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Effects of dietary acidity on calcium balance and mobilisation, bone morphology and 1,25 dihydroxyvitamin D in prepartal dairy cows
Research in Veterinary Science 1994, 56, 310-318
Effects of dietary acidity on calcium balance and mobilisation, bone morphology and 1,25 dihydroxyvitamin D in prepartal dairy cows H. ABU DAMIR, M. PHILLIPPO, Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, B. H. THORP, AFRC Institute of Animal Physiology and Genetics, Edinburgh Research Station, Roslin, Midlothian EH25 9PS, J. S. MILNE, Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB , L. DICK, AFRC Institute of Animal Physiology and Genetics, Edinburgh Research Station, Roslin, Midlothian EH25 9PS, I. M. NEVISON, Scottish Agricultural Statistics Service, Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB
The effect of an acid or alkali diet was investigated in 14 mature dairy cows during the last 28 days of pregnancy. The acid diet reduced the incidence of parturient hypocalcaemia compared with the alkali diet and was associated with higher blood ionised calcium and plasma chloride concentrations and lower blood pH and acid-base excess before parturition. Plasma 1,25-dihydroxyvitamin D concentrations before parturition were increased by the acid diet but the concentrations of parathyroid hormone and intact 1-86 parathyroid-hormone-related peptide were unaffected. The estimated fractional calcium absorption and calcium mobilisation rate during an ethylenediamine tetraacetic acid infusion were increased by the acid diet 14 days before parturition. Cortical bone remodelling occurred in all the animals during late pregnancy but was particularly evident in the cows given the acid diet. The data suggest that an acid diet ameliorates parturient hypocalcaemia by enhancing calcium mobilisation before parturition by increasing calcium absorption and bone resorption, these increases possibly being mediated by increases in the plasma concentration of 1,25-dihydroxyvitamin D. THERE has long been interest in the feeding of an acid diet to reduce the incidence of parturient paresis and hypocalcaemia (Ender et al 1962, 1971, Dishington 1975, Block 1984, Oetzel et al 1988, Gaynor et al 1989, Phillippo et al 1994) and various mechanisms of action have been suggest-
ed. These include increased calcium absorption causing a positive calcium retention both before and after parturition (Ender et al 1971, Verdaris and Evans 1976) which offsets the decreased food intake that occurs just before parturition (Ender et al 1971, Gaynor et al 1989). In addition, acidic diets cause hypercalciuria (Hart et al 1931, Verdaris and Evans 1976, Oetzel et al 1991) which, in conjunction with increased calcium absorption, may increase the turnover of body calcium. Acid diets were also thought to increase bone resorption because they increased plasma hydroxyproline concentrations (Block 1984, Gaynor et al 1989) and therefore would affect the calcium changes occurring in the spongiosa and cortical bone during pregnancy and lactation (Pitkin 1975, Miller et al 1982, Redd et al 1984, Brommage and DeLuca 1985). An involvement of 1,25-dihydroxyvitamin D (1,25-(OH) 2 vitamin D) in the mechanism of action has been proposed by Gaynor et al (1989), Goff et al (1991), and Phillippo et al (1994) but other associated mechanisms have not yet been elucidated. This study was done to elucidate the roles played by the changes in 1,25-(OH) 2 vitamin D and calcium balance, calcium mobilisation and bone resorption occurring before parturition in the prevention of parturient paresis and hypocalcaemia when cows are given an acid diet. Materials and methods
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Fourteen Friesian cows, five to 10 years old,
Acid diet, calcium and vitamin D TABLE 1: Analysis of the acid and alkaline diets fed to dairy COWS
Constituent Calcium Phosphorus Magnesium Anion-cation balance* (mmol)
Concentration (mol kg dry matter-1) Alkali diet Acid diet 0.33 0.13 0.16 +779
0.39 0-13 0-19 -35
* Calculated from (Na + + K+ - C I - - S-) kg dry matter -1
weighing 600 to 800 kg and in their third to eighth parity were randomly allocated by age, parity and lactation yield to two groups which were offered a complete diet of silage and concentrate with either an excess of alkali or of acid ions for the last 28 days of pregnancy. The composition of the concentrate rations and their analyses were reported by Phillippo et al (1994); the dietary analyses and anion-cation balances are given in Table 1. Batches of diet were stored at -20°C and thawed thoroughly before feeding. One cow on the alkali diet had a prolonged gestation (302 days) and produced an oversized calf: its data were excluded because all the measurements were inappropriately timed. The rate of mobilisation of calcium was estimated in 11 animals at the start of the experiment, 14 days before, and one day after parturition if the animal had not developed a profound hypocalcaemia. Disodium ethylenediamine tetra-acetic acid dihydrate (0-1263 mol litre -1) (EDTA; BDH, Poole) in sterile 0-15 mol litre -1 sodium chloride was infused through an intravenous catheter at 10 ml min -1 for a maximum of 120 minutes or until any clinical signs of hypocalcaemia. Blood samples were taken through another catheter every 15 minutes for one hour before, during and for three hours after the end of infusion for the measurement of total plasma calcium and calcium not bound to EDTA. Total plasma calcium was measured by atomic absorption spectrophotometry after dilution (1/20) in 5.4 nmol litre -1 lanthanum chloride; plasma calcium not bound to EDTA was measured by titration against ethylene glycol tetreacetic acid (EGTA;BDH) with calcein as an indicator in 1 mol litre -1 potassium hydroxide, using an automatic titrator (Marius Instrumenten). The amount of calcium mobilised, the rate of calcium mobilisation and the volume of distribution of EDTA were calculated according to Contreras et al (1982). A mineral balance trial was undertaken between
311
21 and 14 days before the expected date of parturition; the daily food intake was measured and the faeces and urine were collected separately, and aliquots of diet, faeces and urine were bulked for analysis. The fractional calcium absorption was estimated after the endogenous calcium loss (ECa) was calculated from the equation (TCORN 1991): ECa (g day -1) = - 0 . 7 9 + 0.0079 bodyweight (kg) + 0.66 dry matter intake (kg day -1) Urine pH and volume were measured daily and the urine was acidified by the addition of 10 per cent (v/v) sulphuric acid for the estimation of minerals and of pyridinoline and deoxypyridinoline (which is a specific marker of bone collagen degradation) by the method of Black et al (1988). Urine collection continued until near to the time of parturition in most animals. Bone biopsies were taken aseptically at the start of the experiment and one day after parturition in 10 of the animals. The animals were lightly sedated with xylazine (Rompun; Bayer) (30 to 40 pg kg bodyweight-1) and an area over the 12th rib was infiltrated with 2 per cent lignocaine hydrochloride (Lignavet; C-Vet). After incising the skin, underlying muscle and periosteum, a 10 mm trephine was used to remove the bone core; the muscle was sutured separately before closing the skin. The second biopsy was taken 8 to 10 cm ventral to the previous site. The biopsy was cut along the long axis of the rib, stored in 80 per cent v/v ethanol/water at 4°C, and embedded in methyl methacrylate resin; 5 gm sections were cut and stained with haematoxylin and eosin, toluidine blue and Masson Goldners trichrome. The sections were examined under polarised light to reveal the architecture of the bone, and the percentage of the cortical bone that consisted of compact lamellar bone was measured by histomorphometry. Details of the blood sampling and analyses, mineral analyses and assays for parathyroid hormone and 1,25-(OH)2 vitamin D have been reported by Phillippo et al (1994). Plasma 25 hydroxyvitamin D (25 OH vitamin D) and 1,25-(OH)2 vitamin D were measured in the same sample by using the high performance liquid chromatography separation described previously (Phillippo et al 1994). The protein binding assay for 25 Oil vitamin D used normal human plasma diluted 1/20 in 4 mmol litre-1 barbitone buffer (pH 8.6) containing 0.15 mol litre -1 sodium chloride and lg litre -1
312
H. Abu Damir, M. Phillippo, B. H. Thorp, J. S. Milne, L. Dick, 1, M. Nevison
gelatine; the mixture was incubated overnight at 4°C and the bound and free hormones were separated as in the 1,25-(OH) 2 vitamin D assay. The sensitivity of the assay was 150 fmol/tube, the inter- and intra-assay coefficients of variation were 9.7 and 9.0 per cent, respectively, and the recovery of an internal standard of 25 OH vitamin D was 55.0 + 1.1 per cent (n=16). The plasma concentrations of bioactive parathyroid hormone were measured by the method of Goltzman et al (1980) and those of 1-86 parathyroid hormone related peptide (PTgrP) by the method of Ratcliffe et al (1991). The data were analysed by analysis of variance, using the Genstat 5 programme (Lawes Agricultural Trust) except where otherwise specified.
