- Email: [email protected]
Biochemical markers of bone turnover in the dairy cow during lactation and the dry period
Research in Veterinary Science 78 (2005) 17–19 www.elsevier.com/locate/rvsc
Biochemical markers of bone turnover in the dairy cow during lactation and the dry period K. Holtenius *, A. Ekelund Kungs€ angen Research Centre, Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, SE-753 23 Uppsala, Sweden Accepted 10 May 2004
Abstract We measured a bone-formation marker recognizing osteocalcin, and a bone-resorption marker recognizing C-telopeptide (CTx) fragments of collagen type I, in a longitudinal study. The levels of these markers in the plasma of dairy cows (n ¼ 11) were recorded over a 12 month postpartum period, including a full lactation and a dry period. The plasma concentration of CTx was highest in the first week after parturition. It then declined slowly over the next 33 weeks and remained low until the next parturition. Osteocalcin concentration was lowest around parturition, reached a plateau during mid-lactation, then fell again towards term. There were large variations in bone metabolism during a lactation, that were not directly related to milk production. These results may be used to facilitate appropriate adjustments to calcium and phosphorous concentrations in the diet, reflecting the specific needs of each stage of the reproductive cycle. Ó 2004 Published by Elsevier Ltd. Keywords: Biochemical bone marker; Bone turnover; Dairy cow; Lactation
In dairy cows, bone metabolism must change to balance the varying demand for calcium and phosphorous depending on lactational and gestational status and dietary supply (Beighle, 1999; Goff, 2000). A thorough understanding of normal bone metabolism facilitates an exploration of the sites of maladaptation of bone metabolism and disease. Bone turnover is characterized by two opposing, but complementary, metabolic activities, the resorption of old bone by osteoclastic cells and the deposition of newly formed bone by osteoblasts. The rate of bone turnover can be assessed in vivo by measuring the serum, plasma or urine concentrations of specific biochemical markers. Most such markers have been developed for studies of human bone metabolism. However, bone accretion has been assessed in cattle by measuring osteocalcin, which is a small non-collagenous protein, synthesized by the osteoblasts. It is primarily deposited in the extracellular matrix of bone and only a
*
Corresponding author. Tel.: +46-18671-629; fax: +46-186-729-46. E-mail address: [email protected] (K. Holtenius).
0034-5288/$ - see front matter Ó 2004 Published by Elsevier Ltd. doi:10.1016/j.rvsc.2004.05.002
small amount of newly synthesized osteocalcin is released directly into the circulation (Davicco et al., 1992; Liesegang et al., 2000). Useful plasma markers of bone resorption are type I collagen degradation molecules (Lepage et al., 2001). As yet, knowledge of the dynamics of such molecules in cattle is limited. However, Liesegang et al. (1998) have developed a bovine assay useful for carboxyterminal telopeptide of type I collagen (ICPT). The dynamics of bone turnover throughout a full lactation have apparently not been reported in cows. Therefore, the objective of the present study was to measure biochemical markers of bone formation and resorption during a complete lactation in dairy cattle. Eleven pluriparous (seven cows in their second lactation and four in later lactations) cows of the Swedish red and white breed were studied during a complete lactation cycle. They were housed in a tie-stall barn and fed silage and concentrate according to their requirements. Five of the cows were supplied with phosphorus equivalent to 75% the recommended amount during the first four months of lactation, while the remaining cows were supplied with 100% of the recommended amount
K. Holtenius, A. Ekelund / Research in Veterinary Science 78 (2005) 17–19 50
Plasma CTx (nmol/l)
