Various methods for cryopreservation of biopsied bovine embryos for sex determination and their effect on pregnancy rate

One slow-freezing method and three vitrification methods were used for cryopreservation of bovine embryos biopsied for sexing and their assessment were made by pregnancy rate In the slow-freezing method, a biopsied embryo was suspended for 10min in 1.5M ethylene glycol(EG) ,0.2M trehalose and 5% polyvinyl pyrolidone in modified Dulbecco's phosphate buffered saline with 4 mg/ml BSA(m-PBS) .The embryo suspension was loaded into a 0.25ml plastic straw. The straw was frozen using a conventional slow-freezing device, and when at -30 deg C plunged and stored in liquid nitrogen(LN2)(SFCP embryo). In the vitrification methods, two types of vitrification solution were used: one was composed of 25% EG and 25% dimethyl sulfoxide(DMSO) in m-PBS (VSED) and another was composed of 20%EG 20%DMSO and 0.6M sucrose(Su) in TCM199 with 20% calf serum(CS)(VSEDS). In one of the vitrification methods, a biopsied embryo was equilibrated for 60sec in solution that VSED was diluted 2-fold in m-PBS. and loaded with in 15 micro 1 VSED into a 0.25ml plastic straws. The straw. 30sec later, was placed for 2min in LN2 vapor and then plunged and stored in LN2(VSED embryo). In the other two vitrification methods, a biopsied embryo was suspended for 90sec in solution that VSEDS was diluted 2-fold in TCM199 with 20% CS and then placed in 1 micro 1 VSEDS. The solution with the embryo was aspirated into a gel-loading tip(GL-tip) or a open pulled straw(OPS) The containers, 30sec later, were plunged and stored in LN2(GL-tip embryo and OPS embryo). SFCP embryos were thawed and transferred. VSED embryos, GL-tip embryos and OPS embryos were thawed and transferred only after their cryoprotectants had been diluted. The results were assessed by pregnancy rate. Pregnancy rate was 27.1%(13/48) for SFCP embryos, 34.8%(23/66) for VSED embryos, 32.0%(8/25) for GL-tip embryos and 45.9%(17/37) for OPS embryos. The difference in the rate between the four types of embryos was not significant (P>0.05).