Parturition (-2.0 to +2.5 days). The plasma total calcium concentration declined at -1 day in both groups to reach nadirs of 2.20 + 0.05 and 1.75 + 0.11 mmol litre -1 in the acid and the alkali groups, respectively, at +1 day. Six of the seven animals on the acid diet had a concentration >2.0 mmol litre -1, compared with one of the six animals on the alkali diet (P=0.024, Fisher's exact test). Minimal blood ionised calcium concentrations (1.10 + 0.03 and 0.86 + 0.05 mmol litre -1, respectively) occurred at +1 day and minimal plasma phosphorus concentrations at +2 days. The alkalifed animals had higher mean plasma magnesium concentrations on the day of parturition but neither the plasma chloride concentrations nor the blood pH were different.
Results
Post partum (+3 to +28 days). No significant differences occurred between the two groups of cows during this period.
Plasma minerals (Table 2) Before parturition (-28 to -2.5 days). Over this period there were no effects on the concentrations of total calcium, phosphorus, magnesium, sodium and potassium but the concentrations of plasma chloride and blood ionised calcium were significantly higher in the acid group than in the alkali group. There were associated lower values for the blood pH and base excess between -28 and -7 days: the pH varied from 7.35 to 7.36 in the acid group and from 7.43 to 7.45 in the alkali groups and the base excess figures were --4.5 to -6.6 and 1.4 to 3.2 mmol litre -1, respectively.
Hormonal changes (Fig 1). 25 OH vitamin D. The concentrations were not affected by the treatments either before or at parturition. 1,25-(oi42) vitamin D. The mean concentrations were not different before the experiment began, but they declined significantly between -28 and -3 days, and to a significantly greater extent in the cows fed the alkali diet. At parturition, however, the mean concentration in the alkali group was higher than in the acid group, a change consistent with the lower calcium concentration in the alkali group.
TABLE 2: Changes in mean plasma mineral concentrations before and after parturition in cows given acid and alkali diets Diet Calcium (mmol litre -1) SED
Number of cows -28
Days from parturition -20 to 2.5 -2-0 t o 0 0-5 to 2.5 3.0 to 4.5 +6 to +26 +28
Acid Alkali
7 6
2"54 2.62 0"09
2"52 2.56 0"04
2"44** 2.29 0"06
2-39** 2.01 0"10
2'49 2.36 0"09
2'57 2.59 0"05
2"70 2.61 0"13
Ionised calcium Acid (mmol [itre-1) Alkali SED
7 6
1.17 1.17 0.03
1-24" 1.18 0.02
1.19"* 1.08 0.03
1.16"* 0.97 0.04
1.14 1.16 0.03
1.17 1.19 0-02
1.18 1.16 0"03
Phosphorus (mmol litre -1) SED
Acid Alkali
7 6
1.67 1.79 0.17
1.64 1.67 0.17
1.41 1.36 0.11
1.49** 1.17 0.12
1 '53 1.31 0.16
1-29 1.32 0"11
1 "38 1-35 0.14
Magnesium (mmol litre -1) SED
Acid Alkali
7 6
0.86 0.84 0.03
0.86 0.84 0.03
0.85 0.95 0.05
0.78 0.78 0.05
0.71 0-73 0.04
0.75 0.71 0.07
0-77 0-70 0-08
Chloride (mmol litre -1) SED
Acid Alkali
7 6
94.4 92-7 3-74
104.1 * 98.2 2'63
104.6 98.4 3-75
100.7 98.0 2"62
96.4 95.9 3"52
96.2 94.0 2"79
95-9 94.2 2"62
* P<0-05,** P<0"01
313
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FIG 1 : Changes in plasma calcium and hormonal concentrations in cows given acid or alkali diets before parturition. SED is for the period until another value is shown
Parathyroid holwzone. There was no significant treatment effect on the plasma amino-terminal PTH concentrations during the period before parturition. At parturition, the concentrations of PTH increased in both groups but were significantly higher in the alkali group. The changes in the carboxyterminal PTH concentrations were similar to those reported for the amino-terminal PTH. Bioassayable PTH measured at 14 days before parturition were not different between treatments: the means were 2.90 + 0.97 (n=5) and 2.92 _+ 1.35 (n=6) ng litre -1 for the acid and alkali groups, respectively.
