Fertilization in Vitro and Development of Mouse Ova

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Abstract
Epididymal sperm were added to embryological watch glasses containing 396 F1, hybrid mouse ova in a simple medium under paraffin oil. The dishes were gently agitated for 8 h in an atmosphere of 5% CO2, 5% O2, and 90% N2. The ova were washed and cultured in glass tubes for 96 h during which time 365 ova cleaved and 347 developed to morulae or early blastocysts. Transferred to the uteri of pseudopregnant hosts were 299 of these embryos, and 67 male and 44 female offspring were born. From these findings, it was concluded that normal fertilization in vitro had occurred. Using first cleavage and embryo development in culture as the criterion, we have confirmed these findings and have examined some of the factors that affect fertilization in vitro. Epididymal sperm fertilized 122 of 131 ova whereas 39 of 122 ova were fertilized by uterine sperm. Agitation of sperm and ova during fertilization resulted in fertilization of 139 of 166 ova compared to fertilization of 84 of 168 ova without agitation. The highest fertilization rate (44 of 45) occurred with ova surrounded by cumulus cells and continuous agitation of sperm and ova in 0.5 ml of medium for 8 h at 37°C.

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