Biochemical Markers of Bone Formation and Resorption Around Parturition and During Lactation in Dairy Cows with High and Low Standard Milk Yields1,2 A. Liesegang,* R. Eicher,† M.-L. Sassi,‡ J. Risteli,‡ M. Kraenzlin,§ J.-L. Riond,* and M. Wanner* *Institute of Animal Nutrition, University of Zurich, 8057 Zurich, Switzerland †Clinic for Food Animals and Horses, University of Berne, 3012 Bern, Switzerland ‡Department of Clinical Chemistry, University of Oulu, 90220 Oulu, Finland §Endocrine Unit, University Hospital, 4031 Basel, Switzerland
ABSTRACT Substantial changes occur in skeletal metabolism during lactation. These dynamic changes are monitored with biochemical bone markers. The goal of the present study was to follow these changes in lactating cows and to investigate whether cows with a higher milk yield have a higher mobilization rate of calcium from bone. Hydroxyproline, deoxypyridinoline, pyridinoline, and the carboxyterminal telopeptide of type I collagen (ICTP) were chosen as markers for bone resorption, whereas osteocalcin was used as a bone formation marker. Urine and blood samples were collected from cows with a mean standard milk yield of 4900 and 6500 kg, respectively, 14 d before, and 14 d, 1 mo, 1.5 mo, and monthly after parturition. Urinary hydroxyproline, deoxypyridinoline, and pyridinoline concentrations increased with time, but no differences between the two groups were evident. Concentrations of 1,25-dihydroxy vitamin D and ICTP of the two groups showed an increase 14 d after parturition. Furthermore, using multivariate regression models with age and milk yield as covariates, ICTP concentrations were higher in the group with a higher milk yield. In contrast, osteocalcin concentrations decreased 14 d after parturition and returned to prepartum values 1 mo after parturition.
Received November 8, 1999. Accepted March 10, 2000. Corresponding author: A. Liesegang; e-mail:
[email protected]. 1 This work was supported by Swiss National Science Foundation Grant Number 32-46791.96. 2 Parts of this work were presented at the Swiss Federal Institute of Technology, Zurich, Switzerland, May 1998 in “Healthy farm animals: New approaches in animal nutrition,” at the 10th International Conference on Production Diseases in Farm Animals, Utrecht, Netherlands, August 1998, at the 2nd joint meeting of the “International Society of Bone and Mineral Research” and “American Society of Bone and Mineral Research,” and at the satellite meeting on “Comparative Endocrinology and Calcium Regulation,” San Francisco, CA, December 1998. 2000 J Dairy Sci 83:1773–1781
The increase of ICTP concentrations in both groups indicates that bone was substantially resorbed. At the same time, probably less Ca was embedded in bone, as indicated by the decrease of the osteocalcin concentrations. In conclusion, cows showed increased bone resorption around parturition, and cows with higher milk yield mobilize calcium more actively from bone than cows with lower milk yield. (Key words: bone markers, lactation, dairy cows, parturition) Abbreviation key: DPD = deoxypyridinoline, HYP = hydroxyproline, ICTP = carboxyterminal telopeptide of type I collagen, MI305 = standard milk yield after 305 d of lactation, MMP = matrix metaloproteinase, OC = osteocalcin, PTH = parathyroid hormone, PYD = pyridinoline, VITD = 1,25-(OH)2 vitamin D. INTRODUCTION The late stages of pregnancy and all stages of lactation represent physiological situations of Ca stress and therefore contribute to dramatic changes in Ca and P metabolism. Increased maternal mineral and bone metabolism is known to occur because of skeletal mineralization of the fetus during pregnancy and milk production during lactation in mammals (6, 15), and marked bone mineral changes are often observed in humans (29, 43), which sometimes even result in severe osteoporosis (19, 28). Intestinal muco-serosal Ca transport and bone resorption increase (23) to meet fetal and neonatal Ca and P requirements as well as subsequent changes in vitamin D and parathyroid hormone (PTH) metabolism and have been described in several studies (21, 22, 23, 28). The PTH and vitamin D endocrine system functions to maintain a normal Ca homeostasis (21). When the Ca concentration in serum falls below the normal range, PTH secretion is stimulated. The increased circulation of PTH stimulates the production of active 1,25-dihydroxy vitamin D (VITD). The elevated VITD concentration stimulates active Ca transport in the intestine to help restore the serum Ca pool. During pregnancy and
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lactation, the flux of Ca to the fetus or into the milk results in a significant decrease in serum calcium. The Ca requirements for milk production have a significant effect on maternal mineral and skeletal homeostasis during lactation. In humans for example, at least about 300 mg of Ca is needed per day for maternal milk production (28). In cows, Ca homeostatic mechanisms are disturbed by a sudden increase in demand for Ca from 10 to 12 g/d to more than 30 g/d because of colostrum production. Biochemical bone markers such as urinary pyridinoline (PYD), deoxypyridnoline (DPD), and serum osteocalcin (OC) have been used in humans and other species to evaluate bone metabolism during lactation and pregnancy (47). The purpose of this study was to investigate which pattern selected bone markers follow, and to determine, by measuring different biochemical bone markers, whether cows with a higher standard milk yield have a higher mobilization of Ca from bone around parturition and during lactation than cows with a lower standard milk yield. The following markers were determined: urinary PYD, DPD, and hydroxyproline (HYP), serum OC, and serum carboxyterminal telopeptide of type I collagen (ICTP).
