PSXIV-17 - Examining mammary gland transcriptomic profiles using RNA-seq in slick and wild-type haired Holstein cattle under tropical conditions

Abstract: In lactating dairy cows, heat stress reduces feed intake, alters the animal’s metabolism, and compromises milk production. Holstein cattle carrying the prolactin receptor gene mutation (SLICK) have been shown to have superior performance under tropical conditions by exhibiting increased milk yield compared with wild-type haired Holstein cattle (WT). Recent research indicated that SLICK cattle have greater mammary gland blood flow and lower vaginal temperatures, while maintaining prolonged voluntary exposure to direct solar radiation than their WT counterparts. Nevertheless, differences in molecular mechanisms between genotypes at the mammary gland level have been unexplored. The objective of the current study was to investigate possible differences in the mammary gland transcriptome from SLICK (n = 7) and WT (n = 6) Puerto Rican Holstein cattle. At 160±3 DIM, ultrasound guided mammary biopsies were collected from the left rear quarter of each animal and samples were snap frozen. Unstranded RNA-seq libraries were created from the frozen mammary tissue and sequenced using Illumina Novaseq 2x150bp. Quantification of expression was done by salmon [v1.9.0] using the Bos taurus (ARS UCD1.3) transcriptome database. Testing for significant differential expression (DE) was done using a pipeline consisting of the R packages tximport [v1.26.1] and edgeR [v3.40.2]. The fry method from edgeR was used to do gene set enrichment analysis (GSEA) for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms. A single gene (GeneID:513329, major allergen Equ c 1 isoform X2) showed significant (B.H. adjusted P-value ≤ 0.05) differential expression. Aligning the protein sequence for this gene using the BLASTp against the non-redundant (NR) database revealed high similarity to two pheromones, salivary lipocalin 2 and salivary lipocalin-like. Both the KEGG pathway and GO term GSEA showed no statistically significant enrichment at B.H. adjusted P-value ≤ 0.05; however, using a less stringent criteria with a normal P-value ≤ 0.05, multiple relevant pathways were identified. For example, the KEGG pathway analysis revealed that the arachidonic acid metabolism pathway (bta00590) was enriched, and the tight junction function pathway (bta4530) was depleted in the SLICK Holstein samples. Among the GO analysis, genes were found to be downregulated in response to cold (GO:0009409), hair follicle development (GO:0001942), and vasoconstriction (GO:0042310), whereas positive regulation of oxytocin production (GO:0140668), and defense response to Gram-positive bacterium (GO:0050830) were upregulated. In conclusion, differences in gene expression were limited, but after enrichment interesting pathways were revealed and deserve further exploration to aid in elucidating different molecular mechanisms between SLICK and WT cattle.