260 HORMONAL PROFILE AND CALCIUM METABOLISM IN

Veterinária e Zootecnia
ISSN 0102-5716
260
HORMONAL PROFILE AND CALCIUM METABOLISM IN BITCHES DURING
GESTATION AND PUERPERAL PERIOD
Luiz Henrique de Araújo Machado 1
Nereu Carlos Prestes2
Marcelo Landim Brisola3
ABSTRACT
Hormone cycle and calcium metabolism ofbitches during gestation and puerperal period were
studied. Ten bitches were submitted to blood sampling on moments MO (beginning of
cytologic diestrus), Ml (20 days after MO), M2 (40 days after MO), M3 (parturition), M4 (15
days after parturition), M5 (45 days after parturition) and M6 (30 days after weaning). PTH
(parathormone), osteocalcin, calcitonin, progesterone, estradiol, total and free T3, total, free
and canine T4, calcitriol, calcium, phosphorus, total protein, albumin and alkaline
phosphatase were analyzed. There was no difference between moments and no involvement
of hormones on calcium and phosphorus metabolismo There was no correlation between litter
size and other parameters. Chemoluminescence kits for total and free T3 and total T4 did not
present enough sensitivity, but canine T4 chemoluminescence
kit demonstrated high
sensitivity and specificity. The methods of RIA used for osteocalcin, calcitonin and calcitriol
demonstrated high sensitivity and efficiency.
Key words: dogs, calcium, endocrinology, gestation, puerperal period.
PERFIL HORMONAL E METABOLISMO DE CÁLCIO EM CADELAS
GESTANTES E DURANTE O PUERPÉRIO
RESUMO
o
ciclo hormonal e o metabolismo de cálcio em cadelas durante a gestação e o puerpério
foram estudados. Dez cadelas foram submetidas a amostragens sangüíneas nos momentos MO
(início do diestro cito lógico ), M 1 (20 dias após MO), M2 (40 dias após MO), M3 (parto), M4
(15 dias após o parto), M5 (45 dias após o parto) e M6 (30 dias após o desmame). Foram
feitas dosagens de PTH (paratormônio), osteocalcina, calcitonina, progesterona, estradiol, T3
total e livre, T4 total, livre e T4 canino, calcitriol, cálcio, fósforo, proteínas totais, albumina e
fosfatase alcalina. Não houve diferença entre os momentos e os hormônios não interferiram
no metabolismo de cálcio e fósforo. Não houve correlação entre o tamanho da ninhada e os
outros parâmetros estudados. Os kits de quimioluminescência para T3 total e livre e T4 total
não apresentaram sensibilidade suficiente, mas o kit de T4 canino demonstrou alta
sensibilidade e especificidade. Os métodos de radioimunoensaio utilizados para osteocalcina,
calcitonina e calcitriol demonstraram alta sensibilidade e eficiência.
Palavras-chave: cães, cálcio, endocrinologia, gestação, puerpério.
1 DVM PhD Departrnent of Clinical Medic - FMVZ - UNESP - Botucatu, Distrito de Rubião Jr SIN. Botucatu
- SP - Brazil, CEP 18618-000, henrique@finvz.unesp.br
2 DVM PhD Departrnent of Animal Reproduction and Veterinary Radiology - FMVZ - UNESP - Botucatu,
Distrito de Rubião Jr SIN. Botucatu - SP - Brazil, CEP 18618-000, nereu@finvz.unesp.br
2 DVM PhD Statistical Analyst - PUC Minas - Poços de Caldas, A V. Padre Francis Cletus Cox, 1661. Poços de
Caldas - MG - Brazil, CEP 37701-355, mlbrisol@pucpcaldas.br
*Projeto financiado pela FAPESP.
Machado, L.H.A. et aI. Horrnonal profile and calcium metabolism in bitches during gestation and puerperal
period. Veto e Zootec. v.14, n.2, dez., p. 260-270, 2007.
Veterinária e Zootecnia
ISSN 0102-5716
261
PERFIL HORMONAL Y METABOLISMO DEL CALCIO EN PERRAS DURANTE
LA GESTACIÓN Y EL PERIODO PUERPERAL
RESUMEN
Se estudió e1 ciclo hormonal y el metabolismo del calcio en hembras caninas durante Ia
gestación y el periodo puerperal. A diez perras se les tomaron muestras sanguíneas en los
momentos MO (comienzo del diestro cito lógico ), M 1 (20 días después del MO), M2 (40 días
después del MO), M3 (parto), M4 (15 días después del parto), M5 (45 días después del parto)
y M6 (30 días después del destete). Se analizó Ia PTH (paratormone), osteocalcina,
calcitonina, progesterona, el estradiol, Ia T3 total y libre, Ia T4 total, libre y canina, el
calcitriol, calcio, fósforo, Ia proteína total, albúmina y Ia fosfatasa alcalina. No hubo
diferencia entre los diferentes momentos, ni tampoco implicación de hormonas en el
metabolismo de calcio y fósforo. No hubo correlación entre el tamaíio de Ia cría y otros
parámetros. Los kits de quimioluminiscencia empleados para Ia determinación del T3 total y
libre y T4 total, no presentaron sensibilidad suficiente, pero el kit utilizado para determinar el
T4 canino demostró gran sensibilidad y especificidad. Los métodos de RIA empleados para
osteocalcina, ca1citonina y calcitriol, demostraron gran sensibilidad y eficiencia.
