth.
As reported by Elliot et al., (2009) who found a significant difference in birth weights
of 1.1 kg between colts and fillies, we found results of higher birth weights in colts of
1.4 kg. Likewise, Elliot et al., (2009) found an increase of the weight at birth with an
increase of the mare’s age up to 12 years. Compared to our results we found an
increase of the weight at birth up to an age of the mare of 9 years, followed by a light
decrease in weight at birth for older mares. Further research is requested to confirm
these promising results.
67
CONCLUSION
Considering the first pregnancy determination after embryo transfer in the recipient
mare, significant effects of the season, the preparation of the recipient mare and the
age of the embryo at time of transfer could be found.
The comparison of embryo transfer foals and non – embryo transfer foals resulted in
a significant difference concerning the length of gestation, but there was no
significance detected for the traits withers height at birth and weight at birth.
The age of the mare at time of birth played in important role, with regard to all three
traits. Younger mares exhibited a shorter gestational length, a smaller withers height
at birth and a lighter weight at birth of the foals.
For the improvement of the procedure embryo transfer and the number of resulting
foals, the management of the recipient is a useful approach in account with the
season and the age of the embryo at time of transfer. No negative influences on
vitality or other traits could be found in ET foals.
A further step should be to monitor the development of the embryo transfer and non
embryo transfer foals, regarding withers height, the weight and fertility, especially for
embryo transfer foals.
REFERENCES
[1] Squires, E. L., McCue, P. M. and Vanderwall, D.; The current status of equine
embryo transfer. Theriogenology 1999; 51: 91-104.
[2] Squires, E. L. and Seidel Jr., G. E.; Collection and transfer of equine embryos.
Animal Reproduction and Biotechnology Laboratory Bulletin No. 11. Fort
Collins CO: Colorado State University, 1995; 7-9, 11-15, 27-32.
[3] McKinnon, A. O., Squires, E. L., Voss, J. L. and Cook, V. M.; Equine embryo
transfer: A review. Comp. Cont. Educ. Pract. Vet. 1988; 10: 343-355.
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[4] Iuliano, M. F., Squires, E. L. and Cook, V. M.; Effect of age of equine embryos and
method of transfer on pregnancy rate. Journal of Animal Science 1985; Vol.
60, No. 1: 258-263.
[5] Squires, E. L., Carnevale, E. M., McCue, P. M. and Bruemmer, J. E.; Embryo
technologies in the horse. Theriogenology 2002; 59: 151-170.
[6] Satué, K., Felipe, M., Mota, J. and Muñoz, A.; Gestational length in Carthusian
broodmares: effects of breeding season, foal gender, age of mare, year of
parturition, parity and sire. Polish Journal of Veterinary Science 2011; Vol. 14,
No. 2: 173-180.
[7] Allen, W. R., Wilsher, S., Turnbull, C., Stewart, F., Ousey, J., Rossdale, P. D. and
Fowden, A. L.; Influence of maternal size on placental, fetal and postnatal
growth in the horse. I. Development in utero. Reproduction 2002; 123: 445453.
[8] Valera, M., Blesa, F., Dos Santos, R. and Molina, A.; Genetic study of gestation
Vol. 95, Issue 1-2: 75-96.
[9] Squires, E. L., Imel, K. J. and Iuliano, M.F.; Factors affecting reproductive
efficiency in an equine embryo transfer program. J. Reprod. Fertil. 1982;
32(Suppl.): 409-414.
[10] Squires, E. L., Garcia, R. H. and Ginther, O.J.; Factors affecting success of
equine embryo transfer. Equine Vet. J. 1985; 3(Suppl.): 92-95.
[11] Squires, E. L., Cook, V. M. and Voss, J. L.; Collection and transfer of equine
embryos. Colorado State University Animal Reproduction and Biotechnology
Lab. Bulletin 1985; 1: 16-25.
[12] Carney, N. J., Squires, E. L., Cook, V. M., Seidel Jr., G. E. and Jasko, D. J.;
Comparison of pregnancy rates from transfer of fresh versus cooled,
transported equine embryos. Theriogenology 1991; Vol. 36, No. 1: 23-32.
[13] Villahoz, M. D., Squires, E. L., Voss, J. L. and Shideler, R. K.; Some
observations on early embryonic death in mares. Theriogenology 1985; 23:
915-924.
[14] Allen, W. R., Wilsher, S., Tiplady, C. and Butterfield, R. M.; The influence of
maternal size on pre- and postnatal growth in the horse: III Postnatal Growth.
Reproduction 2004; 127: 67-77.
[15] Ousey, J. C., Rossdale, P.D., Fowden, A. L., Palmer, L., Turnbull, C. and Allen,
W. R.; Effects of manipulating intrauterine growth on post natal adrenocortical
69
development and other parameters of maturity in neonatal foals. Equine Vet.
J. 2004; 36: 616-621.
[16] Imel, K. J., Squires, E. L., Elsden, R. P., Shideler, R. K.; Collection and transfer
of equine embryos. J. Amer. Vet. Med. Assoc. 1981; 179-987.
[17] Hinrichs, K. and Kenney, R. M.; Effect of timing of progesterone administration
on pregnancy rate after embryo transfer in ovariectomized mares. J. Reprod.
Fertil. 1987; Suppl. 35: 439-443.
[18] Wilson, J. M., Rowley, M. B., Rowley, W. K., Smith, H. A., Webb, R. L. and
Tolleson, D. R.; Successful non-surgical transfer of equine embryos to postpartum mares. Theriogenology 1987; 27: 295. (Abstr.)
[19] Cilek, S.; The survey of reproductive success in Arabian horse breeding from
1976-2007 at Anadolu State farm in Turkey. J. Anim. Vet. Adv. 2009; 8: 389396.
[20] Riera, FL., and McDonough, J.; Commercial embryo transfer in polo ponies in
[21] Vogelsang SG, Bondioli, KR and Massey, JM.; Commercial application of equine
embryo transfer. Equine Vet. J. 1985; (3): 89-91.
