The effects of exogenous xylanase supplementation on the in vivo generation of xylooligosaccharides and monosaccharides in broilers fed a wheat-based diet.

1. This study quantified xylanase-induced changes in soluble monosaccharides, xylooligosaccharides (XOS) and volatile fatty acid (VFA) contents of the different sections of the gastrointestinal tract (GIT) and whether these were related to altered bird performance. 2. An in vitro digestion of the wheat-based diet was carried out with the xylanase (Econase XT at 16,000BXU/kg diet) to compare the in vitro and in vivo generation of these XOS and monosaccharides. For the in vivo study, 80 male Ross 508 b roiler chicks were split into two groups fed a wheat-based diet with or without Econase XT (16,000BXU/kg diet) for 21 days. 3. There were no effects of Econase XT inclusion on growth performance characteristics, likely a result of the high-quality wheat diet, the corresponding high performance of the control group (FCR average of 1.45 in controls) and the relatively young age of the birds (from four to 26 days of age). 4. Econase XT supplementation increased the xylotetraose (X4) content in the colon (P = 0.046, enzyme x GIT section interaction) and the xylose contents in the colon and caeca (P < 0.001, enzyme x GIT section interaction). 5. The trend for increased acetate production in the caeca of Econase XT treated birds (P = 0.062) suggested that the XOS generated were subsequently fermented in the caeca, potentially impacting upon the types of microbiota present. 6. The present study suggested that wheat arabinoxylan degradation was enhanced by xylanase supplementation, which may have increased the production of beneficial volatile fatty acids (VFA) in the caeca, and thereby potentially modulated the caecal microbiome, but without affecting bird performance at this early age.

The association between faecal host DNA or faecal calprotectin and feed efficiency in pigs fed yeast-enriched protein concentrate.

Gut cell losses contribute to overall feed efficiency due to the energy requirement for cell replenishment. Intestinal epithelial cells are sloughed into the intestinal lumen as digesta passes through the gastrointestinal tract, where cells are degraded by endonucleases. This leads to fragmented DNA being present in faeces, which may be an indicator of gut cell loss. Therefore, measuring host faecal DNA content could have potential as a non-invasive marker of gut cell loss and result in a novel technique for the assessment of how different feed ingredients impact upon gut health. Faecal calprotectin (CALP) is a marker of intestinal inflammation. This was a pilot study designed to test a methodology for extracting and quantifying DNA from pig faeces, and to assess whether any differences in host faecal DNA and CALP could be detected. An additional aim was to determine whether any differences in the above measures were related to the pig performance response to dietary yeast-enriched protein concentrate (YPC). Newly weaned (∼26.5 days of age) Large White × Landrace × Pietrain piglets (8.37 kg ±1.10, n = 180) were assigned to one of four treatment groups (nine replicates of five pigs), differing in dietary YPC content: 0% (control), 2.5%, 5% and 7.5% (w/w). Pooled faecal samples were collected on days 14 and 28 of the 36-day trial. Deoxyribonucleic acid was extracted and quantitative PCR was used to assess DNA composition. Pig genomic DNA was detected using primers specific for the pig cytochrome b (CYTB) gene, and bacterial DNA was detected using universal 16S primers. A pig CALP ELISA was used to assess gut inflammation. Dietary YPC significantly reduced feed conversion ratio (FCR) from weaning to day 14 (P<0.001), but not from day 14 to day 28 (P = 0.220). Pig faecal CYTB DNA content was significantly (P = 0.008) reduced in YPC-treated pigs, with no effect of time, whereas total faecal bacterial DNA content was unaffected by diet or time (P>0.05). Faecal CALP levels were significantly higher at day 14 compared with day 28, but there was no effect of YPC inclusion and no relationship with FCR. In conclusion, YPC reduced faecal CYTB DNA content and this correlated positively with FCR, but was unrelated to gut inflammation, suggesting that it could be a non-invasive marker of gut cell loss. However, further validation experiments by an independent method are required to verify the origin of pig faecal CYTB DNA as being from sloughed intestinal epithelial cells.

Epigenetics and developmental programming of welfare and production traits in farm animals.

