Impact of heat stress on the miRNA cargo of pre-ovulatory follicular fluid extracellular vesicles and fertility in dairy cattle

This study aimed to evaluate the impact of heat stress on bovine fertility and extracellular vesicles (EVs) distribution and cargo in the follicular fluid (FF) of pre-ovulatory follicles. Temperature and humidity data recorded on the farm were used to calculate the mean temperature humidity index (THI) during summer (S) and winter (W). Herd fertility was assessed as conception rates following artificial insemination during S and W. The miRNA cargo of the EVs contained in the pre-ovulatory follicle was studied in a representative group of synchronized animals during S and W (n = 10/season). The EVs were isolated by ultracentrifugation, and characterized by Nanosight, TEM, and Western Blot. EVs' miRNAs cargo was obtained by sequencing. The average THI index was 74 and 54 in S and W, respectively. The response to synchronization and conception rates decreased in S (81.5% and 24.4 %, respectively) compared to W (100% and 39.9 %, respectively). The EVs showed similar size distribution (mean size of 218 nm) and particle concentration (2.25e + 10 particles/ml) in both seasons. Seasonal variations of miRNA cargo were observed, with 83 differentially expressed (DE)-miRNAs, the most significant included 6 up-regulated (bta-miR-202, bta-miR-16a, bta-miR-186, bta-miR-532, bta-miR-154c, and bta-miR-19b), and 6 down-regulated (bta-miR-1306, bta-miR-130b, bta-miR-130a, bta-miR-320a, bta-miR-1246, and bta-miR-494) miRNAs during S compared to W. In conclusion, HS significantly alters the miRNA profile of pre-ovulatory FF-EVs in dairy cattle. Most of the DE-miRNA modulate ovarian function and follicular physiology, potentially explaining the reduced fertility observed in heat-stressed cows.