Perspectives on ruminant nutrition and metabolism. II. Metabolism in ruminant tissues.

The discovery of the dominance of short-chain fatty acids as energy sources in the 1940s and 1950s, as discussed in part I of this review (Annison & Bryden, 1998) led to uncertainties concerning the interrelationships of glucose and acetate in ruminant metabolism. These were resolved in the following decade largely by use of 14C-labelled substrates. Although only small amounts of glucose are absorbed in most dietary situations, glucose availability to ruminant tissues as measured by isotope dilution was shown to be substantial, indicating that gluconeogenesis is a major metabolic activity in both fed and fasted states. Studies with 14C-labelled glucose and acetate revealed that in contrast to non-ruminants, acetate and not glucose is the major precursor of long-chain fatty acids in ruminant tissues. Interest in the measurement of energy metabolism in livestock grew rapidly from the 1950s. Most laboratories adopted indirect calorimetry and precise measurements of the energy expenditure of ruminants contributed to the development of new feeding systems. More recently, alternative approaches to the measurement of energy expenditure have included the use of NMR spectroscopy, isotope dilution and the application of the Fick principle to measure O2 consumption in the whole animal and in defined tissues. The refinement of the classical arterio-venous difference procedure in the study of mammary gland metabolism in the 1960s, particularly when combined with isotope dilution, encouraged the use of these methods to generate quantitative data on the metabolism of a range of defined tissues. The recent introduction of new methods for the continuous monitoring of both blood flow and blood O2 content has greatly increased the precision and scope of arterio-venous difference measurements. The impact of data produced by these and other quantitative procedures on current knowledge of the metabolism of glucose, short-chain fatty acids and lipids, and on N metabolism, is outlined. The role of the portal-drained viscera and liver in N metabolism is discussed in relation to data obtained by the use of multi-catheterized animals. Protein turnover, and the impact of stress (physical, social and disease related) on protein metabolism have been reviewed. The growth of knowledge of mammary gland metabolism and milk synthesis since the first quantitative studies in the 1960s has been charted. Recent findings on the regulation of amino acid uptake and utilization by the mammary gland, and on the control of milk secretion, are of particular interest and importance.

Perspectives on ruminant nutrition and metabolism I. Metabolism in the rumen.

Advances in knowledge of ruminant nutrition and metabolism during the second half of the twentieth century have been reviewed. Part I is concerned with metabolism in the rumen: Part II discusses utilization of nutrients absorbed from the rumen and lower tract to support growth and reproduction. The time frame was prompted by the crucial advances in ruminant physiology which arose from the work of Sir Jospeh Barcroft and his colleagues at Cambridge in the 1940s and 50s, and by the brilliant studies of Robert Hungate on rumen microbiology at much the same time. In reviewing the growth of knowledge of the role of bacteria, protozoa, fungi and bacteriophages in the rumen, outstanding developments have included the identification and characterization of fungi and the recognition that the utilization of polysaccharides in the rumen is accomplished by the sequential activities of consortia of rumen microorganisms. The role of protozoa is discussed in relation to the long standing debate on whether or not the removal of protozoa (defaunation) improves the efficiency of ruminant production. In relation to nitrogen (N) metabolism, the predation of bacteria by protozoa increases protein turnover in the rumen and reduces the efficiency of microbial protein production. This may account for the beneficial effects of defaunation where dietary N intakes are low and possibly rate limiting for growth and production. Current approaches to the measurement of rates of production of short chain fatty acids (SCFA) in the rumen based on the mathematical modelling of isotope dilution data are outlined. The absorption of SCFA from the rumen and hindgut is primarily a passive permeation process. The role of microorganisms in N metabolism in the rumen has been discussed in relation to ammonia and urea interrelationships and to current inadequacies in the measurement of both protein degradation in the rumen and microbial protein synthesis. The growth of knowledge of digestion and absorption of dietary lipids has been reviewed with emphasis on the antimicrobial activity of lipids and the biohydrogenation of unsaturated fatty acids. The protection of unsaturated dietary fats from ruminal biohydrogenation is an approach to the manipulation of the fatty acid composition of meat and dairy products. Discussion of the production of toxins in the rumen and the role of microorganisms in detoxification has focused on the metabolism of oxalate, nitrate, mycotoxins, saponins and the amino acid mimosine. Mimosine occurs in the tropical shrub leucaena, which is toxic to cattle in Australia but not in Hawaii. Tolerance to leucaena stems from the presence of a bacterium found in the rumen of Hawaiian cattle, which when transferred to Australian cattle survives and confers protection from mimosine. The genetic modification of rumen microorganisms to improve their capacity to ultilize nutrients or to detoxify antinutritive factors is an attractive strategy which has been pursued with outstanding success in the case of fluoroacetate. A common rumen bacterium has been genetically modified to express the enzyme fluoroacetate dehalogenase. The modified organism has been shown to survive in the rumen at metabolically significant levels and to confer substantial protection from fluoroacetate poisoning.