[22] Elliot, C., Morten, J., Chopin, J.; Factors affecting foal birth weight in
Thoroughbred horses. Theriogenology 2009; 71: 683-689.
70
General Discussion
The aim of the present study was to determine which factors show a significant
influence on the ovulation rate (OR), embryo recovery rate (RE) and the pregnancy
rate in the recipient mare after embryo transfer. Furthermore the estimation of
heritabilities for OR and RE should show whether the considering of fertility
parameters in breeding programs makes sense. The comparison of embryo transfer
foals and non embryo transfer foals should detect, whether embryo transfer foals
have any disadvantages compared to non embryo transfer foals.
In our study we achieved a success rate of 0.54 embryos per flush over all donor
mares (712 mares). This value is in agreement with Douglas [2] (34% vs. 56%),
Squires et al. [1] (28% vs. 80%) and Vogelsang and Vogelsang [24] (29% vs. 53 61%) who got probabilities to flush an embryo ranging from 0.28 to 0.34 in old or
subfertile donor mares in contrast to a recovery rate of 0.53 to 0.80 in young fertile
mares. Several summaries based on data from commercial operations have reported
a 50 % embryo recovery rate from single – ovulating mares, some of which were
subfertile mares [7,38]. There was a wide variety of donor mares at the stud,
regarding the age and fertility. Hence, a success rate of 54% overall donor mares is a
acceptable result.
Despite horses belong to the monovular species, there are reports that multiple
ovulations occur with frequencies ranging between 4 and 43 % [32,33]. In our study
we found a probability to recover at least two ovulations of 1.22 ± 0.50, which was
22%. This is in agreement with the literature, where different percentage of multiple
ovulations for different breeds are reported. The frequency of multiple ovulations in
draft horses and Thoroughbreds ranges from 15 and 30 %, whereas that in light
breeds and ponies showed a lower rate of around 2 to 10 % [32,33]. Another way to
71
increase the number of ovulation is the procedure of superovulation, but it is still
being difficult and generally embryos result from only the normally developing one, or
sometimes two, dominant follicles per cycle. With a view to cattle, successful
superovulating in donor mares would highly increase the efficiency of equine embryo
transfer. The main difficulty is that not every mare responds significantly to
superovulation treatment such as immunization to inhibin, administration of pituitary
extract or pulsatile gonadotropin releasing hormone. Today there is no product for
superovulation that gains widespread acceptance for clinical use in equine embryo
transfer. Thus, most of the mares in an embryo transfer program are currently not
stimulated, and only single embryo collections are attempted. As shown above the
probability for obtaining at least two ovulations were 22 % followed by a recovery rate
of at least two embryos of 10%, so there is a loss of 12% between time of ovulation
and flushing. Despite a higher ovulation rate in the donor mare, more than half of all
ovulated ova do not result in an embryo after insemination. In most breeds, a low
recovery rate due to the lack of multiple ovulation increases the time required to
obtain a pregnancy in the recipient mare. This is associated with raising costs for the
whole procedure of equine embryo transfer. Double or triple ovulations during a given
cycle would result in higher embryo recovery rates than are currently possible from
single – ovulating mares [21].
The age of the donor mare had a significant influence on the ovulation rate (OR).
With an increasing age of the donor mare, the number of ovulations also increased.
Donor mares >15 years showed a decreased probability to recover an embryo; those
donor mares provide significantly smaller numbers of embryos. This is in agreement
with Vogelsang and Vogelsang [24] who reported significant differences in embryo
recovery rates in younger mares (2 to 8 and 9 to 17-year-old) compared with older
mares (18 to 28-year-old), (80/132 [60.6%], 94/183 [51.4 %] and 93/309 [30.1%],
72
respectively). But there is an antagonism between OR and RE regarding the age of
the donor mare: Although there is an increase of OR with an increased age of the
donor mare, there is a lower RE at an increased age of the donor mare. Causes of
reduced embryo recovery from these older mares included uterine and oviductal
pathology, and increased early embryonic death [15]. Carnevale et al. [39] reported
that defective oocytes from aged mares were a cause of reduced fertility. Vidament et
al. [42] reported that the breeding status and the management of the mare during the
estrus period as well as the quality of the semen are the most important factors for
achieving a pregnancy, especially when using frozen semen. Furthermore many
other studies found that the reproductive history is one of the main factors that have
to be considered when using a mare as a donor mare for embryo transfer
[1,19,24,39,38]. Number of embryos decreased with a poor reproductive history and
higher ages of the donor mare. Samper et al., [44] reported that the number of older
maiden mares (>8 years) with a prolonged sport or show career used for breeding is
steadily increasing. Especially when bred with frozen semen, these mares had lower
pregnancy rates due to histories of reproductive problems and were therefore
potential candidates for failure.
In our study, we generated three groups for the breeding status of the donor mare,
showing a different influence on both traits (OR and RE). Whereas the ‘status’ was
significant for the trait OR, there was no significant effect on RE. OR was significantly
lower, when the donor mare was pregnant (‘status’ 1) before flushing. There was no
significant effect on RE, but a slight positive tendency, that donor mares with ‘status’
3 showed a higher probability to recover an embryo than the other ones. This
suggests that a pregnancy before flushing influences a donor mare in a negative way
regarding the number of ovulations. The use of donor mares two years in succession
shows no negative effect concerning the reproductive performance of the mares.
73
Vanderwall [46] reported similar results with very high (>90%) fertilization rates in
young mares under optimal conditions (i.e., fresh, fertile semen) versus older mares
that showed a lower pregnancy rate of 85%. Although high fertilization rates were
achieved, embryo recovery rates on days 6 to 9 after ovulation were distinctly lower
for older mares [47,24], which indicates high embryonic losses during the first week
of gestation. In other studies, the estimated embryonic loss rate between fertilization
and day 14 was lower than 10% for young mares but up to 70% for older mares
[48,15].
