VITRIFICATION OF IN VIVO AND IN VITRO PRODUCED OVINE BLASTOCYSTS

Abstract

Although cryopreservation of bovine embryo has made great progress in recent years, little achievement was obtained in ovine embryo freezing, especially in vitro produced embryos. However, a simple and efficient method for cryopreservation of sheep embryos will be important for application of ovine embryonic techniques such as in vitro fertilization, transgenic, cloning and etc. In this study ovine blastocysts, produced in vivo or in vitro, were cryopreserved by vitrification in EFS40 (40% ethylene glycol (EG), 18% ficoll and 0.5 M sucrose) or GFS40 (40% glycerol (GL), 18% ficoll and 0.5 Mol sucrose). In Vitro produced, early blastocysts were directly plunged into liquid nitrogen (LN₂) after preparation by one of the following procedures at 25°C: (A) equilibration in EFS40 for 1 min; (B) equilibration in EFS40 for 2 min; (C) equilibration in EFS40 for 30 s following pretreatment in 10% EG for 5 min; (D) equilibration in EFS40 for 30s following pretreatment in EFS20 for 2 min (E) equilibration in GFS30 for 30 s following pretreatment in 10% GL for 5 min. The survival rates observed after thawing and in vitro culture for 12 h were A 78.0% (39/50), B 50.0% (26/52), C 93.3% (70/75), D 92.0% (46/50) and E 68.0% (34/50). Survival rates were not significantly different for treatments C and D (p>0.05), but those for groups C and D were significantly higher than for A, B and E (p<0.05). After 24 h in vitro culture, hatched blastocyst rates were A 28.0% (14/50), B 21.1% (11/52), C 49.3% (37/75), D 48.0% (24/50), E 32.0% (16/50) and control 54.0% (27/50). The hatching rates for groups A, B and E were significantly lower than the control (p<0.05) in which early IVF blastocysts were cultured in fresh SOFaaBSA medium following treatment in PBS containing 0.3% BSA for 30 min, but for groups C and D it was similar to the control (p>0.05). The freezing procedures A, B and C were used to vitrify in vivo produced, early blastocysts recovered from superovulated ewes. The survival rates of frozen-thawed in vivo embryos were A 94.7% (72/76), B 75.0% (45/60) and C 96.4% (54/56) and for group B was significantly lower than for the other two treatment groups (p<0.05). Hatched blastocyst rates were A 46.0% (35/76), B 26.6% (16/60), C 51.8% (29/56) and the control 56.7% (34/60) in which early blastocysts from superovulation were cultured in fresh SOFaaBSA medium following treatment in PBS containing 0.3% BSA for 30 min. The hatching rate for treatment B was significantly lower than for the control (p<0.05) but did not differ between groups A, C and the control (p>0.05). Frozen-thawed embryos vitrified by procedure C were transferred into synchronous recipient ewes. Pregnancy and lambing rates were similar for embryos transferred fresh or frozen/thawed for both in vivo and in vitro produced embryos. These rates did not differ between in vivo and in vitro embryos transferred fresh (p>0.05). However, for frozen-thawed embryos, both rates were significantly lower for in vitro than for in vivo produced embryos (p<0.05).

Keywords:

• Blastocyst
• Vitrification
• Ovine
• In vivo
• In vitro

ACKNOWLEDGMENTS

We thank Dr P J Broadbent for his critical review of the manuscript. This work is supported by a grant from Department of Science and Technology, China, “863” Project: Studies on Mammary Gland Bioreactor in Livestock.