land). Serum and urine were stored at −20°C until the analyses were performed.
MATERIALS AND METHODS
Serum concentrations of ICTP were measured using a radioimmunoassay as described by Liesegang et al. (32).
Animals The field trial included 30 purebred Brown Swiss and Brown Swiss crossbred cows. The cows were 5 to 13 yr old and had at least two previous lactations. All cows calved between January and April. The cows were all housed, treated, and fed the same way during the prepartum period. A detailed description of the diets fed to these cows, both pre- and postpartum, is shown in Table 1. During the sampling period, the cows were housed in a free stall. Cows were assigned to two groups: group LOW included 15 cows with a mean standard milk yield (305 d) of 4900 (± 447) kg, and group HIGH consisted of 15 cows with a mean standard milk yield of 6500 (± 620) kg. Blood and urine samples were taken 14 d before parturition (ante partum—ap), 14 d (d), 1 mo (30 d), 1.5 mo (45 d), and monthly until 8 mo after parturition (postpartum—pp).
Laboratory Analyses Urine samples were analyzed for DPD, PYD, HYP, and creatinine and serum was analyzed for Ca, P, Mg, OC, ICTP, and VITD. Blood Determinations of Ca, P, and Mg We determined mineral levels by colorimetry with an autoanalyzer (COBAS MIRA Roche-autoanalyzer, F. Hoffmann-La Roche Ltd., Basle, Switzerland), using commercial kits. Analyses were based on the following methods: methylthymol blue for Ca, phosphomolybdate without precipitation of proteins for P, and calmagite for Mg. Blood Determinations of OC, PTH, and VITD Serum concentrations of OC, PTH, and VITD were measured with a commercially available radioimmunoassay (Nichols Diagnostics, San Juan, Capistrano, CA). Blood Determinations of ICTP
Urinary DPD, PYD, HYP, and Creatinine Determination Deoxypyridinoline and PYD were determined by a HPLC method (4) with a commercial kit (Crosslinks by HPLC, BIO-RAD Laboratories, Munich, Germany). Hydroxyproline was quantified by a colorimetric method (2). The optical density was measured at 558 nm (Shimazu UV-Vis recording spectrophotometer UV160, Shimazu Cooperation, Kyoto, Japan). Urinary creatinine was determined by colorimetry with the COBAS MIRA autoanalyzer, using a commercial kit. The DPD, PYD, and HYP concentrations in urine were corrected for creatinine. Statistical Analysis
Blood and Urine Sampling Blood samples were collected from the jugular vein with a Vacutainer (10 ml, without additives; Aichele Medico AG, Basle, Switzerland). Blood was centrifuged (1580 × g for 10 min, 4°C) within 30 min of sampling. Urine was collected aseptically with a Ru¨sch catheter (external diameter 8 mm; Provet, Lyssach, SwitzerJournal of Dairy Science Vol. 83, No. 8, 2000
A multiple linear regression model was fitted for all the dependent variables at the 14-d pp sample with the factors AGE (in years) and standard milk yield after 305 d of lactation (MI305). The final model was obtained after a backward elimination procedure in which factors of the model were eliminated if the P-value was > 0.1. If significant models were found, corrected values
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BONE MARKERS AROUND PARTURITION AND DURING LACTATION Table 1. Composition of diets as fed to the cows of the experiment in different stages of lactation. First lactation until wk 11; ration balanced for 21 kg of milk/d Ingredient (kg of DM) Hay (barn-dried) 4.9 Grass silage 3.0 Corn silage 6.0 Beet pulp 1.9 Protein complement 1.9 Hay (field-dried) Oat straw Mineral salt 0.31 Total amount fed 18.0 Mineral composition (g of kg DM) Ca 7.4 P 4.4 Mg 2.1 Na 1.3 K 18.3 Cl 5.5 S 1.8 DCAD in meq/kg DM4 256
