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Keywords:

  • parathyroid hormone ;
  • periparturient cow;
  • crosslinked telopeptide of type 1-collagen;
  • magnesium

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

This experiment studied the effect of pre-partal milk removal on calcium metabolism at start of milking and at calving. Nine cows of the Swedish Red breed were milked for 1–7 days pre-partum. The average milk yield at the first milking was 4.8 l, and the average yield the last day prior to calving was 13.4 l. Five cows were used as control cows and were only milked post-partum. Samples of plasma and urine were taken to determine the effect of pre-partum milking and calving on levels of calcium, magnesium, parathyroid hormone and plasma C-terminal crosslinked telopeptide of type 1-collagen (CTx), used as a marker of bone resorption. Pre-partum milking resulted in a decrease in plasma calcium that was evident 2 days after the first milking. Parathyroid hormone increased at the same time, and CTx started to increase from 24 h after the first milking. There were no effects on plasma magnesium or urinary output of calcium or magnesium. The first week after calving, there were no differences between pre-partum milked cows and control cows in plasma or urine variables, or in milk yield. In conclusion, pre-partum milking activated the calcium-restoring mechanisms but did not improve calcium status at calving.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Milk fever in dairy cows is characterised by hypocalcemia which occurs at parturition when milk production is started and calcium requirements increase. The level of calcium in plasma is well regulated, and when the level decreases, the parathyroid gland will excrete parathyroid hormone (PTH). This increases the mobilisation of calcium from the skeleton and also raises the renal threshold for calcium in the kidneys (Goff et al., 1986). During the dry period, the supply of calcium through the diet is usually more than adequate to maintain homoeostasis without activating the calcium mobilisation system (Ramberg et al., 1970), which is thus usually not activated until parturition. It has been suggested that 48 h of PTH stimulus is needed to mobilise calcium (Goff et al., 1986), and this lag phase is believed to be important in the development of milk fever (DeGaris and Lean, 2008). Pre-partum milking has been tried as a means to slowly increase the demand for calcium and thus adapt the calcium metabolism in the cow to the increased losses through milk at parturition. Results from earlier studies on pre-partum milked cows (Smith and Blosser, 1947; Eaton et al., 1949) have not shown any beneficial effects on the plasma calcium homoeostasis at calving. However, results from a field study by Greene et al. (1988) indicate that pre-partum milking prevents milk fever.

Littledike (1976) investigated the changes in plasma calcium during periods of milking and non-milking in lactating dairy cows and found a close relationship between the onset of milking and plasma calcium and PTH. Goff et al. (2002) showed that old mastectomised Jersey cows experienced no hypocalcemia at calving, while intact cows of a similar age all developed milk fever. These studies indicate the importance of onset of lactation and removal of milk in the development of parturient hypocalcemia. However, the results from Littledike (1976) indicated that the decrease in plasma calcium was evident only after some days of milk removal, while the results from other studies have shown that plasma calcium is decreased immediately after parturition, even if no milk has been removed at that time (Melendez et al., 2002; Penner et al., 2008). Stott (1968) cited evidences for a mammary independent cause of hypocalcemia and milk fever in dairy cows, and Smith et al. (1948) could not detect any differences in milk fever incidence or serum calcium in cows completely or partially milked post-partum.

Previous studies using pre-partum milking of cows to decrease the strain on the calcium homoeostasis have started the pre-partum milking at a fixed time before expected calving, often when the udder fill is still low (Smith and Blosser, 1947; Greene et al., 1988). This results in less than a kg of milk at the first milking and a slowly increased yield (Zeliger et al., 1973). To our knowledge, no study has been made where a larger amount of milk is removed from the udder at the first pre-partum milking. To imitate the loss of calcium occurring at the onset of lactation after calving, we aimed to remove a considerable amount of milk at the first pre-partum milking. The objective of this study was to investigate the effects of pre-partum milking on calcium homoeostasis at onset of lactation and at calving.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

The experiment was performed during February to June 2009 in the university herd at Kungsängen Research Centre and was approved by the local ethics committee in Uppsala.

Fifteen cows of the Swedish Red breed (mean body weight 607 kg, SD 68 kg) were used. When the experiment started, the cows were dry, pregnant and about to enter their second to seventh lactation. None of the cows had been treated for milk fever at any of the previous calvings. The cows were dried off approximately 8 weeks before expected calving date. The diets were composed of forage, straw and concentrates according to Swedish feeding recommendations (Spörndly, 2003). The dietary concentrations of calcium and magnesium in the dry period were more than 1.5 times the recommendations of Spörndly (2003). The cows were kept in individual tie stalls, except for during calving which took place in a single-cow calving pen. The calf stayed in the pen with the cow for 1 up to 18 h and was not prevented from suckling. The body weight of the cow was recorded when she was moved from the calving pen after calving. One of the control cows showed clinical signs of hypocalcemia post-partum and was treated with intravenous calcium 7 h after calving. No samples after the treatment were included in the analysis.