(Sp€ orndly, 1999). During the rest of the study, all cows were fed 100% of the recommended amount of phosphorus. The present work was part of a larger study on the effects of a low phosphorus diet in early lactation on P balance and P digestibility (Ekelund et al., 2004). The cows were dried off eight weeks prior to the expected parturition. The average calving interval was 355 days (range 325–375 days). All the cows were milked twice daily and milk yield was recorded weekly. The mean milk yield during the complete lactation was 8701 kg energy corrected milk (ECM). Blood was collected from the tail vein in heparinised tubes. Samples were taken weekly, from parturition to nine weeks postpartum (pp), then every second week until 27 weeks pp, every third week until 45 weeks pp and thereafter every second week until parturition. The blood samples were collected between 10 and 11 h and kept on ice until centrifugation (10 min at 1800g) within one hour. The plasma was stored )80 °C until analysis, which was performed within about two months. Osteocalcin in plasma was analysed with a commercially available kit (Intact Osteocalcin EIA Biomedical Technologies Inc. Stoughton, MA, USA) that measures only intact osteocalcin, which is synthesized de novo by the osteoblast. The assay employs two monoclonal antibodies directed towards the amino- and the carboxy-terminal regions of osteocalcin. It recognizes only intact osteocalcin, requiring the full 49 residue protein for detection, thus eliminating any interference by circulating fragments. The assay reacts with human, bovine, monkey and dog osteocalcin. Bone resorption was studied using a commercial ELISA method validated for cattle (Serum CrossLapsÒ ELISA Nordic Bioscience Diagnostics A/S Herlev, Denmark) (CTx). This assay detects collagen type I fragments generated during osteoclastic bone resorption and employs monoclonal antibodies recognizing CTx fragments of collagen type I a1 chains containing the epitope Glu–Lys–Ala–His–Asp–b–Gly–Gly–Arg in an isomerised form. The results were analysed using a general linear model (Minitab Statistical software Release 13.31). The model included lactation number (second lactation or higher), phosphorus level (75% or 100% during the first four lactation months) and stage of lactation, 0–16, 17– 34 and 35–52 week pp). The experimental design and animal handling procedures were approved by the Uppsala Local Ethics Committee. The effects of lactation number and phosphorus level in diet on the plasma level of the two markers were not significant (P > 0:05) and the differences were small (data not shown). Stage of lactation had significant (P < 0:001) effects on the concentrations of both markers (Fig. 1). The concentration of CTx was highest in the first week after parturition. It then declined
40 30 20 10 0
Plasma osteocalcin (ng/ml)
18
25 20 15 10 Artificial insemination
5
Drying-off
0 0
10
20
30
40
50
Weeks after parturition
Fig. 1. Changes in plasma concentrations of CTx and osteoclacin. Eleven cows were followed during a twelve months period including a complete lactation followed by a dry period. Data are presented as means with their standard errors.
steadily for the next 33 weeks and remained low until the next parturition. The high postparturient level of CTx is consistent with the general view that bone resorption is induced at parturition in dairy cows (Goff, 2000; Liesegang et al., 2000). In contrast to the present study however, Liesegang et al. (2000) found only a brief increase in bone resorption after parturition, reflected by a peak in ICPT. This marker is relatively insensitive to physiological changes in bone turnover (Sassi et al., 2000) and it is possible that subtle increases in bone resorption were not detected. Osteocalcin in plasma showed a different pattern, with the lowest values occurring close to parturition (Fig. 1). This is in agreement with previous observations (Davicco et al., 1992; Liesegang et al., 2000) and indicates suppression of osteoblast function during this period. The concentration then rose and reached a plateau in mid-lactation, followed by a continuous decline until term. It is interesting that neither of the markers appeared to be directly related to the shape of the lactation curve (Fig. 2). This indicates that it is not solely the drain of calcium via the milk that is governing bone turnover. Davicco et al. (1992) found no difference in the osteocalcin plasma levels between cows that were dried off eight weeks before parturition and cows that were milked until the week prior to parturition. They concluded that lactation as such did not have any effects of bone metabolism in late lactating cows. However, the
K. Holtenius, A. Ekelund / Research in Veterinary Science 78 (2005) 17–19
Kemira GrowHow AB, Lantm€annen Animal Feeds Division and Lactamin AB is gratefully acknowledged.
