Diet Impacts Preimplantation Histotroph Proteome in Beef Cattle

In ruminants, the period from oocyte fertilization to implantation is relatively prolonged, and survival of embryos depends on uterine secretions, or histotroph. Diet impacts conception rates following timed artificial insemination (AI) in beef cattle; we thus hypothesized diet affects histotroph. Our objective was to determine if diet fed prior to timed AI treatment impacted histotroph proteome in Angus-Simmental cattle. Cows were assigned to 1 of 4 isocaloric diets: silage-based total mixed ration (CON), high protein (PROT), high fat (OIL), or high protein and fat (PROT+OIL). After ~190d on diets, an intravaginal progesterone implant (CIDR) was inserted into cows for 7 days for timed AI treatment. At 9 days post CIDR removal, animals were selected (n = 16; 4/treatment) for presence of a corpus luteum using ultrasonography. Histotroph was collected by flushing 30cc of a sterile saline solution into the uterine horns using a Foley catheter. The uterus was massaged via palpation, and the saline flush was collected. Samples were frozen, freeze dried, and reconstituted for digestion with Trypsin/Lys-C protease. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was run using a Q Exactive HF. Proteins were identified by comparing against a bovine specific database (UniProt) using MaxQuant software, and differential expression of proteins was determined using analysis of variance (ANOVA) and Tukey adjustments (P-adj) run in ‘R’. DAVID Bioinformatics Resources 6.8 was used for functional analysis. Over 2000 proteins were considered expressed (n≥3 cows in a treatment), with 1239 proteins common among every group. There were 20, 37, 85, and 123 proteins unique to CON, PROT+OIL, PROT, and OIL, respectively. Relative to the CON group, 23, 14, and 51 proteins were found to be differentially expressed (P-adj<0.1), respectively. Functional analysis of histotroph proteins common to all treatments found they highly enriched the gene ontology (GO) Biological Process Cell-cell adhesion (Benjamini score 1.0x10^-21), 53% of all proteins expressed were clustered in Cellular Component GO Extracellular exosome (Benjamini 4.5x10^-261), and 17.4% of expressed proteins were clustered in KEGG pathway Metabolic pathways (Benjamini 7.8x10^-16). Differences among treatments in histotroph proteome supports the hypothesis that diet impacts uterine secretions; an understanding of histotroph proteins and diet differences may improve cattle reproductive programs.