Use of guanidinated dietary protein to measure losses of endogenous amino acids in poultry.

Guanidinated proteins when fed to non-ruminants provide values for both endogenous amino acid losses and amino acid digestibilities, provided that the homoarginine residues in the treated protein are randomly distributed. Earlier studies have established that guanidination has only minor effects on the structure of the protein and, in particular, on its susceptibility to proteolysis. Furthermore, we have confirmed that homoarginine behaves as a typical amino acid in the small intestine. Lysine residues in casein and soya-bean protein, and in the proteins of cotton-seed meal, meat meal, soya-bean meal, maize, sorghum and wheat were converted to homoarginine by guanidination, the extent of conversion ranging from 37-68%. Sequential proteolysis in vitro of these guanidinated materials showed that the ratios of homoarginine to other amino acids remained unchanged for casein and soya-bean protein, indicating random distribution of homoarginine residues, but not for all the amino acids in meals and cereals. The use of guanidinated casein as the sole protein source in diets fed to broiler chickens allowed measurement of endogenous losses of amino acids under normal feeding conditions and calculation of true digestibilities of dietary amino acids at the ileum. Endogenous amino acid losses measured by the use of guanidinated casein (15.3 g/kg dry matter (DM) intake) were significantly higher (P < 0.001) than values obtained by feeding a N-free diet (5.4 g/kg DM intake), or by regression analysis to zero N intake (7.2 g/kg DM intake).

Measurement of endogenous amino acid losses in poultry.

1. Ileal endogenous amino acid losses were determined in broiler chickens and in cannulated cross-bred layer strain cockerels using either a nitrogen-free diet, regression analysis or a 48 h fast. 2. Endogenous amino acid flows to the ileum in fasted cockerels were significantly lower than those obtained both by feeding the nitrogen-free diet, and from regression analysis in either broilers or cockerels. Regression analysis gave the highest flows. 3. The apparent digestibility coefficients of amino acids in a diet containing 200 g/kg crude protein were lower in broilers (0.84) than in cockerels (0.88). When corrected, by regression analysis, for the contribution of endogenous amino acids, the true digestibility coefficients became 0.90 and 0.92 respectively.

Influence of nutritional status on growth hormone-dependent circulating somatomedin-C activity in mature sheep.

The effect of daily administration of ovine GH for a period of 4 weeks on somatomedin-C biological activity in plasma was investigated in mature Merino sheep fed a maintenance energy intake (low plane; LP) or 1.6 times this amount (high plane; HP). The GH treatment resulted in a significant (P less than 0.05) increase in plasma GH levels in blood samples collected 23.5 h after each daily injection in both LP and HP groups. Plasma concentrations of somatomedin-C activity and insulin were significantly stimulated to a maximum level by the third GH injection and remained at this level for 7 days. Subsequently, circulating levels of both hormones fell to 40-50% of the peak response to GH and returned to basal levels within 48 h of the cessation of GH injections. In the HP group the response of plasma insulin and somatomedin-C activity to GH injection was greater than in the LP group.