Pala bras-clave: canes, ca1cio, endocrinología, gestación, período puerperal.
INTRODUCTION
Veterinary nutrition is continuously evoluting, thus contributing to the improvement of
pet longevity and health. Calcium and phosphorus are necessary for mineralization of fetal
bones, thus the ingestion of these minerals is fundamental, specially during the final third of
gestation. The main sources of these minerals to the fetus are maternal nutrition, bone
deposits and ca1ciotrophic hormones (PITKIN, 1985; MOLINA-FONT, 1998).
The increase of osteocytic and osteoclastic activity and of ca1citriol production on
kidneys is a function ofparathormone (CAPEN & MARTIN, 1977; SLATOPOLSKY et al.,
1980; HIGH et al., 1981; KLAHR & SLATOPOLSKI, 1986; BANKS, 1992; CHEW &
DiBARTOLA, 1992; YAPHÉ & FORRESTER, 1994; ROSOL & CAPEN, 1996; PAULINO
& BOLDAN, 1999). Osteoca1cin sinthesys depends on calcitriol which is correlated to
estrogen, growth hormone (GH) and prolactin (POWER et al., 1989; ROSOL & CAPEN,
1996). The presence of the placenta increases calcitriol concentration (PAULINO &
BONDAN, 1999).
Ostecalcin is a specific protein indicator of bone formation (POWER et al., 1989;
ARICAN et al, 1996; ALLEN & RICHARDSON, 1998), more reliable than alkaline
phosphatase as a marker of bone turnover. Its synthesis depends on vitamin KJ stimulated by
calcitriol (POWER et al., 1989; ALLEN & RICHARDSON, 1998).
Thyroid hormones are inversally related to the reduction of intestinal absorption of
calcium (ROSOL & CAPEN, 1996; DAVIDSON, 2000). Tyroxin promotes bone formation,
remodeling and increasing. Feldman and Nelson (1987b) refered estrogen as a facto r of
enhancement of T3 and T4 concentrations. Estrogen decreases bone turnover (CRUESS &
HONG, 1979; SHEN et al., 1993) and increases serum concentrations of calcitriol (SHEN et
aI.,1993).
Progesterone is responsible for pregnancy maintenance in bitches and its concentration
is believed to increase soon after te preovulatory peak ofLH (HADLEY, 1975; GRÀF, 1978;
PURSWELL & PARKER, 2000; VANNUCCHI, 2000; ROOT KUSTRITZ, 2001;
DAVIDSON & FELDMAN, 2004; SCHAEFERS-OKKENS, 2004). The increase in serum
Machado, L.H.A. et al. Horrnonal profile and ca\cium metabolism in bitches during gestation and puerperal
period. Veto e Zootec. v.14, n.2, dez., p. 260-270, 2007.
ISSN 0102-5716
Veterinária e Zootecnia
262
levels of progesterone andlor estrogen is correlated to GH increasing. This hormone presents
catabolic functions, interfering on insulin action on peripheral tissues, and anabolic functions,
leading to the production of somatomedins, which increase cartilage and bone growth
(FELDMAN & NELSON, 1987c).
Human kits ofradioimmuneassay (RIA) for dosage ofserum total and free T4 and free
T3 have been tested and validated for dogs, presenting high sensitivity and specificity (LEE et
al., 1991; NELSON et al., 1991; PANCIERA & POST, 1992; RElMERS et al., 1981;
TORRES et al., 1991; YOUNG et al., 1985).
Nowadays, other methods of hormone assay are being tested in order to substitute
RIA, manly because of biosecurity (JERICÓ et al., 1999; LAZARETTI et al., 1999) and
incubation time (GARNER & LElGHT, 1999). One of these is immunochemoluminescence
(lCMA), which demonstrates high correlation to RIA (EXNER & KOUTTS, 1986; MORITA
et al., 1992). This assay for intact PTH has two antibodies (anti-PTH 1-34 and anti-PTH 3984). Machado (2000) used this method in healthy and renal failure dogs and 66,7% ofhealthy
ones presented values below the limit of detection of the test « 1pg/mL).
The aims of the present study were to stablish hormonal and biochemical pattems
during gestation and lactation in bitches; to study hormone and calcium metabolism; to
correlate all studied parameters; to determine normal values of 1,25 dihydroxi vitamin D,
osteocalcin and calcitonin by RIA in bitches; to study the correlation between the parameters
and litter size and to test the viability of lCMA on hormone assay in bitches.
MATERIAL AND METHODS
Ten adult Springer Spaniel pregnant bitches were used. They were fed with super
premium dry food and, by day 40 of gestation, changed to puppy super premium dry food,
until the end of lactation.