50
Milk (kgECM/day)
19
40 30 20
References
10 0 0
10
20
30
40
50
Weeks after parturition Fig. 2. Milk yield from parturition to dry off in eleven cows. Data are presented as the means of weekly observations with their standard errors.
fetal mineral accretion increases exponentially in late pregnancy (House and Bell, 1993), which also might affect bone metabolism. It is interesting that the dynamics of CTx and osteocalcin in lactating women, show striking similarities to these parameters in the dairy cows in the present study (Holmberg-Martilla, 2003). Holmberg-Martilla et al. suggested that the primary cause of bone loss in postpartum women is hypoestrogenemia and not the calcium drain for lactation. It is well documented that estrogen has a significant influence on the bone metabolism (Manolagas et al., 2002). It is possible that the progressive rise in estrogens during gestation in dairy cows (Patel et al., 1999) inhibits bone resorption during the last four months of pregnancy. Our results show large changes in bone metabolism during lactation that cannot be directly accounted for by the demands of lactation. The knowledge gained might be used to adjust calcium and phosphorous levels in the diet to match the actual needs at different stages of lactation. We hypothesise that variation in estrogens, rather than in calcium demand of lactation, is a prime factor regulating bone dynamics in the dairy cow.
Acknowledgements Financial support from the Swedish Farmer’s Foundation for Agricultural Research (No. 0130017). The Royal Swedish Academy of Agriculture and Forestry,
Beighle, D.E., 1999. The effect of gestation and lactation on bone calcium, phosphorus and magnesium in dairy cows. Journal of the South African Veterinary Association 70, 142–146. Davicco, M.J., Remond, B., Jabet, S., Barlet, J.P., 1992. Plasma osteocalcin concentrations in cows around parturition: the influence of a regular versus a very short dry period. Reproduction, Nutrition and Development 32, 313–319. Ekelund, A., Sp€ orndly, R., Holtenius, K., 2004. Influence of low phosphorus intake during early lactation on apparent digestibility of phosphorus and bone metabolism in dairy cows (submitted). Goff, J.P., 2000. Pathophysiology of calcium and phosphorus disorders: veterinary clinics of North America. Food Animal Practice 16, 319–337. Holmberg-Martilla, D., Leino, A., Sievanen, H., 2003. Bone turnover markers during lactation, postpartum amenorrhea and resumption of menses. Osteoporosis International 14, 103–109. House, W.A., Bell, A.W., 1993. Mineral accretion in the fetus and adnexa during late gestation in Holstein cows. Journal of Dairy Science 76, 2999–3010. Lepage, O.M., Carstanjen, B., Uebelhart, D., 2001. Non-invasive assessment of equine bone: an update. The Veterinary Journal 16, 10–22. Liesegang, A., Eicher, R., Sassi, M.L., Risteli, J., Kraenzlin, M., Riond, J.L., Wanner, M., 2000. Biochemical markers of bone formation and resorption around parturition and during lactation in dairy cows with high and low standard milk yields. Journal of Dairy Science 83, 1773–1781. Liesegang, A., Sassi, M.L., Risteli, J., Eicher, R., Wanner, M., Riond, J.L., 1998. Comparison of bone resorption markers during hypocalcemia in dairy cows. Journal of Dairy Science 81, 2614– 2622. Manolagas, S.C., Kousteni, S., Jilka, R.L., 2002. Sex steroids and bone. Recent Progress in Hormone Research 57, 385–409. Patel, O.V., Takenouchi, N., Takahashi, T., Hirako, M., Sasaki, N., Domeki, I., 1999. Plasma oestrone and oestradiol concentrations throughout gestation in cattle: relationship to stage of gestation and fetal number. Research in Veterinary Science 66, 129–133. Sassi, M.L., Eriksen, H., Risteli, L., Niemi, S., Mansell, J., Gowen, M., Risteli, J., 2000. Immunochemical characterization of assay for carboxyterminal telopeptide of human type I collagen: loss of antigenicity by treatment with cathepsin K. Bone 26, 367–373. Sp€ orndly, R. 1999. Fodertabeller f€ or idisslare Rapport 247. Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, Uppsala, Sweden (in Swedish).
