Presence of follicular fluid extracellular vesicles during in vitro maturation of donor cow (Bos taurus) oocytes increases their ability to in vitro embryo development

In vitro embryo production (IVEP) technology from the oocytes isolated by transvaginal follicle puncture (ovum-pickup, OPU) is widely used to generate a number of descendants from the most valuable donor cows and becomes a routine in cattle breeding programs to replicate and conserve precious genotypes. To increase the efficiency of OPU/IVEP-technology, in this work for the first time, bovine OPU-oocytes were matured in the presence of extracellular vesicles (EVs) from follicular fluid (FF), and the ability of treated oocytes to IVEP after in vitro fertilization was investigated. The aim was to study the effects of FF EVs on oocyte maturation and capacity to develop up to blastocyst of OPU-oocytes, as well as to resistance of such blastocysts to freezing. EVs were obtained from FF by differential centrifugations followed by ultracentrifugation at 100,000 g. The preparations were analyzed by transmission electron microscopy that confirmed the presence of exosome-size EVs. Oocyte donors were sexually mature Yaroslavl breed heifers ( n = 6) following natural cycle. OPU was performed twice a week. The isolated oocytes were in vitro matured in TC-199 medium, supplemented by fetal bull serum (10 %), follicle stimulating and luteinizing (10 mg/ml) hormones, epidermal growth factor (10 ng/ml) in absence (control) or presence of EVs (experiment). Vesicular preparations were added to the in vitro maturation (IVM) medium in the physiological concentration (EVs isolated from 1 ml of FF per 1 ml of medium). After 24 hours of IVM, the oocytes were subjected to in-vitro fertilization and in vitro embryo culture. At day 3 after fertilization, oocyte morphology was checked, and at day 7 of culture, a number of embryos developed to blastocyst (Bl) was determined. The resulting Bl were frozen and stored for some time at -196 °С. Then the blastocysts were thawed and in vitro cultured until hatching to determine their viability. A total of 10 independent experiments were performed, 57 and 56 oocytes were analyzed in control and treatment, respectively. No impact of experimental conditions on nuclear maturation rate was evidenced. The mature oocytes were similar in control and EVs-treated groups and accounted for 90.4±5.6 % and 94.3±3.1 %, respectively. In addition, the presence of EVs during IVM did not change oocyte cleavage rate after vitro fertilization, 78.6±7.3 % and 86.7±4.9 % in control and EV-treated groups, respectively. However, beneficial effect of EVs on blastocyst rate was found. In control, 26.6±5.8 % of OPU oocytes developed to Bl. EVs added during IVM increased blastocyst rate to 41.2±3.2 % (p function show_eabstract() { $('#eabstract1').hide(); $('#eabstract2').show(); $('#eabstract_expand').hide(); }