Based on time to expected calving and udder fill, twice daily pre-partum milking was initiated in nine of the cows, evenly distributed among cows pregnant for the second time and cows in their third or higher pregnancy. The allocation of cows to pre-partum milking or control was made based on age and expected calving date. The cows were milked at 6.30 and 16.00 both before and after calving. All milkings were performed using machines and were preceded by cleaning with a paper tissue and pre-milking to visually inspect the milk. The teat cups were attached until milk flow stopped and the udder felt empty. The volume of the milk was recorded at each milking, and samples were taken for mineral analysis.

Starting approximately 3 week prior to expected calving date, blood samples were drawn from the tail vein twice a week from all cows. A blood sample was drawn 6 and 24 h after initiation of pre-partum milking and then every second day until calving. Blood samples were taken 6, 12 and 24 h, 2, 4 and 7 days after calving in all cows. All blood samples, except for the ones taken 6 or 24 h after initiation of milking in the pre-partum milked cows and 6, 12 and 24 h after calving in all cows, were taken between 7.30 and 8.30. All blood samples were drawn into vacuum tubes with lithium heparin as additive and were centrifuged at 1800 g for 10 min (Jouan 4.11, Saint-Nazaire, France) within 1 h after sampling. Spot samples of urine were taken one per day, at the same days as the blood samples, and were centrifuged at 200 g for 1 min (Jouan 4.11). Plasma and urine samples were stored in triplicates in plastic tubes at −20 °C until analysis.

Plasma, milk and urine samples were analysed for calcium and magnesium using commercial kits based on colorimetric methods with O-Cresophtalein (Randox, Crumlin, UK) and xylidyle blue (Randox), respectively. Plasma C-terminal crosslinked telopeptide of type 1-collagen (CTx) was analysed with an ELISA method (Serum Crosslaps ELISA; Nordic Bioscience, Herlev, Denmark), and plasma PTH was analysed with an ELISA method (Bovine Intact PTH ELISA Kit; Immutopics, San Clemente, CA, USA). Creatinine concentration in urine was determined after dilution 1:20 with distilled water on an auto-analyzer (Technicon, Tarrytown, NY, USA), using Jaffees reaction, a colorimetric method with picric acid (Technicon, 1974).

Daily urine excretion was estimated using the creatinine concentration of the urine and assuming a daily excretion of 29 mg creatinine per kg body weight (Valadares et al., 1999). Correlations between the number of days milked and the production during the last 24 h prior to calving were made using the CORR procedure in sas (SAS 9.1; SAS Institute, Cary, NC, USA). The statistical analyses of the effect of pre-partum milking and time in relation to calving were made using the MIXED procedure in sas. Prior to analysis, the cows were divided into two groups according to parity, with cows calving for the second time and older cows in separate groups. To adjust for time-related differences in, for example, feeds and seasonal variation, the cows were divided into three groups based on calving time. Calving time group (three levels) and parity group (two levels) were included as fixed effects in all models. Cow was included as a random effect, and spatial power covariance structures were used to model repeated measures over time within each cow. Effects were considered significant at p < 0.05, and pairwise comparisons were made to evaluate significant differences. Results are presented as least squares means ± standard error of the mean unless stated otherwise.

Only data from pre-partum milked cows, the last 2 weeks prior to the first pre-partum milking and until calving, were included in the analysis of the effect of pre-partum milking. The statistical models included the effect of time, as a fixed factor with nine levels, representing the time prior to the first pre-partum milking divided into five 3-day periods and the four first sampling times after the first pre-partum milking.

In the analysis of the effect of treatment at calving, only data from the last sample were taken pre-partum, and all post-partum samples were included. The pre-partum milked cows were all milked at the time of the pre-partum sampling. The model included the effect of time, treatment and the interaction treatment*time, where time was a fixed effect with seven levels (prior to calving or 6, 12 or 24 h, 2, 4 or 7 days after calving), and treatment was a fixed effect with two levels, indicating if the cow had been pre-partum milked or not.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

The amount of milk obtained at the first milking pre-partum ranged from 0.5 to 10 l, with a mean value of 4.8 l. Pre-partum milked cows were milked for 1–7 days before calving, on average for 2.5 days (five milkings). The milk production during the 24 h before calving in the pre-partum milked cows ranged from 6.8 to 25.8 l, with an average of 13.4 l. There was an effect of the number of days milked pre-partum on the amount of milk produced during the last 24 h before calving, with a regression coefficient of 1.93 (p = 0.024). There were no effects of treatment on milk production the first week after calving. The concentration of calcium in milk was higher before calving and the first day after calving compared with later (p = 0.021, data not shown).

The pre-partum start of milking was associated with a decrease in plasma calcium that was evident 2 days after the first milking (p = 0.003, Fig. 1). This decrease coincided with a simultaneous increase in PTH (p = 0.041). Plasma CTx was stable prior to milking and started to increase from 24 h after the first milking (p = 0.001). Pre-partum milking did not affect plasma magnesium (p = 0.108, Fig. 1) or the excretion of magnesium (p = 0.918) and calcium (p = 0.481) in urine.

image

Figure 1. Plasma calcium (Ca), magnesium (Mg), parathyroid hormone (PTH) and carboxy-terminal crosslinks of type 1-collagen (CTx) on day relative to the first pre-partum milking, which occur at day 0. Samples after milking are taken 6 h after milking, 24 h after milking, 2 days after milking and 4 days after milking. Data are shown as least squares means ± standard error of the mean.