PTHrP. Intact 1-86 PTHrP was undetectable (<0.23 pmol litre -1) in plasma samples taken daily between seven days before and seven days after parturition. Calcium mobilisation (Table 3) The acid treatment significantly increased the amount of calcium mobilised and the calcium mobilisation rate, expressed absolutely or as a percentage of the bodyweight, at 14 days before parturition, compared to the alkali treatment. The calcium mobilisation rate was further increased
TABLE 3: Changes in calcium mobilisation before parturition and at parturition in cows given acid and alkali diets Time from parturition Diet -28 days
Acid Alkali
Number of DuratJon of Calcium mobilised Mobilisation rate Volume of animals infusion (min) (gmol kg-1) (gmol kg-1 min -1) distribution (litres) 6 5
120 118
98.4 93"8 5,45
0.84 0"78 0.051
68.2 69"3 2'78
- 1 4 days Acid Alkalif SED
6 4
120 118
114.0** 78.5 7.87
0.95"* 0-68 0"066
64-8 69.2 2.63
+1 day
5 3
115 40
124.4 ND 7-31
1-08 ND 0'042
66.8 ND 2"88
SED
SED
Acid Alkali
1- Data from one animal excluded because of negative acid base excess at this period ND No data because clinical hypocalcaemia shortened infusion ** P<0-01
H. Abu Damir, M. PhiIlippo, B. H. Thorp, J. S. Milne, L. Dick, I. M. Nevison
314
TABLE 4: Mineral balance data measured between 21 and 14 days before parturition in cows fed acid and alkali diets
Calcium
Diet
Number of cows
Intake (mol day-1 )
Acid Alkali
7 6
3.413 2.992 0.2136
2-480 2.539 0"1829
0.283** 0.019 0-0344
0-650 0.436 0.1518
Acid Alkali
7 6
1.098 1.149 0.0631
0"873 0.913 0"0740
0.026 0.013 0-0122
0.250 0.254 0.0553
Acid Alkali
7 6
1.701" 1.423 0"0935
1.164 1.148 0'0946
0.046 0.033 0"0062
0.491"* 0.257 0"0792
SED Phosphorus SED Magnesium SED
Faecal excretion Urinary excretion (mol day-1) (mol day-1)
Balance (mol day--1)
* P<0'02, ** P<0"01
one day after parturition in the animals fed the acid diet but insufficient data were available for the alkali group owing to the short periods of EDTA infusion. One day after parturition clinical signs of hypocalcaemia developed in all three alkali-fed animals (at 22, 38 and 60 minutes, respectively) but in only two of the five acid-fed animals (at 106 and 110 minutes); the remaining alkali-fed animals were not infused because their initial plasma total calcium concentration was <1.6 mmol litre-1. The volume of distribution of EDTA was unaffected either by treatment or time.
Balance data (-21 to -14 days prepartum, Table 4). Calcium. The mean dry matter intakes did not vary between treatments (acid diet 8-6 kg day-1, alkali diet 9.0 kg day-t, SED 0.053) but the mean intake of calcium and calcium retention tended to be higher in the acid group than in the alkali group owing to the higher calcium concentration in the acid diet. The acid diet had a significant effect on the urinary calcium excretion. The estimated fractional calcium absorption, after correcting for endogenous loss, was significantly greater in the acid group than in the alkali group (acid diet 0.35, alkali diet 0.25, SEI~0-045, P<0.05, paried t test). Phosphorus. The phosphorus balance was not affected by diet. Magnesium. The magnesium intake and retention were higher on the acid diet than on the alkali diet but neither the faecal nor the urinary output of magnesium were altered. Urinary pyridinoline/deoxypyridinoline excretion There were no significant treatment differences
in the total daily excretion of pyridinoline; between -21 days and - 2 days it varied from 6-0 + 0.9 to 7.7 + 1.20 gmol day -1 on the acid diet and from 6.0 + 0.90 to 5.2 + 0.75 gmol day -1 on the alkali diet. Deoxypyridinoline was present at concentrations that were near to the sensitivity of the method.
Bone histomorphometry There was no difference between the animals from the two groups before the start of the experiment when 64 per cent (range 49 to 76) of the cortical bone consisted of compact lamellar bone. One day after parturition 40 per cent (range 29 to 50) of the cortical bone consisted of compact lamellar bone in the acid group, whereas in the alkali group the proportion was 55 per cent (range 50 to 59; P<0-05, Mann Whitney U-test). The increased remodelling was evident on histological examination (Fig 2), which showed that more of the cortical bone had been replaced by woven bone, sclerotic debris and remnants of lamellar bone in the cows fed the acid diet.
Discussion The changes in plasma 1,25-(OH) 2 vitamin D concentrations, calcium absorption and the rates of calcium mobilisation and calcium resorption from bone give an indication of some of the possible mechanisms involved in the amelioration of parturient hypocalcaemia in dairy cows fed an acid diet before parturition. Plasma 1,25-(OH) 2 vitamin D concentrations were significantly higher in the animals fed the acid diet, in agreement with the results of an earlier study (Phillippo et al 1994). These hormonal changes were associated
Acid diet, calcium and vitamin D
315
FIG 2: Photomicrographs of cortical bone from the 12th rib of adult cows. Samples a) and b) were collected from the same cow at the start of the experiment and at parturition after being fed an alkali diet; sample c) from a parturient cow on the acid diet. In b), O=compact tamellar bone in osteons, <<=area between osteons with disorganised woven bone, sclerotic debris and remnants of lamellar bone. The greatest percentage of osteonal bone is visible in prepartum sample (a); there is a decrease in the osteonal bone and an increase in the disorganised woven bone, sclerotic debris and remnants of lamellar bone in both parturient samples but these changes are more marked in the cow on the acidic diet (c). Undecalcified bone sections, original magnification x 6-5, toluidine blue
only with decreased blood pH and base-excess and increased blood ionised calcium and plasma chloride concentrations; the total plasma calcium,
phosphorus, magnesium and PTH concentrations were not affected. The plasma mineral and hormonal changes observed at parturition resembled
316
H. Abu Damir, M. Phillippo, B. H. Thorp, Y. S. Milne, L. Dick, I. M. Nevison
those described previously (Jorgensen 1974, Hollis et al 1981, Block 1984). However, the lack of detectable levels of intact 1-86 PTHrP during the parturient period raises doubts about the suggestion that this hormone is an important causal factor of bovine parturient paresis (Law et al 1991). The changes in 1,25-(OH)2 vitamin D concentration were not due to different concentrations of its precursor, 25-OH vitamin D, which were not affected either before or at parturition, as has been found by Hollis et al (1981) in control cows and in cows with parturient paresis. Since the PTH concentrations were unaffected in the cows in this and a previous study (Phillippo et al 1994), the control of 1,25-(OH) 2 vitamin D in acidfed animals may be independent of the usual control by PTH of the activity of the 1-c~-hydroxylase enzyme (Fraser and Kodicek 1973, Engstrom et al 1987). Horst and Reinhardt (1983) have also suggested that, in ruminants, PTH stimulates 1,25-(OH)2 vitamin D production only during hypocalcaemia and not at normal calcium concentrations. The other findings suggest the possible mechanisms by which calcium