Effects of growth hormone administration on wool growth in merino sheep.

The effects of daily administration of 10 mg of highly purified ovine growth hormone (GH) for a period of 4 weeks on wool growth have been measured in 12 Merino ewes fed either a calculated maintenance energy intake or 1.6 times this amount (six on each ration). Concentrations of hormones, glucose, urea, alpha-amino N and amino acids in the blood were monitored and faeces and urine collected for measurement of nitrogen balance. Wool growth rate decreased by 20% during the 4 weeks of GH treatment in sheep fed the high energy diet, largely because of reduced wool fibre diameter. This was followed by restoration of normal growth and then an increase of up to 20% above control levels, a response which persisted for 12 weeks following cessation of GH administration, and which was due to increases in both fibre length and diameter. GH administration caused marked increases in plasma concentrations of GH, insulin and somatomedin C, glucose and free fatty acids, all of which returned to basal levels following cessation of GH administration. No consistent changes in plasma concentration of T3, T4, cortisol, prolactin or alpha amino N were detected. Plasma urea and methionine levels decreased during GH treatment and returned to, or were raised above, basal levels after the GH treatment period. GH injection also resulted in a net retention of N during treatment, followed by a transient period of net N loss. The GH-induced changes in wool growth may be caused by a change in the partitioning of amino acids between the muscle mass and the skin. No other contributing factor(s) were identified.

Development of a sheep hind-limb muscle preparation for metabolic studies.

A sheep hind-limb preparation used for the study of muscle metabolism by arteriovenous (AV) difference procedures was validated by identifying the muscles which contribute to venous drainage at different positions along the lateral saphenous vein. Dissection of the hind limbs of six mature sheep (three wethers and three ewes) showed that venous blood from the plantar group (M. gastrocnemius, M. soleus, M. plantaris, M. flexo digitorum profundus), and from M. semitendinosus, M. biceps femoris, M. gracilis, M. pectineus and M. adductor muscles entered the lateral saphenous vein but the position of the tip of the blood sampling catheter was found to be critical. In order to sample venous blood from all of the muscles listed above, and to minimize the contribution of blood from non-muscular tissues, blood samples must be taken 25-26 cm from the junction of the cranial and caudal branches of the lateral saphenous vein (for average size sheep of body length about 108 cm and height at withers about 73 cm). The estimation of sheep hind-limb muscle mass is a necessary concomitant of AV difference studies, and a combined tritiated water and dye-dilution procedure has been used to measure both muscle mass and blood flow. The muscle mass estimated in vivo by this technique was closely similar to the true muscle mass obtained by dissection, the range of values of the difference between true and calculated muscle mass expressed as percentage of the true mass being 0.5-16%. It is concluded that these techniques are sufficiently accurate for use in the quantitation of exchange of metabolites across the hind-limb muscle preparation. Patterns of amino acid uptake and release by muscle need to be related to the amino acid profile of the tissue, and the amino acid content of a representative muscle, M. biceps femoris, was determined, and the results compared with published data.

Metabolism of valine and the exchange of amino acids across the hind-limb muscles of fed and starved sheep.

A combination of the isotope-dilution and arterio-venous (AV) difference techniques was used to study simultaneously the metabolism of valine in the whole body and in the hind-limb muscles of fed and starved (40 h) sheep. The net exchange of gluconeogenic amino acids across hind-limb muscles was also studied. Valine entry rate was unaffected by nutritional status. There was significant extraction of valine by hind-limb muscles in both fed and starved sheep. The percentage of valine uptake decarboxylated was higher (P less than 0.05) in fed sheep but the amount of valine decarboxylated was not significantly different. The proportion of valine uptake that was transaminated was about 30 times higher in starved sheep. About 54% of valine taken up by hind-limb muscle of starved sheep was metabolized. The corresponding value for fed sheep was 21%. The contribution of CO2 from valine decarboxylation to total hind-limb muscle CO2 output was about 0.2%. The output of alanine in both fed and starved sheep was low but the output of glutamine was relatively high and roughly equivalent to the amounts of aspartate, glutamate and branched-chain amino acids that were catabolized. This study has confirmed that valine is catabolized in sheep skeletal muscle, and shown that glutamine is a major carrier of amino nitrogen out of muscle.