Estrus was detected by vaginal cytology and bitches were mated when 80% of
superficial cells were observed. Vaginal cytology was performed daily during heat to
determine the first day of diestrus. Blood sampling was performed always in the moming, on
7 different moments: MO (first day of cytological diestrus, detected by the presence of
neutrophils and abrupt change from superficial to intermediate and parabasal cells, associated
to end of acceptance behaviour), Ml (20 days after MO), M2 (20 days after Ml), M3 (during
parturition), M4 (15 days after parturition), M5 (45 days after parturition) and M6 (30 days
after weaning). Dosages of progesterone ', estradiol", osteocalcin ', total T34, T4 (total ' and
free") and intact PTH7 by RIA were performed at the Endocmology Laboratory of the
Departament of Animal Reproduction and Veterinary Radiology -UNESP - Botucatu - BraziL
Dosages of total and free T389, total and free T41011 and canine T412 by lCMA were
performed at Botucatu Laboratory - Botucatu-Brazil. Dosages of calcitriol':' and calcitonin 14
1
progesterona-DPC-MEDLAB
estradiol-DPC-MEDLAB
3 Osteocalcina -DS L
4 T3 total-DPC-MEDLAB
5 T4 total-DPC-MEDLAB
6 T4 livre-DPC-MEDLAB
7N-tact® PTH SP DiaSorin
8 T3 total Ílnmulite-DPC-MEDLAB
9 T3 livre immulite-DPC-MEDLAB
10 T4 total Ílnmulite-DPC-MEDLAB
11 T4livre immulite-DPC-MEDLAB
12 T4 canino immulite-DPC-MEDLAB
13 1,25 dihidroxivitamina
D-DlASORIN
14calcitonina-DPC-MEDLAB
Machado, L.H.A. et ai. Hormonal profile and calcium metabolism in bitches during gestation and puerperal
period. Vet. e Zootec. v.14, n.2, dez., p. 260-270,2007.
2
ISSN 0102-5716
Veterinária e Zootecnia
263
by RIA were performed at CRIESP - São Paulo- Brazil. Dosages of P 1, total Ca 2, total
proteins 3, albumin 4 and alkaline phosphatase ' were performed at the Service of Clinic
Laboratory ofPUC-MINAS- Poços de Caldas - Brazil.
Statistical analisys was performed at PUC MINAS - Poços de Caldas - Brazil and
consisited on the study ofpossible associations between variables, the Pearson's coeficient of
correlation and the significance associated to this correlation (p value). Multivaried analisys
of repeated measures was performed to compare, in average, the pairs of moments, using
Students t test. Variation of intra-assay tests was determined by repetition of 10% of samples
and was not superior to 5%. The inter-assay precision was determined by a pool of samples.
RESULTS
The average time of gestation, counted after the first day of cytological diestrus was 58
days. There was no correlation between litter size and the other parameters studied.
The values obtained in hormonal and biochemical dos ages are presented in Table 1.
DISCUSSION
Despite literature data suggest that gestation and lactation may lead to mineral
deficiency (MALLUCHE et al., 1982; GRUBER et al., 1984; BROMMAGE & DELUCA,
1985; FUKUDA & IIDA, 1993; COLUSSI et al., 1997;), this was not observed in the present
study, in which a tendency to Ca and P decrease was verified between MO and M2, followed
by an increase. After day 40 of gestation, there was an alteration in the diet, which represented
an increase of23,1 % on calcium intake, that may have contributed to this balance.
Calcitriol, calcitonin and PTH are responsible for Ca and P metabolism (CAPEN &
MARTIN, 1977; CHEW & DIBARTOLA, 1992; YAPHÉ & FORRESTER, 1994).
Comparing consecutive moments, we observed that only PTH and calcitriol presented
significant variation between moments MO and M1. The fact that calcitonin, calcitriol and
PTH, which act together, were not affected by gestation or lactation, may suggest the
efficiency of diet correction or that there is no real need to interfere on mineral homeostasis
during gestation and 1actation, as it is frequently recomended by many veterinarians.
Values of osteocalcin presented no significant variation between any pair of
consecutive moments, whereas alkaline phosphatase presented significant differences between
all moments, showing a tendency of osteocalcin to follow hormonal profiles of mineral
homeostasis. These results are in agreement with previous reports, which suggest osteocalcin
as a better marker of bone tumover than alkaline phosphatase (REIMERS et al., 1981;
ALLEN & RICHARDSON, 1998), as it is a specific protein (POWER et al., 1989; ARICAN
et al, 1996; ALLEN & RICHARDSON, 1998).
The synthesis of osteocalcin is dependent of calcitriol (POWER et al., 1989; ALLEN
& RICHARDSON, 1998), what could be confirmed in the present study by the lack of
variation on both parameters.
As known by literature, tyroid hormones are related to alterations in Ca and P
absorption (ROSOL & CAPEN, 1996; DAVIDSON, 2000). In the present study, the
absorption of these minerals was not tested, but there were some episodes of correlation
Fósforo - ANALISA Diagnóstica S.A.
Cálcio - ANALISA Diagnóstica S.A.