The type and the quality of semen also affect embryo recovery. The use of fresh
semen resulted in a higher recovery rate compared to frozen semen. The fertility of
(frozen) semen is influenced by several factors including semen quality, stallion,
insemination dose, mare selection and mare management. It is well accepted that
there is a variation in semen freezability among service stallions and further between
ejaculates from the same stallion. Pregnancy rates obtained with frozen semen in
commercial programs ranged from 32 to 73% per cycle [49] and from 26% [50,51] to
66% [52] per season with most values vary from 30 to 55% for the seasonal success
rate [54,53,39]. The results of our study agreed with findings reported in the literature.
For the use of fresh semen, we obtained a probability of 0.52 to recover at least one
embryo, whereas probabilities for using frozen semen and frozen semen, which was
gained on different dates were 0.43 and 0.36, respectively. This verifies a significant
influence on the embryo recovery rate, regarding the type of semen used for
insemination of the donor mare. For flushing donor mares, insemination with fresh
semen is always preferable because of significant higher embryo recovery rates. But
if fresh semen is not available it has to pay special attention to the management of
the donor mare for the insemination with frozen semen to get acceptable results.
74
Considering the type of semen used for insemination, differences between fresh
semen and frozen semen with respect to fertility could be found. A Disadvantage of
frozen semen are lower fertility for many stallions [49]. The processing of the semen
is often done by owners and not by professionals, which explained a wide variability
in quality of the transported semen. In contrast to fresh semen, there is more
technical expertise needed for processing frozen semen. Furthermore there are
some stallions producing ejaculates, which are not suitable for cooling or storage.
One explanation for the reduced pregnancy rate after insemination with frozen semen
was reported by Dobrinski et al. [55] and Ellington et al. [56]. They demonstrated that
frozen semen of some stallions does not bind to the oviductal epithelium of the mare
in a normal way, reducing the ability of the mare to create reserves of spermatozoa
within the reproductive tract. These reserves guarantee that survivable spermatozoa
are available in the oviduct at the time of ovulation, when insemination happened
before the mare ovulates.
For investigating the number and motility of spermatozoa only fresh semen was
considered, because there was no information about it for frozen semen. Motility
showed a significant influence on the embryo recovery rate, whereas the number of
spermatozoa did not show a significant influence. The use of high motile
spermatozoa (≥65%) resulted in 59% in at least one embryo, whereas the use of
spermatozoa with a lower motility (≤45%) resulted in a success rate of 47%. Hence,
the important fact to recover at least one embryo is the use of high motile
spermatozoa (≥65 %) for insemination. This is in agreement with the results of Varner
and Blanchard (unpublished) reported by Brinsko [57]. They found a pregnancy rate
per cycle of only 48% for fresh semen, when the progressive sperm motility was
<60%. In the case of using >60 % motile sperm, the pregnancy rate per cycle was
67%.
75
The number of ovulations as well as recovered embryos is higher in spring and early
summer than in late summer and autumn. It is generally known that the reproduction
cycle of horses starts naturally when daylight is extending. The time of mating and
giving birth in the horse is spring and early summer. Our results for OR as well as RE
were in agreement with this facts. Hence, spring is naturally the best time to start
embryo recovery in the year.
The preparation of the donor mare enables a better timing of insemination as well as
a better timing of transfer in the recipient mare. Application of heat- and ovulation
inducing agents makes the recovery and the transfer easier to schedule, because of
knowing a narrow time slot when ovulation will occur. Therefore the number of
recovered embryos are higher in treated mares than in mares that got no treatment at
all.
Considering the running number of flushing within seasons, there is no significant
difference between different flushes concerning the number of recovered embryos.
This is contradictory to the result obtained from ovulation rate; number of ovulations
increases with the increasing of number of flushing, so we would expect a higher
number of recovered embryos. This may fail due to poorer quality of the ovulated
oocytes. There was also no consistency within donor mares over the years. If a donor
mare provided many embryos in one year, it was no guarantee, that she did it again
next season.
Generally the flushing of donor mares ranged from day 6 to day 9 after ovulation [41].
A day nine embryo, however, has grown to a size, where pregnancy rates after
transfer may be decreased, potentially by damaging the embryo during handling. In
this study no significant differences could be detected by flushing donor mares at day
6 to 10 after ovulation. A possible reason for the consistent performance may be the
experienced veterinarians who performed the flushes.
76
Heritabilities and correlation for fertility traits in the horse were not estimated until yet.
In the case of ET, it is important to consider the complexity of various traits and
influencing factors; knowledge about their variance and covariance components for
maternal and paternal effects is of basic interest for the genetic improvement in horse
breeding and was not investigated in previous equine studies. As expected for fertility
parameters heritabilities for both traits were low, although the heritability for OR was
a little bit higher than for RE in the donor mare. The slightly higher heritability of the
stallion on the trait RE was a little bit surprising, we imagined that the donor mares’s
influence would be higher. The correlation between RE and OR showed a slight
positive trend, whereas genetic correlations between maternal and paternal
components show a slightly negative tendency, indicating a genetic antagonism.
Although this data set is very big for the equine domain, high standard errors indicate
that there may be a need for a larger data set. In cattle, genetic correlations between
maternal and paternal effects for most fertility traits are reported to be negative [27].
As König et al. [26] demanded there is a need for studies revealing the physiological
mechanisms for the phenomenon of slightly negative correlations between maternal
and paternal effects for traits related to ET in the future not only for cattle but also for
horses.
A large variation in pregnancy rates was found after non surgical transfer by different
authors ranging from 50 to 75 % [7, 19, 38, 69]. This is in agreement with our results
showing a positive result of PD I of 66.7 % and a success rate of 51.5% regarding the
living born foals. Also lower pregnancy rates were found after performing nonsurgical
transfer (27% vs. 53%, [65]; 45% vs. 72%, [58]) as well as other authors reported
acceptable pregnancy rates after nonsurgical transfer like 75 to 77% (66; 67).