The concept that postnatal health and development can be influenced by events that occur in utero originated from epidemiological studies in humans supported by numerous mechanistic (including epigenetic) studies in a variety of model species. Referred to as the 'developmental origins of health and disease' or 'DOHaD' hypothesis, the primary focus of large-animal studies until quite recently had been biomedical. Attention has since turned towards traits of commercial importance in farm animals. Herein we review the evidence that prenatal risk factors, including suboptimal parental nutrition, gestational stress, exposure to environmental chemicals and advanced breeding technologies, can determine traits such as postnatal growth, feed efficiency, milk yield, carcass composition, animal welfare and reproductive potential. We consider the role of epigenetic and cytoplasmic mechanisms of inheritance, and discuss implications for livestock production and future research endeavours. We conclude that although the concept is proven for several traits, issues relating to effect size, and hence commercial importance, remain. Studies have also invariably been conducted under controlled experimental conditions, frequently assessing single risk factors, thereby limiting their translational value for livestock production. We propose concerted international research efforts that consider multiple, concurrent stressors to better represent effects of contemporary animal production systems.

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) and serine biosynthetic pathway genes are co-ordinately increased during anabolic agent-induced skeletal muscle growth.

We aimed to identify novel molecular mechanisms for muscle growth during administration of anabolic agents. Growing pigs (Duroc/(Landrace/Large-White)) were administered Ractopamine (a beta-adrenergic agonist; BA; 20 ppm in feed) or Reporcin (recombinant growth hormone; GH; 10 mg/48 hours injected) and compared to a control cohort (feed only; no injections) over a 27-day time course (1, 3, 7, 13 or 27-days). Longissimus Dorsi muscle gene expression was analyzed using Agilent porcine transcriptome microarrays and clusters of genes displaying similar expression profiles were identified using a modified maSigPro clustering algorithm. Anabolic agents increased carcass (p = 0.002) and muscle weights (Vastus Lateralis: p < 0.001; Semitendinosus: p = 0.075). Skeletal muscle mRNA expression of serine/one-carbon/glycine biosynthesis pathway genes (Phgdh, Psat1 and Psph) and the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase-M (Pck2/PEPCK-M), increased during treatment with BA, and to a lesser extent GH (p < 0.001, treatment x time interaction). Treatment with BA, but not GH, caused a 2-fold increase in phosphoglycerate dehydrogenase (PHGDH) protein expression at days 3 (p < 0.05) and 7 (p < 0.01), and a 2-fold increase in PEPCK-M protein expression at day 7 (p < 0.01). BA treated pigs exhibit a profound increase in expression of PHGDH and PEPCK-M in skeletal muscle, implicating a role for biosynthetic metabolic pathways in muscle growth.

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.

Increased responses to the actions of fibroblast growth factor 21 on energy balance and body weight in a seasonal model of adiposity.

The present study aimed to investigate the actions of fibroblast growth factor 21 (FGF21) on energy balance in a natural model of relative fatness, the Siberian hamster. Hamsters were studied under long days (LD) to promote weight gain, or short days to induce weight loss, and treated with rhFGF21 (3 mg/kg/day) via s.c. minipumps for 14 days. On days 7-9, detailed assessments of ingestive behaviour, metabolic gas exchange and locomotor activity were made. FGF21 caused substantial (P < 0.0001) weight loss in the fat LD state but not in the lean SD state: at the end of the study, FGF21-treated hamsters in LD lost 18% of body weight compared to vehicle controls, which is comparable to the natural body weight loss observed in SD. Epididymal fat pads, a correlate of total carcass fat content, were reduced by 19% in FGF21 treated hamsters in LD, whereas no difference was found in SD. Body weight loss in LD was associated with a reduction in food intake (P < 0.001) and a decreased respiratory exchange ratio (P < 0.001), indicating increased fat oxidation. Treatment with FGF21 maintained the normal nocturnal increase in oxygen consumption and carbon dioxide production into the early light phase in hamsters in LD, indicating increased energy expenditure, although locomotor activity was unaffected. These data suggest a greater efficacy of FGF21 in hamsters in LD compared to those in SD, which is consistent with both the peripheral and possibly central actions of FGF21 with respect to promoting a lean phenotype. The observed differences in FGF21 sensitivity may relate to day length-induced changes in adipose tissue mass.

Regional variation in angiotensin converting enzyme activity in the human placenta.