Gastrointestinal absorption of vitamin B-6 in the chicken (Gallus domesticus).

The absorption of vitamin B-6 from the gastrointestinal tract of the chicken (Gallus domesticus) was studied by using ligated segments in vivo, everted jejunal sacs in vitro, and in intact birds. [3H]Pyridoxine hydrochloride ([3H]PN . HCl) was absorbed from all sections of the small intestine, from the cecum and the crop, although absorption from the latter two segments was minimal. Absorption was independent of fasting and the vitamin B-6 concentration in the rearing diet. Concentration-dependence was demonstrated for absorption from ligated jejunal segments (24 microM-24 mM) and for 4-min uptake of [3H]PN . HCl by everted sacs (0.01-10,000 microM). Unidirectional flux of 2 microM PN . HCl was reduced by ouabain, iodoacetate and Na+-free media but not by 4-deoxy PN . HCl, D- and DL-penicillamine, anoxia or glucose-free media. Absorption of vitamin B-6 from the lumen to the intestinal epithelium of the chicken occurs by simple diffusion. Free, added vitamin B-6 was almost completely absorbed. In contrast, vitamin B-6 in food ingredients became available only as digestion proceeded and was in no case completely available. The vitamin B-6 concentrations in the contents of the cecum, lower ileum and rectum were similar. Incomplete absorption of dietary vitamin B-6 could explain the presence of the vitamin in the cecum. An enterohepatic route accounting for the recycling of approximately 1% of normal daily vitamin B-6 intake was identified, and the vitamin B-6 concentration in bile increased with vitamin B-6 in the diet. Pyridoxal, pyridoxamine (and their phosphate esters), pyridoxine, and 4-pyridoxic acid were measured in blood, but only the pyridoxal concentration in blood responded noticeably to increases in dietary vitamin B-6.

Acetate metabolism in the mammary gland of the lactating ewe.

Acetate metabolism in the mammary gland of lactating ewes was studied by continuous infusion of radioisotopic [U-14C]sodium acetate and measurement of mammary gland arteriovenous difference and blood flow. Entry rate of acetate into the whole body averaged 75 +/- 7 mumol min-1 kg-1 liveweight and 22.1 +/- 2.7% of total CO2 production was derived from acetate. Acetate was both utilized and produced by the mammary gland. Acetate uptake was related linearly (r2 = 0.94) to arterial concentration and gross utilization of acetate accounted for 16.2 +/- 2.6% of whole-body entry rate. Endogenous acetate production by the mammary gland increased linearly (r2 = 0.90) as milk yield rose, and accounted for 25.6 +/- 2.7% of the gross mammary utilization of acetate. The proportion of mammary CO2 derived from acetate (22.5 +/- 3.9%) was similar to that of the whole body. The uptake of acetate, 3-hydroxybutyrate, esterified fatty acids and plasma free fatty acids accounted for about 25, 13, 60 and 4% of milk fatty acid carbon respectively, after correction for the oxidation of acetate, but not of the other substrates. Metabolism of acetate in the mammary glands of lactating ewes appears quantitatively more important than that in cows, but similar to that in goats.

Partitioning of nutrients in merino ewes. II. Glucose utilization by skeletal muscle, the pregnant uterus and the lactating mammary gland in relation to whole body glucose utilization.

