Gamete co-incubation duration modulates blastocyst developmental competence in buffalo through regulation of growth factors, steroidogenesis and oxidative stress pathways.

BACKGROUND

Gamete (sperm-oocyte) co-incubation duration during in vitro fertilization (IVF) is a key determinant of embryo developmental competence in buffalo. This study evaluated the effect of gamete co-incubation duration (2-h intervals from 2 to 24 h) on embryo development and blastocyst competence.

METHODS

A total of 3680 cumulus-oocyte complexes (COCs) were recovered from 1163 buffalo ovaries. Grade A and B COCs (n = 2390) were matured in vitro, and 2324 matured oocytes were subjected to IVF using different gamete co-incubation durations, followed by culture of 2012 presumptive embryos for 7 days. Cleavage, blastocyst development, and developmental competence were assessed using RT-qPCR.

RESULTS

Cleavage rate was significantly affected (P < 0.05), increasing from 65.2% at 2 h to 100% at 8 h and remaining comparable between 10 and 24 h (81.7-97.1%). Blastocyst development increased progressively, reaching the highest rates at 16-20 h (53.2-54.9%; P > 0.05), before declining significantly at 22 h (32.7%) and 24 h (28.0%). Embryonic arrest was significantly lower in the 16-20 h groups. Blastocysts produced after 16-20 h exhibited higher (P < 0.05) expression of HSPB1, SOD2, GPX3, GDF9, BMP15, STAR, and BCL2, with lower BAX and CASP3 expression, indicating enhanced developmental competence.

CONCLUSIONS

Although blastocysts developed following as little as 2 h of gamete co-incubation, a duration of 16 h produced outcomes comparable to the conventional 20-h protocol. Therefore, 16-20 h represents the optimal gamete co-incubation window for maximizing blastocyst yield and developmental competence in buffalo IVF.