Differential effects of short-term ß agonist and growth hormone treatments on expression of myosin heavy chain IIB and associated metabolic genes in sheep muscle.

Growth hormone (GH) and ß agonists increase muscle mass, but the mechanisms for this response are unclear and the magnitude of response is thought to vary with age of animal. To investigate the mechanisms driving the muscle response to these agents, we examined the effects of short-term (6 day) administration of GH or cimaterol (a ß2-adrenergic agonist, BA) on skeletal muscle phenotype in both young (day 60) and mature (day 120) lambs. Expression of myosin heavy chain (MyHC) isoforms were measured in Longissimus dorsi (LD), Semitendinosus (ST) and Supraspinatus (SS) muscles as markers of fibre type and metabolic enzyme activities were measured in LD. To investigate potential mechanisms regulating the changes in fibre type/metabolism, expression or activity of a number of signalling molecules were examined in LD. There were no effects of GH administration on MyHC isoform expression at either the mRNA or protein level in any of the muscles. However, BA treatment induced a proportional change in MyHC mRNA expression at both ages, with the %MyHCI and/or IIA mRNA being significantly decreased in all three muscles and %MyHCIIX/IIB mRNA significantly increased in the LD and ST. BA treatment induced de novo expression of MyHCIIB mRNA in LD, the fastest isoform not normally expressed in sheep LD, as well as increasing expression in the other two muscles. In the LD, the increased expression of the fastest MyHC isoforms (IIX and IIB) was associated with a decrease in isocitrate dehydrogenase activity, but no change in lactate dehydrogenase activity, indicating a reduced capacity for oxidative metabolism. In both young and mature lambs, changes in expression of metabolic regulatory factors were observed that might induce these changes in muscle metabolism/fibre type. In particular, BA treatment decreased PPAR-γ coactivator-1ß mRNA and increased receptor-interacting protein 140 mRNA. The results suggest that the two agents work via different mechanisms or over different timescales, with only BA inducing changes in muscle mass and transitions to a faster, less oxidative fibre type after a 6-day treatment.

Examination of myosin heavy chain isoform expression in ovine skeletal muscles.

The contractile and associated metabolic characteristics of muscles are determined by their myosin heavy chain (MHC) isoform expression. In large mammals, the level of MHCIIB expression, which is associated with fast glycolytic-type muscle fibers, has not been fully characterized. In this study, quantitative reverse transcription-PCR and SDS-PAGE methodologies were developed for the analyses of adult ovine MHC isoform expression and used to characterize MHC expression in 3 skeletal muscles [LM, semitendinosus, and supraspinatus) from 66-d-old lambs. Three MHC isoforms (MHCI, MHCIIA, and MHCIIX) were detected at both the protein and messenger RNA levels in all 3 muscles, with greater proportions of type II than type I MHC. The expression of MHCIIB could not be detected at the protein level in any of the muscles and was detectable (in semitendinosus muscle) only at the messenger RNA level by using semiquantitative reverse transcription-PCR, indicating that MHCIIX is the predominant fast glycolytic fiber type in the sheep muscles studied. The methodologies developed are suitable for studying fiber type transformations at the molecular level, as well as allowing analyses of very small samples, including biopsies, when histochemical analysis may not be possible.

Effect of maternal dietary restriction during pregnancy on lamb carcass characteristics and muscle fiber composition.