The net uptake and oxidation of glucose by leg muscle, pregnant uterus, and lactating mammary gland, together with the rate of irreversible loss and oxidation of glucose in the whole body of Merino ewes are reported. The ewes were fed on either chaffed oaten hay (OH), chaffed lucerne hay (L), or a mixture of chaffed oaten and lucerne hays (OHL). Measurements were made during five different physiological states: dry (nonpregnant), at 94 and 125 days of pregnancy, and at 20 and 50 days after lambing. Whole body glucose irreversible loss was related significantly to intake of metabolizable energy and fleece-free maternal body weight and this relation was the same in dry, pregnant and lactating ewes. The proportion of glucose oxidized in the whole body was unaffected by diet, but was lower in pregnant than in dry or lactating ewes. Some 6% of whole body carbon dioxide (CO2) production was derived from oxidation of glucose, and in ewes eating the OH diet this proportion was lower than for ewes fed on other diets. The proportion of CO2 derived from glucose was lower in pregnant ewes than in dry and lactating ewes. Leg (muscle) glucose uptake was lower in ewes fed on the OH diet than in ewes given the other diets. This arose partly because of decreased blood flow to the leg in ewes fed OH. Muscle glucose uptake, corrected for lactate output, accounted for 20, 44 and 34% of glucose irreversible loss in ewes fed OH, OHL and L respectively. There was no significant effect of physiological state on glucose uptake by leg muscle. The maximum contribution glucose uptake, corrected for output of lactate, could make to leg muscle oxygen consumption was 31% and there were no differences due to diet or physiological state. Uterine glucose uptake was 10.5 mg min-1 kg-1, and was unaffected by diet and stage of pregnancy. Glucose uptake was maintained, despite a decline in blood flow per kilogram of uterus from 399 to 237 ml min-1 kg-1, between 94 and 125 days of pregnancy by an increase in arteriovenous difference of glucose over the same period from 2.8 to 4.4 mg 100 ml-1. Total uptake of glucose by the uterus increased from 26 to 47 mg min-1 between 94 and 125 days of pregnancy. The proportion of glucose irreversible loss accounted for by uterine uptake increased from 46 to 65% between 94 and 125 days, and was greater for ewes fed OH (84%) than L (46%) at 125 days of pregnancy. A maximum of 71% of milk lactose could have been derived directly from glucose; 17% of glucose taken up by the mammary gland was oxidized, contributing to 20% of mammary CO2 output. Mammary glucose uptake was lower in ewes fed OH than in ewes fed the other diets.(ABSTRACT TRUNCATED AT 400 WORDS)

Partitioning of nutrients in Merino ewes. I. Contribution of skeletal muscle, the pregnant uterus and the lactating mammary gland to total energy expenditure.

The contribution of leg muscle, pregnant uterine tissue and lactating mammary gland to overall energy utilization was determined in Merino ewes. Ewes were offered one of three diets based on chaffed oaten hay (7.9 MJ metabolizable energy per kilogram dry matter); chaffed lucerne hay (8.6 MJ/kg); or a 50:50 (w/w) mixture of chaffed oaten and lucerne hays (8.2 MJ/kg). Measurements were made during five different physiological states: dry (non-pregnant), at 94 and 125 days after mating, and at 20 and 50 days after lambing. Tissue energy use was calculated from oxygen uptake and carbon dioxide output obtained from measurement of blood flow and arteriovenous difference. Whole-body energy use was calculated from carbon dioxide energy rate. Energy use by leg muscle was 144 +/- 8 (mean +/- s.e.) kJ kg-1 day-1, and unrelated to metabolizable energy intake, but leg energy use increased with ewe body weight. On the basis that leg muscle was representative of all muscle, total muscle energy use accounted for 26 +/- 4% of whole-body energy expenditure in dry ewes. Uterine energy use per unit weight was respectively 348 +/- 53 and 254 +/- 23 kJ kg-1 day-1 at 94 and 125 days after mating. Milk production was highly correlated with weight of secretory tissue, and with blood flow to the mammary gland. The ratio of blood flow to milk produced was 473:1 in ewes producing from 200 to 1000 ml of milk per day. The mammary gland used energy to produce milk with an efficiency of 0.90 +/- 0.01, a value close to the theoretical estimate of 0.89. On the basis that metabolic rate does not increase during lactation, the efficiency of use of metabolizable energy for milk production was 0.51 +/- 0.05. Examination of energy use by different tissues indicated that energy use by muscle was related to weight, but energy use by remaining tissues (whole body less muscle, uterus and mammary gland) was related to metabolizable energy intake. The results reveal an increase in energy use by the remaining tissue in lactating ewes (8500 +/- 569 kJ/day) compared with dry (5634 +/- 216 kJ/day) and pregnant ewes (5815 +/- 393 kJ/day).

