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Lysine metabolism in beef cattle
Comp. Biochem. Physiol., 1968, Vol. 25, pp. 349 to 353. Pergamon Press. Printed in Great Britain
LYSINE METABOLISM IN BEEF CATTLE P. H. S P R I N G E L L Division of Animal Genetics, C.S.I.R.O., Cattle Research Laboratory, Rockhampton, Queensland 4700, Australia (Received 26 September 1967)
Abstract--1. The metabolism of intravenously injected tritiated L-lysine was compared in Bos taurus and Bos indicus x Bos taurus cattle. 2. A number of characteristics were examined, including the loss of label from the blood-stream, its appearance in the urine, and the rate of incorporation and elimination from plasma proteins. 3. The fate of the lysine in the two types of cattle appears to be very similar, so that performance differences could be attributable to other than postabsorptive factors. 4. The level of free plasma amino-acid nitrogen remained unaltered in cattle even after 4 days' starvation. INTRODUCTION THE REASONSwhy European and tropical breeds of cattle have different growth rates in the tropics could be due to one or more of a number of causes (Post, 1963 ; Turner, 1964; Evans & Turner, 1965; Springell, 1966). Although factors other than nutrition may be implicated, differential nitrogen intake, digestion, absorption, anabolism, catabolism or excretion must be involved. Certain aspects of nitrogen intake, digestion and excretion have already been studied in our laboratory (Ashton, 1962, 1963; Vercoe, 1966, 1967), and as an extension of these studies, an aspect of post-absorptive nitrogen metabolism was examined.
MATERIALS AND METHODS One-year-old steers were used in these experiments. In the main trial, three representatives of Bos taurus belonging to British breeds (namely Hereford x Shorthorn crosses) were examined together with two Bos indicus × Bos taurus steers, represented by Brahman × British crosses. A preliminary experiment designed to test the effect of nutrition on plasma amino-acid nitrogen levels was carried out on four of these animals, two coming from each breed. The cattle were first fed normally (2.5 kg high-quality lucerne hay, twice daily) for 2 days, starved for 3 days and then allowed to recover on normal feed for a further day. Plasma protein was precipitated with 10% trichloroacetic acid, the supernatant was made alkaline and boiled to remove urea and ammonia. The remaining nitrogen, consisting mainly of free plasma amino acid, but also including nitrogen from other minor components such as creatinine, etc., was determined by the micro-Kjeldahl procedure. 349
350
P.H. SPRINGELL
A month later, the five animals were placed in metabolic cages and were main rained on a normal diet. At 10 a.m. of the first day, 5 mC L-lysine-4,5-T (Radiochemical Centre, Amersham) was injected into the jugular vein of each. At least eighteen jugular blood and urine samples were collected at selected intervals from each steer over a 4-day period, during which the animals were kept caged. The steers were then released from their cages, kept group fed under similar nutritional conditions in the yards, and bled at various intervals up to 46 days after the lysine administrations. At the time of injection, the body weight of the Brahman x British cattle (227 + 16 kg) was typically higher ( P < 0.05) than that of the B . taurus steers at 168 + 8 kg. The plasma volume was assumed to be 3-3 per cent of the body weight (Springell, 1968a), and was estimated at 7.49 _4-0"53 and 5.56 -+ 0-26 1. for the two breeds respectively. Water (from plasma and urine) and the supernatant (1 ml) obtained following plasma protein precipitation with 10 per cent trichloroacetic acid were mixed with 10 ml naphthalene-dioxane scintillator (Butler, 1961). Whole plasma (1 ml) was first soluhilized with 1 ml 1 M Hyamine 10-X (Herberg, 1960), while the urine residue, after removal of water by sublimation, was dissolved in 1 ml water, and suspension counted with Cab-O-Sil (Gordon & Wolfe, 1960).
RESULTS AND DISCUSSION The free amino-acid nitrogen was found to be 19.1_+0.4 mg/100ml plasma under normal feeding, and 18.0_+0.4 during starvation (Table I). It was considered desirable that plasma amino-acid levels remain reasonably constant after T A B L E 1 - - E F F E C T OF N U T R I T I O N ON THE FREE PLASMA AMINO-ACID NITROGEN I N STEERS
Breed
Nutritional status
n*
mg N/IO0 ml plasma + S.E.M.
