Introduction
Subclinical hypocalcaemia (SCH) is characterised by a rapid decline in blood calcium (Ca) concentrations resulting from the relatively rapid loss of Ca associated with the formation of colostrum. Hypocalcaemia is a metabolic disorder in which homoeostatic mechanisms fail to maintain normal blood Ca concentrations at the onset of lactation. SCH as well as clinical hypocalcaemia (CH) are risk factors for many of the important diseases during lactation including mastitis, ketosis, retained placenta, displaced abomasum and uterine prolapse (Degaris & Lean, Reference Degaris and Lean2008; Heppelmann et al. Reference Heppelmann, Krach, Krueger, Benz, Herzog, Piechotta, Hoedemaker and Bollwein2015). However, cows with SCH usually do not show any observable clinical signs of hypocalacemia, therefore, it is difficult to diagnose SCH in on-farm practice. Some studies have suggested that SCH occurs more frequently than CH in periparturient dairy cows (Degaris & Lean, Reference Degaris and Lean2008; Reinhardt et al. Reference Reinhardt, Lippolis, Mccluskey, Goff and Horst2011). The involvement of calcitropic hormones in hypocalacemia has not been well documented and little is known about calcitonin, parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] concentrations in dairy cows with SCH. Furthermore, other mineral elements may also play prominent roles in regulating Ca homoeostasis and may be involved in the pathogenesis of SCH in dairy cows, however, these hypotheses have not been verified.
The goals of this study were to measure the serum concentrations of macrominerals and major calcitropic hormones in periparturient dairy cows and to assess these measures with the state of energy metabolism in SCH and healthy cows. We sought to identify potential mechanisms involved in the pathogenesis of SCH by detecting relevant elements and metabolites with altered concentrations in dairy cows with SCH and in healthy cows and to provide strategies for the prevention and early detection of SCH.
Materials and methods
This study was conducted in a commercial dairy farm located in Heilongjiang Province of China. The experimental procedures of this study were in accordance with the Chinese law on animal protection and were conducted with the approval of the Institutional Animal Care and Use Committee of Northwest A&F University, Yangling, People's Republic of China.
Cows
Initially a total of 310 nonlactating multiparous Chinese Holstein dairy cows in late gestation were used. They were housed in a free-stall barn from 4 wk before anticipated calving date through to 4 wk after parturition. The cows were 3–6 years old with 1–4 parities and similar initial body condition scores (BCS). Prepartum and postpartum diets were formulated to meet the nutrient requirements recommended by the Dairy Cows Breeding Standard of China. Details of the diet are presented in online Supplementary Table S1. All cows were fed ad libitum during the perinatal period.
Based on the criteria from Roche & Berry (Reference Roche and Berry2006), serum Ca levels of 2·0 and 1·4 mmol/l at calving day were proposed as thresholds of SCH and CH, respectively. We selected 51 cows with blood Ca concentration falling in the range of 1·4–2·0 mmol/l at calving as the SCH group. We also selected another 51 cows with the total blood Ca levels within the range of 2·1–2·5 mmol/l at calving, similar lactation number and BCS and no obvious signs or symptoms of other diseases, and used them as the normocalcemic control group. Daily milk production and dry matter intake (DMI) over the first 3 d postpartum are shown in Table 1. The parity and BW at calving are shown in online Supplementary Table S2.
Serum concentrations of glucose, NEFA, BHBA, calcitropic hormones and macromineral elements in cows at calving (mean ± se, n = 51)
Means with different superscription letters within the row differ significantly (lowercase letter, P < 0·05, uppercase letters, P < 0·01). Blood was collected within 24 h of parturition. Milk production and DMI were recorded for the first 3 d postpartum.
Serum samples and analyses
Blood samples from the control group were collected weekly from 4 wk antepartum to 4 wk postpartum, while from cows in SCH group blood samples were taken weekly before calving, and on the calving day. All the blood samples were withdrawn from the coccygeal vein before the morning feeding. Anticoagulants were not used during blood collection. According to the management system of the farm, those cows at parturition with SCH were immediately given an intravenous (IV) injection of calcium (commonly Ca borogluconate) to prevent the catastrophic consequence, afterwards no blood sample was taken from those cows. Periparturient dairy cows with the normal blood Ca concentration were not administered with the Ca injection at any time. Within 1 h of the collection, blood samples were carried to the laboratory, stood at room temperature for 30 min, then centrifuged at 4000 × g for 10 min to obtain serum. The serum was packaged in sterile tubes and stored at −80 °C until further analysis.
