Effects of an inadequate dietary intake of magnesium on osteogenesis in dairy cows during the dry period

Research in Veterinary Science /990, 48, 280-287

Effects of an inadequate dietary intake of magnesium on osteogenesis in dairy cows during the dry period M. VAN MOSEL, A. Th. VAN'T KLOOSTER, A. MALESTEIN, Department of Large Animal Medicine and Nutrition, Veterinary Faculty, State Universitty of Utrecht, Yalelaan 16, NL-3584 cu Utrecht, The Netherlands The osteogenic activity in the rib bones of 33 dairy cows (14 of parity 1 or 2, and 19 of parity 3 or more) during their last two months of pregnancy, and the effects upon it of a deficient supply of dietary magnesium, were studied by means of intravenous infusions of the f1uorochromes oxytetracycline hydrochloride, calcein and chlortetracycline given nine weeks, five weeks and one week, respectively, before the expected time of parturition. Seven weeks before this date the cows' ration was changed to one containing either O· 22 per cent magnesium (LMg) or 0·82 per cent magnesium (HMg) in the dry matter, and the potassium content of both rations was increased to approximately 4·1 per cent in the dry matter to reduce the absorption of magnesium. Four of the 10 LMg-high parity cows showed signs of periparturient hypocalcaemia. A significant decrease in the number of labelled osteons towards parturition was demonstrated in both the low parity (P < O' OS) and high parity cows (P
The rate of flow of calcium into the blood plasma of a cow depends principally on the rate of absorption of calcium from the gut and the rate of resorption of calcium from bone (Allen and Sansom 1985). When these two processes are insufficient to balance the rate of loss of calcium to the fetus, the skeleton and the udder, hypocalcaemia will develop and the cow may succumb to milk fever. Van de Braak et al (I987b) found that at parturition the cellular resorptive activity of the bones of cows which had been fed inadequate magnesium during the dry period was significantly less than in cows receiving the same ration supplemented with magnesium. Decreased bone formation (Jones et al 1980) and mineralisation (Schwartz and Reddi 1979, Jones et al 1980) and reduced bone resorption (Rayssiguier and Larvor 1978, Jones et al 1980) have also been observed in magnesium deficient rats. These observations indicate that there is a decrease in the rate of bone turnover in magnesium deficient animals. There are thus good reasons why it is likely that magnesium deficient cows would be more susceptible to peri parturient hypocalcaemia and milk fever. On the basis of indirect evidence it is generally assumed that bone remodelling activity in cows decreases during the period between the end of lactation and parturition, but direct evidence is lacking. By using fluorescent bone 'markers' (fluorochromes) it is possible to study osteogenesis because these substances are deposited with the bone mineral at the calcification front where active mineralisation of new osteoid takes place (Frost 1969). The technique has been used in rats (Tam et al 1978) and in sheep (Van de Braak and van't Klooster 1987c) to investigate the effect of a low calcium intake on bone remodelling. It was shown that the amount of label per osteon tended to be higher in rats and sheep fed a low calcium diet than in animals fed a high calcium diet. The aim of the present experiment was to use a similar technique to study the changes in the rate of bone remodelling in dairy cows during the dry period, and to see whether a deficient magnesium supply had any effect on these changes.

280

Effects of low dietary magnesium on osteogenesis Materials and methods

281

TABLE 1: Compositions of the concentrates fed to the high Mg and low Mg groups of cows

Experimental design

High Mg

Thirty-three dairy cows were used; 20 were Friesian-Holstein cross Holstein-Friesian, eight were Friesian-Holsteins and five were of the Meuse-RhineYssel breed. They were allocated at random to two groups containing 16 and 17 cows which were subsequently fed diets containing either a low or a high amount of magnesium, respectively. As the rate of bone turnover is greater in young cows than old cows these two groups were subdivided into groups containing cows of parity I and 2 (low parity) and cows of parity 3 and more (high parity). Nine weeks before their expected calving date they were dried off.