Sensitivity of chorionic plate arteries to angiotensin II (AngII) is greatest at the placental periphery. Angiotensin converting enzyme (ACE) is central to the synthesis of AngII and is present in the placental vasculature. We measured vascular ACE activity/mg protein at 8-9 sites between the cord insertion and the periphery in 12 term placentae from normotensive, vaginally-delivered women. ACE increased from insertion to the periphery (P = 0.015); median ACE for each placenta was positively correlated with placental weight (P < 0.05) and placental:birthweight ratio (P < 0.02). We speculate that this may be related to fetal programming since placental:birthweight ratio is related to long-term health.

Advances in research on the prenatal development of skeletal muscle in animals in relation to the quality of muscle-based food. I. Regulation of myogenesis and environmental impact.

Skeletal muscle development in vertebrates - also termed myogenesis - is a highly integrated process. Evidence to date indicates that the processes are very similar across mammals, poultry and fish, although the timings of the various steps differ considerably. Myogenesis is regulated by the myogenic regulatory factors and consists of two to three distinct phases when different fibre populations appear. The critical times when myogenesis is prone to hormonal or environmental influences depend largely on the developmental stage. One of the main mechanisms for both genetic and environmental effects on muscle fibre development is via the direct action of the growth hormone-insulin-like growth factor (GH-IGF) axis. In mammals and poultry, postnatal growth and function of muscles relate mainly to the hypertrophy of the fibres formed during myogenesis and to their fibre-type composition in terms of metabolic and contractile properties, whereas in fish hyperplasia still plays a major role. Candidate genes that are important in skeletal muscle development, for instance, encode for IGFs and IGF-binding proteins, myosin heavy chain isoforms, troponin T, myosin light chain and others have been identified. In mammals, nutritional supply in utero affects myogenesis and the GH-IGF axis may have an indirect action through the partitioning of nutrients towards the gravid uterus. Impaired myogenesis resulting in low skeletal myofibre numbers is considered one of the main reasons for negative long-term consequences of intrauterine growth retardation. Severe undernutrition in utero due to natural variation in litter or twin-bearing species or insufficient maternal nutrient supply may impair myogenesis and adversely affect carcass quality later in terms of reduced lean and increased fat deposition in the progeny. On the other hand, increases in maternal feed intake above standard requirement seem to have no beneficial effects on the growth of the progeny with myogenesis not or only slightly affected. Initial studies on low and high maternal protein feeding are published. Although there are only a few studies, first results also reveal an influence of nutrition on skeletal muscle development in fish and poultry. Finally, environmental temperature has been identified as a critical factor for growth and development of skeletal muscle in both fish and poultry.

Advances in research on the prenatal development of skeletal muscle in animals in relation to the quality of muscle-based food. II--Genetic factors related to animal performance and advances in methodology.

Selective breeding is an effective tool to improve livestock. Several selection experiments have been conducted to study direct selection responses as well as correlated responses in traits of skeletal muscle growth and function. Moreover, comparisons of domestic with wild-type species and of extreme breeds provide information on the genetic background of the skeletal muscle phenotype. Structural muscular components that differed with increasing distance in lean growth or meat quality in mammals were found to be myofibre number, myofibre size, proportions of fibre types as well as the numbers and proportions of secondary and primary fibres. Furthermore, markers of satellite cell proliferation, metabolic enzyme activities, glycogen and fat contents, the expression of myosin heavy chain isoforms, of activated AMPKα and other proteins in skeletal muscle tissue and circulating IGF1 and IGF-binding proteins have been identified to be involved in selection responses observed in pigs, cattle and/or chicken. The use of molecular methods for selective breeding of fish has only recently been adopted in aquaculture and studies of the genetic basis of growth and flesh quality traits are scarce. Some of the molecular markers of muscle structure/metabolism in livestock have also been identified in fish, but so far no studies have linked them with selection response. Genome scans have been applied to identify genomic regions exhibiting quantitative trait loci that control traits of interest, for example, muscle structure and meat quality in pigs and growth rate in chicken. As another approach, polymorphisms in candidate genes reveal the relationship between genetic variation and target traits. Thus, in large-scale studies with pigs' associations of polymorphisms in the HMGA2, CA3, EPOR, NME1 and TTN genes with traits of carcass and meat quality were detected. Other studies revealed the significance of mutations in the IGF2 and RYR1 genes for carcass lean and muscle fibre traits in pigs. Mutations in the myostatin (MSTN) gene in fish were also examined. Advances in research of the genetic and environmental control of traits related to meat quality and growth have been made by the application of holistic 'omics' techniques that studied the whole muscle-specific genome, transcriptome and proteome in relation to muscle and meat traits, the development of new methods for muscle fibre typing and the adaptation of biophysical measures to develop parameters of muscle fibre traits as well as the application of in vitro studies. Finally, future research priorities in the field are defined.