British Brahman cross British and Brahman cross British and Brahman cross British and Brahman cross
Fed and starved Fed and starved Fed Starved Fed and starved
24 24 28 20 48
18.6 + 0.4 18.7 + 0.4 19"1 + 0"4 18"0 + 0"4 18"6 + 0"3
* Number of observations. labelled lysine administration, but since no breed, animal or nutritional difference was detected statistically, the animals could be fed normally during the main experiment. The fate of the H s label was essentially similar to the classic pattern previously observed with N tS- and Ct4-amino acids in monogastric animals (Schoenheimer, 1942; Miller et al., 1949). Less than 10 per cent of the injected radioactivity could be recovered in the trichloroacetic acid supernatant 10 min after the tritiated lysine injection (Table 2). The half-life (tj) of the label was estimated at 2.8 + 0-1 min.
351
LYSINE METABOLISM I N BEEF CATTLE
Subsequently there was a slower loss of label over the next 11.2+ 1"9 hr (tj = 123 + 16 rain) in the non-water component of the trichloroacetic acid supernatant fraction. Tritiated water gradually built up in the plasma, reaching a m a x i m u m (1.0-1.3 per cent of the initial dose) within 24-30 hr. Plasma protein TAm.~ 2~SUMMARY OF EARLYOBSERVATIONS Parameter
Units
Dose left in TCA supernatant at 10 rain
(%)
Half-life in TCA supernatant, 0-10 rain
(rain)
Half-life of non-HzO in TCA supernatant, 10 m i n - l l hr Time for non-H20 to disappear from TCA supernatant Maximum dose in plasma protein
(rain)
Time of peak activity in plasma protein Maximum specific activity of plasma protein
(hr) (%) (hr) QtC/g)
British 8"2 +0.7 2"7 +0"1 143 +18 13"3 + 2"3 4'9 + 0"4 7"7 + 0'9 0'56 +_0.06
Brahman ×
Mean
9.2 +0.4 2.9 +_0-1 94 ±18 8"0 + 1.5 4"8 + 0"5 6'5 + 0"0 0"42 + 0.08
8'6 +0"5 2"8 ±0"1 123 ±16 11"2 _+1"9 4'9 ± 0"3 7.2 _+0.6 0.50 + 0.08
activity was detectable within I hr, and reached a m a x i m u m (4.9 + 0.3 per cent of the initial dose) within 7.2 + 0.6 hr. T h e peak specific activity was 0.50 + 0.08/zC/g. After 4 days 0-81 + 0-04 per cent of the initial dose was left in the trichloroacetic acid supernatant (Table 3), all as tritiated water. By this time the plasma TABLE 3~SUMMARY OF OBSERVATIONSAT 4 DAYS'POST-INJECTION Parameter
Units
British
Brahman ×
Dose left in TCA supernatant
(%)
Dose left in plasma protein
(%)
Cumulative dose loss in urine as water
(°/o)
Cumulative dose loss in urine as residue
(%)
Half-life of plasma protein, 7 hr-4 days
(days)
Half-life in urine, 0--4 days
(days)
0.75 + 0.04 3-3 +0.5 5"3 ±0"5 10.6 + 1.5 8.9 + 2.9 16"4 ± 1-9
0.91 * +_0.01 2.8 ±0.5 7-5 +0"8 10.9 ± 1"0 5-1 + 0-9 13"2 + 1"4
* This value is significantly higher (P < 0"05) than that for the British steers.