metabolism may be affected by acid diets. The animals on the acid diet had a higher estimated fractional absorption of calcium than those on the alkali diet (0.35 vs 0.25); Verdaris and Evans (1976) observed similar changes in calcium retention in dry pregnant cows and Ender et al (1971) in cows in the period immediately after parturition. Braithwaite (1972) and Freeden et al (1988) also showed that induced acidosis increased calcium absorption in sheep and goats, respectively, although Abu Damir et al (1991) found that there was no effect in lambs. A definitive radiotracer experiment would be needed to confirm this effect on calcium absorption. During the last month of pregnancy cows absorb calcium according to their requirement, and the diets in this experiment contained more than the estimated calcium requirement for this period (TCORN1991). The acid diet therefore appeared to override the mechanisms which, at the end of pregnancy, normally cause a reduction of intestinal absorption which is the predominant factor affecting calcium homeostasis at that time (Ramberg et al 1970). Although Verdaris and Evans (1976) suggested that the effect on calcium absorption could be caused by the solubilisation of calcium salts, it is more likely that it is due to an alteration in the concentration of 1,25-(OH)2 vita-
min D, the hormone that is known to regulate intestinal calcium absorption. The acid diet increased the ability of the pregnant cows to mobilise calcium during late pregnancy, in contrast to the finding of Mosel et al (1993) in younger animals. The calcium mobilisation rate in the acid-fed animals had increased by 13 per cent 14 days before parturition and by 28 per cent at the time of parturition, whereas it had declined by 14 per cent at 14 days before parturition in the alkali-fed group. The difference in the mobilisation rates 14 days before parturition was equivalent to an extra 0.225 tool calcium day -1 which could have been met directly by the estimated increase in calcium absorption and retention of 0-250 mol day -1 on the acid diet. Factors other than intestinal absorption would seem to have a role in calcium homeostasis immediately before parturition because the increase in mobilisation rate in the acid animals exceeded the increase in absorption and retention, especially as the cows' voluntary food intake had declined. It was not possible to identify the source of the extra calcium, because intestinal absorption, bone resorption and the availability of calcium from the extravascular pool may all contribute to the calculated figures, and radiotracer studies would be needed to resolve them. The results provide the first direct histological evidence in cows that acid diets mobilise calcium from the skeleton. The histomorphometric analysis showed that pregnancy had a general effect upon bone remodelling, but that the animals fed the acid diet had reorganised much more of their compact lamellar cortical bone into woven bone at parturition than had the animals fed the alkali diet. Woven bone is a temporary tissue that is formed rapidly and has a higher calcium content than compact bone, and could therefore act as a readily mobilisable reserve of calcium during late pregnancy and lactation. Vagg and Payne (1970), Braithwaite (1972) and Freeden et al (1988) also found that acidosis increase bone resorption in radioactive tracer studies and Block (1984) and Oetzel et al (1988) inferred the same conclusion from changes in plasma hydroxyproline concentrations. Although the excretion of pyridinoline showed a tendency to increase just before parturition in the cows fed the acid diet there was no increase in the excretion of deoxypyridinoline, the specific marker of bone matrix turnover (Black et al 1988).
Acid diet, calcium and vitamin D
These results therefore show that the ameliorat-
ing effects of aciddiets on parturient hypocalcaemia were accompanied by an increase in the plasma concentration of 1,25-(OH) 2 vitamin D in the period before parturition. Although no direct causal connection was established, it is likely that the increased concentrations of 1,25(OH)2 vitamin D were responsible for the stimulation of both intestinal calcium absorption and bone resorption, which together could account for the increase mobilisation of calcium observed, and thus helped to prevent severe parturient hypocalcaemia. Acknowledgements The authors would like to thank the staff of the Duthie Farm, especially John McIntosh, for the care of the experimental animals and the Analytical Department for the chemical analyses. We are indebted to Dr Wendy Ratcliffe for the P T H r P analyses.
References ABU DAMIR, H., SCOTT, D., LOVERIDGE, N., BUCHAN, W. & MILNE, J. (1991) The effects of feeding diets containing either NaHCO 3 or NH4CI on indices of bone formation and resorption and on mineral balance in the lamb. Experimental Physiology 76, 725-732 BLACK, D.. DUNCAN, A. & ROBINS, S. P. (1988) Quantitative analysis of the pyfidinium crosslinks of collagen in urine using ion paired reversed phase high performance liquid chromatography. Analytical Biochemistry 169, 197-203 BLOCK, E. (1984) Manipulating dietary anions and cations for preparturn dairy cows to reduce the incidence of milk fever. Journal of Dairy Science 67, 2939-2948 BRAITHWAITE, G. D. (1972) The effect of ammonium chloride on calcium metabolism in sheep. British Journal of Nutrition 27, 201-209 BROMMAGE, R. & DeLUCA, H. F. (1985) Regulation of bone mineral loss during lactation. American Journal of Physiology 248, E182E187 CONTRERAS, P. A., MANSON, R. & SANSOM, B. F. (1982) Calcium mobilisation in hypomagnesaemic cattle. Research in Veterinary Science 33, 10-16 DISHINGTON, I. W. (1975) Prevention of milk fever hypocalcaemic paresis puerperalis by dietary salt supplements. Acta Veterinaria Scandinaviea 16, 503-512 ENDER, F., DISHINGTON, 1. W. & HELGEBOSTAD, A. (1962) Parturient paresis and related forms of hypocalcaemic disorders induced experimentally in dairy cows. II Studies on the etiological importance of feeding prepartai diets high in calcium and low or normaI in phosphorus in relation to development of milk fever. Effect of high and low alkalinity of diets. Acta Veterinaria Scandinavica Supplement 3, 1-52 ENDER, F., DISHINGTON, I. W. & HELGEBOSTAD, A. (1971) Calcium balance studies in dairy cows under experimental induction and prevention of hypocalcaemic paresis puerperalis. Zeitschrift fiir Tierphysiologie, TierernOTwtmg und Futterm#telkunde 28, 233-256
