Effect of Maternal Nutrition and Sex on Skeletal Muscle Gene Expression in Angus Cattle during Immune Challenge

Maternal nutrition during gestation has long-term effects on skeletal muscle development of the offspring. The objective of this work was to identify differentially expressed (DE) genes and their biological functions as an effect of maternal energy restriction in Angus steers and heifers during immune challenge. Twenty-four purebred Angus pregnant cows were fed one of two diets: 70% (restricted; REST) or 100% (control, CTRL) of the net energy requirements for maintenance of a pregnant beef cow. Three weeks after weaning, and 3 days after vaccination for bovine viral diarrhea virus, Longissimus dorsi muscle biopsies were collected from 12 steers and 12 heifers and snap frozen in liquid nitrogen. Total RNA was extracted and sequenced for 150 bp paired-end reads. A total of 2,076,680,240 reads were generated. Reads were checked for quality with FASTQC software. Sequence reads were mapped to Bos taurus UMD3.1 reference genome using Bowtie2. For statistical analysis, raw counts were normalized by Trimmed Mean of M values (TMM) procedure and then log2-transformed. A linear model including the fixed effects of maternal diet, sex, diet-by-sex interaction, sequencing lane (2 lanes), and RIN score (as a covariate) was used. Statistical analyses were performed in R using the gls function from the nlme package. Multiple testing correction was conducted using the Benjamini & Hochberg false discovery rate (FDR) method. For the DE genes, an enrichment analysis was performed in DAVID Bioinformatics Resources 6.8. A total of 719, 658, and 1041 DE genes (p<0.10) were identified for the effects of diet, sex and diet-by-sex interaction, respectively. The most significant DE gene (p<0.0001) for each of these effects were: RSPH3, DDX3Y, and LRRFIP1, respectively. For diet, DE genes were enriched for protein synthesis and degradation activity. For sex, most DE genes were involved in the apoptotic process, indicating a potential difference in homeostatic potential during challenge. For sex-by-diet interaction, a greater range of biological processes was identified for DE genes, such as glycolysis and gluconeogenesis processes, myofibril assembly, actin filament elongation, and fatty acid metabolism. Differences in protein metabolism between steers and heifers are expected due to effects of sex hormones. Maternal energy restriction also can alter protein metabolism due to lack of nutrients for embryo muscle development. Therefore, these results suggest that the expression profiles of genes controlling protein turnover differ in the skeletal muscle of animals born from dams with or without energy restriction during pregnancy under immunological challenge.