Mean 0"81 +_0.04 3-1 +0.3 6"2 ±0.6 10-7 + 0.9 7.4 +_1-8 15"1 + 1-4
352
P.H. SPRtNGELL
protein contained 3.1 + 0.3 per cent of the initial radioactivity. The half-life of plasma proteins was calculated at 7.4 + 1-8 days, by taking into account all the data collected in 4 days. In view of the many protein fractions present, the decay pattern was understandably complex, and tt values ranging from 3.9-19.2 days could be obtained by selecting observation periods of between 1.5 and 46 days respectively. The 4-day urinary excretion amounted to 16.9 _+1.1 per cent of the administered dose. Of this 6.2 + 0.6 per cent was accounted for as labelled water, and 10.7 +_0-9 per cent as the residue. Initially only 2.5 per cent of the urinary radioactivity was found in tritiated water, but by 24-48 hr more label was excreted as tritiated water than in the form of the residue. The overall half-life of the label during the first 96 hr, as measured by the total urinary losses, was 15.1 + 1.4 days. Of the thirteen characteristics listed in Tables 2 and 3, only the percentage of tritiated water left in the 4-day trichloroacetic acid supernatant (Table 3) showed a significant breed difference. This may in small part be associated with the faster water turnover generaUy found in British cattle (Bianca, 1965; Macfarlane & Howard, 1966; Springell, 1968b). Over the first 4 days there would, nevertheless, appear to be a somewhat slower rate of tritiated water loss via the urine in British steers, which is presumably associated with a correspondingly longer plasma protein half-life. This trend was, however, not maintained during longer observation periods, when the half-life of plasma proteins in the British steers actually became the shorter. Differences such as those discussed above could be accounted for in terms of differences in the amounts of plasma protein fractions of varying lability. It is concluded that the metabolic fate of tritiated lysine in the two breeds of cattle appears to be essentially indistinguishable. If the effect observed with lysine is found with other amino acids, then factors other than post-absorptive metabolism may be responsible for the observed breed performance differences. Small differences in the metabolic fate can not be ruled out, as the number of animals was too small to exclude anything but gross differences. .4cknowledgements--I thank Mr. A. K. Duffield and Miss S. J. Shepherd for technical assistance. This work was supported in part by the Australian Meat Research Committee.
REFERENCES ASHTON G. C. (1962) Comparative nitrogen digestibility in Brahman, Brahman × Shorthorn, Africander × Hereford, and Hereford steers..7, agric. Sci. 58, 333-342. ASHTONG. C. (1963) Weight gain and faecal nitrogen excretion in grazing British and Zebu cross steers. Aust. ft. agric. Res. 14, 898-908. BXANCAW. (1965) Reviews of the progress of dairy science. Section A: Physiology. Cattle in a hot environment, ft. Dairy Res. 32, 291-345. BUTLER F. E. (1961) Determination of tritium in water and urine. Liquid scintillation counting and rate-of-drift determination, tlnalyt. Chem. 33, 409-414. EVANSJ. V. & TURNERH. G. (1965) Interrelationships of erythrocyte characters of British and Zebu crossbred beef cattle. Aust.jT. biol. Sd. 18, 124-139. GORDON C. F. & WOLFEA. L. (1960) Liquid scintillation counting of aqueous samples. Analyt. Chem. 32, 574.
LYSINE METABOLISM I N BEEF CATTLE
353
HE~ERG R. J. (1960) Determination of carbon-14 and tritium in blood and other whole tissues. Liquid scintillation counting of tissues. Analyt. Chem. 32, 42-46. MACFARLA~mW. V. & Howmm B. (1966) Water content and turnover of identical twin Bos indicus and B. taurus in Kenya. ft. agric. S d . Camb. 66, 297-302. MILLER L. L., B^LS W. F., YuIIm C. L., MAsaxR R. E., TISHKOFF G. H. & WmPPIm G. H. (1949) Use of radioactive lysine in studies of protein metabolism. Synthesis and utilization of plasma proteins..7, exp. Med. 90, 297-313. POST T. B. (1963) Plasma protein-bound iodine and growth rates of beef cattle. Aust. J. agrie. Res. 14, 572-579. SCHOEI~HEIMERR. (1942) The Dynamic State of the Body Constituents. Harvard University Press, Cambridge, Mass. SPmNCELL P. H. (1966) Sulphur requirements for cattle hair growth. Proc. Aust. Soc. Animal Prod. 6, 399--402. SPRIIq~ELL P. H. (1968a) Red cell volume and blood volume in beef cattle. Aust..~. agric. Res. 19, 145-160. SPRINOELL P. H. (1968b) Water content and water turnover in beef cattle. Aust. jY. agric. Res. 19, 129-144. TURNER H. G. (1964) Coat characteristics of cattle in relation to adaptation. Proc. Aust. Soc. Animal Prod. 5, 181-187. VERCOE J. E. (1966) Some aspects of nitrogen metabolism of British and Zebu-type cattle. Proc. Aust. Soe. Animal Prod. 6, 370-377. VERCOB J. E. (1967) Breed and nutritional effects on the composition of faeces, urine and plasma from Hereford and Brahman x Hereford steers fed high and low quality diets. Aust. J. agric. Res. 8, 1003-1013.