3 Proti AJG CELM Cia Equipadora de Laboratórios Modernos
4 Proti AJG CELM Cia Equipadora de Laboratórios Modernos
5 Fosfatase alcalina cinética otimizada - CELM Cia Equipadora de Laboratórios Modernos
Machado, L.H.A. et al. Hormonal profile and calcium metabolism in bitches during gestation and puerperal
period. Veto e Zootec. v.14, n.2, dez., p. 260-270,2007.
1
2
Veterinária e Zootecnia
ISSN 0102-5716
264
between these hormones and the concentrations of Ca and/or P, that may be also correlated to
their absorption.
Table
1
Average
values
of hormonal
(radioimmune
assay
RlA
or
immunechemoluminescent
assay - ICMA) and biochemical parameters of bitches on 7
moments during gestation and the puerperal period (MO - first day of cytological diestrus, Ml
- 20 days after MO, M2 - 20 days after Ml, M3 - during parturition, M4 - 15 days after
parturition, M5 - 45 days after parturition and M6 - 30 days after weaning).
lntact PTH
(pg/dL)
Osteocalcin
(nz/ml.)
Calcitonin
(pg/dL)
Progesterone
(ng/mL)
MO*
Ml
68,2 ± 43,6
a
33,1±
15,5 a
1,1 ±0,1
1,1 ±0,1
M2
M3
11,1 ± 0,3 11,5 ± 1,4 13 ± 2,7 14,7 ± 3,3
9,3 ± 1-1 15,7 ± 2-1 108
, ± TI
a,b
b,c
c.d
1 ± 2·'.1g.a
Total T4 RlA
(/-Lg/dL)
Total T4 ICMA
(uz/dl.)
Free T4 RlA
(ng/mL)
Free T4 ICMA
(nz/ml.)
Canine T4 ICMA
(uz/dl.)
Calcitriol RlA
(pg/dL)
P (mg/dL)
Ca (mg/dL)
Total Protein
(mg/dL)
Albumin (mg/dL)
M5
48,1 ± 84 ± 60,2 62,7 ± 59,9 55 ± 23,1
21,4
1,0 ± 0,1
1,0 ± 0,1 1,1±0,1
1,0±0,1
Estradiol (pg/dL) 43, ± 1,1 g.a 2,2 ± 1,3 a 1,3±1,0
Total T3 RlA
(nz/ml.)
Total T3 ICMA
(nz/ml.)
Free T3 ICMA
M4
100,1 ±
63,6±
35 1
349c
< limit a 50,2 ± 2,7 < limit
1,2±0,3d
97,1 ±9,9
b
M6
77,4±
59,7
1,2 ± 0,1
14,1 ± 3,5 13,1 ± 3,6
15,4 ± 3,5
49±3, ld
5 ,1 ± 2-1 58, ± 4-1g
0,8 ± 0,4 d
0,7 ± 0,5 0,5 ± 0,4g
198,3 ±
191 9 c
< limit
174,5 ±
141
< lirnit
°
110,0 ±
656
< limit
145,1 ±
148,1
< limit
< limit
< limit
< limit
< limit
a.b
<limit
< limit
24, ± 09,
g.a
1,2 ± 0,2
<limit
3,2 ± 1,1 a 2,9 ± 1,5 2,8 ± 1,2
1,8 ± 1,2
1,2 ± 0,6 1,2±0,6g
1,2 ± 0,1 b 1,1 ± 0,1
1,1 ± 0,1 e
1,0 ± 0,0
b
1,1 ± 0,2
1,1 ± 0,1 t
e.f
0,4 ± <0,1
°
°,
g
0,3 ± <0,1 0,2 ± <0,1 0,1 ± <0,1 0,1 ± <0,1
d
b
b
1,3 ± ,1 g.a 1,4 ± 0,1 0,8 ± 0,1
1,3 ±
0,1 ± <0,1 0,1±<0,1
g
0,8 ± 0,1 d 0,9 ± 0,1 o
0,9 ± 0,1 0,9 ±O,l g
0,9 ± 0,2 c 0,9 ± 0,3
0,9 ± 0,2 1,0 ± 0,2g
a.b
1 g,a 1,I±O,la
26±3,Oa
b
d
27±2,7a
1,I±O,lc
27 ± 3,3
28 ± 3,1
33±06
, g,a,
3,0 ± 0,6 a 3,0 ± 0,4 c 3,3 ± 0,6
cd
10,2 ± 0,8 a 9,2 ± 0,4 8,7 ± 0,3 9,9±0,4c
ab
bc
6,6 ± 0,4 a 6,2 ±0,4 5,7±0,4b 5,9±0,7d
30 ± 2,3
28 ± 2,4
27 ± 2,7
g
3,4 ± 0,5 d
3,5 ± 0,5 3,7 ± 0,4
9,9 ± 0,3
9,6±0,4t
6,3 ± 0,3 d
6,3 ± 0,2 r 6,9 ±0,3'
10,6 ± 0,5
f
a.b
3,2 ± 0,2 a 3,1 ± 0,2 2,9 ± 0,1 3,0± 0,2 3,2 ± 0,2 o 3,1 ± 0,1 3,2 ± 0,2
ab
bc
cd
73,2 ± 17,3 78,5 ± 7,3 89,7 ± 7,2 97,3 ± 4,9 112,9 ± 9,8 129,5 ± 90 ± 27,9'
b,c
b
c.d
d.e
162, e,f
Alkaline
Phosphatase
(mg/dL)
ln each line, equal letters represent significant difference between consecutive moments
(p<0,05).