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At the sampling before calving, pre-partum milked cows had a lower level of plasma calcium compared with control cows (p = 0.004), but there were no differences between treatment groups after calving. Plasma PTH increased at calving in control cows (p = 0.005), but the level in pre-partum milked cows remained at the same level as before calving. At 4 days after calving, plasma PTH decreased in all cows. There was no effect of pre-partum milking on plasma CTx at calving (p = 0.087), but the level was increased at the sampling 6 h after calving and remained higher than pre-partum levels (Fig. 2). There was also no effect of pre-partum milking on plasma magnesium (Fig. 2) or the urinary excretion of calcium and magnesium after calving.

image

Figure 2. Plasma calcium (Ca), magnesium (Mg), parathyroid hormone (PTH) and carboxy-terminal crosslinks of type 1-collagen (CTx) in cows milked pre-partum (○) and control cows (●). Time is related to calving, occurring at day 0. Samples after calving are taken 6, 12 and 24 h after calving and 2, 4 and 7 days after calving. Data are shown as least squares means ± standard error of the mean.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Pre-partum milking affected plasma calcium, with decreased values from 2 days after onset of milking. The levels of PTH increased when plasma calcium decreased, in accordance with earlier results (Kurosaki et al., 2007). However, plasma CTx was increased already at 24 h after the first milking and continued to increase until calving. This indicates that the mobilisation of calcium from bone was rapidly activated as a response to the removal of milk, before any significant differences in plasma calcium or the excretion of PTH could be seen. The decrease in plasma calcium was much slower than what was found in control cows after calving, and the levels remained low during the rest of the pre-partum milking period, while plasma calcium after calving was restored to pre-partum levels after 4 days. Littledike (1976) found a nadir in plasma calcium approximately 3 days after milking was started, and four of the pre-partum milked cows in the present study were milked <3 days. From these results, it is suggested that the losses of calcium through milk decreases plasma calcium, which could affect the extent of hypocalcemia and the risk of milk fever.

Plasma calcium was decreased 6 h after calving compared with pre-partum levels in all cows and reached a nadir 24 h after calving. Even if the increase in both PTH and CTx indicated that the pre-partum milked cows had activated their calcium homoeostatic mechanisms prior to calving, the lack of treatment effect after calving suggests that they were as susceptible to the calcium losses at parturition, because they were not more able to mobilise calcium. Also, they did not maintain the calcium level in plasma to a higher extent compared with control cows. This is in accordance with Andersen et al. (2005), who showed that omitting the dry period resulted in lower milk yield after calving but did not affect plasma calcium levels. We found that the changes in plasma calcium, PTH and CTx were more rapid at calving, compared with at onset of milking, in the pre-partum milked cows. In most of the cows, the lowest measured level of plasma calcium and the peak in PTH was within 24 h after calving, and CTx was increased already at the first sampling 6 h after calving. Eleven cows in the present study experienced some degree of hypocalcemia, defined as plasma calcium below 1.88 mm (Goff et al., 1996) on at least one sampling after parturition, and the cows that managed to maintain normal values in plasma calcium were from both treatment groups (data not shown). The rapid increase in CTx indicates that the assumed lag phase between the increase in PTH and the mobilisation of calcium from bones is shorter than suggested by Goff et al. (1986).

Four days after calving, plasma calcium levels were restored to pre-partum levels. In contrast, 4 days after milking in the pre-partum milked cows, plasma calcium was at its lowest. This indicates that cows in late gestation have a lower capacity to cope with increased losses of calcium compared with cows after calving. The hormones related to pregnancy have been shown to suppress bone mobilisation in humans (Clowes et al., 2009), and earlier studies have shown a decreased rate of calcium mobilisation as a response to a hypocalcemic challenge in late gestation compared with in lactating or non-pregnant dry cows (van de Braak et al., 1984). However, in the present study, plasma CTx did increase 24 h after the first pre-partum milking, although plasma Ca was not significantly decreased at that time. This suggests that there may be other factors than a suppression of bone mobilisation that caused the decreased ability to restore plasma calcium in the pre-partum milked cows. There are however other processes for bone catabolism than the ones where CTx is formed (Garnero et al., 2003), and it may be possible that these processes are more suppressed by the hormones related to pregnancy.

The transient increase in plasma magnesium that occurred after calving is consistent with what have been found previously (Thilsing-Hansen et al., 2002) and has been suggested to result from a decrease in urine excretion of magnesium as a result of the PTH-induced increase in the renal threshold (Goff, 2008). However, in the present study, urinary excretion of magnesium did not decrease after calving, even if calcium excretion decreased rapidly.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Pre-partum milking for 1–7 days decreased plasma calcium and resulted in an activation of the calcium homoeostatic mechanisms prior to calving. However, the response in plasma calcium, PTH and bone mobilisation was slower compared with at calving. Pre-partum milking did not improve the calcium status in periparturient dairy cows.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Håkan Wallin, Kungsängen Research Centre, is acknowledged for analytical work.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
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