I2
LYSINE METABOLISM IN BEEF CATTLE P. H. S P R I N G E L L Division of Animal Genetics, C.S.I.R.O., Cattle Research Laboratory, Rockhampton, Queensland 4700, Australia (Received 26 September 1967)
Abstract--1. The metabolism of intravenously injected tritiated L-lysine was compared in Bos taurus and Bos indicus x Bos taurus cattle. 2. A number of characteristics were examined, including the loss of label from the blood-stream, its appearance in the urine, and the rate of incorporation and elimination from plasma proteins. 3. The fate of the lysine in the two types of cattle appears to be very similar, so that performance differences could be attributable to other than postabsorptive factors. 4. The level of free plasma amino-acid nitrogen remained unaltered in cattle even after 4 days' starvation. INTRODUCTION THE REASONSwhy European and tropical breeds of cattle have different growth rates in the tropics could be due to one or more of a number of causes (Post, 1963 ; Turner, 1964; Evans & Turner, 1965; Springell, 1966). Although factors other than nutrition may be implicated, differential nitrogen intake, digestion, absorption, anabolism, catabolism or excretion must be involved. Certain aspects of nitrogen intake, digestion and excretion have already been studied in our laboratory (Ashton, 1962, 1963; Vercoe, 1966, 1967), and as an extension of these studies, an aspect of post-absorptive nitrogen metabolism was examined.
MATERIALS AND METHODS One-year-old steers were used in these experiments. In the main trial, three representatives of Bos taurus belonging to British breeds (namely Hereford x Shorthorn crosses) were examined together with two Bos indicus × Bos taurus steers, represented by Brahman × British crosses. A preliminary experiment designed to test the effect of nutrition on plasma amino-acid nitrogen levels was carried out on four of these animals, two coming from each breed. The cattle were first fed normally (2.5 kg high-quality lucerne hay, twice daily) for 2 days, starved for 3 days and then allowed to recover on normal feed for a further day. Plasma protein was precipitated with 10% trichloroacetic acid, the supernatant was made alkaline and boiled to remove urea and ammonia. The remaining nitrogen, consisting mainly of free plasma amino acid, but also including nitrogen from other minor components such as creatinine, etc., was determined by the micro-Kjeldahl procedure. 349
350
P.H. SPRINGELL
A month later, the five animals were placed in metabolic cages and were main rained on a normal diet. At 10 a.m. of the first day, 5 mC L-lysine-4,5-T (Radiochemical Centre, Amersham) was injected into the jugular vein of each. At least eighteen jugular blood and urine samples were collected at selected intervals from each steer over a 4-day period, during which the animals were kept caged. The steers were then released from their cages, kept group fed under similar nutritional conditions in the yards, and bled at various intervals up to 46 days after the lysine administrations. At the time of injection, the body weight of the Brahman x British cattle (227 + 16 kg) was typically higher ( P < 0.05) than that of the B . taurus steers at 168 + 8 kg. The plasma volume was assumed to be 3-3 per cent of the body weight (Springell, 1968a), and was estimated at 7.49 _4-0"53 and 5.56 -+ 0-26 1. for the two breeds respectively. Water (from plasma and urine) and the supernatant (1 ml) obtained following plasma protein precipitation with 10 per cent trichloroacetic acid were mixed with 10 ml naphthalene-dioxane scintillator (Butler, 1961). Whole plasma (1 ml) was first soluhilized with 1 ml 1 M Hyamine 10-X (Herberg, 1960), while the urine residue, after removal of water by sublimation, was dissolved in 1 ml water, and suspension counted with Cab-O-Sil (Gordon & Wolfe, 1960).
RESULTS AND DISCUSSION The free amino-acid nitrogen was found to be 19.1_+0.4 mg/100ml plasma under normal feeding, and 18.0_+0.4 during starvation (Table I). It was considered desirable that plasma amino-acid levels remain reasonably constant after T A B L E 1 - - E F F E C T OF N U T R I T I O N ON THE FREE PLASMA AMINO-ACID NITROGEN I N STEERS
Breed
Nutritional status
n*
mg N/IO0 ml plasma + S.E.M.