1%1

Composition of concentrates

Soya bean meal, 44 per cent crude protein Isolvent extract) Tapioca meal Soyabeans (ground) Grass meal Palm kernel meal Molasses Calcium phosphate Potassium carbonate Magnesium oxide Sodium chloride Premix "

31·7 12·3 8·7 15·2 8·7 11·7 0·83

Low Mg

1%)

33·2 13·2

9 14·2

9 12 0·8

8

8

2·5 0·66 0·012

0·6 0·012

• Premix per 1 kg of concentrates: 115 mg zinc sulphate, 2 mg cobalt sulphate, 1500 iu vitamin A, 700 iu 9 - 1 vitamin D 500 (Roche)

Rations Seven weeks before their expected calving date the cows were tied in stalls and changed to the experimental rations. They consisted of 6 kg grass hay and 4 kg concentrates daily, the concentrates containing either O·23 per cent magnesium (LMg) or I· 58 per cent magnesium (HMg). The energy provided by this ration was sufficient, on average, for l : 3 times maintenance (high parity cows) to I· 5 times maintenance (low parity cows). The concentrates consisted as far as possible of ingredients with a low magnesium content, and 8 per cent potassium carbonate was added to try to reduce the absorption of magnesium. For the HMg group the concentrates were supplemented with 2· 5 per cent magnesium oxide. After parturition the cows were given grass hay ad libitum and the allocation of the high or low magnesium concentrates was increased by I kg d - I on each of the first two days. The composition of the concentrate part of the ration is shown in Table I. The mean daily intakes of calcium, phosphorus, magnesium and potassium by the cows during the experimental period before calving are shown in Table 2.

Blood and urine samples During the dry period blood samples were taken from a jugular vein once a week, between 08.30 and

09.30, into evacuated tubes (Venoject; Terumo) containing sodium heparin as anticoagulant. From approximately one week before parturition samples were taken daily. A blood sample was then taken immediately after parturition and on the first and second day after parturition. Until approximately one week before parturition urine samples were collected once a week between 10.00 and 11.00, usually from spontaneously voided urine, but occasionally by means of a steel catheter. From one week before parturition all the urine was collected daily by means of urinals attached to the cows with leather harnesses; the urine was weighed, mixed thoroughly and sampled daily. The blood samples were centrifuged for 10 to 15 minutes at approximately 2700 g and the plasma was separated at once.s'I'he samples of plasma and urine were stored at - 20°C until analysed.

Intravenous infusion of the jluorochromes Twenty-two cows were injected intravenously with calcein (Sigma C-0875) nine weeks before the expected calving date and then with oxytetracycline hydrochloride (Terramycin; Pfizer) one week before that date. The II other cows received intravenous injections of oxytetracycline hydrochloride nine weeks before calving, of calcein five weeks before

TABLE 2: Mineral composition of the ration and the mean daily intakes of calcium. phosphorus. magnesium and potassium during the dry period Mean intake Ig d High Mg 6 kg grass hay 4 kg concentrates TOlal

Ca

P

31 ·0 30·0 61·0

17 ·0 20·0 37·0

'1

Low Mg

Mg

K

Ca

P

Mg

K

11 ·0 63·0 74·0

126 244 370

31 ·0 31 ·0 62·0

17'0 20'0 37'0

11 ·0 9·0 20·0

126 244 370

282

M. van Mosel, A. T. van't Klooster, A. Malestein

calving and of chlortetracycline (Kombivet) one week before calving. Calcein (10 g per cow) was infused over a period of two hours; oxytetracycline hydrochloride (10 mg kg - I bodyweight) over a period of 10 to 15 minutes on two successive days and chlortetracycline (10 mg kg - I bodyweight) over a period of two hours on two successive days. The 10 g of calcein was dissolved in 17 ml of 5 M potassium hydroxide and 742 ml of distilled water was added to make a total of 759 ml. Chlortetracycline was dissolved in 17 ml of 4 M sodium hydroxide and 1983 ml of 0·9 per cent sodium chloride.

Surgical procedure The middle of the 12th rib was resected, under local anaesthesia, between three and eight hours after parturition. A 12 cm incision was made through the skin over the middle of the rib, followed by a 7 em incision through the subcutaneous tissues and the periosteum. Using a periosteum elevator the periosteum was lifted away to the right and the left and, using a specially made device, the periosteum behind the rib was also lifted. Using a wire saw a section of the entire rib was resected.