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ENGSTROM,G. W., GOFF, J. P., HORST, R. L. & REINHARDT, J. A. (1987) Regulation of calf renal 25-hydroxyvitamin D-hydroxyIase activities by calcinm-regulating hormones. Journal of Dairy Science 70, 2266-2271 FRASER, D. A. & KODICEK, E. (1973) Regulation of 25-hydroxycholecalcfferol-l-hydroxylase activity by parathyroid hormone. Nature 241, 163-165 FREEDEN, A. H., De PETERS, E. J. & BALDWIN, R. L. (1988) Characterisation of acid-base disturbances and effects on calcium and phosphorus balances of dietary fixed ions in pregnant or lactating does. Journal of AnimaI Science 66, 159-173 GAYNOR, D. J., MUELLER, F. J., MILLER, L K, RAMSEY, N., GOFF, J. P. & HORST, R. L. (1989) Parturient hypocaicaemia in Jersey cows fed alfalfa haylage-based diets with different cation to anion ratios. Journal of Dairy Science 72, 2525-2531 GOFF, J. P., HORST, R. L., MUELLER, F. J., MILLER, J. K., KREISS, G. A. & DOWLEN, H. H. (1991) Addition of chloride to prepartal diet high in cations increases 1,25-dibydroxyvitamin D response to hypocalcemia preventing milk fever. Journal of Dairy Science 74, 3863-3871 GOLTZMAN, D., HENDERSON, B. & LOVER1DGE. N. (1980) Cytochemical bioassay of parathyroid hormone: characteristics of the assay and analysis of cJxculating hormonal forms. Journal of Clinical Investigation 65, 1309-1317 HART, E. B., STEENBOCK, H. & KLINE, O. L. (1931) Dietary factors affecting calcium assimiIation. XIV The influence of mineral acids and sugar on the calcium metabolism of milking cows. Journal of Dairy Science 14, 307-321 HOLLIS, B. W., DRAPER, H. H., BURTON, J. H. & ETCHES, R. J. (1981) A hormonal assessment of bovine parturient paresis: evidence for a role of oestrogen. Journal of Endocrinology 88, 161-171 HORST, R. L. & REINHARDT, T. A. (1983) Vitamin D metabolism in ruminants and its relevance to the periparturient cow. Journal of Dairy Science 66, 661-678 JORGENSEN, N. A. (1974) Combating milk fever. Journal of Dairy Science 57, 933-944 LAW, F. M. K., MOATE, P. J., LEAVER, D. D., DIEFENBACHJAGGER, J. GRILL, V., HO, P. W. M. & MARTIN, J. J. (1991) Parathyroid hormone-related protein in milk and its correlation with bovine milk calcium. Journal of Endocrinology 128, 21-26 MILLER, S. C., OMURA, T. H. & SMITH, L. C. (1982) Role of vitamin D in maternal skeletal changes during pregnancy and lactation: a histomorphological study. Calcified Tissue Research 34, 245-252 MOSEL, M. VAN, KLOOSTER, A. Th. VAN'T, MOSEL, F. VAN & KUILEN, J. VAN DER (1993) Effects of reducing dietary [(Na+ + K+) - (CI- + SO4=)] on the rate of calcium mobilisation by dairy cows at parturition. Research in Veterinary Science 54, 1-9 OETZEL, G. R., FETTMAN, M. J., HAMAR, D. W. & OLSON, J. D. (1991 ) Screening of anionic salts for palatability, effects on acid-base status and urinary calcium excretion in dairy cows. Journal of Dairy Science 74, 965-971 OETZEL, G. R., OLSON, J. D., CURTIS, C. R. & FETTMAN, M. J. (1988) Ammonium chloride and ammonium sulfate for prevention of parturient paresis in dairy cows. Journal of Dairy Science 71, 33023309 PHILLIPPO, M., REID, G. W. & NEVISON, I. M. (1994) Parturient hypocalcaemia in dairy cows: effects of dietary acidity on plasma minerals and calciolxophic hormones. Research in Veterinary Science 56. 303-309 PITKIN, R. M. (1975) Calcium metabolism in pregnancy: a review. American Journal of Obstetrics 121, 724-737 RAMBERG, C. F., MAYER, G. P., KRONFELD, D. S., PHANG. J. M. & BURMAN, M. (1970) Calcium kinetics in cows during late pregnancy, parturition and early lactation. American Journal of Physiology 219, 1166-1177 RATCLIFFE, J. G., RATCLIFFE, W. A., HEATH, D. A. & NORBURY, S. (1991) PTH related protein (PTI-IrP) immunoreactivity in plasma. Journal of Endocrinology Supplement 129, 118 REDD, E. H., MILLER, E. C. & JEE, W. S. S. (1984) Changes in endochondral bone elongation rates during pregnancy and lactation: a histomorphometric study. Calcified Tissue Research 36, 697-701
318
H. Abu Damir, M. Phillippo, B. H. Thorp, J. S. Milne, L. Dick, I. M. Nevison
TCORN (1991) AFRC Technical Committee on Responses to Nutrients. Report 6. A reappraisal of the calcium and phosphorus requirements of sheep and cattle. NutritionAbstracts& Reviews,SeriesB 61,573-612 VAGG, M. J. & PAYNE, J. M. (1970) The effect of ammonium chloride induced acidosis on calcium metabolism in ruminants. British VeterinaryJournal 126, 531-537
VERDARIS, J. N. & EVANS, J. L. (1976) Diet calcium and pH versus mineral balance in Holstein cows 84 days pre- to 2 days postpartum. Journal of Dairy Science 59, 1271-1277
ReceivedJune 22, 1992 AcceptedNovember4,1993
Effects of dietary acidity on calcium balance and mobilisation, bone morphology and 1,25 dihydroxyvitamin D in prepartal dairy cows H. ABU DAMIR, M. PHILLIPPO, Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, B. H. THORP, AFRC Institute of Animal Physiology and Genetics, Edinburgh Research Station, Roslin, Midlothian EH25 9PS, J. S. MILNE, Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB , L. DICK, AFRC Institute of Animal Physiology and Genetics, Edinburgh Research Station, Roslin, Midlothian EH25 9PS, I. M. NEVISON, Scottish Agricultural Statistics Service, Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB
The effect of an acid or alkali diet was investigated in 14 mature dairy cows during the last 28 days of pregnancy. The acid diet reduced the incidence of parturient hypocalcaemia compared with the alkali diet and was associated with higher blood ionised calcium and plasma chloride concentrations and lower blood pH and acid-base excess before parturition. Plasma 1,25-dihydroxyvitamin D concentrations before parturition were increased by the acid diet but the concentrations of parathyroid hormone and intact 1-86 parathyroid-hormone-related peptide were unaffected. The estimated fractional calcium absorption and calcium mobilisation rate during an ethylenediamine tetraacetic acid infusion were increased by the acid diet 14 days before parturition. Cortical bone remodelling occurred in all the animals during late pregnancy but was particularly evident in the cows given the acid diet. The data suggest that an acid diet ameliorates parturient hypocalcaemia by enhancing calcium mobilisation before parturition by increasing calcium absorption and bone resorption, these increases possibly being mediated by increases in the plasma concentration of 1,25-dihydroxyvitamin D. THERE has long been interest in the feeding of an acid diet to reduce the incidence of parturient paresis and hypocalcaemia (Ender et al 1962, 1971, Dishington 1975, Block 1984, Oetzel et al 1988, Gaynor et al 1989, Phillippo et al 1994) and various mechanisms of action have been suggest-
ed. These include increased calcium absorption causing a positive calcium retention both before and after parturition (Ender et al 1971, Verdaris and Evans 1976) which offsets the decreased food intake that occurs just before parturition (Ender et al 1971, Gaynor et al 1989). In addition, acidic diets cause hypercalciuria (Hart et al 1931, Verdaris and Evans 1976, Oetzel et al 1991) which, in conjunction with increased calcium absorption, may increase the turnover of body calcium. Acid diets were also thought to increase bone resorption because they increased plasma hydroxyproline concentrations (Block 1984, Gaynor et al 1989) and therefore would affect the calcium changes occurring in the spongiosa and cortical bone during pregnancy and lactation (Pitkin 1975, Miller et al 1982, Redd et al 1984, Brommage and DeLuca 1985). An involvement of 1,25-dihydroxyvitamin D (1,25-(OH) 2 vitamin D) in the mechanism of action has been proposed by Gaynor et al (1989), Goff et al (1991), and Phillippo et al (1994) but other associated mechanisms have not yet been elucidated. This study was done to elucidate the roles played by the changes in 1,25-(OH) 2 vitamin D and calcium balance, calcium mobilisation and bone resorption occurring before parturition in the prevention of parturient paresis and hypocalcaemia when cows are given an acid diet. Materials and methods