British Brahman cross British and Brahman cross British and Brahman cross British and Brahman cross
Fed and starved Fed and starved Fed Starved Fed and starved
24 24 28 20 48
18.6 + 0.4 18.7 + 0.4 19"1 + 0"4 18"0 + 0"4 18"6 + 0"3
* Number of observations. labelled lysine administration, but since no breed, animal or nutritional difference was detected statistically, the animals could be fed normally during the main experiment. The fate of the H s label was essentially similar to the classic pattern previously observed with N tS- and Ct4-amino acids in monogastric animals (Schoenheimer, 1942; Miller et al., 1949). Less than 10 per cent of the injected radioactivity could be recovered in the trichloroacetic acid supernatant 10 min after the tritiated lysine injection (Table 2). The half-life (tj) of the label was estimated at 2.8 + 0-1 min.
351
LYSINE METABOLISM I N BEEF CATTLE
Subsequently there was a slower loss of label over the next 11.2+ 1"9 hr (tj = 123 + 16 rain) in the non-water component of the trichloroacetic acid supernatant fraction. Tritiated water gradually built up in the plasma, reaching a m a x i m u m (1.0-1.3 per cent of the initial dose) within 24-30 hr. Plasma protein TAm.~ 2~SUMMARY OF EARLYOBSERVATIONS Parameter
Units
Dose left in TCA supernatant at 10 rain
(%)
Half-life in TCA supernatant, 0-10 rain
(rain)
Half-life of non-HzO in TCA supernatant, 10 m i n - l l hr Time for non-H20 to disappear from TCA supernatant Maximum dose in plasma protein
(rain)
Time of peak activity in plasma protein Maximum specific activity of plasma protein
(hr) (%) (hr) QtC/g)
British 8"2 +0.7 2"7 +0"1 143 +18 13"3 + 2"3 4'9 + 0"4 7"7 + 0'9 0'56 +_0.06
Brahman ×
Mean
9.2 +0.4 2.9 +_0-1 94 ±18 8"0 + 1.5 4"8 + 0"5 6'5 + 0"0 0"42 + 0.08
8'6 +0"5 2"8 ±0"1 123 ±16 11"2 _+1"9 4'9 ± 0"3 7.2 _+0.6 0.50 + 0.08
activity was detectable within I hr, and reached a m a x i m u m (4.9 + 0.3 per cent of the initial dose) within 7.2 + 0.6 hr. T h e peak specific activity was 0.50 + 0.08/zC/g. After 4 days 0-81 + 0-04 per cent of the initial dose was left in the trichloroacetic acid supernatant (Table 3), all as tritiated water. By this time the plasma TABLE 3~SUMMARY OF OBSERVATIONSAT 4 DAYS'POST-INJECTION Parameter
Units
British
Brahman ×
Dose left in TCA supernatant
(%)
Dose left in plasma protein
(%)
Cumulative dose loss in urine as water
(°/o)
Cumulative dose loss in urine as residue
(%)
Half-life of plasma protein, 7 hr-4 days
(days)
Half-life in urine, 0--4 days
(days)
0.75 + 0.04 3-3 +0.5 5"3 ±0"5 10.6 + 1.5 8.9 + 2.9 16"4 ± 1-9
0.91 * +_0.01 2.8 ±0.5 7-5 +0"8 10.9 ± 1"0 5-1 + 0-9 13"2 + 1"4
* This value is significantly higher (P < 0"05) than that for the British steers.