Fixation and preparation of the rib The bone was fixed in Burckhardt's fixative (Burckhardt 1966) for 48 hours at 0 to 4°C. It was then stored in 80 per cent ethanol for 14 days at 0 to 4°C. Thereafter it was further dehydrated at room temperature by daily transfer to increasing concentrations of ethanol (96 per cent and two changes of 100 per cent). The bone was infiltrated with methylbenzoate for one day followed by methylmethacrylate for two days (changed daily). For polyrnerisation the bone was put in a mixture of 10 ml of methymethacrylate, I ml plastoid and O· 39 g benzoylperoxide. Polymerisation took place within about two or three days at 40°C. Sections 50 to 70.ttm in thickness were cut on a Leitz 1600 Sagemicrotome, transversely to the long axis of the rib. Three sections were prepared from each rib, with 4 mm between each of them.

Bone histomorphometry The sections were viewed under ultraviolet light with a fluorescence microscope (Zeiss) with a 50 HIlO lamp. The filters used were an exciter filter G 365, a dichromatic beam splitter FT 395 and a barrier filter LP 420. A magnification of x 160 was used when counting the numbers of osteons and a magnification of x 320 when the distance between the fluorochrome labels were being measured. The measurements were made in the cortex of the three sections of each rib sampled.

Oxytetracycline hydrochloride produces a yellow fluorescence, calcein a green fluorescence and chlortetracycline a yellow-brown fluorescence in bone sections viewed under ultraviolet light. However, when a biopsy is taken too soon after the administration of the last label, this label may elute out while in the fixative (Frost 1983). As a result, when the time between the administration of the last label and the biopsy was less than four days, the rib sections were not examined. In each section the total numbers of labelled secondary osteons, that is, osteons growing within a resorption cavity, and the numbers of secondary osteons with one, two or three fluorescent markers were counted. The bone apposition rate, expressed as ttm d - I, was calculated by dividing the average distance between the centres of the labels by the period in days between the application of the labels plus half the period of labelling. The distance between the labels was measured, perpendicular to the labelled surface, by means of a calibrated Zeiss eyepiece micrometer, at four points spaced approximately equally apart around the osteonal circumference.

Chemical analysis Calcium and magnesium in plasma, urine and feedstuffs were estimated by atomic absorption spectrophotometry by modifications of the methods of Trudeau and Freier (1967). Inorganic phosphorus in plasma, and phosphorus in feedstuffs were determined by the method of Quinlan and DeSesa (1955). Urinary inorganic phosphorus was estimated by the molybdenum-blue method. The potassium content of feedstuffs was determined by atomic absorption spectrophotometry. Urinary creatinine was determined by a modification of the method of Folin (1914).

Statistical analyses Histomorphometric data were subjected to twoway analysis of variance (magnesium supply and parity) with interaction. Differences of means were evaluated by Student's t test. When variances were unequal an approximation of the t test, based on separate variance estimation, was used. The data of the number of osteons labelled nine weeks and one week before parturition were subjected to repeated measures analysis of variance for the evaluation of time effects. P values for time effects were determined using Wilks' criterion. P values are reported when less than 0·05 and are one-sided for Student's t test. Results The real times of injections with fluorochromes

Effects of low dietary magnesium on osteogenesis

283

TABLE 3: Mean (iiI and standard deviation (SO) of urinary magnesium:creatinine ratios of high (> 2nd parity) and low (,,; 2nd parity) parity cows fed rations containing O' 22 per cent magnesium (lMg) or O· 82 per cent magnesium (HMg) during the dry period

,,;2

Parity group Mg level Number of cows

Urinary magnesium:creatinine ratio

x

Time ante partum

X 1 ·85 2·16 1 ·65

SD 0'16 0·07 0-24

0,35* 0,24* 0,34*

7-4 weeks 4-2 weeks 180-24 h

>2

HMg n=8

LMg n=6

HMg n=9

LMg n=10 SD 0·56 0'45 0-46

X 0,35* 0,16* 0,06*

SD 0·35 0·14 0·07

x 1·70 1 - 56 1·21

SD 0·65 0·68 0·70

* Significantly different from HMg cows in the same period IP< 0·0011

were for the low parity cows, on average, 8·5 ± 1·0 (so) weeks and I· 5 ± O' 5 (SD) weeks before the calving date and for the high parity cows, on average, 9'7 ± 1'1 (SD) weeks, 5·9 ± 1·0 (so) weeks and 1·8 ± O: 9 (so) weeks before that date. Of the 10 high parity cows fed the LMg diet, four showed signs of hypocalcaemia after calving and were unable to rise although the mean plasma calcium concentration was above 2· a mmol litre - I. They were

treated successfully with a commercial calcium/ magnesium solution (Aescaldyn; Aesculaap). None of the cows fed the HMg diet showed signs of hypocalcaemia.

