288 EFFECT OF IN VITRO MATURATION PERIOD AND OOCYTE ACTIVATION BY CALCIUM IONOPHORE ON MONOSPERMIC PORCINE EMBRYO RATES

Abstract

It has already been reported that polyspermy in prepubertal pig oocytes is higher than in mature oocytes. Oocyte activation by calcium ionophore may help cortical granule exocytose. Normally, pig zygotes stay in maturation medium for 44 h, but at 36 h oocytes are already at metaphase II stage (MII). Arrest at MII for a longer period may cause deterioration of various cytoskeletal components. The aim of this study was to evaluate the effect of maturation period (36 or 44 h) and 0 or 50 ¼M calcium ionophore on monospermic pig embryo rates. Prepubertal pig oocytes were matured in TCM-199 with 10 IU eCG + 10 IU hCG + 50 IU epidermal growth factor (eGF) + 90 ¼L porcine follicular fluid (pFF) for 20 h and in the same medium without hormones for the last 16 or 24 h. In vitro fertilization was performed in mTBM medium with 1 × 10⁵ spermatozoa/mL in three different periods (2, 4, and 6 h). For nuclear evaluation, the zigotes were stained by Hoechst 33342 18 h after fertilization. Results were analyzed by PRC GLM of SAS (ANOVA and Tukey test) at a 5% level. Data were expressed as mean ± standard error of the mean non transformed. Independent variables were maturation period, effect of calcium ionophore, interaction among calcium*maturation period, and manipulations. In non-fertilized oocytes, there was no effect of maturation period, manipulation, or interaction calcium*maturation, but there was effect of calcium ionophore, having 43.5 ± 2.5 more non-fertilized oocytes at 36 h and 43.1 ± 5.1 more at 44 h of maturation (P < 0.01). At 36 h oocytes not activated by calcium ionophore showed 29.7 ± 4.9 more monospermic embryos than activated oocytes (P < 0.01). However, at 44 h of maturation, activation with calcium ionophore produced 47.4 ± 7.2 more monospermic embryos than non activated oocytes. From this study we conclude that monospermic embryo rates were influenced by maturation period, with the best results at 36 h in the absence and at 44 h in the presence of 50 ¼M of calcium ionophore.

This work was supported by CAPES.