Two experiments were conducted to determine whether the decreased proportion of fast muscle fibers seen previously in 2-wk-old lambs from ewes that were dietary restricted from d 30 to 70 of gestation are still evident in older lambs and what the consequences may be in terms of growth rates and carcass composition. Throughout both experiments, ewes were fed on an individual basis according to the recommended dietary allowance throughout pregnancy relative to metabolic BW (BW(0.73)). Control groups were fed as above, and the treatment groups had their nutrient supply reduced to 50% of this recommended allowance from d 30 to 70 (Exp. 1) or d 30 to 85 (Exp. 2) of gestation, after which they were returned to the same level of nutrition as the control group. All twin lambs were kept with their dams, and at 2 wk were given access to creep. After weaning, lambs were individually housed and fed ad libitum to 24 or 17 wk of age for Exp. 1 and 2, respectively. Although not significant (P = 0.18), growth to 24 wk (Exp. 1) resulted in a small decrease in the protein content and therefore an increase in the fat:lean ratio in the carcass of lambs subjected to maternal dietary restriction. This was not apparent when animals were slaughtered at 17 wk (Exp. 2; P > 0.68). Few significant effects of maternal dietary restriction on the fiber type composition of muscles were observed. In Exp. 1 the number of fast fibers increased (P < 0.008) with no effect on slow fiber number in LM. In Exp. 2 an increase in the total number of fibers in male lambs and an increase in type II (A and B) fibers in female lambs were observed in the LM, and an increase in IIB fiber number was observed in semitendinosus (ST) muscle from male lambs. Prenatal maternal dietary restriction during the time of muscle differentiation demonstrated an increase in type IIB muscle fibers and increase in intramuscular fat; although significant, effects on subsequent carcass quality of lambs were relatively small. These data suggest that the lambs adapted to changes in muscle fiber composition previously observed at 2 wk. However, lambs in this study were well fed during postnatal growth. Whether offspring would still have been able to compensate if they had received poor nutrition postnatally and whether that failure to compensate would have influenced carcass composition remain to be determined.

Factors affecting birth weight in sheep: maternal environment.

Knowledge of factors affecting variation in birth weight is especially important given the relationship of birth weight to neonatal and adult health. The present study utilises two large contemporary datasets in sheep of differing breeds to explore factors that influence weight at term. For dataset one (Study 1; n=154 Blue-faced Leicester x Swaledale (Mule) and 87 Welsh Mountain ewes, 315 separate cases of birth weight), lamb birth weight as the outcome measure was related to maternal characteristics and individual energy intake of the ewe during specified periods of gestation, i.e. early (1-30 days; term ~147 days gestation), mid (31-80 days) or late (110-147 days) pregnancy. For dataset two (Study 2; n=856 Mule ewes and 5821 cases of birth weight), we investigated using multilevel modelling the influence of ewe weight, parity, barrenness, lamb sex, litter size, lamb mortality and year of birth on lamb birth weight. For a subset of these ewes (n=283), the effect of the ewes' own birth weight was also examined. Interactions between combinations of variables were selectively investigated. Litter size, as expected, had the single greatest influence on birth weight with other significant effects being year of birth, maternal birth weight, maternal nutrition, sex of the lamb, ewe barrenness and maternal body composition at mating. The results of the present study have practical implications not only for sheep husbandry but also for the increased knowledge of factors that significantly influence variation in birth weight; as birth weight itself has become a significant predictor of later health outcomes.

Effect of feeding rumen-protected conjugated linoleic acid on carcass characteristics and fatty acid composition of sheep tissues.

Two experiments were conducted to determine the effectiveness of a rumen-protected CLA (pCLA) supplement and the impact of feeding this pCLA on carcass characteristics and tissue fatty acid composition of lambs. In Exp. 1, a CLA-80 preparation (80% pure CLA; contained similar proportions of cis-9, trans-11, and trans-10, cis-12 CLA), protected against rumen degradation, was fed to sheep, and the proportion of CLA reaching the duodenum was determined. A 3 x 3 Latin square design was used with 3 diets (1.4 kg of concentrate-based control diet, the same control diet plus 22 g of CLA-80, or the same control diet plus 110 g of pCLA/d), 3 feeding periods, and 3 rumen and duodenally cannulated sheep (Mule x Charolais males, 10 mo of age, BW 55.3 +/- 1.8 kg). After 7 d of feeding, sheep were ruminally infused with chromium EDTA and Yb acetate for 7 d, after which samples of duodenal digesta were collected every 6 h for 48 h to determine the quantity of CLA reaching the small intestine each day. The amounts of CLA cis-9, trans-11 and trans-10, cis-12, and combined isomers, flowing through the duodenum each day were greater (P = 0.01) in sheep fed pCLA. Approximately 65% of the pCLA avoided rumen biohydrogenation, with the ratio of the 2 main isomers remaining similar. In Exp. 2, 36 Mule x Charolais ewe lambs (approximately 13-wk old, average initial BW 29.3 kg) were fed 3 levels of the pCLA or Megalac, which were fed to provide an equivalent energy content at each pCLA level. Lambs were randomly assigned to 1 of 7 treatment groups, which were fed for 10 wk to achieve a growth rate of 180 g/d. Treatments included the basal diet and the basal diet plus 25, 50, or 100 g of pCLA/kg of diet or the equivalent amount of Megalac. In liver (P < 0.001) and all adipose tissue depots studied, the proportions of both CLA isomers increased (P = 0.02) with the amount of pCLA fed but were not altered with increasing of Megalac. Although there was no effect of treatment on cis-9, trans-11 CLA content, accumulation (P < 0.001) in the LM with increasing of pCLA supplementation was observed for the trans-10, cis-12 isomer. Although tissues had been enriched with CLA, there was no evidence of a reduction in adipose tissue or an increase in muscle mass in these sheep. However, an effect of pCLA on tissue fatty acid composition was consistent with an inhibition of stearoyl-CoA desaturase.