Serum non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHBA) levels were measured with the BHBA kit and the NEFA kit (enzymatic method, Randox Laboratories Ltd., Ibach, Switzerland), respectively. Serum glucose concentrations were measured with a commercial kit (glucose oxidase method, Yulan Biotechnology Research Institute, Shanghai, China). Those assays were analysed using a Hitachi 7170 auto-analyzer (Hitachi Co., Tokyo, Japan). The serum concentrations of Ca, potassium (K), sodium (Na), magnesium (Mg), phosphorus (P) and chloride (Cl) were measured with a Beckman Synchron CX system (Beckman Instruments Inc., Fullerton, CA, USA). Calcitonin, PTH and 1,25(OH)2D3 levels were measured using bovine calcitonin, PTH and 1,25(OH)2D3 ELISA kits specifically for bovine (Cusabio Biotech Co. Ltd., Wuhan, China; Calcitonin: CSB-E17374B; PTH: CSB-EL018987BO; 1,25(OH)2D3: CSB-EQ027278BO), respectively. All of the analyses with the kits were performed according to the manufacturer's instructions.
Data analysis
The statistical analyses were performed using SAS software (release 9·3; SAS Institute, Cary, NC). The weekly changes in serum parameters in healthy cows during the peripartal period were analysed as one-way ANOVA using the PROC MIXED procedure. For the serum concentration of Ca between SCH and the healthy control over the peripartum period, one-way ANOVA for repeated-measures was performed using the PROC MIXED procedure, where the group, time, and their interactions were set as the fixed effects, and animal was set as a random effect. In addition, the slice option of MIXED procedure in the SAS software was used to test the difference between two groups at each time point. The differences in the serum index on calving day and in milk production and DMI for the first 3 d postpartum between SCH and the healthy control were compared using the unpaired t-test. Results were expressed as the mean ± se. P < 0·05 was considered as statistically significant.
Results and discussion
Changes in serum parameters in healthy cows
The present study showed clearly that dairy cows underwent substantial changes in macrominerals during the peripartal period (Fig. 1). Serum Ca and K concentrations were significantly higher during late pregnancy than those during parturition (P< 0·01), and their concentrations were lowest at parturition (Fig. 1a, b). After calving the Ca concentrations remained at this low level over the first 3 wk postpartum, and then rose in wk 4 (Fig. 1a). Serum K levels increased gradually and reached their baseline levels, defined as the average concentrations of serum parameters over 3–4 wk prepartum, at wk 4 postpartum (P < 0·01, vs. at parturition) (Fig. 1b). Serum Na levels peaked at parturition, and then decreased gradually to their baseline levels (P < 0·01, vs. at parturition) (Fig. 1c). The Mg serum concentrations were lowest in wk 1 postpartum (P < 0·01, vs. at parturition) and subsequently returned to their baseline levels (Fig. 1d). Serum Cl concentrations peaked in the first week prepartum, then decreased gradually and remained at low levels during early lactation (P < 0·01, vs. at parturition) (Fig. 1e). The serum P concentrations were significantly higher during wk 1 and wk 2 prepartum than at parturition (P < 0·05 and P < 0·01, respectively), and returned to the baseline levels from wk 1 to wk 4 postpartum (Fig. 1f). The changes of these macrominerals during the peripartal period coincide with previous findings (Chan et al. Reference Chan, West and Bernard2006), which can be explained by the fact that lactation starts at parturition, and macrominerals are used for forming the colostrum.
Weekly changes in serum analytes for dairy cows during peripartum period A-K, Weekly changes in serum analytes in healthy cows, * and ** indicate significant differences (P < 0·05, P < 0·01) vs. parturition (week 0). L, Weekly changes in serum Ca levels in SCH and the healthy control. Data are mean ± se; n = 51 for A-K; n = 10 for L.