Urinary magnesium excretion The mean ratios of the concentrations of magnesium and creatinine in urine were calculated for

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FIG 1: Mean plasma concentrations (mmollitre - , I (vertical bars give standard deviations) of calcium, magnesium and inorganic

phosphorus during the dry period. at parturition (PI and 12 to 36 hours post partum (ppl in low parity cows fed rations containing O· 22 per cent magnesium (L. n = 61or 0·82 per cent magnesium IH. n=81 during the dry period. *P<0·05. **P
HL HL HL HL 7-4 weeks 180-100 hours 4-2 weeks 100-36 hours P Weeks or hours ante partum FIG 2: Mean plasma concentrations (mmol litre : 1) (vertical bars give standard deviations I of calcium. magnesium and inorganic phosphorus during the dry period. at parturition IPI and 12 to 36 hours post partum (ppl in high parity cows fed rations containing O· 22 per cent magnesium (L, n = 101 or O· 82 per cent magnesium IH. n = 9) during the dry period. * P< O· 05, * * P < O· 01, ***P
M. van Mosel, A. T. van't Klooster, A. Malestein

284

TABLE 4: Sum of squares-, F and P values for the number of osteons labelled and Wilks' criterion. F (exact}- and P values for the hypothesis of no time effect in the number of osteons labelled nine weeks and one week before parturition Source

df

Sum of squares

Mg level Parity Mg level x parity Error

1 1 1 24

495133 38916 76356 5871179

F value

2·02 0·16 0·31

Probability> F

0·1677 0·6935 0·5816

Wilks' criterion

df

F (exact)

Probability> F

0·431 0·922 0·930 0·997

1.24 1,24 1,24 1,24

31 ·65 2·04 1 ·80 0·07

0·0001 0·1665 0·1928 0·7959

Time Time x Mg level Time x parity Time x Mg level x parity

the periods from seven to four weeks, four to two weeks and 180 to 24 hours before parturition (Table 3). In each period the mean ratio was significantly lower (P
Concentrations ofcalcium, magnesium and inorganic phosphorus in plasma The mean concentrations of calcium, magnesium and inorganic phosphorus in the plasma of the low parity group and the high parity group on five occasions before calving, at calving and 12 to 36 hours after calving are shown in Figs I and 2, respectively. The mean plasma concentration of calcium decreased significantly between 36 and 12 hours before parturition and parturition in both the LMghigh parity group (P
inorganic phosphorus was lower but not significantly so. The mean inorganic phosphorus concentrations in the LMg-low parity group were significantly lower than in the HMg-Iow parity group only in the periods from seven to four weeks, four to two weeks and 180 to 100 hours before parturition.

Histomorphometric examination of rib sections The rib sections from one cow in the LMg-high parity group and one cow in the HMg-high parity group were not examined because the biopsies were taken too soon after the administration of the last label. In young bone, which is growing rapidly in width by laying down bone on the periosteal surface, primary osteons may be formed. When apposition is slow, that is, in older animals, no primary osteons appear. Primary and secondary osteons differ in their formation and distribution. All secondary osteons begin with the tunnelling of a resorption cavity while primary osteons do not. Secondary osteons are produced at different stages of formation while primary osteons will be added to the periosteal surface by apposition in stages (Vanderhoeft et al 1962). Successive cortical surface lines, induced at each injection of a fluorochrome, separate these different stages or' primary osteons. In the rib sections from the low parity cows, and particularly in those from the first parity cows, it was difficult to count the secondary osteons, especially at the anterior and posterior sites of the rib, where the distribution between primary periosteal and secondary osteons was not always clear. Because the primary osteons are anatomically and functionally distinct from the secondary osteons, the primary osteons must be excluded from the measurements. Besides this, the transition between cortical and spongeous bone was not always distinct. For these reasons it was decided not to count the labelled osteons in the rib sections from cows 3, 4 and 6 in the HMg-low parity group.