No significant differences in PD I were detected depending on the age of the
recipient mare at time of transfer. This is in contrast to the influence of the age on
77
embryo recovery rate in donor mares founded in previous studies, where mares older
than 16 years donate fewer numbers of embryos. Hence, the ability to become
pregnant is independent of the age of the recipient mare in contrast to the production
of an embryo, which depends on the age of the donor mare.
Regarding the season, there is a significant influence on PD I. Higher numbers of
positive PD I were achieved in spring and early summer, but generally decreased in
midsummer. In spite of the fact, there is no clear trend between the years. It is
generally known that the reproduction cycle of horses starts naturally when daylight is
extending. Hence, spring and early summer are the best time to achieve acceptable
pregnancy rates after embryo transfer. For the years 2005, 2006 and 2008 a rise of
positive pregnancy determinations in late summer and autumn was found. This
increase may be depending on the weaning of the foals, because mares with no foal
at foot are often more likely to become pregnant.
The treatment of the recipient mare differs only between recipient mares that got no
treatment at all and processed mares independent of the treatment were heat
inducing agents or ovulation inducing agents. Treated mares showed higher numbers
of positive PD I than untreated mares. The management of the recipient mare plays
an important role and therefore the timing and detection of the ovulation is of great
importance for the success of embryo transfer. In treated mares this is more
predictable than in untreated mares.
Regarding the age of the embryo at time of transfer, best pregnancy rates were
obtained when transferring Day 7 embryos or Day 8 embryos. Day 9 embryos
resulted in significant lower pregnancy rates due to potential damage during the
flushing and handling before transfer. This result is in agreement with Squires et al.,
[63] and Iuliano et al., [58], who reported reduced pregnancy rates after the transfer
of large embryos. Important for the freezing of embryos (flushing at day 6 after
78
ovulation), day 6 embryos showed no significant differences regarding the pregnancy
rate compared to day 7, day 8 and day 9 embryos. Especially for frozen embryos the
viability and therefore acceptable pregnancy rates plays an important role, because
of the forces of the freezing and thawing process.
Due to individual variation of the heat length, the exact timing of ovulation in donor
and recipient is, however, difficult. An approach to handle estrus synchrony of donor
and recipient is the employment of induced ovulation in the recipient once an
ovulation has been detected in the donor mare. The highest initial pregnancy rates of
>75% at day 12 and around 65% at day 50 will be achieved when the recipient
mare’s ovulation occurs within a time range of two days before to three days after the
donor mare’s ovulation (19; 64). Even though some investigators found a
substantially influence of estrus synchrony on the success of embryo transfer, i.e. the
pregnancy rate (1; 19; 38; 62; 63), in our study no significant influence of synchrony
of donor and recipient could be detected. Recipient mares ovulated in a range of zero
days up to five days after the donor mare, followed by comparable pregnancy rates
after embryo transfer.
No variation between the recipient mares could be found, due to the failure of
generating a value for the covariance parameter estimates.
Comparing colts and fillies revealed in significant differences in all analyzed traits.
The comparison of embryo transfer and non embryo transfer foals showed only a
significant influence with respect to the length of gestation but not to withers height at
birth and weight at birth. No interaction was found between the type of the foal (ET
foal/Non – ET foal) and the gender.
Concerning withers height at birth no significant difference was detected between ET
and Non – ET foals. However, there was a significant difference regarding the gender
of the foal. Colts were 0.7 cm taller than fillies, maybe due to a significant longer
79
length of gestation. Satuè et al., [59] found also a significant influence of the mare’s
age on withers height at birth, agreeing with our results, showing that younger mares
(≤ 6 years old) and mares, older than 16 years, give birth to smaller foals. There is a
significant influence of the mare’s age with an increasing height until the age of mare
of 9, 10, 11, but decreasing in mares older than 11 years old.
Cilek [68] investigated Purebred Arabian foals and found a significant influence of
gender of the foal on the birth weight as well. The weight at birth was 46.27 ± 0.40 kg
for colts, whereas fillies weighted 45.22 ± 0.40 kg at birth. Satuè et al., [59] found
younger mares (≤ 6 years) foaling offspring with little weights compared with mares
older than eight years foaling heavier offspring. As reported by Elliot et al., [70] who
found a significant difference in birth weights of 1.1 kg between colts and fillies, we
found results of higher birth weights in colts of 1.4 kg. Likewise, Elliot et al., [70]
found an increase of the weight at birth with an increase of the mare’s age up to 12
years. Compared to our results we found an increase of the weight at birth up to an
age of the mare of 9 years, followed by a light decrease in weight at birth for older
mares.
The length of gestation is influenced by the gender of the foal as well as the type of
the foal (embryo transfer/non embryo transfer). Allen et al., (60) found a mean
duration of gestation of 339.0 ± 3.0 days in Thoroughbred mares. Valera et al., [61]
found mean length of gestation of 336.8 ± 0.48 days in Andalusians or Spanish
Purebreds and 340.3 ± 0.63 days in Arabian mares. They found that two of the main
factors significantly affecting gestational length are the age of the mother and the sex
of the foal. Satuè et al., [59] reported a mean gestational length of 332.4 ± 12.1 days
in Carthusian broodmares, which was significantly prolonged (5.7 days) when mares
give birth to colts (336.8 ± 11.0 days) rather than fillies (331.1 ± 12.8 days). In our
study where we found a longer length of gestation for colts of 2 days. The range is
80
not that big as in the study above, but showed a significant longer length of gestation
for colts rather than fillies as well. Satuè et al., [59] also found a 5.3 days shorter
pregnancy in mares 8 to 12 years (330.8 ± 11.0 days) old in contrast to those of 13 to
17 years (336.1 ± 8.9 days). This is also in agreement with our results, that mares
(>10 years old) are significantly longer in foal than younger mares. Younger mares
(≤7 years old) exhibit a significantly shorter gestational length (- 4 days) than mares
older than 17 years.
