Sexually dimorphic changes in liver and muscle transcriptome of 4-month-old dairy calves derived from artificial insemination or the transfer of a frozen-thawed in vitro produced embryo

More than 1.2 million in vitro produced (IVP) bovine embryos are transferred annually worldwide, significantly outnumbering those from traditional superovulation, but there are limited reports on the birth and postnatal development of such calves. The conditions imposed during in vitro embryo culture may cause modifications of the embryo genome, including the transcriptome, potentially affecting postnatal organ function. This study compared the liver and muscle transcriptome in 4-month-old male and female dairy calves conceived by artificial insemination (AI) or IVP (n = 4 per sex per treatment). Biopsy tissue samples were collected and processed for RNA sequencing. Analysis of the RNAseq data revealed a distinct separation between the liver transcriptomes of female and male calves, regardless of method of production. Moreover, within the cohort of female calves, a strong separation between female calves derived from IVP vs AI was observed. Analysis of differentially expressed genes (DEGs) indicated downregulation of oxidative phosphorylation pathways and upregulation of pathways strongly enriching immune system processes, specifically Th17 cell differentiation and antigen processing and presentation. For the muscle transcriptome, the separation between male and female calves was less apparent compared with liver transcriptome, but there was still a clear separation between female calves derived from IVP vs AI, with downregulated genes enriching the p53 signalling pathway and upregulated genes enriching muscle structure development pathways. These results suggest that female embryos derived from IVP vs AI have developmental differences that manifest as differential transcriptomic profiles postnatally. In conclusion, these findings demonstrate that the IVP process induces significant alterations in the liver and muscle transcriptome of female postnatal calves. Further exploration is necessary to understand the potential implications for lifetime growth, health and production characteristics. Overall design: Heifers were randomly assigned to one of the two groups; those receiving timed AI with frozen thawed semen (AI, n=8) and those transferred a single frozen-thawed Grade 1 Day 7 or Day 8 in vitro produced embryo (IVP, n=8). Resulting calves were divided into two groups based on their origin, AI (n=8; 4 male, 4 female) or IVP (n=8; 4 male, 4 female). Calves in both groups were raised in similar conditions up to 4 months of age, at which point biopsies were taken from the liver and muscle using a percutaneous punch technique. RNA extraction, quality control, library preparation, and sequencing was performed on the liver and muscle tissue samples. The subsequent read count files were employed to determine the differentially expressed genes (DEG) with the edgeR package for the R software and the gene expression counts were normalized by library size with edgeR methods. The DEG between groups were defined as those with false discovery rate (FDR) <0.05. The EntrezID corresponding to the DEG, was interrogated for enriched Gene Ontology and KEGG pathways using ShinyGO V0.77.