Control of gluconeogenesis in the lactating sheep.

Ewes which had been lactating for 3--4 weeks and which had been milked by hand from the day of parturition were subjected to food restriction for 4 days. One group of three ewes was fed ad libitum and a second group of four ewes was fed to meet calculated requirements for maintenance and milk production. Over 4 days food intake was reduced by 80% in both groups of ewes. In response to food restriction, milk yields and body weight decreased. Blood amino acids, plasma glucose, glucose pool size, glucose irreversible loss, insulin, thyroxine and the insulin: glucagon molar ratio decreased. In contrast, plasma glucagon remained relatively unaffected and plasma free fatty acids and growth hormone increased. These changes were similar for both groups of ewes and were reversed when food intake was restored. The results suggest that the hormonal control of gluconeogenesis in the ruminant is similar to that in the non-ruminant.

Comparison of glucose biokinetics in parturient ewes and ewes induced to lactate artificially.

Glucose biokinetics of six normal pregnant/lactating ewes during the periparturient period were compared with those of five non-pregnant ewes induced to lactate artificially by treatment with oestrogen and progesterone, followed by a series of infusions of oxytocin. Normal ewes produced large amounts of milk (800-900 g/day) on day 1, and yields remained relatively constant until day 8 post partum. Milk production of induced ewes, however, was negligible on day 1 (30 g/day) but increased progressively until day 8 (540 g/day) after the start of milking. The glucose irreversible loss per minute 2-8 days before (6.4 v. 5.6 mg per kilogram body weight 0.75), and 2 days after the onset of lactation (9.1 v. 5.5 mg/min per kg0.75) was significantly greater (P less than 0.05) in normal pregnant/lactating ewes than in ewes induced to lactate artificially. By the eighth day of lactation rates of glucose irreversible loss per minute (7.4 v. 6.4 mg per kilogram body weight 0.75) were not significantly different (P greater than 0.05). The data were consistent with the hypothesis that glucose supply is rate limiting for milk production for several days after the initiation of lactation in non-pregnant, hormone-treated ewes. In parturient ewes, the rate of glucose irreversible loss was significantly increased 1-2 days post partum.

The influence of tungsten on the molybdenum status of poultry.

1. Large doses of tungsten, administered to the chick either by injection or by feeding, increased tissue concentrations of tungsten and decreased tissue concentrations of molybdenum and tissue activities of xanthine dehydrogenase. 2. The rate of loss of large doses of tungsten from the liver occurred in an exponential manner with a half-life of 27 h. 3. When tungsten was administered to chicks fed on a semi-synthetic diet containing abnormally low concentrations of molybdenum, the activity of hepatic xanthine dehydrogenase was reduced to negligible levels. 4. The alterations in molybdenum metabolism resulting from the administration of large doses of tungsten to the chick appears to be the result of tungsten toxicity and not of molybdenum deficiency. 5. Deaths from tungsten toxicity occurred when tissue concentrations of tungsten were increased to approximately 25 micrograms/g liver. At this tissue tungstencon centration the activity of xanthine dehydrogenase was zero.

Molybdenum requirements of chickens.

1. The requirements of all classes of chickens for molybdenum are 0.03 mg/kg diet or less. 2. Dietary sulphate and tungstate both reduce the availability of dietary molybdate. 3. No evidence was obtained that either the clubbed down, ginger hair syndrome in chicks or the scabby hip syndrome in broilers is due to a simple molybdenum deficiency.