Biochemical markers of bone turnover in the dairy cow during lactation and the dry period K. Holtenius *, A. Ekelund Kungs€ angen Research Centre, Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, SE-753 23 Uppsala, Sweden Accepted 10 May 2004
Abstract We measured a bone-formation marker recognizing osteocalcin, and a bone-resorption marker recognizing C-telopeptide (CTx) fragments of collagen type I, in a longitudinal study. The levels of these markers in the plasma of dairy cows (n ¼ 11) were recorded over a 12 month postpartum period, including a full lactation and a dry period. The plasma concentration of CTx was highest in the first week after parturition. It then declined slowly over the next 33 weeks and remained low until the next parturition. Osteocalcin concentration was lowest around parturition, reached a plateau during mid-lactation, then fell again towards term. There were large variations in bone metabolism during a lactation, that were not directly related to milk production. These results may be used to facilitate appropriate adjustments to calcium and phosphorous concentrations in the diet, reflecting the specific needs of each stage of the reproductive cycle. Ó 2004 Published by Elsevier Ltd. Keywords: Biochemical bone marker; Bone turnover; Dairy cow; Lactation
In dairy cows, bone metabolism must change to balance the varying demand for calcium and phosphorous depending on lactational and gestational status and dietary supply (Beighle, 1999; Goff, 2000). A thorough understanding of normal bone metabolism facilitates an exploration of the sites of maladaptation of bone metabolism and disease. Bone turnover is characterized by two opposing, but complementary, metabolic activities, the resorption of old bone by osteoclastic cells and the deposition of newly formed bone by osteoblasts. The rate of bone turnover can be assessed in vivo by measuring the serum, plasma or urine concentrations of specific biochemical markers. Most such markers have been developed for studies of human bone metabolism. However, bone accretion has been assessed in cattle by measuring osteocalcin, which is a small non-collagenous protein, synthesized by the osteoblasts. It is primarily deposited in the extracellular matrix of bone and only a
*
Corresponding author. Tel.: +46-18671-629; fax: +46-186-729-46. E-mail address: [email protected] (K. Holtenius).
0034-5288/$ - see front matter Ó 2004 Published by Elsevier Ltd. doi:10.1016/j.rvsc.2004.05.002
small amount of newly synthesized osteocalcin is released directly into the circulation (Davicco et al., 1992; Liesegang et al., 2000). Useful plasma markers of bone resorption are type I collagen degradation molecules (Lepage et al., 2001). As yet, knowledge of the dynamics of such molecules in cattle is limited. However, Liesegang et al. (1998) have developed a bovine assay useful for carboxyterminal telopeptide of type I collagen (ICPT). The dynamics of bone turnover throughout a full lactation have apparently not been reported in cows. Therefore, the objective of the present study was to measure biochemical markers of bone formation and resorption during a complete lactation in dairy cattle. Eleven pluriparous (seven cows in their second lactation and four in later lactations) cows of the Swedish red and white breed were studied during a complete lactation cycle. They were housed in a tie-stall barn and fed silage and concentrate according to their requirements. Five of the cows were supplied with phosphorus equivalent to 75% the recommended amount during the first four months of lactation, while the remaining cows were supplied with 100% of the recommended amount
K. Holtenius, A. Ekelund / Research in Veterinary Science 78 (2005) 17–19 50
Plasma CTx (nmol/l)