Second lactation and following until wk 8; ration balanced for 27.3 kg of milk/d
First lactation from wk 12 of lactation; ration balanced for 18.2 kg of milk/d
4.9 3.0 6.0 1.9 2.3
Second lactation and following from wk 9 of lactation; ration balanced for 24.2 kg of milk/d
5.5 3.4 6.8 2.2 0.5
1
5.5 3.4 6.8 2.2 0.6
2
1.6 5.3 2.2 0.1753 9.3
2
0.3 18.4
0.5 18.9
0.5 19.0
7.2 4.3 2.1 1.2 17.9 5.1 2.1 237
8.4 5.2 2.2 1.4 19.7 5.0 2.3 280
8.4 5.2 2.2 1.4 19.6 5.0 2.3 277
Dry cows
5.8 5.6 1.8 0.9 21.0 8.0 3.6 124
1
Ca-rich salt with (per kg) 170 g of Ca, 50 g of P, 40 g of Mg, and 40 g of Na. Ca/P-balanced salt with (per kg) 150 g of Ca, 75 g of P, 30 g of Mg, and 40 g of Na. 3 P-rich salt with (per kg) 75 g of Ca, 150 g of P, 20 g of Mg, and 20 g of Na. 4 DCAD: dietary cation anion difference calculated as (Na+ + K+) − (Cl− − S2−) in meq/kg DM: 1 g of Na+ + 43.5 meq, 1 g of K+ = 25.6 meq, 1 g of Cl− = 28.2 meq, 1 g of S2 = 62.5 meq. 2
were generated and used for further analyses (graphics and nonparametric comparisons). The time-dependent patterns of the variables are presented as box-plots. The length of each box shows the range of the central 50% of the values, with the box hinges at the first and third quartiles (25 to 75% of the values) and the line within the box showing the median value (50%). The whiskers show the range of values within the inner fences (= hinge ± 1.5 × (hinge − median)). Values between the inner and the outer fences (= hinge ± 3 × (hinge − median)) are plotted with asterisks (“outside values”), values outside the outer fences are plotted as empty circles (“far outside values”). A multivariate analysis of variance for repeated measures (MANOVA) was performed with GROUP as a cofactor included in the model to test differences of the time-dependent patterns between groups. To avoid false conclusions due to a violation of the assumption of compound symmetry, a Huynh-Feldt correction was performed. Furthermore, the difference between GROUPS at 14 d pp was tested with the Mann-Whitney U-test (nonparametric) to limit the influence of extreme values. The level of significance was set at α = 0.05 for all tests. All statistical analyses were performed by use of SYSTAT for Windows (Version 7.0, SPSS Inc. Chicago, IL).
RESULTS Results of the general linear model analysis for the four markers with significant models 14 d pp are presented in Table 2. The median ICTP concentration was significantly influenced by GROUP and MI305, whereas median OC and VITD concentrations were only nearly significantly influenced by AGE. The ICTP to OC ratio was neither AGE nor MI305 dependent, but GROUP was significantly included in the regression model. The multiple r-square values indicate how much Table 2. Results of the general linear model analysis for carboxyterminal telopeptide of type I collagen (ICTP), osteocalcin (OC), 1,25dihydroxy vitamin D (VITD), and ICTP to OC ratio at 14 d postpartum. The initial model included the factors GROUP (high/low standard 305-d milk yield1), AGE (in years) and standard 305-d milk yield (MI305). The final model was obtained after a backward elimination procedure (factors eliminated of the model if their P-value was > 0.10). Parameter
Factors
P-value
Multiple r-squared
ICTP
GROUP MI305 AGE AGE GROUP
