87 HIGH HYDROSTATIC PRESSURE TREATMENT IMPROVES THE QUALITY OF IN VITRO-PRODUCED OVINE BLASTOCYSTS

Abstract

High hydrostatic pressure (HHP) has been introduced into the field of embryology recently. It has been reported that a sublethal HHP could induce the synthesis of molecular chaperons to protect the embryos from other environmental stresses. The possible beneficial effect of HHP was examined on embryo vitrification, and improved cryotolerance has been achieved in HHP-treated mouse and bovine embryos. In the present study we evaluated, first, the behavior of in vitro-produced ovine blastocysts after treatment with 2 different HHP conditions. In the second part of the study, optimized pressure treatment was combined with a cryopreservation protocol. Day 7 in vitro-produced ovine blastocysts were loaded in 0.5-mL straws and pressure treated in a custom-made hydrostatic pressure chamber (Cryo-Innovation Ltd., Budapest, Hungary) according to the following conditions: -60 MPa, 70 min, 38°C; and -40 MPa, 70 min, 38°C. After HHP treatment, embryos were cultured for 24 h and their survival evaluated upon morphological appearance, re-expansion of the blastocoel, and hatching from the zona pellucida. Blastocysts were fixed and stained, observed with confocal microscope to evaluate blastocyst cell number, and mitotic and picnotic index. Untreated blastocysts were used as controls. On the basis of results obtained in this experiment, groups of blastocysts were exposed to 40 MPa for 70 min at 38°C and, following 2 h incubation after HHp treatment, were vitrified using cryotops. After warming, embryos were checked for re-expansion and hatching and finally fixed and stained as described for fresh embryos. Vitrified untreated blastocysts were used as controls. Results showed that the hatching rate of embryos treated at 40 MPa was significantly higher than that of 60 MPa-treated group (87.5 v. 0%; P < 0.01, chi-squared test) and similar to that of control embryos (85.3%) after 24 h culture. Blastocysts exposed at 40 MPa showed higher cell number compared with control embryos (161.3 ± 8.7 v. 123.9 ± 9.4, P < 0.01, ANOVA ± SD) and a lower picnotic index (PI: 1.3 ± 0.4% v. 3.8 ± 0.6%, P < 0.05). Similar re-expansion rate has been recorded between HHP treated and untreated (86.6 v. 89.6%) vitrified blastocysts as well as hatching rates (53.3 v. 48.3) and embryo cell number (131 ± 4.6 v. 139 ± 5.8). Conversely, HHP treatment significantly decreased picnotic index of vitrified treated embryos compared with control (2.3 ± 0.9 v. 4.8 ± 0.5, P < 0.05). We concluded that HHP treatment significantly improved the quality of in vitro-produced ovine blastocysts by increasing their cell number and reducing the proportion of picnosis. The HHP treatment exerted a positive effect in vitrified blastocysts, decreasing the number of picnotic nuclei. Further studies are needed to evaluate the possible beneficial effect at the molecular level of HHP treatment after vitrification and also to test if different interval times between HHP treatment and embryo cryopreservation could affect embryo response.