Mean 0"81 +_0.04 3-1 +0.3 6"2 ±0.6 10-7 + 0.9 7.4 +_1-8 15"1 + 1-4
352
P.H. SPRtNGELL
protein contained 3.1 + 0.3 per cent of the initial radioactivity. The half-life of plasma proteins was calculated at 7.4 + 1-8 days, by taking into account all the data collected in 4 days. In view of the many protein fractions present, the decay pattern was understandably complex, and tt values ranging from 3.9-19.2 days could be obtained by selecting observation periods of between 1.5 and 46 days respectively. The 4-day urinary excretion amounted to 16.9 _+1.1 per cent of the administered dose. Of this 6.2 + 0.6 per cent was accounted for as labelled water, and 10.7 +_0-9 per cent as the residue. Initially only 2.5 per cent of the urinary radioactivity was found in tritiated water, but by 24-48 hr more label was excreted as tritiated water than in the form of the residue. The overall half-life of the label during the first 96 hr, as measured by the total urinary losses, was 15.1 + 1.4 days. Of the thirteen characteristics listed in Tables 2 and 3, only the percentage of tritiated water left in the 4-day trichloroacetic acid supernatant (Table 3) showed a significant breed difference. This may in small part be associated with the faster water turnover generaUy found in British cattle (Bianca, 1965; Macfarlane & Howard, 1966; Springell, 1968b). Over the first 4 days there would, nevertheless, appear to be a somewhat slower rate of tritiated water loss via the urine in British steers, which is presumably associated with a correspondingly longer plasma protein half-life. This trend was, however, not maintained during longer observation periods, when the half-life of plasma proteins in the British steers actually became the shorter. Differences such as those discussed above could be accounted for in terms of differences in the amounts of plasma protein fractions of varying lability. It is concluded that the metabolic fate of tritiated lysine in the two breeds of cattle appears to be essentially indistinguishable. If the effect observed with lysine is found with other amino acids, then factors other than post-absorptive metabolism may be responsible for the observed breed performance differences. Small differences in the metabolic fate can not be ruled out, as the number of animals was too small to exclude anything but gross differences. .4cknowledgements--I thank Mr. A. K. Duffield and Miss S. J. Shepherd for technical assistance. This work was supported in part by the Australian Meat Research Committee.
REFERENCES ASHTON G. C. (1962) Comparative nitrogen digestibility in Brahman, Brahman × Shorthorn, Africander × Hereford, and Hereford steers..7, agric. Sci. 58, 333-342. ASHTONG. C. (1963) Weight gain and faecal nitrogen excretion in grazing British and Zebu cross steers. Aust. ft. agric. Res. 14, 898-908. BXANCAW. (1965) Reviews of the progress of dairy science. Section A: Physiology. Cattle in a hot environment, ft. Dairy Res. 32, 291-345. BUTLER F. E. (1961) Determination of tritium in water and urine. Liquid scintillation counting and rate-of-drift determination, tlnalyt. Chem. 33, 409-414. EVANSJ. V. & TURNERH. G. (1965) Interrelationships of erythrocyte characters of British and Zebu crossbred beef cattle. Aust.jT. biol. Sd. 18, 124-139. GORDON C. F. & WOLFEA. L. (1960) Liquid scintillation counting of aqueous samples. Analyt. Chem. 32, 574.
LYSINE METABOLISM I N BEEF CATTLE
353
HE~ERG R. J. (1960) Determination of carbon-14 and tritium in blood and other whole tissues. Liquid scintillation counting of tissues. Analyt. Chem. 32, 42-46. MACFARLA~mW. V. & Howmm B. (1966) Water content and turnover of identical twin Bos indicus and B. taurus in Kenya. ft. agric. S d . Camb. 66, 297-302. MILLER L. L., B^LS W. F., YuIIm C. L., MAsaxR R. E., TISHKOFF G. H. & WmPPIm G. H. (1949) Use of radioactive lysine in studies of protein metabolism. Synthesis and utilization of plasma proteins..7, exp. Med. 90, 297-313. POST T. B. (1963) Plasma protein-bound iodine and growth rates of beef cattle. Aust. J. agrie. Res. 14, 572-579. SCHOEI~HEIMERR. (1942) The Dynamic State of the Body Constituents. Harvard University Press, Cambridge, Mass. SPmNCELL P. H. (1966) Sulphur requirements for cattle hair growth. Proc. Aust. Soc. Animal Prod. 6, 399--402. SPRIIq~ELL P. H. (1968a) Red cell volume and blood volume in beef cattle. Aust..~. agric. Res. 19, 145-160. SPRINOELL P. H. (1968b) Water content and water turnover in beef cattle. Aust. jY. agric. Res. 19, 129-144. TURNER H. G. (1964) Coat characteristics of cattle in relation to adaptation. Proc. Aust. Soc. Animal Prod. 5, 181-187. VERCOE J. E. (1966) Some aspects of nitrogen metabolism of British and Zebu-type cattle. Proc. Aust. Soe. Animal Prod. 6, 370-377. VERCOB J. E. (1967) Breed and nutritional effects on the composition of faeces, urine and plasma from Hereford and Brahman x Hereford steers fed high and low quality diets. Aust. J. agric. Res. 8, 1003-1013.
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