The comparison of ET and Non – ET foals resulted in a difference of gestational
length. ET foals showed a shorter length of gestation than Non – ET foals. Although
there are high standard errors in the present study, it is obvious, that the season (=
month of birth) shows a significant influence on length of gestation, which decreases,
when the foals are born in late summer or autumn. But there is no clear trend
between the years. Maybe there is a hormonal influence on the foal due to
decreasing daylight, triggering the birth.
There are a lot of factors that influence the procedure embryo transfer. It would be
desirable, if there will be further research, especially with regard to the development
of the foals, to confirm these results.
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[39] Carnevale, E. and O. Ginther. Defective oocytes as a cause of subfertility in old
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[41] Squires, E. L., Mc Cue, P.M. and Vanderwall, D. The current status of equine
embryo transfer. Theriogenology 1999;51:91-104.
[42] Vidament, M., Dupere, A. M., Julienne, P., Evain, A., Noue, P. and Palmer, E.;
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[44] Samper, J. C., Hearn, P. H. and Ganheim, A. (1994). Pregnancy rates and effect
of extender and motility and acrosome status of frozen-thawed stallion
spermatozoa. Proceedings of the 40th Ann. Conv. Amer. Assoc. Equine Pract.
[45] Sanchez, R., Gomez, I. and Samper, J. C. (2008). Artificial insemination with
frozen semen. Equine Breeding Management and Artificial Insemination J. C.
Samper, Saunders: 175-185.
[46] Vanderwall, D.; Early Embryonic Loss in the Mare. Journal Of Equine Veterinary
Science 2008; 28(11): 691-702.
[47] Woods, G. L., Hillman, R. B. and Schlafer, D. H.; Recovery And Evaluation Of
Embryos From Normal And Infertile Mares. Cornell Veterinarian 1986; 76:
386-394.
[48] Ball, B. A., Little, T. V., Hillman, R. B. and Woods, G. L.; Pregnancy Rates At
Day-2 And Day-14 And Estimated Embryonic Loss Rates Prior To Day 14 In
Normal And Subfertile Mares. Theriogenology 1986; 26: 611-619.
[49] Loomis, P. R.; The equine frozen semen industry. Animal Reproduction Science
2001; 68: 191-200.
[50] Klug, E., Treu, H., Hillmann, H. and Heinze, H.; Results Of Insemination Of
Mares With Fresh And Frozen Stallion Semen. Journal of Reproduction and
Fertility 1975: 107-110.
[51] Tischner, M.; Results Of Artificial-Insemination Of Horses In Poland In Post-War
Period. Journal of Reproduction and Fertility 1975: 111-114.
[52] Salazar-Valencia, F.; Embryo recovery rates in mares of the pasofino
Colombiano breed and deep freezing stallion semen in the tropics.
Theriogenology 1983; 19: 146-146.
[53] Palmer, E. and Magistrini, M.; Automated analysis of stallion semen post-thaw
motility. Acta veterinaria Scandinavica. Supplementum 1992; 88: 137-52.
[54] Samper, J. C., Hellander, J. C. and Crabo, B. G.; Relationship between the
fertility of fresh and frozen stallion semen and semen quality. Journal of
reproduction and fertility. Supplement 1991; 44: 107-14.
[55] Dobrinski, I., Thomas, P. G. A. and Ball, B. A.; Cryopreservation reduces the
ability of equine spermatozoa to attach to oviductal epithelial cells and zonae
pellucidae in vitro. Journal of Andrology 1995; 16: 536-542.
[56] Ellington, J. E., Samper, J. C. and Wright, W. R. (2000). Stallion sperm
interactions with oviduct cells in vitro as an indicator of field fertility.
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Stockholm, Sweden.
[57] Brinsko, S. P.; Insemination doses: How low can we go? Theriogenology 2006;
66: 543-550.
[58] Iuliano, M. F., Squires, E. L. and Cook, V. M.; Effect of age of equine embryos
and method of transfer on pregnancy rate. Journal of Animal Science 1985;
Vol. 60, No. 1: 258-263.
[59] Satué, K., Felipe, M., Mota, J. and Muñoz, A.; Gestational length in Carthusian
broodmares: effects of breeding season, foal gender, age of mare, year of
parturition, parity and sire. Polish Journal of Veterinary Science 2011; Vol. 14,
No. 2: 173-180.
[60] Allen, W. R., Wilsher, S., Turnbull, C., Stewart, F., Ousey, J., Rossdale, P. D.
and Fowden, A. L.; Influence of maternal size on placental, fetal and postnatal
growth in the horse. I. Development in utero. Reproduction 2002; 123: 445453.
[61] Valera, M., Blesa, F., Dos Santos, R. and Molina, A.; Genetic study of gestation
Vol. 95, Issue 1-2: 75-96.
[62] Squires, E. L., Garcia, R. H. and Ginther, O.J.; Factors affecting success of
equine embryo transfer. Equine Vet. J. 1985; 3(Suppl.): 92-95.
[63] Squires, E. L., Cook, V. M. and Voss, J. L.; Collection and transfer of equine
embryos. Colorado State University Animal Reproduction and Biotechnology
Lab. Bulletin 1985; 1: 16-25.
[64] Carney, N. J., Squires, E. L., Cook, V. M., Seidel Jr., G. E. and Jasko, D. J.;
Comparison of pregnancy rates from transfer of fresh versus cooled,
transported equine embryos. Theriogenology 1991; Vol. 36, No. 1: 23-32.
[65] Imel, K. J., Squires, E. L., Elsden, R. P., Shideler, R. K.; Collection and transfer
of equine embryos. J. Amer. Vet. Med. Assoc. 1981; 179-987.
[66] Hinrichs, K. and Kenney, R. M.; Effect of timing of progesterone administration
on pregnancy rate after embryo transfer in ovariectomized mares. J. Reprod.
Fertil. 1987; Suppl. 35: 439-443.