Effect of anabolic agents on calpastatin promoters in porcine skeletal muscle and their responsiveness to cyclic adenosine monophosphate- and calcium-related stimuli.

The calpain proteinases and their specific inhibitor calpastatin have been proposed to influence both the rates of myofibrillar protein turnover in vivo and meat tenderization postmortem. Elevated calpastatin concentrations in particular are associated with certain forms of hypertrophic growth and meat toughness. In the 5'region of the porcine calpastatin gene, there are 3 calpastatin promoters upstream of exons 1xa, 1xb, and 1u, respectively, each of which contain transcription factor-binding motifs, suggesting sensitivity to a variety of growth-promoting stimuli. This study examined the effect of the beta-adrenergic agonist clenbuterol and porcine ST (pST) treatment on calpastatin promoter usage in porcine LM in vivo using real-time PCR and also the responsiveness of transfected calpastatin promoter sequences to cyclic adenosine monophosphate (cAMP) and calcium (Ca2+)-related stimuli in reporter gene systems in cell studies. The effect of clenbuterol and pST on potential signaling pathways in vivo was also assessed by monitoring protein phosphatase 2B (calcineurin), NFATc3, calpain 3, IkappaB alpha, and NFkappaB by quantitative immunoblotting. Total calpastatin mRNA was increased by 52% (P < 0.05) after treatment with clenbuterol for 1 d and reduced by 35% (P < 0.01) after pST treatment for 7 d. Whereas clenbuterol had no significant differential effects on individual mRNA transcripts (types 1 to 3) derived from the 3 upstream promoters, pST significantly reduced all of these by 51, 39, and 40% (P < 0.001, 0.05, and 0.05), respectively. Promoter activity was increased in rat L6G8 cells transfected with a construct derived from exon 1u after treatment with dibutyryl cAMP (68%, P < 0.05) or forskolin (43%, P < 0.05), whereas 1xa activity was reduced by both of these agents (47 and 33%, respectively, P < 0.05). Treatment of cells with the calcium ionophore calcimycin reduced the activity of the 1u promoter by 40% (P < 0.01), with no effect on the other promoter constructs. Cyclosporin A had no effect on any promoter construct. The only signaling pathway component to be significantly altered by the in vivo treatments was calcineurin, which was decreased by 24% (P < 0.05) in clenbuterol-treated animals. In conclusion, 2 types of growth promoter in pigs had contrasting effects on calpastatin expression in LM. Transfected calpastatin promoters were differentially sensitive to cAMP- and Ca2+-related stimuli, in agreement with the proposed mode of action of the 2 growth promoters.

Fat and protein metabolism in growing steers fed either grass silage or dried grass.

