Improvement of normal fertilization rate and embryo development by reduction of sperm: Oocyte ratio during in vitro fertilization in pig

Abstract

The present study was undertaken to determine the influence of sperm:oocyte ratio at fertilization in vitro using frozen boar semen on fertilization rates and subsequent embryo development and quality in pig. Cumulus-oocyte complexes (COCs) were collected from porcine ovaries and then matured in vitro. After 44 hours of culture, matured oocytes were fertilized for 6 hours with three different sperm:oocyte ratios (1000:1, 2000:1, and 4000:1). Presumptive zygotes were fixed at 18-20 hours post-fertilization and then examined for sperm penetration and monospermy rates. The developmental competence, in terms of cleavage and blastocyst rates, and the blastocyst quality were evaluated at 48 and 168 hours post-fertilization, respectively. Sperm penetration rate significantly increased when the oocytes were fertilized with 2000 (90.23±2.5%) and 4000 (93.46±3.7%) sperm: oocyte, compared with those fertilized with 1000 (74.08±1.2%) sperm:oocyte (p=0.005). The oocytes inseminated with 1000 sperm per oocyte had a significantly higher rate of monospermic zygotes (81.79±2.9%) than those inseminated with 2,000 and 4,000 sperm per oocyte (48.07±6.0 and 31.51±4.9%, respectively; p=0.001). The development of blastocysts increased significantly (p<0.05) in the group fertilized with 1,000 sperm:oocyte (29.02±1.8%) compared to those of 4,000 sperm:oocyte (14.00±3.0%). However, there was no significant difference in the mean number of blastocyst cells among the groups but blastocysts derived from 1000:1 oocyte/sperm ratio had the highest ICM cell numbers. Our results indicated that optimization of sperm:oocyte ratio at fertilization in vitro improved fertilization rates, in particular, the monospermic penetration rate and blastocyst rate.