[67] Wilson, J. M., Rowley, M. B., Rowley, W. K., Smith, H. A., Webb, R. L. and
Tolleson, D. R.; Successful non-surgical transfer of equine embryos to postpartum mares. Theriogenology 1987; 27: 295. (Abstr.)
84
[68] Cilek, S.; The survey of reproductive success in Arabian horse breeding from
1976-2007 at Anadolu State farm in Turkey. J. Anim. Vet. Adv. 2009; 8: 389396.
[69] Riera, FL., and McDonough, J.; Commercial embryo transfer in polo ponies in
[70] Elliot, C., Morten, J., Chopin, J.; Factors affecting foal birth weight in
Thoroughbred horses. Theriogenology 2009; 71: 683-689.
85
86
General Summary
Equine embryo transfer includes the insemination and the following embryo recovery
of the donor mare as well as the transfer into the recipient mare. The recipient takes
over the pregnancy and the raising of the foal. Due to numerous process steps,
there are several influencing factors, concerning the success of embryo transfer in a
positive or negative way. In our study we investigated possible influencing factors,
affecting the traits “number of ovulations” and “number of recovered embryos” in the
donor mare. The influencing factors on the pregnancy rates after transfer in the
recipient mare were investigated as well as the number of living born foals. Embryo
transfer foals were compared to non embryo transfer foals regarding the length of
gestation, withers height at birth and weight at birth. Also a comparison between colts
and fillies was done, with respect to this traits. The influence of the age of the
(recipient) mare with regard to the same traits was also investigated. Additionally, the
influence of the season was tested for the trait “length of gestation”. Furthermore
heritabilities and correlations for fertility parameters “number of ovulations” and
“number of recovered embryos” were estimated.
In Chapter One the following influencing factors with regard to the probability for a
successful flushing (recovery of at least one embryo) were investigated: age of the
donor mare at time of flushing, the month, where flushing is performed, the
preparation of the donor mare prior to the flushing (application of heat-/ovulation
inducing agents) as well as the running number of flushing within donor mare and
season. No significant differences could be detected at an donor mare’s age between
2 and 15 years. Only donor mares (>15 years old) showed a significant lower number
of recovered embryos.
87
With respect to the season (month of flushing) the highest probability for a successful
flushing was found in spring and early summer. The success rate decreased, if the
flushing was performed in late summer and autumn.
Regarding the preparation of the donor mare, a significant higher number of
recovered embryos were obtained after application of ovulation inducing agents, with
or without a prior application of heat inducing agents.
The running number of flushes within donor mare and season showed no significant
influence on the embryo recovery rate.
As expected for fertility parameters, heritabilities the for embryo recovery rate were
low (3.7% for the donor mare and 4.5% for the service stallion). The service stallion
showed a slightly higher influence on the success of flushing than the donor mare.
Additionally following influencing factors with regard to the probability of a successful
flushing were investigated in Chapter Two: type of semen (fresh or frozen semen),
motility of the spermatozoa as well as the number of spermatozoa and the breeding
status of the donor mare. The use of fresh semen resulted in a significant higher
number of recovered embryos, than insemination with frozen semen. Furthermore
utilization of spermatozoa with a motility of >65% resulted in significant higher
embryo recovery rates as well. The number of spermatozoa as well as the breeding
status of the donor mare showed no significant influence on the success of flushing.
Chapter Three contains the investigation of the following influencing factors with
respect to positive pregnancy determination around 9 days after transfer in the
recipient mare. The age of the recipient mare at time of transfer showed no
significant influence on the pregnancy rate.
Despite of some differences between the years, spring was the most suitable time to
obtain a positive pregnancy result after embryo transfer.
88
The preparation of the recipient mare showed significant higher pregnancy rates in
treated recipients, than in recipients that got no treatment at all.
The age of the embryo at time of transfer resulted in significant higher pregnancy
rates for 7 and 8 day old embryos, than for those who were 9 days old.
The comparison of embryo transfer to non embryo transfer foals resulted only in a
significant shorter length of gestation (0.7 days) for embryo transfer foals. Withers
height at birth and weight at birth showed no significant differences between both
groups. Also there was no interaction between the “type” of the foal (embryo
transfer/non embryo transfer) and the gender of the foal. However, the gender of the
foal showed a significant influence on length of gestation, withers height at birth as
well as weight at birth. Length of gestation was in colts 2 days longer, colts were 0.7
cm taller and 1.4 kg heavier at birth.
The age of the mares at time of birth resulted in the following significant differences:
Mares (>16 years old) had a longer pregnancy than younger mares. Mares (<6years
old and >16 years old) gave birth to smaller foals (lower withers height at birth).
Furthermore the foals of mares (<8 years old) showed lower weight at birth.
Due to low heritabilities for fertility parameters on the one hand and breeding goals
for sport horses (improvement of dressage and/or jumping ability) on the other hand,
the practical use for breeding is limited. The improvement of the management of
equine embryo transfer should be the focus of future action .
89
90
Zusammenfassung
Das Verfahren Embryo Transfer beim Pferd setzt sich aus der Besamung und der
folgenden
Gewinnung
der
Embryonen
aus
der
Spenderstute
sowie
dem
anschließenden Transfer auf die Empfängerstute zusammen, die dann die
Trächtigkeit und die Aufzucht des Fohlens übernimmt. Aufgrund der zahlreichen
Verfahrensschritte gibt es viele Faktoren, die den Erfolg des Embryo Transfer in
positiver oder negativer Weise beeinflussen. In dieser Arbeit wurden mögliche
Faktoren untersucht, die die Merkmale „Anzahl der Ovulationen“ und Anzahl der
gewonnen Embryonen“ in der Spenderstute beeinflussen können. Für die
Empfängerstuten wurden sowohl die Trächtigkeitswahrscheinlichkeiten nach dem
Transfer, inklusive der einwirkenden Faktoren ermittelt als auch die Anzahl
lebendgeborener Fohlen. Die Fohlen aus Embryo Transfer wurden mit den Fohlen,
die von ihren genetischen Müttern selbst ausgetragen wurden, hinsichtlich
Trächtigkeitsdauer, Widerristhöhe bei Geburt und Geburtsgewicht verglichen.