Cattle fed grass silage diets have been reported to have high carcass fat:protein ratios. The effect of grass silage and dried grass diets, fed at different levels of intake to ensure a range of equivalent metabolisable energy intakes (MEI) from 1 .1 x metabolisable energy requirement for maintenance to ad libitum, on fat and protein metabolism in twenty-four Hereford x Friesian steers was investigated. After about 84 d of dietary treatment rates of whole-body fat and protein metabolism were measured, as were rates of lipogenesis in omental, perirenal and subcutaneous adipose tissue. Carcass composition was determined. Animals fed silage had greater (P<0 .001) carcass fat:protein ratios than animals fed dried grass at equivalent levels of MEI. Animals fed silage had lower (P<0 .001) rates of protein gain. Rates of leucine entry and oxidation were lower (P<0 .001) in animals fed silage, but there was no dietary difference in the rate of whole-body protein synthesis. There was no dietary difference in the rate of carcass fat gain, but rates of lipogenesis in perirenal adipose tissue were significantly (P=0 .007) higher in animals fed silage. There was no dietary difference in the rate of palmitate and glycerol entry or palmitate oxidation. There were no interactions between MEI and diet, indicating that increments of energy were utilised with the same efficiency from both diets. It was concluded that the high carcass fat:protein ratios of young growing steers was due to limited rates of protein accretion and not to elevated rates of carcass fat accretion.

Comparison of the relative expression of caspase isoforms in different porcine skeletal muscles.

The present study investigated the relationship between muscle type and components of the caspase protease system in porcine trapezius (TZ), psoas (PS), longissimus dorsi (LD) and semitendinosus (ST) muscles. Muscles were classified according to slow and fast myosin heavy chain (MHC) content determined by western blotting. MHC slow, but not MHC fast protein expression was significantly different between muscles (p<0.001). Protein levels of caspases 3, 8 and 12 and the caspase inhibitor apoptosis repressor with caspase recruitment domain (ARC) were determined. In addition the level of caspase 3 mRNA and activity levels of caspase 3/7 were determined. There was a significant difference in protein levels and activity between muscles (p<0.01), although no difference was observed in mRNA abundance. The data show that multiple components of the caspase system are expressed in porcine skeletal muscle and that their levels are variable, but there is not a distinct association of expression with a particular muscle.

The effect of maternal undernutrition before muscle differentiation on the muscle fiber development of the newborn lamb.

There is a need to improve the lean tissue content of ruminant animals destined for meat production. Muscle fiber number is set during fetal development. The effect of undernutrition of pregnant ewes on subsequent muscle fiber characteristics of their offspring was investigated. The trial involved 32 pregnant ewes carrying twins. The ewes were allocated randomly to one of four groups: three different treatment groups (n = 8) and a control group (n = 8). The diet of the treatment groups was dropped to 50% of their daily requirement to support the ewe and allow for conceptus growth for varying periods before being returned to 100% of their daily requirement until term. Group d 30-70 ewes were fed 100% of their daily requirement until d 30, the diet was then decreased to 50% until d 70; it was then returned to 100% of their daily requirement until term. Group d 55-95 ewes were similarly restricted from d 55 through 95, and Group d 85-115 ewes were restricted from d 85 through 115. The control group was fed 100% of their daily requirement to support the ewe and allow for conceptus growth throughout gestation. After parturition, the lactating ewes were fed a normal commercial diet. On d 14 (after parturition), the lambs were slaughtered and the LM, semitendinosus (ST), and vastus lateralis (VL) were dissected and snap frozen. The immunochemical determination of myosin heavy-chain slow (MHC-slow) and myosin heavy-chain fast (MHC-fast) proteins was measured by immunoprobing of Western blots. The number of fast and slow fibers and the diameter of these fibers also were measured in each muscle sample by histochemical techniques. Decreased maternal nutrition before fiber formation (d 30 through 70) was observed to change the muscle characteristics of the newborn lambs. These lambs had significantly fewer fast fibers (P < 0.001) and significantly more slow fibers (P < 0.001) in both the LM and VL compared with the other groups. Maternal nutrient restriction at the other periods had no effect on the number of muscle fibers in the newborn lambs; however, a decrease (LM, P < 0.05; VL, P < 0.01; ST, P = 0.08) in muscle weight was observed in the lambs born to the ewes restricted between d 85 and 115 of gestation compared with the other groups. This study has shown that decreased maternal diet before muscle fiber formation will alter the muscle fiber development in the fetus.

Ontogeny of factors associated with proliferation and differentiation of muscle in the ovine fetus.

