139 Effect of follicle characteristics on bovine in vitro embryo development

Individual in vitro culture systems allow for the follow-up of the development of immature oocytes to the blastocyst stage. As such, the associations between follicle characteristics and oocyte developmental competence can be examined. This study aimed to evaluate follicular factors associated with embryo development using a bovine in vitro model. To do so, slaughterhouse-derived genital tracts were collected, and ovaries derived from nonpregnant uteri without signs of gross pathology were selected. Ovarian selection criteria included the presence of a corpus luteum in the contralateral ovary and the absence of follicles ≥15 mm in the ovary from which oocytes were collected. Individual follicles were dissected (DF), measured, and categorised as small (S; <4 mm, n = 118) or large (L; >5 mm, n = 32), and as atretic (n = 39) or non-atretic (n = 111), based on their vascularisation and colour. Cumulus–oocyte complexes (COCs) were liberated by rupturing the follicles, and only COCs with ≥5 cumulus layers, homogeneous ooplasm, and compact cumulus investment (COC-A, n = 117) or with minor granulation of the ooplasm and less compact cumulus cells (COC-B, n = 33) were selected for in vitro maturation-fertilisation-culture in individual 20-µL droplets under paraffin oil. Furthermore, COC areas were measured before and after maturation. In each replicate (n = 12), group and individual culture (IC) were performed as control. In the IC, COCs (n = 189) were aspirated from the same ovaries used to collect the dissected follicles. Group culture COCs (n = 517) were selected from a random pool of ovaries. The effects of culture methods (DF, IC, and group culture), follicle and COC parameters on cleavage and Day 8 blastocyst rates were fitted in generalised mixed-effects models in which the cow was set as random. The cleavage rate was greater (P = 0.04) in L than in S (85.4 ± 6.6% and 65.5 ± 5.2%, respectively) and L yielded more blastocysts (P = 0.003) than S (50.3 ± 9.4% and 21.3 ± 4.2% respectively). No differences were detected among other follicle parameters (P > 0.05). The blastocyst rate in group culture (38.0 ± 2.1%) was greater than that in the IC and DF groups (14.2 ± 2.5% and 28.0 ± 3.6%; P < 0.05). However, the blastocyst rate was lower in IC compared to DF (P = 0.02). Interestingly, COCs collected after follicle dissection had higher (P = 0.01) proportions of COC-B (78.0 ± 3.3%) than aspirated follicles for IC (51.6 ± 3.6%). Moreover, the area of immature and mature COCs was four times greater in the DF group than in the IC group (P = 0.03). The present results suggest that follicle size is a key element for selecting competent oocytes and that follicle aspiration causes loss of cumulus cells, impairing oocyte developmental competence in an individual culture system. These results provide the basis for the study of follicular characteristics associated with oocyte competence. Future studies are essential to identify molecular factors within follicles associated with developmental capacity.