198 Assessment of biphasic CAPA-IVM for improving equine oocyte quality and developmental potential

IVM of immature oocytes retrieved by ovum pickup or from slaughterhouse ovaries is a standard procedure for the production of in vitro equine embryos. However, the developmental competence of oocytes matured in vitro is still inferior to that of oocytes matured in vivo. Capacitation-IVM (CAPA-IVM) includes an extra step of prematuration culture with c-type natriuretic peptide (CNP) as a meiotic arrestor to synchronize cytoplasmic and nuclear maturity in oocytes. It has shown promising clinical outcomes in human patients. This study aimed to evaluate the effect of CAPA-IVM on equine oocyte development competence and blastocyst quality. For this, immature cumulus–oocyte complexes (COCs) were retrieved from slaughterhouse ovarian follicles ranging from 5 to 30 mm in diameter. Suitable COCs (n = 335) were either cultured for 6 or 24 h in prematuration culture with 200 nM CNP before IVM (referred to as short and long CAPA-IVM, respectively) or directly transferred to IVM culture (control). During IVM, COCs were cultured in a group of 10–25 in 500 µL of TCM-199 with Earle’s salts, 10% fetal bovine serum, 50 µg mL⁻¹ gentamicin, 9.4 µg mL⁻¹ FSH, and 1.88 µg mL⁻¹ LH for 30 h at 38.2°C and 5% CO₂ in air. To test developmental competence, mature oocytes from all groups were fertilized by piezo-drilled intracytoplasmic sperm injection (ICSI), cultured in standard embryo culture conditions for 7 to 10 days, and the resulting blastocysts were snap-frozen for shallow whole genome sequencing. Our experimental findings delineate a comparable survival rate in the long CAPA-IVM group (n = 61) as compared with the control (n = 138; 70% vs. 60%; P = 0.163). A significant increase in maturation rates was observed in the long CAPA-IVM group compared with controls (69% vs. 51%; P = 0.017). Nonetheless, discernible features such as bigger perivitelline space and granular cytoplasm in these long CAPA-IVM oocytes suggested signs of aging. Following ICSI, developmental competency in these oocytes was compromised, as shown by the lower cleavage (41% vs. 60%) and blastocyst rates (3% vs. 13%) compared with the control group (P = 0.050 and P = 0.114, respectively). Separate experiments with short CAPA-IVM (n = 96) showed higher, albeit statistically nonsignificant survival (63% vs. 56%; P = 0.280) and maturation rates (61% vs. 53%; P = 0.417) in comparison with the control (n = 72). Moreover, developmental potential tended to be improved in short CAPA-IVM, with higher cleavage (80% vs. 63%; P = 0.048) and blastocyst rates (25% vs. 15%; P = 0.177) compared with the control. Genetic analysis of the developed blastocysts demonstrates that different maturation conditions did not affect normal euploidy rates (i.e. 1/1 in long CAPA-IVM, 15/16 in short CAPA-IVM, and 19/21 in the control group). Our results show that CAPA-IVM holds the potential to improve the viability and developmental competency of immature oocytes in horses. However, data should be interpreted cautiously owing to the limited sample size. Moreover, future transcriptomic and proteomic studies would be beneficial in understanding the underlying mechanism for the effective use of these IVM systems.