Effect of 9-cis retinoic acid, and, colony-stimulating factor 2 and interleukin 6 cytokines on in vitro maturation and embryo development of OPU-derived Murrah buffalo (Bubalus bubalis) oocytes

Reproductive inadequacy in buffaloes remains a major constraint to the efficacy of assisted reproductive technologies (ART), particularly in vitro embryo production (IVEP). This study investigated the effects of 9-cis retinoic acid (9-cisRA) during in vitro maturation (IVM) and cytokines (CSF2 and IL6) during in vitro culture (IVC) on enhancing the developmental competence of ovum pick-up (OPU) derived buffalo oocytes. In Experiment 1, oocytes were subjected to IVM supplemented with or without 9-cisRA. In Experiment 2, presumptive zygotes were cultured with CSF2, IL6, or their combination. Oocyte developmental competence was comprehensively evaluated based on cumulus cell expansion, cleavage rate, blastocyst yield, embryo quality grade, and developmental stage distribution. Data from Experiment 1 were analyzed using an independent t -test, while Experiment 2 data were subjected to one-way ANOVA followed by Tukey’s post-hoc test. The results demonstrated that 9-cisRA supplementation during IVM significantly enhanced ( P  < 0.05) cumulus expansion (84.6% vs. 61.0%), cleavage rate (65.7% vs. 52.1%), blastocyst yield (31.4% vs. 19.1%), and the proportion of excellent/good quality embryos (54.4% vs. 30.0%). Furthermore, 9-cisRA-treated groups yielded a higher proportion of advanced-stage blastocysts, including expanded (54.5%) and hatched (27.3%) blastocysts. In contrast, cytokine supplementation yielded only modest and non-significant improvements across all measured parameters. These findings identify 9-cisRA supplementation during IVM as a highly effective strategy to improve buffalo IVEP outcomes, while the application of CSF2 and IL6 during embryo culture requires further dose and timing optimization to ascertain their potential benefits.