Use of the Affymetrix Human GeneChip array and genomic DNA hybridisation probe selection to study ovine transcriptomes.

Affymetrix GeneChip microarrays are a powerful tool to study global gene expression profiles and have been used on many species. However, no sheep-specific Affymetrix GeneChip is currently available and the bovine array is fairly limited in its coverage and annotation. Previously, a probe-selection method based on hybridisation of genomic DNA (gDNA) was developed, which enables GeneChips to be used for species that they were not designed for. This approach can greatly increase the number of potential annotated genes that can be studied beyond that which is currently available, particularly for species that do not have comprehensively characterised genomes. In this study, we demonstrate that gDNA-based probe selection on the Affymetrix Human U133+2 GeneChip array can be used to study gene expression profiles in sheep tissues. More than 20 000 transcripts were detected in triplicate ovine skeletal muscle and liver samples, which is more than would be possible using the commercially available sheep-specific microarray. The majority of the top 15 differentially expressed genes for each tissue were known to either be expressed in a tissue-specific manner or relate to specific functions of that tissue. Gene ontology analysis of the differentially expressed genes revealed the expected differences in gene expression profiles between the two tissues. Besides demonstrating that the probe selection method can be used to study the ovine transcriptome, the benefits of this approach are that it can greatly increase the number of annotated and novel genes that can be studied beyond those currently possible using ovine- or bovine-specific microarrays. This same method also has the potential to allow the study of other species where species-specific microarrays are not available or whose genomes have not been comprehensively characterised.

Regulation of ovine and porcine stearoyl coenzyme A desaturase gene promoters by fatty acids and sterols.

Stearoyl CoA desaturase (SCD) is responsible for converting SFA into MUFA and plays an important role in regulating the fatty acid composition of tissues. Although the number of SCD isoforms differs among species, SCD-1 is the predominant isoform expressed in the major lipogenic tissues of all species studied. The SCD-1 gene promoter region has been cloned for several species, including the human, mouse, pig, and recently, the cow. In this study, we cloned and partially characterized the ovine SCD promoter region. Sequence alignment showed a high degree of similarity with published bovine (94%) and porcine (92%) sequences. This included a highly conserved PUFA response region, which was also similar to that found in the human SCD and mouse SCD-1 promoters. Previous studies have indicated that there may be species differences in the regulation of SCD promoter activity by fatty acids. Using promoter-reporter gene (luciferase) constructs transfected into both HEK 293 and McA-RH7777 cells (kidney- and liver-derived cell lines, respectively), we showed the activity of the SCD promoter from 4 different species (mouse, human, pig, and sheep) to be reduced in a dose-dependent manner by addition of unsaturated fatty acids to the media, with linoleic acid being more potent than oleic acid after a 24-h treatment at 60 microM. This effect was dependent on the presence of the PUFA response region. In each of the species studied, the PUFA response region of the SCD promoter was shown to have an active sterol response element, which responded to treatment of cells with sterol or overexpression of the truncated active form of sterol regulatory element binding protein-1c. Thus, any species differences in previously reported regulation of SCD expression by fatty acids are not due to differences in promoter structure between species, but are more likely to depend on the cell type being studied or the relative concentrations and distribution of sterol regulatory element binding proteins or other transcription factors.

Short-days induce weight loss in Siberian hamsters despite overexpression of the agouti-related peptide gene.