310
Fourteen Friesian cows, five to 10 years old,
Acid diet, calcium and vitamin D TABLE 1: Analysis of the acid and alkaline diets fed to dairy COWS
Constituent Calcium Phosphorus Magnesium Anion-cation balance* (mmol)
Concentration (mol kg dry matter-1) Alkali diet Acid diet 0.33 0.13 0.16 +779
0.39 0-13 0-19 -35
* Calculated from (Na + + K+ - C I - - S-) kg dry matter -1
weighing 600 to 800 kg and in their third to eighth parity were randomly allocated by age, parity and lactation yield to two groups which were offered a complete diet of silage and concentrate with either an excess of alkali or of acid ions for the last 28 days of pregnancy. The composition of the concentrate rations and their analyses were reported by Phillippo et al (1994); the dietary analyses and anion-cation balances are given in Table 1. Batches of diet were stored at -20°C and thawed thoroughly before feeding. One cow on the alkali diet had a prolonged gestation (302 days) and produced an oversized calf: its data were excluded because all the measurements were inappropriately timed. The rate of mobilisation of calcium was estimated in 11 animals at the start of the experiment, 14 days before, and one day after parturition if the animal had not developed a profound hypocalcaemia. Disodium ethylenediamine tetra-acetic acid dihydrate (0-1263 mol litre -1) (EDTA; BDH, Poole) in sterile 0-15 mol litre -1 sodium chloride was infused through an intravenous catheter at 10 ml min -1 for a maximum of 120 minutes or until any clinical signs of hypocalcaemia. Blood samples were taken through another catheter every 15 minutes for one hour before, during and for three hours after the end of infusion for the measurement of total plasma calcium and calcium not bound to EDTA. Total plasma calcium was measured by atomic absorption spectrophotometry after dilution (1/20) in 5.4 nmol litre -1 lanthanum chloride; plasma calcium not bound to EDTA was measured by titration against ethylene glycol tetreacetic acid (EGTA;BDH) with calcein as an indicator in 1 mol litre -1 potassium hydroxide, using an automatic titrator (Marius Instrumenten). The amount of calcium mobilised, the rate of calcium mobilisation and the volume of distribution of EDTA were calculated according to Contreras et al (1982). A mineral balance trial was undertaken between
311
21 and 14 days before the expected date of parturition; the daily food intake was measured and the faeces and urine were collected separately, and aliquots of diet, faeces and urine were bulked for analysis. The fractional calcium absorption was estimated after the endogenous calcium loss (ECa) was calculated from the equation (TCORN 1991): ECa (g day -1) = - 0 . 7 9 + 0.0079 bodyweight (kg) + 0.66 dry matter intake (kg day -1) Urine pH and volume were measured daily and the urine was acidified by the addition of 10 per cent (v/v) sulphuric acid for the estimation of minerals and of pyridinoline and deoxypyridinoline (which is a specific marker of bone collagen degradation) by the method of Black et al (1988). Urine collection continued until near to the time of parturition in most animals. Bone biopsies were taken aseptically at the start of the experiment and one day after parturition in 10 of the animals. The animals were lightly sedated with xylazine (Rompun; Bayer) (30 to 40 pg kg bodyweight-1) and an area over the 12th rib was infiltrated with 2 per cent lignocaine hydrochloride (Lignavet; C-Vet). After incising the skin, underlying muscle and periosteum, a 10 mm trephine was used to remove the bone core; the muscle was sutured separately before closing the skin. The second biopsy was taken 8 to 10 cm ventral to the previous site. The biopsy was cut along the long axis of the rib, stored in 80 per cent v/v ethanol/water at 4°C, and embedded in methyl methacrylate resin; 5 gm sections were cut and stained with haematoxylin and eosin, toluidine blue and Masson Goldners trichrome. The sections were examined under polarised light to reveal the architecture of the bone, and the percentage of the cortical bone that consisted of compact lamellar bone was measured by histomorphometry. Details of the blood sampling and analyses, mineral analyses and assays for parathyroid hormone and 1,25-(OH)2 vitamin D have been reported by Phillippo et al (1994). Plasma 25 hydroxyvitamin D (25 OH vitamin D) and 1,25-(OH)2 vitamin D were measured in the same sample by using the high performance liquid chromatography separation described previously (Phillippo et al 1994). The protein binding assay for 25 Oil vitamin D used normal human plasma diluted 1/20 in 4 mmol litre-1 barbitone buffer (pH 8.6) containing 0.15 mol litre -1 sodium chloride and lg litre -1
312
H. Abu Damir, M. Phillippo, B. H. Thorp, J. S. Milne, L. Dick, 1, M. Nevison
gelatine; the mixture was incubated overnight at 4°C and the bound and free hormones were separated as in the 1,25-(OH) 2 vitamin D assay. The sensitivity of the assay was 150 fmol/tube, the inter- and intra-assay coefficients of variation were 9.7 and 9.0 per cent, respectively, and the recovery of an internal standard of 25 OH vitamin D was 55.0 + 1.1 per cent (n=16). The plasma concentrations of bioactive parathyroid hormone were measured by the method of Goltzman et al (1980) and those of 1-86 parathyroid hormone related peptide (PTgrP) by the method of Ratcliffe et al (1991). The data were analysed by analysis of variance, using the Genstat 5 programme (Lawes Agricultural Trust) except where otherwise specified.
Parturition (-2.0 to +2.5 days). The plasma total calcium concentration declined at -1 day in both groups to reach nadirs of 2.20 + 0.05 and 1.75 + 0.11 mmol litre -1 in the acid and the alkali groups, respectively, at +1 day. Six of the seven animals on the acid diet had a concentration >2.0 mmol litre -1, compared with one of the six animals on the alkali diet (P=0.024, Fisher's exact test). Minimal blood ionised calcium concentrations (1.10 + 0.03 and 0.86 + 0.05 mmol litre -1, respectively) occurred at +1 day and minimal plasma phosphorus concentrations at +2 days. The alkalifed animals had higher mean plasma magnesium concentrations on the day of parturition but neither the plasma chloride concentrations nor the blood pH were different.
Results
Post partum (+3 to +28 days). No significant differences occurred between the two groups of cows during this period.
Plasma minerals (Table 2) Before parturition (-28 to -2.5 days). Over this period there were no effects on the concentrations of total calcium, phosphorus, magnesium, sodium and potassium but the concentrations of plasma chloride and blood ionised calcium were significantly higher in the acid group than in the alkali group. There were associated lower values for the blood pH and base excess between -28 and -7 days: the pH varied from 7.35 to 7.36 in the acid group and from 7.43 to 7.45 in the alkali groups and the base excess figures were --4.5 to -6.6 and 1.4 to 3.2 mmol litre -1, respectively.