Machado, L.H.A. et aI. Hormonal profile and calcium metabolism
period. Veto e Zootec. v.14, n.2, dez., p. 260-270, 2007.
in bitches during gestation and puerperal
ISSN 0102-5716
Veterinária e Zootecnia
265
Significant differences (p<0,05) between moments were verified on tyroid hormones
concentrations by any of the assay methods, what may represent an increase in energy needs
during gestation and lactation, but none of the concentrations were above or below normal
values.
Studies from other research groups (SCHAEFERS-OKKENS, 2004) concluded that
T4 is increased in bitches during diestrus or gestation and suggested that this may occur
because of the elevation in progesterone during these fases. What was seen in the present
study was a real increase in T4 concentration during gestation, but the correlation with
progesterone concentration was not verified.
Previous studies (RElMERS et al., 1981; ECKERSALL & WlLLIANS, 1983)
reported problems with human kits of RIA for T4 dosage in dogs, because of low limits of
detection, what could lead to fail in 17% of animals. This problem was not observed in this
experiment, which is in agreement with most authors (YOUNG et al., 1985; LEE et al., 1991;
NELSON et al., 1991; RElMERS et al., 1991; TORRES et al., 1991; PANCIERA & POST,
1992). This method, however, can be replaced by immunechemoluminescent assay specific
for dogs, which was tested and presented good sensitivity,demonstrating similarity with the
elevation in T4 concentrations in the presence of progesterone previously reported in
literature (RElMERS et al., 1984).
There are two hypothesis for estradiol behaviour during gestation in bitches: the first is
that its concentrations remain stable (HADLEY, 1975; AUSTAD et aI, 1976; VANNUCCHl,
2000) and the second refers significant increase during gestation, with a peak before
parturition (FELDMAN & NELSON, 1987a; CONCANNON et al., 1989). The results ofthis
study are similar to those of the first hypothesis, as there was a decline on estradiol
concentrations (p<0,05) at the beginning of gestation, specially between moments MO and
M1, followed by the maintenance of low concentrations until the end of the study.
Radioimmune assay kit for estradiol used on this experiment presented a slight fail on
sensitivity, thus the calibration curve was corrected by a computer programo
The results of the present study agree with the literature (HADLEY, 1975; GRÂF,
1978; PURSWELL & PAEKER, 2000; VANNUCCHl, 2000) which reports that progesterone
presents crescent concentrations during estrus, maintaining high serum levels during gestation
and decreasing before parturition, suggesting that this hormone is responsible for gestation
maintenance, as proposed by many authors (HADLEY, 1975; GRÂF, 1978; PURSWELL &
PARKER, 2000, VANNUCCHl, 2000; ROOT KUSTRITZ, 2001; SCHAEFERS-OKKENS,
2004; DAVlDSON & FELDMAN, 2004). This partem was evidenced by the curve of
progesterone, in which there is a decrease in serum concentrations between moments M2 and
M3.
The correlation between the hormones progesterone and estradiol and phosphorus
proposed by some authors (FELDMAN & NELSON, 1987c; ORTOLANl, 1999; PAULINO
& BONDAN, 1999) was not verified in this work, what may explain the fact that PTH
remained stable, as this mineral is fundamental for PTH increase, as confirmed by a previous
study of the same author (MACHADO, 2000). The increase of 64,3% on P intake promoted
by diet change may have contributed to PTH stability, as the maintenance ofP concentrations
stabilizes PTH, preventing osteoclastic and osteocytic actions of this hormone (CAPEN &
MARTIN, 1977; HlGH et al., 1981; BANKS, 1992; CHEW & DlBARTOLA, 1992; YAPHÉ
& FORRESTER, 1994; ROSOL & CAPEN, 1996; PAULINO & BONDAN, 1999).
This experiment demonstrated that some lCMA kits developed for humans don't
present good sensitivity and specificity for dogs, giving many values below the limit of
detection (80% total T3, 100% free T3 and 28% total T4), as previously reported for intact
PTH in healthy dogs (MACHADO, 2000). The same was not verified on free T4 and canine
T4 assays, indicating that the failure may not be due to the method, but to the kit.
Machado, L.H.A. et aI. Hormonal profile and calcium metabolism
period. Veto e Zootec. v.14, n.2, dez., p. 260-270, 2007.
in bitches during gestation and puerperal
Veterinária e Zootecnia
ISSN 0102-5716
266
ln the present study, the authors opted for the most practical and cheapest method of
estrus monitoring for c1inicians. Although the literature refers association of vaginal cytology
with sequential progesterone dosages (WILDT et al., 1979; HAY et al., 2000; BETINI et al.,
2001; ROOT KUSTRITZ, 2001), the control of estrus cyc1e by vaginal cytology alone was
effective, as all bitches became pregnant and the length of gestation was according to
literature, which refers 56,9 days (JOHNSTON et al., 2001). Behavioral and cytological
changes caused by estrogen during estrus and the transition to diestrus proposed in the
literature (FELDMAN & NELSON, 1996) were observed in this experiment and were
fundamental to correctly define the moments.
