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EFFECT of CUMULUS CELLS and MATURATION CULTURE PERIOD of OOCYTES on THE SEX RATIO of IN VITRO-FERTILIZED BOVINE EMBRYOS

Studies have reported a high proportion of males from IVP of embryos systems. Factors such as the stage of oocyte maturation at the time of insemination and conditions of the IVC may influence the proportion of sex. Although cumulus cells (CC) improve the fertilization ability of sperm cells, there are some evidence that exposure of sperm to CC increases the proportion of male embryos produced in vitro. The objective of this study was to investigate the effects and interaction between CC and IVM culture period of bovine oocytes on the sex ratio of in vitro-produced embryos. COCs (n = 781) were collected from the ovaries of slaughtered cows, and then matured in vitro for various periods (18, 20, 22, and 24 h). Maturation medium consisted of TCM-199 with 0.2 mM pyruvate, 25 mM sodium bicarbonate, 75 μg mL-1 gentamicin, 0.5 μg mL-1 FSH, 100 IU mL-1 hCG, and 10% FBS. After maturation culture, oocytes were inseminated with frozen-thawed spermatozoa. Oocytes were inseminated included in CC (COC) or denuded of such cells (denuded oocytes, DO). All zygotes were then co-cultured in vitro with CC in SOF medium with 0.5% BSA and 2.5% FBS. Cultures were carried out at 38.5°C and 5% CO2 in air. Cleavage and blastocyst development rates were observed, respectively, at 48 (Day 2) and 168 (Day 7) h post-insemination (hpi). Blastocysts were harvested on Day 7, and the sex of the embryos was examined using the pair of primers S4B (Kageyama S et al. 2004 Theriogenology 66, 509-514). The data (mean ± SEM) were analyzed by ANOVA, in which the effects of the existence of CC (COC v. DO) and IVM culture periods (18 h, 20 h, 22 h v. 24 h), and interaction between these variables were evaluated. Multiple comparisons of means were followed using Tukey’s test (P 0.05) on cleavage (81.5 to 94.8% for COC; and 71.7 to 75.8% for DO) and blastocyst rates (20.0 to 35.9% for COC; and 10.0 to 15.5% for DO). But independently of IVM culture duration, the comparison between COC and DO groups revealed that absence of CC adversely affected (P < 0.05) the cleavage (87.0 and 73.3%, respectively; averaged results) and blastocyst rates (24.5 and 13.5%, respectively; averaged results). In COC, the proportion of male blastocysts increased with the progression in maturation culture period from 18 up to 22 h (54.2, 65.9, and 83.3%, respectively). But for DO, the proportion of females was higher, with exception of 22 h IVM group (65.4, 32.3, 53.3, and 60.0%, respectively from 18 to 24 h of IVM). These results indicate that the sex ratio of in vitro-fertilized embryos is apparently influenced by the maturation culture period of the oocytes and by the presence of CC. FAPESP.