(Sp€ orndly, 1999). During the rest of the study, all cows were fed 100% of the recommended amount of phosphorus. The present work was part of a larger study on the effects of a low phosphorus diet in early lactation on P balance and P digestibility (Ekelund et al., 2004). The cows were dried off eight weeks prior to the expected parturition. The average calving interval was 355 days (range 325–375 days). All the cows were milked twice daily and milk yield was recorded weekly. The mean milk yield during the complete lactation was 8701 kg energy corrected milk (ECM). Blood was collected from the tail vein in heparinised tubes. Samples were taken weekly, from parturition to nine weeks postpartum (pp), then every second week until 27 weeks pp, every third week until 45 weeks pp and thereafter every second week until parturition. The blood samples were collected between 10 and 11 h and kept on ice until centrifugation (10 min at 1800g) within one hour. The plasma was stored )80 °C until analysis, which was performed within about two months. Osteocalcin in plasma was analysed with a commercially available kit (Intact Osteocalcin EIA Biomedical Technologies Inc. Stoughton, MA, USA) that measures only intact osteocalcin, which is synthesized de novo by the osteoblast. The assay employs two monoclonal antibodies directed towards the amino- and the carboxy-terminal regions of osteocalcin. It recognizes only intact osteocalcin, requiring the full 49 residue protein for detection, thus eliminating any interference by circulating fragments. The assay reacts with human, bovine, monkey and dog osteocalcin. Bone resorption was studied using a commercial ELISA method validated for cattle (Serum CrossLapsÒ ELISA Nordic Bioscience Diagnostics A/S Herlev, Denmark) (CTx). This assay detects collagen type I fragments generated during osteoclastic bone resorption and employs monoclonal antibodies recognizing CTx fragments of collagen type I a1 chains containing the epitope Glu–Lys–Ala–His–Asp–b–Gly–Gly–Arg in an isomerised form. The results were analysed using a general linear model (Minitab Statistical software Release 13.31). The model included lactation number (second lactation or higher), phosphorus level (75% or 100% during the first four lactation months) and stage of lactation, 0–16, 17– 34 and 35–52 week pp). The experimental design and animal handling procedures were approved by the Uppsala Local Ethics Committee. The effects of lactation number and phosphorus level in diet on the plasma level of the two markers were not significant (P > 0:05) and the differences were small (data not shown). Stage of lactation had significant (P < 0:001) effects on the concentrations of both markers (Fig. 1). The concentration of CTx was highest in the first week after parturition. It then declined
40 30 20 10 0
Plasma osteocalcin (ng/ml)
18
25 20 15 10 Artificial insemination
5
Drying-off
0 0
10
20
30
40
50
Weeks after parturition
Fig. 1. Changes in plasma concentrations of CTx and osteoclacin. Eleven cows were followed during a twelve months period including a complete lactation followed by a dry period. Data are presented as means with their standard errors.
steadily for the next 33 weeks and remained low until the next parturition. The high postparturient level of CTx is consistent with the general view that bone resorption is induced at parturition in dairy cows (Goff, 2000; Liesegang et al., 2000). In contrast to the present study however, Liesegang et al. (2000) found only a brief increase in bone resorption after parturition, reflected by a peak in ICPT. This marker is relatively insensitive to physiological changes in bone turnover (Sassi et al., 2000) and it is possible that subtle increases in bone resorption were not detected. Osteocalcin in plasma showed a different pattern, with the lowest values occurring close to parturition (Fig. 1). This is in agreement with previous observations (Davicco et al., 1992; Liesegang et al., 2000) and indicates suppression of osteoblast function during this period. The concentration then rose and reached a plateau in mid-lactation, followed by a continuous decline until term. It is interesting that neither of the markers appeared to be directly related to the shape of the lactation curve (Fig. 2). This indicates that it is not solely the drain of calcium via the milk that is governing bone turnover. Davicco et al. (1992) found no difference in the osteocalcin plasma levels between cows that were dried off eight weeks before parturition and cows that were milked until the week prior to parturition. They concluded that lactation as such did not have any effects of bone metabolism in late lactating cows. However, the
K. Holtenius, A. Ekelund / Research in Veterinary Science 78 (2005) 17–19
Kemira GrowHow AB, Lantm€annen Animal Feeds Division and Lactamin AB is gratefully acknowledged.
