Paul L. Greenwood

Gene expression patterns during intramuscular fat development in cattle.

Deposition of intramuscular fat, or marbling, in beef cattle contributes significantly to meat quality variables, including juiciness, flavor, and tenderness. The accumulation of intramuscular fat is largely influenced by the genetic background of cattle, as well as their age and nutrition. To identify genes that can be used as early biomarkers for the prediction of marbling capacity, we studied the muscle transcriptome of 2 cattle crossbreeds with contrasting intramuscular fat content. The transcriptomes of marbling LM tissue of heifers from Wagyu x Hereford (WxH; n = 6) and Piedmontese x Hereford (PxH; n = 7) crosses were profiled by using a combination of complementary DNA microarray and quantitative reverse transcription-PCR. Five biopsies of LM were taken from each animal at approximately 3, 7, 12, 20, and 25 mo from birth. Tissue was also collected from the LM of each animal at slaughter (approximately 30 mo). Microarray experiments, conducted on the first 3 biopsies of 2 animals from each crossbreed, identified 97 differentially expressed genes. The gene expression results indicated that the LM transcriptome of animals with high marbling potential (WxH) could be reliably distinguished from less marbled animals (PxH) when the animals were as young as 7 mo of age. At this early age, one cannot reliably determine meaningful differences in intramuscular fat deposition. We observed greater expression of a set of adipogenesis- and lipogenesis-related genes in the LM of young WxH animals compared with their PxH contemporaries. In contrast, genes highly expressed in PxH animals were associated with mitochondrial oxidative activity. Further quantitative reverse transcription-PCR experiments revealed that the messenger RNA of 6 of the lipogenesis-related genes also peaked at the age of 20 to 25 mo in WxH animals. The messenger RNA expression of ADIPOQ, SCD, and THRSP was highly correlated with intramuscular fat content of an individual in WxH animals. Our study provides clear evidence of early molecular changes associated with marbling and also identifies specific time frames when intramuscular fat development in cattle muscle can be detected by using gene expression. This information could be used by animal scientists to design optimal nutrition for high marbling potential. In addition, the genes found to be highly expressed during development of marbling could be used to develop genetic markers or biomarkers to assist with beef production strategies.

Gene expression studies of developing bovine longissimus muscle from two different beef cattle breeds

BackgroundThe muscle fiber number and fiber composition of muscle is largely determined during prenatal development. In order to discover genes that are involved in determining adult muscle phenotypes, we studied the gene expression profile of developing fetal bovine longissimus muscle from animals with two different genetic backgrounds using a bovine cDNA microarray. Fetal longissimus muscle was sampled at 4 stages of myogenesis and muscle maturation: primary myogenesis (d 60), secondary myogenesis (d 135), as well as beginning (d 195) and final stages (birth) of functional differentiation of muscle fibers. All fetuses and newborns (total n = 24) were from Hereford dams and crossed with either Wagyu (high intramuscular fat) or Piedmontese (GDF8 mutant) sires, genotypes that vary markedly in muscle and compositional characteristics later in postnatal life.ResultsWe obtained expression profiles of three individuals for each time point and genotype to allow comparisons across time and between sire breeds. Quantitative reverse transcription-PCR analysis of RNA from developing longissimus muscle was able to validate the differential expression patterns observed for a selection of differentially expressed genes, with one exception. We detected large-scale changes in temporal gene expression between the four developmental stages in genes coding for extracellular matrix and for muscle fiber structural and metabolic proteins. FSTL1 and IGFBP5 were two genes implicated in growth and differentiation that showed developmentally regulated expression levels in fetal muscle. An abundantly expressed gene with no functional annotation was found to be developmentally regulated in the same manner as muscle structural proteins. We also observed differences in gene expression profiles between the two different sire breeds. Wagyu-sired calves showed higher expression of fatty acid binding protein 5 (FABP5) RNA at birth. The developing longissimus muscle of fetuses carrying the Piedmontese mutation shows an emphasis on glycolytic muscle biochemistry and a large-scale up-regulation of the translational machinery at birth. We also document evidence for timing differences in differentiation events between the two breeds.ConclusionTaken together, these findings provide a detailed description of molecular events accompanying skeletal muscle differentiation in the bovine, as well as gene expression differences that may underpin the phenotype differences between the two breeds. In addition, this study has highlighted a non-coding RNA, which is abundantly expressed and developmentally regulated in bovine fetal muscle.

