260 HORMONAL PROFILE AND CALCIUM METABOLISM IN
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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, [email protected] 2 DVM PhD Departrnent of Animal Reproduction and Veterinary Radiology - FMVZ - UNESP - Botucatu, Distrito de Rubião Jr SIN. Botucatu - SP - Brazil, CEP 18618-000, [email protected] 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, [email protected] *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. 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