50
Milk (kgECM/day)
19
40 30 20
References
10 0 0
10
20
30
40
50
Weeks after parturition Fig. 2. Milk yield from parturition to dry off in eleven cows. Data are presented as the means of weekly observations with their standard errors.
fetal mineral accretion increases exponentially in late pregnancy (House and Bell, 1993), which also might affect bone metabolism. It is interesting that the dynamics of CTx and osteocalcin in lactating women, show striking similarities to these parameters in the dairy cows in the present study (Holmberg-Martilla, 2003). Holmberg-Martilla et al. suggested that the primary cause of bone loss in postpartum women is hypoestrogenemia and not the calcium drain for lactation. It is well documented that estrogen has a significant influence on the bone metabolism (Manolagas et al., 2002). It is possible that the progressive rise in estrogens during gestation in dairy cows (Patel et al., 1999) inhibits bone resorption during the last four months of pregnancy. Our results show large changes in bone metabolism during lactation that cannot be directly accounted for by the demands of lactation. The knowledge gained might be used to adjust calcium and phosphorous levels in the diet to match the actual needs at different stages of lactation. We hypothesise that variation in estrogens, rather than in calcium demand of lactation, is a prime factor regulating bone dynamics in the dairy cow.
Acknowledgements Financial support from the Swedish Farmer’s Foundation for Agricultural Research (No. 0130017). The Royal Swedish Academy of Agriculture and Forestry,
Beighle, D.E., 1999. The effect of gestation and lactation on bone calcium, phosphorus and magnesium in dairy cows. Journal of the South African Veterinary Association 70, 142–146. Davicco, M.J., Remond, B., Jabet, S., Barlet, J.P., 1992. Plasma osteocalcin concentrations in cows around parturition: the influence of a regular versus a very short dry period. Reproduction, Nutrition and Development 32, 313–319. Ekelund, A., Sp€ orndly, R., Holtenius, K., 2004. Influence of low phosphorus intake during early lactation on apparent digestibility of phosphorus and bone metabolism in dairy cows (submitted). Goff, J.P., 2000. Pathophysiology of calcium and phosphorus disorders: veterinary clinics of North America. Food Animal Practice 16, 319–337. Holmberg-Martilla, D., Leino, A., Sievanen, H., 2003. Bone turnover markers during lactation, postpartum amenorrhea and resumption of menses. Osteoporosis International 14, 103–109. House, W.A., Bell, A.W., 1993. Mineral accretion in the fetus and adnexa during late gestation in Holstein cows. Journal of Dairy Science 76, 2999–3010. Lepage, O.M., Carstanjen, B., Uebelhart, D., 2001. Non-invasive assessment of equine bone: an update. The Veterinary Journal 16, 10–22. Liesegang, A., Eicher, R., Sassi, M.L., Risteli, J., Kraenzlin, M., Riond, J.L., Wanner, M., 2000. Biochemical markers of bone formation and resorption around parturition and during lactation in dairy cows with high and low standard milk yields. Journal of Dairy Science 83, 1773–1781. Liesegang, A., Sassi, M.L., Risteli, J., Eicher, R., Wanner, M., Riond, J.L., 1998. Comparison of bone resorption markers during hypocalcemia in dairy cows. Journal of Dairy Science 81, 2614– 2622. Manolagas, S.C., Kousteni, S., Jilka, R.L., 2002. Sex steroids and bone. Recent Progress in Hormone Research 57, 385–409. Patel, O.V., Takenouchi, N., Takahashi, T., Hirako, M., Sasaki, N., Domeki, I., 1999. Plasma oestrone and oestradiol concentrations throughout gestation in cattle: relationship to stage of gestation and fetal number. Research in Veterinary Science 66, 129–133. Sassi, M.L., Eriksen, H., Risteli, L., Niemi, S., Mansell, J., Gowen, M., Risteli, J., 2000. Immunochemical characterization of assay for carboxyterminal telopeptide of human type I collagen: loss of antigenicity by treatment with cathepsin K. Bone 26, 367–373. Sp€ orndly, R. 1999. Fodertabeller f€ or idisslare Rapport 247. Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, Uppsala, Sweden (in Swedish).