Hormonal changes (Fig 1). 25 OH vitamin D. The concentrations were not affected by the treatments either before or at parturition. 1,25-(oi42) vitamin D. The mean concentrations were not different before the experiment began, but they declined significantly between -28 and -3 days, and to a significantly greater extent in the cows fed the alkali diet. At parturition, however, the mean concentration in the alkali group was higher than in the acid group, a change consistent with the lower calcium concentration in the alkali group.
TABLE 2: Changes in mean plasma mineral concentrations before and after parturition in cows given acid and alkali diets Diet Calcium (mmol litre -1) SED
Number of cows -28
Days from parturition -20 to 2.5 -2-0 t o 0 0-5 to 2.5 3.0 to 4.5 +6 to +26 +28
Acid Alkali
7 6
2"54 2.62 0"09
2"52 2.56 0"04
2"44** 2.29 0"06
2-39** 2.01 0"10
2'49 2.36 0"09
2'57 2.59 0"05
2"70 2.61 0"13
Ionised calcium Acid (mmol [itre-1) Alkali SED
7 6
1.17 1.17 0.03
1-24" 1.18 0.02
1.19"* 1.08 0.03
1.16"* 0.97 0.04
1.14 1.16 0.03
1.17 1.19 0-02
1.18 1.16 0"03
Phosphorus (mmol litre -1) SED
Acid Alkali
7 6
1.67 1.79 0.17
1.64 1.67 0.17
1.41 1.36 0.11
1.49** 1.17 0.12
1 '53 1.31 0.16
1-29 1.32 0"11
1 "38 1-35 0.14
Magnesium (mmol litre -1) SED
Acid Alkali
7 6
0.86 0.84 0.03
0.86 0.84 0.03
0.85 0.95 0.05
0.78 0.78 0.05
0.71 0-73 0.04
0.75 0.71 0.07
0-77 0-70 0-08
Chloride (mmol litre -1) SED
Acid Alkali
7 6
94.4 92-7 3-74
104.1 * 98.2 2'63
104.6 98.4 3-75
100.7 98.0 2"62
96.4 95.9 3"52
96.2 94.0 2"79
95-9 94.2 2"62
* P<0-05,** P<0"01
313
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FIG 1 : Changes in plasma calcium and hormonal concentrations in cows given acid or alkali diets before parturition. SED is for the period until another value is shown
Parathyroid holwzone. There was no significant treatment effect on the plasma amino-terminal PTH concentrations during the period before parturition. At parturition, the concentrations of PTH increased in both groups but were significantly higher in the alkali group. The changes in the carboxyterminal PTH concentrations were similar to those reported for the amino-terminal PTH. Bioassayable PTH measured at 14 days before parturition were not different between treatments: the means were 2.90 + 0.97 (n=5) and 2.92 _+ 1.35 (n=6) ng litre -1 for the acid and alkali groups, respectively.
PTHrP. Intact 1-86 PTHrP was undetectable (<0.23 pmol litre -1) in plasma samples taken daily between seven days before and seven days after parturition. Calcium mobilisation (Table 3) The acid treatment significantly increased the amount of calcium mobilised and the calcium mobilisation rate, expressed absolutely or as a percentage of the bodyweight, at 14 days before parturition, compared to the alkali treatment. The calcium mobilisation rate was further increased
TABLE 3: Changes in calcium mobilisation before parturition and at parturition in cows given acid and alkali diets Time from parturition Diet -28 days
Acid Alkali
Number of DuratJon of Calcium mobilised Mobilisation rate Volume of animals infusion (min) (gmol kg-1) (gmol kg-1 min -1) distribution (litres) 6 5
120 118
98.4 93"8 5,45
0.84 0"78 0.051
68.2 69"3 2'78
- 1 4 days Acid Alkalif SED
6 4
120 118
114.0** 78.5 7.87
0.95"* 0-68 0"066
64-8 69.2 2.63
+1 day
5 3
115 40
124.4 ND 7-31
1-08 ND 0'042
66.8 ND 2"88
SED
SED
Acid Alkali
1- Data from one animal excluded because of negative acid base excess at this period ND No data because clinical hypocalcaemia shortened infusion ** P<0-01
H. Abu Damir, M. PhiIlippo, B. H. Thorp, J. S. Milne, L. Dick, I. M. Nevison
314
TABLE 4: Mineral balance data measured between 21 and 14 days before parturition in cows fed acid and alkali diets
Calcium
Diet
Number of cows
Intake (mol day-1 )
Acid Alkali
7 6
3.413 2.992 0.2136
2-480 2.539 0"1829
0.283** 0.019 0-0344
0-650 0.436 0.1518
Acid Alkali
7 6
1.098 1.149 0.0631
0"873 0.913 0"0740
0.026 0.013 0-0122
0.250 0.254 0.0553
Acid Alkali
7 6
1.701" 1.423 0"0935
1.164 1.148 0'0946
0.046 0.033 0"0062
0.491"* 0.257 0"0792
SED Phosphorus SED Magnesium SED
Faecal excretion Urinary excretion (mol day-1) (mol day-1)
Balance (mol day--1)
* P<0'02, ** P<0"01
one day after parturition in the animals fed the acid diet but insufficient data were available for the alkali group owing to the short periods of EDTA infusion. One day after parturition clinical signs of hypocalcaemia developed in all three alkali-fed animals (at 22, 38 and 60 minutes, respectively) but in only two of the five acid-fed animals (at 106 and 110 minutes); the remaining alkali-fed animals were not infused because their initial plasma total calcium concentration was <1.6 mmol litre-1. The volume of distribution of EDTA was unaffected either by treatment or time.
Balance data (-21 to -14 days prepartum, Table 4). Calcium. The mean dry matter intakes did not vary between treatments (acid diet 8-6 kg day-1, alkali diet 9.0 kg day-t, SED 0.053) but the mean intake of calcium and calcium retention tended to be higher in the acid group than in the alkali group owing to the higher calcium concentration in the acid diet. The acid diet had a significant effect on the urinary calcium excretion. The estimated fractional calcium absorption, after correcting for endogenous loss, was significantly greater in the acid group than in the alkali group (acid diet 0.35, alkali diet 0.25, SEI~0-045, P<0.05, paried t test). Phosphorus. The phosphorus balance was not affected by diet. Magnesium. The magnesium intake and retention were higher on the acid diet than on the alkali diet but neither the faecal nor the urinary output of magnesium were altered. Urinary pyridinoline/deoxypyridinoline excretion There were no significant treatment differences
in the total daily excretion of pyridinoline; between -21 days and - 2 days it varied from 6-0 + 0.9 to 7.7 + 1.20 gmol day -1 on the acid diet and from 6.0 + 0.90 to 5.2 + 0.75 gmol day -1 on the alkali diet. Deoxypyridinoline was present at concentrations that were near to the sensitivity of the method.