Ebenso fand ein Vergleich in diesen Merkmalen zwischen Hengst- und Stutfohlen
statt. Der Einfluss des Alters der (Empfänger-) Stute auf diese Merkmale wurde
gleichfalls untersucht. Das Merkmal Trächtigkeitsdauer wurde zusätzlich noch auf
den Einfluss der Saison untersucht. Weiterhin wurden Heritabilitäten und
Korrelationen für die Fruchtbarkeitsmerkmale „Anzahl der Ovulationen“ und „Anzahl
der gewonnenen Embryonen“ geschätzt.
In
Kapitel
Eins
wurden
folgende
signifikante
Einflussfaktoren
auf
die
Wahrscheinlichkeit einer erfolgreichen Spülung (Gewinnung von mindestens einem
Embryo) untersucht: Alter der Spenderstute zum Zeitpunkt der Spülung, der Monat,
in dem die Spülung durchgeführt wurde, die Vorbereitung der Spenderstute auf die
Spülung (Gabe von rosse- bzw. ovulationsindizierenden Wirkstoffen) sowie die
91
Anzahl der Spülungen einer Spenderstute innerhalb einer Saison. Keine signifikanten
Unterschiede in der Anzahl der gewonnenen Embryonen konnten bei Spenderstuten
im Alter von 2 bis 15 Jahren festgestellt werden. Lediglich Spenderstuten, die älter
als 15 Jahre waren, zeigten eine signifikant geringe Anzahl an gewonnenen
Embryonen. Die höchste Wahrscheinlichkeit einer erfolgreichen Spülung wurde in
den Frühlings- und frühen Sommermonaten festgestellt. Die Erfolgsrate sank, wenn
die Spülungen im späten Sommer und Herbst durchgeführt wurden. Die Vorbereitung
der Spenderstute auf die Spülung zeigte, dass Spenderstuten, die einen
ovulationsinduzierenden
rosseinduzierenden
Wirkstoff,
Wirkstoffs,
mit
oder
appliziert
ohne
vorheriger
bekamen
Gabe
signifikant
einen
höhere
Wahrscheinlichkeiten einer erfolgreichen Spülung aufwiesen, als Spenderstuten, die
nur rossestimuliert wurden bzw. gar nicht stimuliert wurden. Die Anzahl der
durchgeführten Spülungen pro Stute/Jahr zeigte keinen signifikanten Einfluss auf die
Embryonengewinnungsrate. Wie für Fruchtbarkeitsparameter zu erwarten war, lag
die Heritabilität für das Merkmal „Anzahl gewonnener Embryonen“ in
einem
niedrigen Bereich (3,7% für die Spenderstute und 4,5% für den Besamungshengst).
Der Besamungshengst zeigte somit einen etwas höheren Einfluss auf den Erfolg der
Spülung als die Spenderstute. Zusätzlich wurden in Kapitel Zwei folgende
Einflussfaktoren auf die Wahrscheinlichkeit einer erfolgreichen Spülung untersucht:
Art
des
zur
Besamung
der
Spenderstute
eingesetzten
Spermas
(Frisch-
/Tiefgefriersperma), die Vorwärtsbeweglichkeit der Spermien sowie die Anzahl der
Spermien und der Zuchtstatus der Spenderstute. Der Einsatz von Frischsperma
führte zu einer signifikant höheren Anzahl gewonnener Embryonen, als der
Gebrauch von Tiefgefriersperma. Weiterhin ergab der Einsatz von Sperma mit einer
Motilität von >65% eine signifikant höhere Wahrscheinlichkeit für eine erfolgreiche
Spülung. Keinen signifikanten Einfluss auf die Embryonengewinnungsrate zeigten
92
die
Anzahl
der
Spermien
und
der
Zuchtstatus
der
Spenderstute.
In Kapitel Drei wurden folgende Einflussfaktoren auf die Wahrscheinlichkeit einer
erfolgreichen
Trächtigkeit
der
Empfängerstute
untersucht:
Das
Alter
der
Empfängerstute zum Zeitpunkt des Transfers zeigte keinen signifikanten Einfluss auf
die Trächtigkeitsrate. Trotz gewissen Unterschieden zwischen den Jahren, zeigten
sich, die Frühlingsmonate am geeignetsten für eine erfolgreiche Trächtigkeit nach
Embryo Transfer. In der Vorbereitung der Empfängerstute auf den Transfer zeigte
sich, dass Empfängerstuten, die rosse- und/oder ovulationsstimuliert wurden,
signifikant höhere Trächtigkeitsraten aufwiesen, im Gegensatz zu denen, die nicht
stimuliert wurden. Das Alter des Embryos zum Zeitpunkt des Transfers zeigte
signifikant höhere Trächtigkeitsraten bei der Nutzung von sieben bzw. acht Tage
alten
Embryonen,
als
bei
Embryonen,
die
neun
Tage
alt
waren.
Der Vergleich zwischen „Embryo Transfer Fohlen“ und „Nicht Embryo Transfer
Fohlen“ ergab eine etwas kürzere Trächtigkeitsdauer (0,7 Tage) für die Embryo
Transfer Fohlen. Das Geburtsgewicht und die Größe bei der Geburt zeigten keine
signifikanten Unterschiede zwischen den beiden Gruppen. Ebenfalls konnte keine
Interaktion zwischen dem „Typ“ des Fohlens (Embryo Transfer/Nicht Embryo
Transfer) und dem Geschlecht des Fohlens festgestellt werden. Der Einfluss des
Geschlechts des Fohlens auf die Merkmale Trächtigkeitsdauer, Größe bei Geburt
und Geburtsgewicht war hingegen signifikant. Die Trächtigkeitsdauer war bei
Hengstfohlen im Mittel 2 Tage länger, die Hengstfohlen waren bei Geburt 0,7 cm
größer und 1,4 kg schwerer, als die Stutfohlen. Das Alter der Stuten zum Zeitpunkt
der Geburt zeigte folgende signifikante Unterschiede: Stuten (>16 Jahre alt) waren
länger tragend als jüngere Stuten. Stuten (<6 Jahre alt und >16 Jahre alt) bekamen
kleinere Fohlen (geringer Widerristhöhe bei Geburt). Weiterhin bekamen Stuten (<8
Jahre alt) Fohlen mit geringeren Geburtsgewichten.