Effects of low dietary magnesium on osteogenesis

285

TABLE 5: Mean (x) and standard deviation (SOl of the bone apposition rate (I'm d-'l measured between the first and last marker in three sections of the 12th rib of 14 cows of low parity and 17 cows of high parity fed rations containing 0·22 per cent magnesium (LMg) or O· 82 per cent magnesium (HMg) during the dry period Bone apposition rate (I'm d HMg Number of measure-

Cow 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Parity

ments

x

SO

1st 1st 1st 1st 1st 1st 2nd 2nd 3rd 3rd 3rd 4th 4th 5th 5th 7th

60 60 60 60 60 60 60 60 90 90 90 26 90 90 44 10

0·91 0·73 0·90 1 ·10 0·95 0·99 0·80 1 ·04 0·66 0·85 0·52 0·81 0·82 0·81 0·44 0·63

0·47 0·39 0·20 0·37 0·35 0·34 0·35 0·31 0·33 0·34 0·30 0·40 0·32 0·30 0·23 0·28

There were wide variations between the numbers of osteons labelled in individual cows at the three times of observation. However, a significant time effect was demonstrated in the number of osteons labelled nine weeks and one week before parturition in both the low parity group (P < O' 05) and in the high parity group (P
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COW 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Parity

LMg Number of measurements

x

SO

1st 1st 1st 1st 1st 2nd 3rd 4th 4th 5th 5th 5th 5th 5th 6th

60 60 60 33 60 60 90 44 90 46 90 55 90 74 90

0·84 1 ·02 0·75 0·95 1·03 0·91 0·69 0·60 0·86 0·92 0·63 0·59 0·73 O' 71 0·81

0·41 0·37 0·32 0·27 0·33 0·33 0·24 0·29 0·32 0·26 0·25 0·25 0·24 0·25 O' 21

In the low parity group the mean (± so) bone apposition rates were 0·93 ± 0·12 Jlm d~1 for the HMg cows and 0'92 ± 0·11 Jlm d~' for the LMg cows. In the high parity group the mean bone apposition rates were 0·69 ± 0·15 Jlm d- I for the HMg cows and 0·73 ± 0·12 Jlm d- I for the LMg cows. Discussion The use of the three fluorochrome bone markers made it possible to measure the bone apposition rates and the numbers of active osteons which were present when the fluorochrornes were administered. It was shown that parity had a significant effect (P
286

M. van Mosel, A. T. van't Klooster, A. Malestein

variations it is possible to demonstrate only large changes in osteogenic activity with this technique, and the wide variations may have been responsible for the failure to demonstrate any effect of magnesium deficiency. However, the decrease in bone formation rate during the last two months of pregnancy, suggests that there may have been a reduction in the amount of bone remodelling. Such a reduction would decrease the amount of calcium which could be mobilised from bone at parturition, when the cow depends heavily on the reserves of calcium in bone. There are two other reasons why a deficient magnesium supply failed to produce a detectable effect on osteogenesis. First, the observed decrease in the bone formation rate would have made it more difficult to detect changes in bone formation due to magnesium deficiency. Secondly, any change in bone remodelling due to a dietary change would require a minimum period before a new steady state is established; this minimum time is known as sigma and the normal value of sigma in dogs and rabbits is about two months and in man about four months (Frost 1969). Because one can be misled by measurements made during this transient phase, measurements would need to have been made at least one sigma after the change in bone remodelling. In the present experiment it is possible that the cows were still in a transitional state. It is not known by how much, or for how long, plasma magnesium concentration must decrease before there is any change in bone remodelling. In the LMg-high parity cows plasma magnesium did not fall below O: 85 mmol litre -I, the lower limit of the normal range (Rowlands and Manston 1976), before two weeks before parturition, and decreased to a mean of 0·53 mmol litre-I at parturition. These changes were possibly too small and, or, too short lived for any effect on osteogenesis to be observed. In all the LMg cows the mean urinary magnesium: creatinine ratio in the dry period was O· 35 or less, indicating that less than 1 g of magnesium was being excreted daily in urine (Van de Braak et al 1987a) and that the cows were probably in. negative magnesium balance (Kemp et al 1961). Between four and two weeks before parturition and between 180 and 24 hours before parturition the magnesium: creatinine ratio in LMg-high parity cows was lower than in the LMg-low parity cows; in the latter group the mean plasma magnesium concentration remained above 0·80 mmollitre- t during the dry period, whereas it decreased to O· 53 mmol litre - 1 at parturition in the LMg-high parity group. These results are similar to earlier data which showed that young cows can mobilise magnesium from body reserves more easily than old cows (Blaxter and McGill 1956). There was no clear difference between the plasma calcium concentrations of the cows fed the high or low

