Cell Counts and Survival to Vitrification of Bovine In Vitro Produced Blastocysts Subjected to Sublethal High Hydrostatic Pressure

Abstract

This work analyses the effects of a high hydrostatic pressure (HHP) treatment on in vitro survival of in vitro produced (IVP) bovine embryos vitrified with the Cryologic Vitrification Method (CVM). Consequences on embryo quality in terms of cell proliferation and differentiation, and levels of embryonic Heat Shock Protein 70 (Hsp-70) were also examined. Day 7 and 8 bovine in vitro-produced blastocysts were submitted to an HHP treatment (60 MPa, at 32 °C for 1 h) and allowed to recover for 1 or 2 h in culture medium. The HHP treatment did not improve blastocyst survival rates after vitrification/warming. Survival (24 h post-warming) and hatching (48 h post-warming) rates were 79.3 ± 4.9 and 51.8 ± 4.2 vs 73.9 ± 4.2 and 44.7 ± 4.1 for untreated controls and HHP-treated embryos, respectively. Total cell numbers measured in fresh embryos were reduced after 1 h at 32 °C, with or without HHP treatment, indicating that cell proliferation was stopped as a result of stress. Vitrified HHP-treated embryos that hatched at 48 h after warming showed increased cell numbers in their ICM compared with untreated controls (50.2 ± 3.1 vs 38.8 ± 2.7), indicating higher embryo quality. Treatment of blastocysts with HHP did not alter the level of the Hsp-70 protein. In our conditions, HHP treatment did not affect the cryoresistance of these embryos. However, combination of HHP treatment and vitrification in fibreplugs resulted in an increase in the ICM cell number of hatched embryos 48 h post-warming.