The number of muscle fibers within a muscle has been found to be of high importance for the growth potential of an animal, and this number is set during fetal development. The objective of this study was to identify the ontogeny of muscle cell differentiation and fiber formation by observing the changes in expression of factors known to influence myoblast proliferation and differentiation. Twenty-one Swaledale x Leicester Blue Face ewes carrying twins were allotted to this trial. From d 40 of gestation, three ewes were killed every 15 d until term. At each time point, the fetuses were located, removed, and total muscle from both hind limbs was dissected from each fetus and snap frozen in liquid N2. Ribonuclease protection assays were used to quantify transcripts for IGF-I, IGF-II, GH receptor (GHR), and myostatin genes in the muscle samples, whereas quantitative real-time PCR was used to quantify myogenin transcripts. Histological sections also were taken from the fetal muscle samples and observed for evidence of muscle differentiation resulting in fiber formation. The abundance of mRNA for ovine IGF-II and ovine myogenin peaked at d 85 of gestation (P < 0.001). The abundance of ovine IGF-I transcripts peaked at d 100 of gestation, whereas the abundance of ovine GHR mRNA increased throughout gestation (P < 0.05). No change (P = 0.87) in the abundance of myostatin mRNA was observed. The histological sections from the muscle samples demonstrated a clear change in the appearance of the muscle tissue at each time period. Major fiber formation was observed around d 85. The results obtained from the analysis of gene expression and the histological sections suggest that the majority of muscle differentiation and fiber formation takes place around d 85, with myoblast proliferation mainly occurring before this time. It may be possible to manipulate the number of muscle fibers formed by targeting treatments during this proliferation stage immediately before the period of major fiber formation.

The relationship between slow and fast myosin heavy chain content, calpastatin and meat tenderness in different ovine skeletal muscles.

The present study investigated the relationship between fibre type distribution and slow (MHC-s) and fast (MHC-f) myosin heavy chain content on calpastatin and meat tenderness in longissimus dorsi (LD), tensor fasciae latae (TFL), semitendinosus (ST), trapezius (TZ) and supraspinatus (SS) muscles from six Mule×Charolais rams. Samples taken at slaughter were frozen either in liquid N(2) for analysis of MHC-s and MHC-f by immunoblotting, or in cooled isopentane for histochemical fibre typing. Calpastatin activity and an immunoreactive 135 kDa calpastatin band were analysed in samples taken 24 h postmortem. Shear force was determined on muscle chops taken at 24 h postmortem and conditioned until day 14. The intensity of MHC-s and MHC-f immunopositive bands correlated with %Type I and %Type II fibres identified histochemically (r(2)=0.612 and 0.366, respectively, p<0.001). Muscle specific differences were observed in MHC-s and MHC-f immunoreactivity, fibre type distribution, calpastatin activity, calpastatin 135 kDa immunoreactivity and shear force. MHC-s correlated positively with calpastatin activity (r(2)=0.725, p<0.001) and 135 kDa calpastatin (r(2)=0.228, p<0.01) across all muscle types. The data show that detection of MHC-s can be used to identify fibre type differences between ovine muscles and that this correlates with differences in calpastatin content and inhibitory activity, but not tenderness.

Differing effects of forage and concentrate diets on the oleic acid and conjugated linoleic acid content of sheep tissues: the role of stearoyl-CoA desaturase.

Feeding sheep concentrate-based diets increases the oleic acid content of their tissues, whereas the cis-9, trans-11 conjugated linoleic acid (CLA) content is increased by feeding forage diets. Both these metabolic transformations could be attributable to increased activity of stearoyl-CoA desaturase (SCD). Therefore, the effect of forage or concentrate feeding regimens on the fatty acid composition of sheep tissues were investigated to determine whether any changes are related to an alteration of SCD mRNA levels. Twenty-four ewe lambs were randomly allotted to one of three dietary treatment groups: 1) dehydrated grass pellets, 2) concentrate diet fed to achieve a growth rate similar to that of the dehydrated grass pellets, and 3) the same concentrate diet approaching ad libitum intake. As expected, animals fed ad libitum concentrates grew at a greater (P = 0.001) rate (280 g/d) than those fed either of the other two diets (180 g/d), which were similar. In samples of liver and the three adipose tissue depots studied, the concentration of oleic acid from sheep fed either level of the concentrate diet was greater (P < 0.001) than from animals fed forage. This was associated with an increase (P < 0.05) in the ratio of SCD to acetyl-CoA carboxylase mRNA in adipose tissue and liver. Compared with concentrate-fed, the forage-fed lambs had increased (P < 0.05) levels of the cis-9, trans-11 isomer of CLA and C18:1, trans-11 in all their tissues, although the levels of SCD mRNA were lower. It therefore seems that the increased oleic acid content of sheep tissues in response to concentrate-rich diets is associated with an increase in SCD gene expression. By contrast, the increased concentration of CLA in animals fed forage-based diets is associated with an increase in substrate (C18:1 trans-11) availability.

