GENE EXPRESSION STUDIES IN BOVINE SKELETAL MUSCLE: INVESTIGATIONS INTO THE EFFECTS OF HORMONE GROWTH PROMOTANT TREATMENT, ENVIRONMENT AND TENDERNESS GENOTYPE ASSOCIATED WITH TENDERNESS AND MARBLING

Tenderness (shear force) and intramuscular fat (marbling) are economically important production traits in AustraliaA¢â‚¬â„¢s beef industry. Improving the predictability of these traits could potentially overcome undesirable variation. Growth rate and efficiency are also important production traits and are increased through the use of steroid hormone growth promotant (HGP) treatment. Evidence suggests that HGPs have deleterious effects on tenderness and marbling. To improve tenderness, commercially available tenderness markers exist and are used to select cattle with favoured single nucleotide polymorphisms in the genes encoding Calpain 1, 3 and Calpastatin. The molecular mechanisms involved in the tenderness and marbling traits and interactions between them and the tenderness genotype, environment or HGP treatment are unclear. This thesis aimed to improve the understanding of molecular mechanisms and interactions involved in these traits by studying global gene expression changes in skeletal muscle of cattle. Microarrays were used to measure the gene expression changes in muscle biopsy samples (collected 2 weeks prior to slaughter) from 48 Brahman cattle in response to tenderness genotype (favoured (n=14), intermediate (n=16) and unfavoured (n=18)), HGP treatment (trenbolone-estradiol) (n=24) and an environmental contrast between two Australian sites (New South Wales, NSW (n=26) and Western Australia, WA (n=22)). Objective shear force measures and intramuscular fat levels for the tenderness and marbling traits were also collected from these cattle to enable correlation analysis to be carried out. We found that the expression levels of previously identified lipid storage genes (for example: PCK1, FABP4, ADIPOQ, AGPAT2, DGAT2, CIDEC and TUSC5) to be among the genes with the most consistent correlation with marbling in any of the contrasts investigated. This suggests that the storage of lipid, as opposed to lipid synthesis or inhibition of lipid breakdown in muscle is a better measure of marbling. The favoured tenderness genotype was associated with a decrease in expression of the lipid storage genes, an effect more pronounced in the NSW site. In addition, the cattle from the WA site had higher marbling levels compared to NSW cattle and also higher expression level of lipid storage genes, suggesting that a favourable situation for marbling exists in WA. We found that pyruvate dehydrogenase kinase 4 (PDK4) was the gene with the largest increase in expression in WA compared to NSW. The expression of this gene is known to increase in response to increased circulating long chain fatty acids (LCFA) which switches the muscle to using this as fuel source for maintenance and contraction. Since a large overlap was found between those genes with increased expression in the muscle of WA cattle and those genes with increased expression in the muscle of cattle going from grass to feedlot rations, we hypothesise that there may be a nutritional difference between the NSW and WA sites, which is having an effect on marbling. Shear force was found to be weakly, but consistently negatively correlated with the expression levels of genes enriched for those involved in the ubiquitin-protease system. The association of this system with muscle remodelling is well established; however its involvement in tenderness is not clear. There are hints of this association in previous studies where the expression levels of ubiquitin-proteasome related genes in the muscle of both cattle and sheep were correlated with shear force. We suggest that the activity of this system (two weeks prior to slaughter) may be a measure of tenderness. Chronic exposure of cattle to the HGP treatment was associated with an increased muscle growth rate, an increased expression of oxytocin (21-fold by array and 97-fold by qPCR), as well as an increased circulating level of oxytocin (50-fold). Oxytocin was recently shown to be an anabolic bone hormone in addition to reports illustrating its association with in vitro induction of myogenic cell differentiation and fusion. Therefore, we hypothesise that oxytocin is involved in the steroid mediated muscle growth response in cattle. In order to test the oxytocin hypothesis in a related ruminant species, analogous data from sheep treated with an equivalent HGP treatment was analysed. Similar to cattle, but to a lesser extent, the treatment was associated with increased muscle growth and an increase in oxytocin expression (4.4 fold by qPCR) was found. Control sheep unexpectedly had much higher circulating levels of oxytocin potentially highlighting an important species difference. In conclusion, this thesis has provided evidence that interactions may exist between HGP treatment, calpain-calpastatin genotype and site which could have effects on marbling. Furthermore, the genes identified that had expression levels showing similar patterns of changes in response to HGP treatment in both cattle and sheep may also be of future interest for understanding the mechanisms involved in HGP treatment. However, potentially the most industry relevant finding from this study may be that diet composition could be an area to explore as a means for reducing the negative effects of HGP treatment on marbling.