CONCLUSION
The present data allowed us to conc1ude that there is no tendency to hypocalcemia
during gestation and puerperal period; osteocalcin is more specific as bone marker than
alkaline phosphatase; values of Ca, P, total protein and albumin are corrected by increase on
their intake; estradiol remains stable during gestation while progesterone increases at the
beginning then decreases at the end of gestation; tyroid hormones correlate to other
parameters because of their metabolic functions; human kits of ICMA for total and free T3
and total T4 do not present enough sensitivity and specificity for dogs; the kit of ICMA for
canine T4 presents good sensitivity and specificity; the human kits of RIA for osteocalcin,
ca1citonin and calcitriol present good sensitivity and specificity.
REFERENCES
ALLEN, MJ.; RICHARDSON, D.e.
Veto Res., v.59, p.250-254, 1998.
Serum Markers of bone metabolism in dogs. Am. J.
ARICAN, M.; CARTER, S.D.; BENNETT, D. Osteoca1cin in canine joint diseases. Br. Veto
J., v.142, pAll-423, 1996.
AUSTAD, R.; LUNDE, A.; SJAASTAD, V. Peripheral plasma levels of oestradiol 17-beta
and progesterone in the bitch during the oestros cyc1e, in normal pregnancy and after
dexamethasone treatment. J. Reprod. Fertil., vA6, p.129-136, 1976.
BANKS, WJ. Sistema endócrino. ln:
São Paulo: Manole, 1992. cap.25, p.521-545.
o
Histologia veterinária
aplicada.
2.ed.
BETINI, C.M.; MORAES, G.F.; RIGOLON, L.P. lnseminação artificial de cadelas com
sêmen fresco diluído em meios formulados com água de coco e leite em pó desnatado. Rev.
Bras. Reprod. Anim.,v.25, p.375-377, 200l.
BROMMAGE, R.; DELUCA, H.F. Regulation
Endocrinol. Metab., v.11, p.E182-187, 1985.
of bone mineral loss during lactation.
CAPEN, e.e.; MARTIN, S.L. Calcium metabolism and disorders of parathyroid glands.
Veto Clin. North Am., v.7, p.513-548, 1977.
CHEW, DJ.; DIBARTOLA, S.P. Diagnóstico e fisiopatologia da moléstia renal. ln:
ETTINGER, E.J. Tratado de medicina interna veterinária.
3.ed. São Paulo: Manole,
1992. vA, cap.107, p.1975-2046.
Machado, L.H.A. et ai. Honnonal profile and calcium metabolism in bitches during gestation and puerperal
period. Veto e Zootec. v.14, n.2, dez., p. 260-270, 2007.
ISSN 0102-5716
Veterinária e Zootecnia
267
COLUSSI, G. et al. The changes in plasma diffusible levels and renal tubular handling of
magnesium during pregnancy: a longitudinal study. Bone Miner., v.2, p.311-319, 1997.
CONCANNON, P.W. et al. . Biology and endocrinology of ovulation, pregnancy
parturition in the dog. J. Reprod. Fertil., Supp1.39, p.3-25, 1989.
CRUESS, R.L.; HONG, K.e. The effect of long term estrogen administration
metabolism in the female rat. Endocrinology, v.l04, p.1188-1193, 1979.
and
on bone
DAVIDSON, A. Thyroid dysfunction - diagnosis, treatment, and effect on reproductive
performance. In: EQUINE SYNPOSIUM AND ANNUAL CONFERENCE 2000 SOCIETY
FOR THERIOGENOLOGY, 2000, San Antonio. Proceedings ...San Antonio, 2000. p.333338.
DAVIDSON, A.P.; FELDMAN, E.C.
Alterações ovarianas e do ciclo estral.
In:
ETTINGER, SJ.; FELDMAN, E.e. Tratado de medicina interna veterinária - doenças do
cão e do gato. 5.ed. Rio de Janeiro: Guanabara Koogan, 2004. v.2, cap.158, p.1602-1609.
ECKERSALL,
P.D.; WILLIANS, M.E.
Thyroid function tests
radioimmunoassay kits. J. Small Anim. Pract., v.24, p.525-532, 1983.
EXNER, T.; KOUTTS, J. Simple immunochromometric
Clin. Med., v.l 07, pA05-411, 1986.
in
dogs
using
assay for protein C activity. J. Lab.
FELDMAN, E.e.; NELSON, R.W. Canine female reproduction. In:
. Canine and
feline endocrinology and reproduction.
Philadelphia: WB Saunders, 1987a. cap.14, p.399480.
FELDMAN, E.e.; NELSON, R.W. Hypothyroidism.
In:
. Canine and feline
endocrinology and reproduction.
Philadelphia: WB Saunders, 1987b. cap.3, p.55-90.