Many vertebrates express profound annual cycles of body fattening, although it is not clear whether these represent differential activity of the central pathways known to mediate homeostatic control of food intake and energy expenditure, or whether the recent discovery of a major role for pars tuberalis-ependymal signalling points towards novel mechanisms. We examined this in the Siberian hamster (Phodopus sungorus) by using gene transfection to up-regulate a major orexigenic peptide, agouti-related peptide (AgRP), and then determined whether this increased anabolic drive could prevent the short-day induced winter catabolic state. Infusions of a recombinant adeno-associated virus encoding an AgRP construct into the hypothalamus of hamsters in the long-day obese phase of their seasonal cycle produced a 20% gain in body weight over 6 weeks compared to hamsters receiving a control reporter construct, reflecting a significant increase in food intake and a significant decrease in energy expenditure. However, all hamsters showed a significant, prolonged decrease in body weight when exposed to short photoperiods, despite the hamsters expressing the AgRP construct maintaining a higher food intake and lower energy expenditure relative to the control hamsters. Visualisation of the green fluorescent protein reporter and analysis of AgRP-immunoreactivity confirmed widespread expression of the construct in the hypothalamus, which was maintained for the 21-week duration of the study. In conclusion, the over-expression of AgRP in the hypothalamus produced a profoundly obese state but did not block the seasonal catabolic response, suggesting a separation of rheostatic mechanisms in seasonality from those maintaining homeostasis of energy metabolism.

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.

Thyrotrophin-releasing hormone decreases feeding and increases body temperature, activity and oxygen consumption in Siberian hamsters.

Thyrotrophin-releasing hormone (TRH) is known to play an important role in the control of food intake and energy metabolism in addition to its actions on the pituitary-thyroid axis. We have previously shown that central administration of TRH decreases food intake in Siberian hamsters. This species is being increasingly used as a physiological rodent model in which to understand hypothalamic control of long-term changes in energy balance because it accumulates fat reserves in long summer photoperiods, and decreases food intake and body weight when exposed to short winter photoperiods. The objectives of our study in Siberian hamsters were: (i) to investigate whether peripheral administration of TRH would mimic the effects of central administration of TRH on food intake and whether these effects would differ dependent upon the ambient photoperiod; (ii) to determine whether TRH would have an effect on energy expenditure; and (iii) to investigate the potential sites of action of TRH. Both peripheral (5-50 mg/kg body weight; i.p.) and central (0.5 microg/ml; i.c.v.) administration of TRH decreased food intake, and increased locomotor activity, body temperature and oxygen consumption in the Siberian hamster, with a rapid onset and short duration of action. Systemic treatment with TRH was equally effective in suppressing feeding regardless of ambient photoperiod. The acute effects of TRH are likely to be centrally mediated and independent of its role in the control of the production of thyroid hormones. We conclude that TRH functions to promote a catabolic energetic state by co-ordinating acute central and chronic peripheral (thyroid-mediated) function.

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.

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.

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.

Glucose and amino acids interact with hormones to control expression of insulin-like growth factor-I and growth hormone receptor mRNA in cultured pig hepatocytes.

Nutrients and hormones are major determinants of animal growth, but the mechanisms of how nutrients influence the growth process are still unclear. A primary pig hepatocyte culture system was used to investigate possible direct effects of glucose and individual amino acids on the expression of growth hormone receptor (GHR) and insulin-like growth factor-I (IGF-I) mRNA. The removal of glucose from the culture medium for 40 h resulted in significant reductions (to 45% of control, P = 0.013) in the expression of GHR in the presence of growth hormone (GH), dexamethasone (DEX) and tri-iodothyronine (T3). The decrease in GHR expression with removal of glucose from the culture medium resulted in a decreased response in class 1 (22% of control, P = 0.011) and 2 (5% of control P = 0. 068) transcripts of IGF-I to any GH added. The effects of glucose on GHR and IGF-I expression were dose-dependent, appearing to plateau at approximately 1-2 g/L (P = 0.031, for quadratic trend). Removal of arginine, proline, threonine, tryptophan or valine inhibited the stimulation of IGF-I expression that was induced by the combination of T3, DEX and GH (to 15, 6, 11, 16 and 16% of control, respectively, P < 0.05), with significant decreases in GHR expression also observed in some cases. The stimulatory effect of some of these amino acids (arginine, proline, threonine and tryptophan) was dose-dependent for expression of class 1 transcripts of IGF-I (P = 0. 041, 0.022, 0.016 and 0.097, respectively, for linear trends), but there was no effect on GHR or class 2 transcripts of IGF-I. Whether the observed effects of nutrients on mRNA levels are due to direct effects on gene transcription or differences in mRNA stability remains to be established. Energy, in the form of glucose, appears to control GHR expression, interacting with the effects of glucocorticoids and thyroid hormones, whereas protein, in the form of certain individual amino acids, appears to control GH-stimulated IGF-I expression.