Freezing of in Vivo Derived and in Vitro Pre-Cultured Porcine Blastocysts: Differences of the Cryoprotective Effect between Glycerol and Ethylene Glycol

Abstract

The present study was conducted to compare glycerol and ethylene glycol as cryoprotectants for porcine in vivo derived and in vitro pre-cultured peri-hatching blastocysts. Embryos were surgically collected from superovulated gilts on Day 6. In vivo derived hatched blastocysts (in vivo HB) were frozen with 1.5 M glycerol (1.5 G) or 1.5 M ethylene glycol (1.5 EG). In vitro pre-cultured peri-hatching blastocysts (in vitro pHB), obtained by culturing Day 6 embryos for 18 h in Whitten's medium supplemented with 1.5% bovine serum albumin and with or without 5% fetal calf serum (FCS), were frozen with 1.5 G or 1.8 EG. All embryos were frozen by a slow cooling method using a programmable freezer. The survival of in vivo HB frozen with 1.5 G (82%) was higher (P<0.01) compared with embryos frozen with 1.5 EG (36%). More blastocysts cultured with FCS developed to the hatched blastocyst stage compared with those cultured without FCS (73% vs 33%, P<0.01) within 18 h. The survival of embryos pre-cultured with or without FCS however was not different following freezing with 1.5 G (23% vs 34%) or 1.8 EG (74% vs 81%). The survival rates for embryos frozen with 1.8 EG were significantly higher (P<0.01) than those for the 1.5 G groups. These results demonstrate that more than 80% of in vivo HB can be successfully frozen with 1.5 G and that similar survival rates for in vitro pHB can be achieved by using 1.8 EG. The study also indicates inherent differences in the post-thaw survival of in vivo derived and in vitro pre-cultured peri-hatching blastocysts frozen with 1.5 G.