Ovarian follicle extracellular vesicle-microRNAs mediated response of beef cows to environmental heat stress

Elevated ambient temperature during the summer season is a main cause of stress in dairy and beef cows, leading to impaired reproductive function and fertility. Follicular fluid extracellular vesicles (FF-EVs) play an important role in intrafollicular cellular communication by in part mediating the deleterious effects of heat stress (HS). Here we aimed to investigate the changes in FF-EV miRNA cargoes in beef cows in response to HS during the summer (SUM) compared to the winter (WIN) season using high throughput sequencing of FF-EV-coupled miRNAs. Overall design: dry beef cows (n=11) were subjected to synchronization and stimulation for follicular dynamics. Ovum pick-up (OPU) was conducted on all animals in the summer (August 2021) and winter (February 2021) seasons by ultrasound-guided transvaginal follicle aspiration. Rectal temperatures were collected on each OPU and environmental data were collected daily for three weeks before each OPU. The FFs were collected from each target group (4 biological replicates/group; 8 mL FF/replicate) and subjected to differential centrifugation and high-speed ultracentrifugation (120,000xg for 70 min x2) to isolate EVs. Isolated EVs were characterized using a western blot, nanosight tracking analysis, and transmission electron microscopy. MiRNA-enriched total RNA was isolated from EVs and small-RNA library preparation and RNAseq (NovaSeq6000; Illumina) were performed.