Variations of follicular fluid extracellular vesicles miRNAs content in relation to development stage and season in buffalo (original) (raw)

In buffalo (Bubalus bubalis) reproductive seasonality, causing cycles of milk production, is one of the major factors affecting farming profitability. Follicular fluid (FF) contains extracellular vesicles (EVs) playing an important role in modulating oocyte developmental competence and carrying microRNAs (miRNAs) essential for in vitro fertilization outcomes. The aim of this work was to characterize the FF-EVs-miRNA cargo of antral (An) and preovulatory (pO) follicles collected in the breeding (BS) and non-breeding (NBS) seasons, to unravel the molecular causes of the reduced oocyte competence recorded in buffalo during the NBS. In total, 1335 miRNAs (538 known Bos taurus miRNAs, 324 homologous to known miRNAs from other species and 473 new candidate miRNAs) were found. We identified 413 differentially expressed miRNAs (DE-miRNAs) (FDR < 0.05) between An and pO groups. A subset of the most significant DE-miRNAs between An and pO groups targets genes which function is related to the lipid and steroid metabolism, response to glucocorticoid and oestradiol stimulus. Comparison between BS and NBS showed 14 and 12 DE-miRNAs in An-FF-EVs and pO-FF-EVs, which regulate IL6 release and cellular adhesion, respectively. In conclusion, these results demonstrated that the miRNA cargo of buffalo FF-EVs varies in relation to both follicular development and season. The importance of buffalo (Bubalus bubalis) breeding is clearly indicated by the positive growth trend all over the world (Faostat.fao.org/faostat), due to specific features making this species a valuable protein source, particularly for tropical countries. A peculiar situation is described in Italy, where the success of buffalo breeding is closely related to the production of mozzarella cheese, highly requested around the world. A major limiting factor is reproductive seasonality, impeding continuity of milk production throughout the year, and hence not allowing to meet the market demand. Buffalo is a short-day breeder, with an increased reproductive activity observed during decreasing day length months 1,2. In Italy, where the Italian Mediterranean buffalo breed has been selected, the seasonality pattern shows an opposite trend to the market request. Therefore, the out of breeding mating strategy (OBMS), has been efficiently applied to distribute calving more evenly during the year 2. However, forcing buffalo cows to conceive during the non-breeding season (NBS) may lead to extended post-partum anestrus, higher rates of embryonic mortality and overall reduced fertility 1,3,4. In previous studies it was demonstrated that embryonic mortality is in part caused by impaired luteal function, and consequently reduced progesterone secretion 5. This in turn interferes with embryo growth that is accompanied by transcriptomic and proteomic changes at the level of embryos and chorioamnios/caruncles 6,7 , definitively hampering embryo attachment. Embryonic mortality during the NBS is also in part due to reduced oocyte developmental competence, as shown by the decreased cleavage and blastocyst rates obtained after in vitro fertilization during increasing daylight months in Italian Mediterranean buffaloes 8,9. A seasonal effect on follicular population and embryo outcomes was also reported by other authors 10,11. A poorer oocyte quality was observed in Murrah buffalo heifers during