Optimization of porcine embryo vitrification

Abstract:

The purpose was to optimize the vitrification for porcine embryos cryopreservation. Blastocyst/Morula(5-6th day-embryos) were collected from superovulated Bama mini-pigs(sows/gilts). We compared different cryopreservation methods, cryopreservation tools, thining of zona pellucida(ZP) and recipient breeds on the efficiency of porcine embryo cryopreservation. The results showed that: in embryo survival rate and blastocyst cell number, there were no significant differences between cryopreservation method I [embryos were vitrified by two step method with open pulled straw (OPS) and glass micropipette (GMP) in solution 1(TCM199 + 20% FBS + 10% EG + 10% DMSO) for 3 min, and solution 2(TCM199 + 20% FBS + 20% EG + 20% DMSO + 0.4 mol/L SUC)for 1 min, stored in liquid nitrogen] and method II[Blastocysts were cultured for 25 min in NCSU23 + 7.5 mg/mL cytochalasin B, centrifuged at approximately 13 000 ×g for 12-13 min, and recovered back into pNCSU23. They were then equilibrated for 5 min in 2 mol/L ethylene glycol in pNCSU23, washed quickly in the vitrification medium, 8 mol/L ethylene glycol, 7% polyvinylpyrrolidone (PVP) in pNCSU23, loaded into OPS/GMP, and plunged into liquid nitrogen). GMP vitrification method was more suitable and efficient than OPS method (P<0.05) in embryo survival rate (83.8% vs 77.6%) and blastocyst cell number (53.1 vs 47.5) after thawing. Thining of ZP did not increase the survival rate, but significantly improved blastocyst cell number in the survival blastcysts (60.1 and 46, P<0.01). Local pig breeds (Fengjing sows) were more suitable as recipients for embryo transfer of vitrified/warmed blastcysts, which can improve pregnant rate and embryo efficiency.