Serum PTH concentrations peaked at calving, and then returned to the baseline levels during wk 4 postpartum (P < 0·01, vs. at parturition) (Fig. 1g). The serum concentration of 1,25(OH)2D3 peaked in the first week postpartum, and these concentrations were significantly higher at parturition and in wk 3 postpartum than those of late pregnancy (P < 0·01) (Fig. 1h). Serum calcitonin concentration did not undergo marked fluctuations during the peripartal period (Fig. 1i). Overall, our data suggest that the increasing concentrations of PTH and 1,25(OH)2D3 play key roles in maintaining the stabilisation of blood Ca concentrations of periparturient dairy cows. We found that the serum NEFA and BHBA concentrations peaked at parturition and in wk 1 postpartum, respectively (Fig. 1j, k). Compared with the healthy cows, the cows with SCH had significantly lower serum glucose concentrations (P < 0·05) and higher serum NEFA and BHBA concentrations (P < 0·01) (Table 1), which were resulted from the higher milk production and lower DMI, suggesting a negative energy balance status (Wang et al. Reference Wang, Zhu, Chen, Li, Gao, Li, Zhang, Long, Wang and Liu2012). These results are consistent with those of previous studies (Reinhardt et al. Reference Reinhardt, Lippolis, Mccluskey, Goff and Horst2011). Large amounts of NEFA may be incompletely oxidised to ketones or esterified to triacylglycerides (TAG) when the uptake of NEFA exceeds the oxidative capacity of the liver, which can potentially lead to the development of ketosis or fatty liver (Song et al. Reference Song, Li, Gu, Fu, Peng, Zhao, Zhang, Li, Wang, Li and Liu2016; Du et al. Reference Du, Shi, Peng, Zhao, Zhang, Wang, Li, Liu and Li2017). These results could explain why cows with SCH are more susceptible to ketosis, fatty liver and other metabolic diseases. Consequently, SCH represents a potential unhealthy risk to the periparturient dairy cow.
Serum analytes in subclinically hypocalcaemic and normocalcemic cows
As shown in Table 1, serum P concentrations were significantly higher in the dairy cows with SCH (P < 0·05); in contrast,.Ca, K, Na, Mg and Cl concentrations were significantly lower in the dairy cows with SCH than in the normocalcemic control (P < 0·05). The results of weekly changes in serum Ca levels over the peripartum period showed that the Ca level in SCH cows in 1 week before calving was significant lower than those in healthy cows (Fig. 1l). These results suggest that diets with high levels of K or Na could reduce cow's ability to maintain Ca homoeostasis, thereby increasing the risk of hypocalcaemia. It is believed that the reduced concentrations of serum K and Na may be associated with prolonged inappetence in cows with SCH around calving or with a decreased intake. However, the relationship between K and Na blood concentrations and the incidence of SCH needs to be further investigated.
As the most important calcitropic hormone, PTH is a part of the negative feedback loop to maintain Ca homoeostasis. Moreover, when blood Ca concentration decreases, PTH is rapidly activated (within minutes) and has regulatory effects on Ca homoeostasis via stimulating osteoclastogenesis or promoting production of 1,25(OH)2D3. Studies also showed that cows with CH have higher PTH and 1,25(OH)2D3 concentrations than healthy cows (Degaris & Lean, Reference Degaris and Lean2008). In the present study, serum 1,25(OH)2D3 levels were lower for the cows with SCH than those for the healthy cows (P < 0·01), whereas the serum calcitonin and PTH concentrations were not significantly different between the two groups (P > 0·05). We suggest that the cows with SCH may fail to respond to PTH. This view is consistent with a previous study (Goff, Reference Goff2008). On the other hand, lower serum concentrations of Mg, critical for the release of PTH and the synthesis of 1,25(OH)2D3, may partly result in the blunted response to PTH in cows with SCH. Very recently, Rodroguez and coworkers reported that blood calcitonin concentration was not affected by the severity of SCH (Rodriquez et al. Reference Rodriquez, Bach, Devant and Aris2016). However, under metabolic acidosis, the activity of calcitonin is impaired, and the activity of PTH could be increased at the same time, thereby maintaining Ca homoeostasis. Therefore, it could be a potential strategy to prevent hypocalcaemia by lowering blood pH in the future.
Conclusion
In summary, the present study shows that the levels of macrominerals and major calcium-regulating hormones (with the exception of calcitonin) changed dramatically in dairy cows over the periparturient period, particularly around calving. These changes complicate the maintenance of Ca homoeostasis. The cows with SCH display a blunted PTH response to the decline in blood Ca levels and a reduced production of 1,25(OH)2D3, resulting in the suppression of bone resorption and the renal reabsorption of Ca. Furthermore, the cows with SCH exhibited more severe NEB, which confirms that SCH can predispose dairy cows to other metabolic diseases such as ketosis and fatty liver.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029918000031.
The project was supported by the National Natural Science Foundation of China (Grant No. 31502129), China Postdoctoral Science Foundation funded project (No. 2014M560811), and Programs for Science and Technology Shaanxi (No. 2016NY-100).