Insulin and dexamethasone regulate stearoyl-CoA desaturase mRNA levels and fatty acid synthesis in ovine adipose tissue explants.

Sheep adipose tissue explants were maintained in culture for 24 h in the presence of insulin, dexamethasone, or insulin and dexamethasone, and stearoyl-CoA desaturase (SCD) messenger RNA (mRNA) levels and fatty acid synthesis were measured. Insulin increased SCD mRNA levels (P = 0.008) and synthesis of both saturated (P = 0.07) and unsaturated (P < 0.001) fatty acids but had the greatest effect on unsaturated fatty acid synthesis, resulting in the overall production of a greater (P < 0.001) proportion of monounsaturated fat. Dexamethasone, alone, had the opposite effect but actually potentiated the effect of insulin in stimulating SCD expression and both saturated and monounsaturated fatty acid synthesis, without affecting the relative proportions of each. Across adipose tissue depots, the effect of hormones was similar, although the increase in SCD mRNA levels (P = 0.008) and monounsaturated fatty acid synthesis (P < 0.001) was greater in subcutaneous adipose tissue than in the internal (omental and perirenal) depots. These data clearly show that, in ovine adipose tissue, changes in SCD gene expression in response to insulin and dexamethasone are associated with changes in monounsaturated fatty acid synthesis and suggest that it may be possible to develop strategies to manipulate sheep tissues to produce a less-saturated fatty acid profile.

The effect of altered growth rates on the calpain proteolytic system and meat tenderness in cattle.

The present study was conducted to determine the effects of growth pattern on the calpain system and meat tenderization. Twenty-four Friesian calves were randomly allocated to three treatment groups: FAST (fast growth rate), SLOW (severely restricted growth rate) and ALTER (restricted growth for 30 days followed by fast growth rate). Four animals from each group were slaughtered on day 32 or 45 after altering the growth rates. Samples of M. longissimus dorsi were rapidly frozen at slaughter for protein analysis by Western blotting. Restricted growth reduced the immunoreactivity of a calpastatin band (135 kDa) measured at 24 h postmortem. Immunoreactivity associated with the large subunit of µ- or m-calpain appeared to be unaffected by growth patterns. Shear force measurements taken after 14 days of conditioning were positively related to 135 kDa calpastatin at 24 h postmortem. In this study there was no clear relationship between shear force and growth pattern.

Calpastatin expression in porcine cardiac and skeletal muscle and partial gene structure.

The expression in porcine skeletal and cardiac muscle of calpastatin, the specific endogenous inhibitor of the calpain proteolytic system, was examined 16 h after a single dose of a specific beta(2)-agonist. Immunoblotting of extracts indicated that treatment increased skeletal calpastatin 135-kDa band intensity (P < 0.01), while in cardiac combined 145- and 135-kDa band intensity decreased (P < 0.05). Treatment increased skeletal (P < 0.01) but not cardiac calpastatin mRNA steady-state levels. Three types of cardiac calpastatin mRNA transcripts were identified by 5'-RACE. Types I and II encoded a putative XL region that originated either from exon 1x(A) or exon 1x(B), arranged in tandem. Type III predominated in skeletal muscle and originated from exon 1u, which was located 40-50 kb 3' to exons 1x(A) and 1x(B). The region 5' to exon 1u may act as an independent promoter regulated by a cAMP-dependent mechanisms, thereby explaining the differential response of calpastatin to adrenergic stimulation in cardiac and skeletal muscle.

In vivo measurement of lipogenesis in ruminants using [1-(14)C]acetate.