93
Aufgrund der geringen Heritabilität für Fruchtbarkeitsmerkmale und der Zuchtziele
für Sportpferde (Verbesserung der Dressur- und /oder Springleistung) ist der
praktische, züchterische Nutzen begrenzt. Die Optimierung des Embryo Transfers
sollte im Management des Verfahrens liegen.
94
Danksagung
An dieser Stelle sollen die Personen erwähnt werden, die mich im Rahmen meiner
Promotion begleitet haben und zum Gelingen dieser Arbeit beigetragen haben.
Mein Dank gilt Herrn Prof. Dr. Georg Thaller für die Überlassung des sehr
interessanten Dissertationsthemas, die wissenschaftliche Unterstützung und die
Chance meine Ergebnisse auf nationalen und internationalen Workshops, Tagungen
und Kongressen zu präsentieren.
Mein besonderer Dank gilt Herrn Paul Schockemöhle für die Bereitstellung der Daten
dieser Arbeit, die kooperative Zusammenarbeit und die Möglichkeit ein dreiviertel
Jahr lang den Alltag auf Gestüt Lewitz erleben zu können.
Weiterhin bin ich Herrn Prof. Dr. Dr. h.c. mult. Ernst Kalm sehr verbunden für die
Begleitung durch die Zeit der Promotion und die Möglichkeit jederzeit „hippologische
Fragen“ stellen zu können.
Zu größtem Dank bin ich Dr. Eckhard Stamer verpflichtet, der wirklich jederzeit für
Fragen zur Verfügung stand und immer eine Lösung für statistische Probleme parat
hatte. Seine motivierende Art war stets ein großes Vorbild für mich.
Herrn Dr. Jens Tetens sei herzlich gedankt für sein „sehr spontanes Einspringen“
beim Korrigieren, für seine guten Englischkenntnisse, die zum Gelingen der Arbeit
wesentlich beigetragen haben und sein Einsatz für das Veröffentlichen der Artikel in
den Fachjournals.
Für die finanzielle Unterstützung im Rahmen eines Stipendiums danke ich sehr
herzlich der H. Wilhelm Schaumann Stiftung.
Herrn Dr. Marc Lämmer, Dr. Roberto Sanchez und „Gyn-Team“, sowie dem ganzen
Team des Gestütes Lewitz danke ich sehr für die tolle Zeit und die herzliche
Aufnahme.
95
Ich danke meinen „Wintermitbewohnerinnen“ Karina, Wiebke und Kristina für die
grandiose Zeit, die Planung der „Worldtour“, die zahlreichen Kinobesuche und die
tolle Freundschaft, die sich daraus entwickelt hat.
Ganz besonders bedanken möchte ich mich bei meinen beiden Bürokollegen
Gesche und Tobias. Es war eine phantastische Zeit mit Euch, die ich wirklich
vermisse.
Vielen Dank an alle Mitglieder des Instituts für Tierzucht und Tierhaltung; ich habe
mich im Haus immer sehr wohl gefühlt und es hat mir großen Spaß gemacht dort zu
arbeiten.
Der Campus Suite danke ich für das „rund-um-die-Uhr“ Bereitstellen von
Kaffespezialitäten und leckeren Snacks.
Für das „Cat and Cavia Sitting“ möchte ich mich ganz herzlich bei Marita und
Christiane bedanken; ohne Euch wäre das alles nicht so unkompliziert möglich
gewesen.
Meiner Familie gehört der größte Dank, insbesondere meinen Eltern, die mich immer
selbstverständlich unterstützt haben und so maßgeblich an dem Gelingen der Arbeit
beteiligt waren.
Zum Schluss möchte ich mich noch Maren, Christian, Claudia, Thekla-Karina,
Simone, Steffi, Kristina, Jens und Marco für eine tolle, sehr offene Freundschaft
bedanken.
96
GREFFENER STRASSE 1 • 33775 VERSMOLD
MOBIL 0173/2888505 • E-MAIL HENRYVDAHE@WEB.DE
HENRIK VON DER AHE
PERSÖNLICHE INFORMATIONEN
Familienstand: ledig
Nationalität: deutsch
Alter: 33
Geburtsort: Münster
Eltern: Horst von der Ahe (Apotheker)
Marianne von der Ahe (Pharmazeutisch-technische Assistentin)
AUSBILDUNG
1985 bis 1989 Kreuzschule (Grundschule), Münster
1989 bis 1998 Pascalgymnasium, Münster
1998 bis 1999 Zivildienst Raphaelsklinik GmbH, Münster
1999 bis 2001 Ausbildung zum Pferdewirt Schwerpunkt Zucht und Haltung,
Hessisches Landgestüt Dillenburg
2001 bis 2002 Studium der Pharmazie an der CAU Kiel (2 Semester)
2002 bis 2008 Studium der Agrarwissenschaften an der CAU Kiel,
2007 Abschluss Bachelor of Science
2008 Abschluss Master of Science
2008 bis 2011 Promotion am Institut für Tierzucht und Tierhaltung der
CAU Kiel
2011 bis heute Nachwuchsführungskraft bei Deutsche Reiterliche
Vereinigung e.V. (FN), Warendorf
97

Untersuchung zur Optimierung des Embryotransfers beim Pferd

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