magnesium rations either before or at parturition. Previous work showed that calcium concentrations tended to be lower in cows fed a low magnesium diet in the dry period (Van de Braak et al 1987a). Of the four cows in the LMg-high parity group which showed clinical signs of hypocalcaemia at calving, only one had a plasma calcium concentration less than 2'0 mrnol litre "! while the others had plasma calcium concentrations of about 2· 2 mmol litre-I, within the normal range. The mean plasma magnesium concentration in these four cows was 0·44 mmol litre- 1 at calving. This suggests that only a slight degree of hypocalcaemia at calving, coupled with a greater degree of hypomagnesaemia, can lead to clinical signs similar to those of severe peri parturient hypocalcaemia. Acknowledgements The authors thank Hugo Wouterse, Jan van der Kuilen and Fred van Mosel for the chemical analyses. Gerard ten Broeke, Gerrit Ekkelenkamp, Jan van Mourik, Fred van Mosel and Henk Obbink are thanked for the care of the cows used in this study. References ALLEN. W. M. & SANSOM. B. F. (1985) Journal of Veterinary Pharmacology and Therapeutics 8, 19-29 BLAXTER. K. L. & McGILL, R. F. (1956) Veterinary Reviews and Annotations 2,35-54 BRAAK, A. E. VAN DE, KLOOSTER, A. Th. VAN'T & MALESTEIN, A. (l987a) Research in Veterinary Science 42,101-108 BRAAK, A E. VAN DE, KLOOSTER, A. Th. VAN'T, GOEDEGEBUURE, S. A. & FABER, 1. A. J. (1987b) Research in Veterinary Science 43,7-12 BRAAK, A. E. VAN DE & KLOOSTER, A. Th. VAN'T (1987c) Veterinary Research Communications 11,101-108 BRAITHWAITE, G. D. (1976) Journal oj Dairy Research 43, 501-520 BURCKHARDT, R. (1966) !JIul 14, 30-41i CONTRERAS, 1'. A., MANSTON, R. & SANSOM, B. F. (1982) Research in Veterinary Science 33, 10-16 FOLIN, O. (1914) Journal of Biological Chemistry 17,463 . FROST, 11. M. (1963) Canadian Journal oj Biochemistry and Physiology 4I, 31-42 FROST, H. M. (1969) Calcijied Tissue Research 3, 211-237 . FROST, H. M. (1983) Bone Histomorphometry: Techniques and Interpretation. Ed R. Recker. Boca Raton, Florida, cnc Press. PI' 37-52 JONES. J. E., SCHWARTZ, R. & KROOK, L. (1980) Calcijied Tissue tntcrnationat si, 231-238 JONSSON, G. (1978) Veterinary Record 102, 165-169 KEMP, A., DEYS, W. B., HEMKES, O. J. & ES, A. J. H. VAN (1961) Netherlands Journal of Agricultural Science 9, 134-149 LEE, W. R., MARSHALL, J. H. &SISSONS, H. A. (J965) Journal oj Bone and Joint Surgery 47-11, 157-180 QUINLAN, K. P. & DESESA, M. A. (1955) Analytical Chemistry 27, 1626-1629 RAMAN, A. (1969) Acla Orlhopaedica Scandinavica 40,193-197 RAYSSIGUIER, Y. & LARVOR, P. (1978) Annates de Biologie Animate, Biochimie et Biophysique 18, 157-166

Effects of /011' dietary magnesium on osteogenesis ROWl.ANDS, o. L & ~lANSTON, R. (1Y76) Livestock Production Science 3.2.19-256 SANS()~I, B. I'., ~lANST()N, R., VA(;(i, xt. L, ~lALLINSON, C. B. '" CONTR£:RAS, 1'. A. (1982) Proceedings, Xllth World Congress 011 Diseases of Cattle, Amsterdam. PP 574-578 SCH\\'ART/, R. '" REDDI, A. II. (1979) Culcilicd Tissuc lnternational I'), 15-20 TA~I, C. S., HARRISON,.I. E., REED, R. & CRUICKSHANK, B. (197S) Metabolism 27, 14.1·· 150

287

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Received February 13, 1989 Accepted August 16, 1989