A method for the measurement of the rate of lipogenesis in ruminants using a continuous intravenous infusion of [1-(14)C]acetate and measuring the rate of [1-(14)C]acetate incorporation into adipose tissue lipid was evaluated. Subcutaneous adipose tissue samples obtained by biopsy over the course of a 6 h continuous intravenous infusion of [1-(14)C]acetate into a wether and a steer maintained in a 'metabolic steady state' demonstrated that the incorporation of [1-(14)C]acetate into subcutaneous adipose tissue lipid was linear for the duration of the infusion period. Subsequent measures of rates of [1-(14)C]acetate incorporation into adipose tissue lipid were made on adipose tissue samples taken at a single time point during the infusion period. The technique was used to measure rates of lipogenesis in the subcutaneous adipose tissue of fourteen Hereford x Friesian steers that had been fed a pelleted diet of dried grass at a range of metabolizable energy (ME) intakes from 1.1 x ME requirement for maintenance to ad libitum for 11 weeks. Rates of lipogenesis increased linearly with increasing ME intake. It was concluded that the method is an effective technique for measuring rates of lipogenesis in specific adipose tissue depots in vivo in ruminants.

Effects of EGF receptor ligands on fetal ovine myoblasts.

Epidermal growth factor (EGF) receptors are widely distributed in mammalian tissues, including muscle. One ligand of these receptors, heparin-binding epidermal growth factor-like growth factor (HB-EGF) is also strongly expressed in adult muscle. However, in vitro studies of EGF action in cultured muscle cells of different species have yielded conflicting results. The purpose of this study was to investigate the potential role of EGF and related factors in the growth and development of fetal ovine muscle. High affinity EGF receptors were detected on clonally purified ovine fetal myoblasts, using [(125)I] human EGF as a ligand (K(d) values of 47 and 54 pM in separate experiments). Competitive binding studies in mixed secondary cultures showed that EGF had the highest affinity for the fetal ovine receptor, followed by HB-EGF and transforming growth factor alpha (TGF-alpha). These ligands all stimulated DNA synthesis in clonally purified ovine myoblasts, with their relative potencies at 0.1 nM reflecting their receptor binding affinities. Maximal effects were seen at 1-10 nM. EGF (10 nM) did not significantly inhibit the differentiation of clonally purified fetal ovine myoblasts, although there was increased proliferation of nondifferentiating cells. Hence a variety of EGF receptor ligands have the potential to influence the proliferation ovine muscle cell precursors in utero, but it is unlikely that they promote differentiation.

Maternal nutrition alters the expression of insulin-like growth factors in fetal sheep liver and skeletal muscle.

We investigated the influence of maternal dietary restriction between days 28 and 80 of gestation followed by re-feeding to the intake of well-fed ewes up to 140 days of gestation (term is 147 days) in sheep, on expression of mRNA for insulin-like growth factor (IGF)-I, IGF-II and growth hormone receptor (GHR) in fetal liver and skeletal muscle. Singleton bearing ewes either consumed 3.2-3.8 MJ/day of metabolisable energy (ME) (i.e. nutrient restricted - approximately 60% of ME requirements, taking into account requirements for both ewe maintenance and growth of the conceptus) or 8.7-9.9 MJ/day (i.e. well fed - approximately 150% of ME requirements) between days 28 and 80 of gestation. All ewes were then well fed (150% of ME requirements) up to day 140 of gestation and consumed 8-10.9 MJ/day. At days 80 and 140 of gestation, five ewes were sampled from each group and fetal tissues taken. There was no difference in fetal body weight or liver weights between groups at either sampling date, or skeletal muscle (quadriceps) weight at 140 days. IGF-I mRNA abundance was lower in livers of nutrient-restricted fetuses at day 80 of gestation (nutrient restricted 2.35; well fed 3.70 arbitrary units), but was higher than well-fed fetuses at day 140 of gestation, after 60 days of re-feeding (restricted/re-fed 4.27; well fed 2.83;s.e.d. 0.98 arbitrary units, P=0.061 for dietxage interaction). IGF-II mRNA abundance was consistently higher in livers of nutrient-restricted fetuses (80 days: nutrient restricted 7.78; well fed 5.91; 140 days: restricted/re-fed 7.23; well fed 6.01;s.e.d. 1.09 arbitrary units, P=0.061 for diet). Nutrient restriction had no effect on hepatic GHR mRNA abundance, but re-feeding of previously nutrient-restricted fetuses increased GHR mRNA compared with continuously well-fed fetuses (80 days: nutrient restricted 70.6; well fed 75.1; 140 days: restricted/re-fed 115.7; well fed 89.4;s.e.d. 10.13 arbitrary units, P=0.047 for dietxage interaction). In fetal skeletal muscle, IGF-I mRNA abundance was not influenced by maternal nutrition and decreased with gestation age (P<0.01). IGF-II mRNA abundance was higher in skeletal muscle of nutrient-restricted fetuses compared with well-fed fetuses at day 80 of gestation (nutrient restricted 16.72; well fed 10.53 arbitrary units), but was lower than well-fed fetuses after 60 days of re-feeding (restricted/re-fed 7.77; well fed 13.72;s.e.d. 1.98 arbitrary units, P<0.001 for dietxage interaction). There was no effect of maternal nutrition or gestation age on fetal skeletal muscle GHR expression. In conclusion, maternal nutrient restriction in early to mid gestation with re-feeding thereafter results in alterations in hepatic and skeletal muscle expression of IGF-I, IGF-II and/or GHR in the fetus which may subsequently relate to altered organ and tissue function.