Inferring the transcriptional landscape of bovine skeletal muscle by integrating co-expression networks

Background Despite modern technologies and novel computational approaches, decoding causal transcriptional regulation remains challenging. This is particularly true for less well studied organisms and when only gene expression data is available. In muscle a small number of well characterised transcription factors are proposed to regulate development. Therefore, muscle appears to be a tractable system for proposing new computational approaches. Methodology/Principal Findings Here we report a simple algorithm that asks “which transcriptional regulator has the highest average absolute co-expression correlation to the genes in a co-expression module?” It correctly infers a number of known causal regulators of fundamental biological processes, including cell cycle activity (E2F1), glycolysis (HLF), mitochondrial transcription (TFB2M), adipogenesis (PIAS1), neuronal development (TLX3), immune function (IRF1) and vasculogenesis (SOX17), within a skeletal muscle context. However, none of the canonical pro-myogenic transcription factors (MYOD1, MYOG, MYF5, MYF6 and MEF2C) were linked to muscle structural gene expression modules. Co-expression values were computed using developing bovine muscle from 60 days post conception (early foetal) to 30 months post natal (adulthood) for two breeds of cattle, in addition to a nutritional comparison with a third breed. A number of transcriptional landscapes were constructed and integrated into an always correlated landscape. One notable feature was a ‘metabolic axis’ formed from glycolysis genes at one end, nuclear-encoded mitochondrial protein genes at the other, and centrally tethered by mitochondrially-encoded mitochondrial protein genes. Conclusions/Significance The new module-to-regulator algorithm complements our recently described Regulatory Impact Factor analysis. Together with a simple examination of a co-expression modules contents, these three gene expression approaches are starting to illuminate the in vivo transcriptional regulation of skeletal muscle development.

Influences on fetal and placental weights during mid to late gestation in prolific ewes well nourished throughout pregnancy.

This study investigated associations between fetal and placental weights from 85 to 130 days gestation in 49 fetuses from 21 ewes of a prolific genotype used as an experimental model of intrauterine growth retardation. The proportion of variation in fetal weight explained by placental weight increased from zero at 85 days to 91% (residual standard deviation (RSD) = 260 g) at 130 days. Overall, stage of pregnancy plus placental weight accounted for 96% of fetal weight variation (RSD = 212 g). Litter size and number of fetuses per uterine horn also influenced individual fetal weights. Gestational age, litter size, placental weight per ewe, and liveweight and condition score of ewes during early to mid gestation (initial LW and CS) explained 99.5% of the variation in fetal weight per ewe (RSD = 236 g). Most variation (86%) in placental weight was explained by stage of pregnancy, litter size, number of placentomes, and initial LW and CS (RSD = 53 g). Placental weight per ewe was influenced by stage of pregnancy, litter size and initial ewe LW and CS (R2 = 0.97; RSD = 89 g). The association of fetal and placental weights with initial ewe LW was positive, and with initial CS was negative. The results show that in the absence of overt nutritional restriction of pregnant ewes, fetal and placental weights are tightly coupled during late gestation and ewe fatness during early pregnancy is inversely related to placental and fetal weights. They demonstrate that placental weight explains most of the variation in fetal weight in the present intrauterine growth retardation model.

Chronic exposure to anabolic steroids induces the muscle expression of oxytocin and a more than fiftyfold increase in circulating oxytocin in cattle

Molecular mechanisms in skeletal muscle associated with anabolic steroid treatment of cattle are unclear and we aimed to characterize transcriptional changes. Cattle were chronically exposed (68 ± 20 days) to a steroid hormone implant containing 200 mg trenbolone acetate and 20 mg estradiol (Revalor-H). Biopsy samples from 48 cattle (half treated) from longissimus dorsi (LD) muscle under local anesthesia were collected. Gene expression levels were profiled by microarray, covering 16,944 unique bovine genes: 121 genes were differentially expressed (DE) due to the implant (99.99% posterior probability of not being false positives). Among DE genes, a decrease in expression of a number of fat metabolism-associated genes, likely reflecting the lipid storage activity of intramuscular adipocytes, was observed. The expression of IGF1 and genes related to the extracellular matrix, slow twitch fibers, and cell cycle (including SOX8, a satellite cell marker) was increased in the treated muscle. Unexpectedly, a very large 21- (microarray) to 97 (real time quantitative PCR)-fold higher expression of the mRNA encoding the neuropeptide hormone oxytocin was observed in treated muscle. We also observed an ∼50-fold higher level of circulating oxytocin in the plasma of treated animals at the time of biopsy. Using a coexpression network strategy OXTR was identified as more likely than IGF1R to be a major mediator of the muscle response to Revalor-H. A re-investigation of in vivo cattle LD muscle samples during early to mid-fetal development identified a >128-fold increased expression of OXT, coincident with myofiber differentiation and fusion. We propose that oxytocin may be involved in mediating the anabolic effects of Revalor-H treatment.

