Development of Ovine Embryos Derived from Oocytes Matured in vitro in the Absence of C02

Abstract

The purpose of the study was to determine the effect of C02 during In Vitro Maturation (IVM) and subsequent development into morula/blastocyst stages in sheep and to develop a method of in vitro maturation in ruminant animals. Oocytes from abattoir ovaries were colected by methods of aspiration and spraying of media using a^-18-G needle with aspiration medium of hepes-199 (H199) + 0.4% BSA + 50 i^g/ml Heparin. The oocytes were divided into 3 groups and treated separately as follows: T1) oocytes were cultured in an eppendorf containing maturation medium (Bicarbonate-199 + 10% FCS + 10|ig/ml FSH + 10 jag/ml hCG +1 i^g/ml Estradiol) overlaid with mineral oil. The IVM medium was equilibrated in 5% C02 incubator prior to culture of the oocytes then matured in the absence of C02; T2) oocytes were; T3) oocytes were cultured in a petri dish containing maturation drops (as T1 medium) overlaid with mineral oil it was equilibrated in 5% C02 incubator prior to culture then matures in 5% C02 incubator. All cultures were maintained at 38°C in a humaditified incubator for 24 hours. The mature oocytes were fertilized in BO (Brackett and Oliphant) medium with concentration of approximately 12 x 106 sperm/ml for 6 hours and cultured in systhetic oviductal fluit (SOF) supplemented with amino acids (AA) and bovine serum albumin (BSA) at 38°C in humaditified incubator with 5%C02 for 7 days. The result showed that there was a highly significant effect of C02 during oocytes maturation on the percentage of cleaved oocytes (P<0.01). However, oocytes matured in the absence of C02 did not affect on either the percentages of morula/blastocyst or the rates of morula/blastocyst. This study suggests that ovine embryos derived from oocytes matured in vitro in the absence of C02 can be developed in vitro morula or blastocyst stages.