FELDMAN, E.e.; NELSON, R.W. Growth hormone. In:
. Canine and feline
endocrinology and reproduction.
Philadelphia: WB Saunders, 1987c. cap.2, p.29-54.
FELDMAN, E.e.; NELSON, R.W. Ovarian cycle and vaginal cytology. In:
_
Canine and feline endocrinology and reproduction.
Philadelphia: WB Saunders, 1996.
cap.17, p.526-546.
FUKUDA, S., IIDA, H. Histomorphometric changes in iliac trabecular bone during
pregnancy and lactation in beagle dogs. J. Veto Med. Sei. v.55, p.565-569, 1993.
GARNER, S.e.; LEIGTH, G.S. Initial experience with intraoperative PTH determinations in
the surgical management of 130 consecutive cases ofprimary hyperparathyroidism. Surgery,
v.126, p.1l32-1138, 1999.
GRÀF, KJ. Serum oestrogen, progesterone and prolactin concentrations in cyclic, pregnant
and lactating beagle dogs. J. Reprod. Fertil., v.52, p.9-14, 1978.
Machado, L.H.A. et ai. Honnonal profile and ca\cium metabolism in bitches during gestation and puerperal
period. Veto e Zootec. v.14, n.2, dez., p. 260-270, 2007.
Veterinária e Zootecnia
ISSN 0102-5716
268
GRUBER, H.E.; GUTTERRlDGE, D.H.; BA YLINK, D.l. Osteoporosis associated with
pregnancy and lactation: bone biopsy and skeletal features in three patients. Metab. Bone
Dis. Relat. Res., v.5, p.159-165, 1984.
HADLEY, J.C. Total unconjugated oestrogen and progesterone concentrations in peripheral
blood during pregnancy in the dog. J. Reprod. Fertil., v.44, p.453-460, 1975.
HA Y, M.A. et al. Correlation of periovulatory serum and fecal progestins in the domestic
dog. Cano J. Veto Res., v.64, p.59-63, 2000.
HIGH, W.B.; CAPEN, c.c., BLACK, H.E. The effects of 1,25-dihydroxycholecalciferol,
parathyroid hormone, and thyroxine on trabecular bone remodeling in adult dogs. Am. J.
Pathol., v.l05, p.279-287, 1981.
lERICÓ, M.M. et al. Avaliação dos valores séricos de paratormônio intacto, em cães hígidos,
utilizando-se o método imunofluoromérico.
CONGRESSO BRASILEIRO DE CLÍNICOS
VETERINÁRIOS DE PEQUENOS ANIMAIS, 20., 1999, Águas de Lindóia. Anais ...Águas
de Lindóia, 1999. p.19.
lOHNSTON, S.D.; ROOT KUSTRITZ, M.V.; OLSON,
theriogenology. Philadelphia: WB Saunders, 2001. 592p.
KLAHR, S.; SLATOPOLSKY,
Med., v.37, p.71-78, 1986.
P.N.S.
Canine
E. Toxicity of parathyroid hormone in uremia.
and
feline
Ann. Rev.
LAZARETTI, P. et al. Avaliação da função tireóidea na espécie canina: padronização e
comparação
entre imunoensaios.
CONGRESSO
BRASILEIRO
DE CLÍNICOS
VETERINÁRIOS
DE PEQUENOS
ANIMAIS,
20., 1999, Águas de Lindóia.
ANAIS ...Águas de Lindóia, 1999. p.19.
LEE, D.E.; LAMB, S.V.; REIMERS, r.r. Effects ofhyperlipemia on radioimmunoassays for
progesterone, testosterone, thyroxine, and cortisol in serum and plasma samples from dogs.
Am. J. Veto Res., v.52, p.1489-1491, 1991.
MACHADO, L.H.A. Estudo do hiperparatireoidismo
secundário à insuficiência renal
crônica em cães. 2000. 110f. Dissertação (Mestrado). Faculdade de Medicina Veterinária e
Zootecnia, Universidade Estadual Paulista, Botucatu.
MALLUCHE, H.H. et al. A new semiautomatic method for quantitative static and dynamic
bone histology. Calcif. Tissue Int., v.34, p.439-448, 1982.
MOLINA-FONT,
1998.
l.A. Nutrition and fetal growth.
Early Rum. Dev., SuppI.53, p.s51-s60,
MORITA, A. et al. A two-site immunochemiluminometric
assay for intact parathyroid
hormone and its clinical utility in hemodialysis patients. Clin. Nephrol., v.38, p.154-157,
1992.
Machado, L.H.A. et ai. Honnonal profile and calcium metabolism in bitches during gestation and puerperal
período Veto e Zootec. v.l4, n.2, dez., p. 260-270, 2007.
Veterinária e Zootecnia
ISSN 0102-5716
NELSON, R.W. et al. Serum free thyroxine concentration
hypothyroidism, and euthyroid dogs with concurrent illness.
v.198, p.1401-1407, 1991..
269
in healthy dogs, dogs with
J. Am. Vet. Med. Assoc.,
ORTOLANl, E.L. Macro e microelementos.
ln: SPINOSA, H.S.; GÓRNlAK, S.L.;
BERNARDl, M.M. Farmacologia
aplicada à medicina veterinária.