Bone histomorphometry There was no difference between the animals from the two groups before the start of the experiment when 64 per cent (range 49 to 76) of the cortical bone consisted of compact lamellar bone. One day after parturition 40 per cent (range 29 to 50) of the cortical bone consisted of compact lamellar bone in the acid group, whereas in the alkali group the proportion was 55 per cent (range 50 to 59; P<0-05, Mann Whitney U-test). The increased remodelling was evident on histological examination (Fig 2), which showed that more of the cortical bone had been replaced by woven bone, sclerotic debris and remnants of lamellar bone in the cows fed the acid diet.
Discussion The changes in plasma 1,25-(OH) 2 vitamin D concentrations, calcium absorption and the rates of calcium mobilisation and calcium resorption from bone give an indication of some of the possible mechanisms involved in the amelioration of parturient hypocalcaemia in dairy cows fed an acid diet before parturition. Plasma 1,25-(OH) 2 vitamin D concentrations were significantly higher in the animals fed the acid diet, in agreement with the results of an earlier study (Phillippo et al 1994). These hormonal changes were associated
Acid diet, calcium and vitamin D
315
FIG 2: Photomicrographs of cortical bone from the 12th rib of adult cows. Samples a) and b) were collected from the same cow at the start of the experiment and at parturition after being fed an alkali diet; sample c) from a parturient cow on the acid diet. In b), O=compact tamellar bone in osteons, <<=area between osteons with disorganised woven bone, sclerotic debris and remnants of lamellar bone. The greatest percentage of osteonal bone is visible in prepartum sample (a); there is a decrease in the osteonal bone and an increase in the disorganised woven bone, sclerotic debris and remnants of lamellar bone in both parturient samples but these changes are more marked in the cow on the acidic diet (c). Undecalcified bone sections, original magnification x 6-5, toluidine blue
only with decreased blood pH and base-excess and increased blood ionised calcium and plasma chloride concentrations; the total plasma calcium,
phosphorus, magnesium and PTH concentrations were not affected. The plasma mineral and hormonal changes observed at parturition resembled
316
H. Abu Damir, M. Phillippo, B. H. Thorp, Y. S. Milne, L. Dick, I. M. Nevison
those described previously (Jorgensen 1974, Hollis et al 1981, Block 1984). However, the lack of detectable levels of intact 1-86 PTHrP during the parturient period raises doubts about the suggestion that this hormone is an important causal factor of bovine parturient paresis (Law et al 1991). The changes in 1,25-(OH)2 vitamin D concentration were not due to different concentrations of its precursor, 25-OH vitamin D, which were not affected either before or at parturition, as has been found by Hollis et al (1981) in control cows and in cows with parturient paresis. Since the PTH concentrations were unaffected in the cows in this and a previous study (Phillippo et al 1994), the control of 1,25-(OH) 2 vitamin D in acidfed animals may be independent of the usual control by PTH of the activity of the 1-c~-hydroxylase enzyme (Fraser and Kodicek 1973, Engstrom et al 1987). Horst and Reinhardt (1983) have also suggested that, in ruminants, PTH stimulates 1,25-(OH)2 vitamin D production only during hypocalcaemia and not at normal calcium concentrations. The other findings suggest the possible mechanisms by which calcium metabolism may be affected by acid diets. The animals on the acid diet had a higher estimated fractional absorption of calcium than those on the alkali diet (0.35 vs 0.25); Verdaris and Evans (1976) observed similar changes in calcium retention in dry pregnant cows and Ender et al (1971) in cows in the period immediately after parturition. Braithwaite (1972) and Freeden et al (1988) also showed that induced acidosis increased calcium absorption in sheep and goats, respectively, although Abu Damir et al (1991) found that there was no effect in lambs. A definitive radiotracer experiment would be needed to confirm this effect on calcium absorption. During the last month of pregnancy cows absorb calcium according to their requirement, and the diets in this experiment contained more than the estimated calcium requirement for this period (TCORN1991). The acid diet therefore appeared to override the mechanisms which, at the end of pregnancy, normally cause a reduction of intestinal absorption which is the predominant factor affecting calcium homeostasis at that time (Ramberg et al 1970). Although Verdaris and Evans (1976) suggested that the effect on calcium absorption could be caused by the solubilisation of calcium salts, it is more likely that it is due to an alteration in the concentration of 1,25-(OH)2 vita-
min D, the hormone that is known to regulate intestinal calcium absorption. The acid diet increased the ability of the pregnant cows to mobilise calcium during late pregnancy, in contrast to the finding of Mosel et al (1993) in younger animals. The calcium mobilisation rate in the acid-fed animals had increased by 13 per cent 14 days before parturition and by 28 per cent at the time of parturition, whereas it had declined by 14 per cent at 14 days before parturition in the alkali-fed group. The difference in the mobilisation rates 14 days before parturition was equivalent to an extra 0.225 tool calcium day -1 which could have been met directly by the estimated increase in calcium absorption and retention of 0-250 mol day -1 on the acid diet. Factors other than intestinal absorption would seem to have a role in calcium homeostasis immediately before parturition because the increase in mobilisation rate in the acid animals exceeded the increase in absorption and retention, especially as the cows' voluntary food intake had declined. It was not possible to identify the source of the extra calcium, because intestinal absorption, bone resorption and the availability of calcium from the extravascular pool may all contribute to the calculated figures, and radiotracer studies would be needed to resolve them. The results provide the first direct histological evidence in cows that acid diets mobilise calcium from the skeleton. The histomorphometric analysis showed that pregnancy had a general effect upon bone remodelling, but that the animals fed the acid diet had reorganised much more of their compact lamellar cortical bone into woven bone at parturition than had the animals fed the alkali diet. Woven bone is a temporary tissue that is formed rapidly and has a higher calcium content than compact bone, and could therefore act as a readily mobilisable reserve of calcium during late pregnancy and lactation. Vagg and Payne (1970), Braithwaite (1972) and Freeden et al (1988) also found that acidosis increase bone resorption in radioactive tracer studies and Block (1984) and Oetzel et al (1988) inferred the same conclusion from changes in plasma hydroxyproline concentrations. Although the excretion of pyridinoline showed a tendency to increase just before parturition in the cows fed the acid diet there was no increase in the excretion of deoxypyridinoline, the specific marker of bone matrix turnover (Black et al 1988).
Acid diet, calcium and vitamin D
These results therefore show that the ameliorat-
ing effects of aciddiets on parturient hypocalcaemia were accompanied by an increase in the plasma concentration of 1,25-(OH) 2 vitamin D in the period before parturition. Although no direct causal connection was established, it is likely that the increased concentrations of 1,25(OH)2 vitamin D were responsible for the stimulation of both intestinal calcium absorption and bone resorption, which together could account for the increase mobilisation of calcium observed, and thus helped to prevent severe parturient hypocalcaemia. Acknowledgements The authors would like to thank the staff of the Duthie Farm, especially John McIntosh, for the care of the experimental animals and the Analytical Department for the chemical analyses. We are indebted to Dr Wendy Ratcliffe for the P T H r P analyses.
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ReceivedJune 22, 1992 AcceptedNovember4,1993