Effects of gastric inhibitory polypeptide, somatostatin and epidermal growth factor on lipogenesis in ovine adipose explants.

Feeding raises the plasma concentrations of a number of gut-related hormones that may, in turn, influence the metabolism of peripheral tissues. This study investigated the effects of gut-related hormones on lipogenesis in explants from three differing adipose depots in lambs (aged 4-9 months). Incorporation of [14C]-acetate into lipid was measured over a 2-h period, following 24 h pre-incubation in the presence of hormone combinations. In perirenal fat explants, gastric inhibitory polypeptide (GIP) in the concentration range 0.01-10 nM stimulated lipogenesis. Maximal effects were seen at 1 nM (an average increase of 64% over basal values). In contrast, in the presence of insulin (0.1 nM), a dose-dependent decrease in lipogenesis was seen with increasing GIP concentration (P < 0.001 for the insulin x GIP interaction). Epidermal growth factor (EGF) and somatostatin in the same concentration range each inhibited lipogenesis. both in the presence and the absence of insulin (P < 0.001 in each case). Subcutaneous (back) fat and intermuscular (popliteal) fat responded similarly to each other, but significantly differently from the perirenal depot (P < 0.001). Here GIP, somatostatin or EGF (each at 1 nM) all separately stimulated lipogenesis.

Ovine adipose tissue monounsaturated fat content is correlated to depot-specific expression of the stearoyl-CoA desaturase gene.

The basis for the variation in fatty acid composition in different ovine adipose tissue depots was investigated. The proportion of stearic (C18:0) and oleic (C18:1) acids vary in a site-specific fashion; abdominal depots (omental and perirenal) contain relatively more C18:0 than C18:1, and carcass depots, especially sternum, have a markedly higher proportion of C18:1. Additionally, expression of a number of lipogenic enzyme genes (stearoyl-CoA desaturase [SCD], acetyl-CoA carboxylase-alpha [ACC-alpha], lipoprotein lipase [LPL]) and the cytoskeletal protein gene alpha-tubulin vary among depots, although the pattern of variation differs for each mRNA. When these expression data were related to the mean cell volume of adipocytes pooled from all depots, a significant pattern emerged: expression of the ACC-alpha, LPL, and alpha-tubulin genes was highly correlated with the size of adipocytes. In contrast, when the expression of SCD mRNA was assessed as a function of mean cell volume, two populations of adipocytes emerged: no significant correlation was found between the expression of SCD mRNA per adipocyte and mean cell volume for the abdominal depots, although a highly significant correlation was observed between SCD gene expression and mean cell volume for the carcass and epicardial depots. Similarly, a highly significant correlation was found for the amount of C18:1 per adipocyte and the abundance of SCD mRNA per adipocyte for the carcass and epicardial depots, whereas no significant correlation was observed for these traits for the omental and perirenal depots. Thus, the SCD gene seems to be regulated in a depot-specific fashion and in a manner distinct from that of the ACC and LPL genes.