Rio de Janeiro:
Guanabara Koogan, 1991. cap.52, p.555-565.
PANCIERA, D.L., POST, K. Effect of oral administration of sulfadiazine and trimethoprim
in combination on thyroid function in dogs. Cano J. Veto Res. v.56, p.349-352, 1992.
PAULINO, C.A.; BONDAN, E.F. Metabolismo de cálcio e fósforo. ln: SPINOSA, H.S.;
GÓRNlAK, S.L.; BERNARDl, M.M. Farmacologia aplicada à medicina veterinária.
Rio
de Janeiro: Guanabara Koogan, 1999. cap.30, p.319-330.
PITKIN, R.M. Calcium metabolism in pregnancy and the perinatal period. Am. J. Obstet.
Gynecol., v.151, p.99-109, 1985.
POWER, M.J.; GOSLING, 1.G.; FOTTRELL, P.F. Radioimmunoassay of osteocalcin with
polyclonal and monoclonal antibodies. Clin. Chem., v.35, p.1408-1413, 1989.
PURSWELL, B.J.; PARKER, N.A. Modem breeding management in dogs. Veto Med., sept.,
p.708-725, 2000.
RElMERS, T.J. et al. Validation of radioimmunoassays for triiodothyronine, thyroxine, and
hydrocortisone (cortisol) in canine, feline, and equine sera. Am. J. Veto Res., v.42, p.20162021,1981.
RElMERS, T.J. et al. Effects of reproductive state on concentrations of thyroxine, 3,5,3'triiodothyronine and cortisol in serum of dogs. Biol. Reprod., v.31, p.148-154, 1984.
RElMERS, T.J. et al. Effects of hemolysis and storage on quantification of hormones in
blood samples from dogs, cattle, and horses. Am. J. Veto Res., v.52, p.1075-1080, 1991.
ROSOL, T.J.; CAPEN, c.c. Pathophysiology of calcium, phosphorus, and magnesium
metabolism in animais. Veto Clinics North Am. Small Anim. Pract., v.26, p.1155-1184,
1996.
ROOT KUSTRITZ, M.V. Uso de kits comerciales para ensayos de hormona luteinizante y de
progesterone en el manejo reproductivo canino. ln: CONCANNON, P.W.; ENGLAND, G.;
VERSTEGEN,1. Recent Advances in Small Animal Reproduction, 2001. Disponível em:
<http://www.ivis.org>. Acesso em: 30 jun. 2002.
SCHAEFERS-OKKENS, A.C. Ciclo estral e manejo reprodutivo da cadela sadia. ln:
ETTINGER, S.1.; FELDMAN, E.C. Tratado de medicina interna veterinária - doenças do
cão e do gato. 5.ed. Rio de Janeiro: Guanabara Koogan, 2004. v.2, cap.157, p.1592-1602.
SHEN, V. et al. Loss of cancellous bone mass and connectivity in ovariectomized rats can be
restored by combined treatment with parathyroid hormone and estradiol. J. Clin. Invest.,
v.91, p.2479-2487, 1993.
Machado, L.H.A. et ai. Hormonal profile and calcium metabolism
period. Veto e Zootec. v.14, n.2, dez., p. 260-270, 2007.
in bitches during gestation and puerperal
ISSN 0102-5716
Veterinária e Zootecnia
270
SLATOPOLSKY, E.; MARTIN, K.; HRUSKA, K. Parathyroid hormone metabolism and its
potential as a uremic toxin. Am. J. Physiol., v.239, p.F1-F12, 1980.
TORRES, S.M.F.; MCKEEVER, PJ.; JOHNSTON, S.D. Effect of oral administration
prednisolone on thyroid function in dogs. Am. J. Veto Res., v.52, p.416-421, 1991.
of
VANNUCCHI, c.I. Perfil das proteínas plasmáticas e de fase aguda durante a gestação e
diestro: utilização no diagnóstico precoce de gestação em cadelas.
2000.
95f.
Dissertação (Mestrado). Faculdade de Medicina Veterinária e Zootecnia, Universidade de
São Paulo, São Paulo.
WILDT, D.E. et al. Relationship of serum estrone, estradiol-I'rf and progesterone to LH,
sexual behavior and time ofovulation in the bitch. Biol. Reprod., v.20, p.648-658, 1979.
YAPHÉ, W., FORRESTER, SD. Renal secondary hyperparathyroidism: pathophysiology,
diagnosis, and treatment. Compend. Contin. Educ. Pract. Vet., v.16, p.173-181, 1994.
YOUNG, D.W.; SARTIN, lL.; KEMPPAINEN, RJ. Abnormal canine triiodothyroninebinding factor characterized as a possible triiodothyronine autoantibody. Am. J. Veto Res.,
v.46, p.1346-1350, 1985.
Recebido em: 14/02/2006
Aceito em: 23/12/2006
Machado, L.H.A. et al. Hormonal profile and calcium metabolism
período Veto e Zootec. v.14, n.2, dez., p. 260-270, 2007.
in bitches during gestation and puerperal