Prediction of stage of pregnancy in prolific sheep using ultrasound measurement of fetal bones

The use of ultrasound to estimate stage of pregnancy was assessed in 32 ewes of a prolific genotype carrying 7 singleton fetuses and 9 twin, 10 triplet and 6 quadruplet litters that were scanned on six occasions from 60 to 120 days of gestation. At least one ultrasound measurement per ewe of fetal metacarpal bone length (MCL), biparietal diameter (BPD), or of both bones was made on over 90% of attempts (n = 152). Measurement of MCL was made on 78% of attempts (n = 371), of BPD on 73% of attempts, and of both bones on 62% of attempts. The equation developed from BPD (mean absolute error (MAE) = 3.2 days) was similar to that developed from measurement of MCL (MAE = 3.3 days) in its capacity to predict stage of pregnancy. Accuracy of prediction was improved using equations that included mean values within litters for BPD (MAE = 2.5 days) and MCL (MAE = 2.6 days). Further improvement in predictive capacity was achieved using multiple regression equations developed from measurement of both bones (individual fetuses: MAE = 2.6 days; equations including mean values within litters: MAE = 2.2 days). The results demonstrate that ultrasound can be used to estimate stage of pregnancy in prolific ewes, and that the use of mean values for bone measurements from different fetuses within litters and/or measurement of bones with different growth allometry can increase the reliability of estimates. The utility of the procedure depends on the number of fetuses measured per ewe, the number of bones measured per fetus and, hence, the time required to measure bones and the degree of accuracy required.

Inferring the in vivo cellular program of developing bovine skeletal muscle from expression data

We outline an in vivo cellular program of bovine longissimus muscle development inferred from expression data from 60 days post conception to 3months postnatal. Analytic challenges included changes in cellular composition, ambiguous diagnostic markers of cell type and contrasts between cattle human and mouse myogenesis. Nevertheless, the expression profiles of the myosin isoforms support slow and fast muscle fibres emanating from primary and secondary myogenesis respectively, while expression of the prenatal myosin subunits is down regulated prior to birth. Of the canonical pro-myogenic transcription factors (TF), MYF6 and MYF5 are negatively co-expressed, with MYF6 displaying higher expression in the post-natal samples and MYF5, MYOG, HES6 and PAX7 displaying higher expression in early development. A set of TFs (SIX1, EYA2 and DACH2) considered important in undifferentiated murine cells were equally abundant in differentiated bovine cells. An examination of mammalian regulators of fibre composition, muscle mass and muscle metabolism, underscored the roles of PPARGC1A, TGFβ signalling and the NHR4 Nuclear Hormone Receptors on bovine muscle development. Enriched among the most variably expressed genes from the entire data set were molecules regulating mitochondrial metabolism of carbohydrate (PDK4), fat (UCP3), protein (AGXT2L1) and high energy phosphate (CKMT2). The dramatic increase in the expression of these transcripts, which may enable the peri-natal transition to metabolic independence critical for new-born herbivores, provides surprising evidence for substantial developmental remodelling of muscle mitochondria and reflects changes in nutrient availability. Overall, despite differences in size, metabolism and physiology, the muscle structural subunit expression program appears very similar in ruminants, rodents and humans.

Current situation and future prospects for the Australian beef industry — A review

Beef production extends over almost half of Australia, with about 47,000 cattle producers that contribute about 20% (

Biological Mechanisms of Fetal Development Relating to Postnatal Growth, Efficiency and Carcass Characteristics in Ruminants

A12.7 billion gross value of production) of the total value of farm production in Australia. Australia is one of the world’s most efficient producers of cattle and was the world’s third largest beef exporter in 2016. The Australian beef industry had 25 million head of cattle in 2016–17, with a national beef breeding herd of 11.5 million head. Australian beef production includes pasture-based cow-calf systems, a backgrounding or grow-out period on pasture, and feedlot or pasture finishing. Feedlot finishing has assumed more importance in recent years to assure the eating quality of beef entering the relatively small Australian domestic market, and to enhance the supply of higher value beef for export markets. Maintenance of Australia’s preferred status as a quality assured supplier of high value beef produced under environmentally sustainable systems from ‘disease-free’ cattle is of highest importance. Stringent livestock and meat quality regulations and quality assurance systems, and productivity growth and efficiency across the supply chain to ensure price competiveness, are crucial for continued export market growth in the face of increasing competition. Major industry issues, that also represent research, development and adoption priorities and opportunities for the Australian beef industry have been captured within exhaustive strategic planning processes by the red meat and beef industries. At the broadest level, these issues include consumer and industry support, market growth and diversification, supply chain efficiency, productivity and profitability, environmental sustainability, and animal health and welfare. This review provides an overview of the Australian beef industry including current market trends and future prospects, and major issues and opportunities for the continued growth, development and profitability of the industry.

Nutrition-Gene Interactions (Post-Genomics)

Over recent years there has been a lot of interest in the effects of prenatal environment on subsequent development of tissues and the postnatal consequences. In farm animal species this has particularly related to muscle and fat development and the later consequences in terms of body composition at slaughter. Studies have been carried out in a variety of species, including rats, guinea pigs, pigs, sheep and, more recently, cattle. This chapter will concentrate on the evidence for effects of prenatal environment on development of muscle and adipose cells in ruminant species, the possible mechanisms for these effects and the long-term consequences relating to postnatal growth and body composition.