117 EFFECTS OF PREVIOUS PRESSURE TREATMENT ON THE SURVIVAL AND DEVELOPMENTAL SPEED OF EXPANDED MOUSE BLASTOCYSTS FROZEN RAPIDLY (PILOT STUDY)

Abstract

It has been demonstrated that embryos can survive exposure to a substantial amount of pressure. (Pribenszky et al., 2003 Theriogenology 59, 329, and 2002 Theriogenology 57, 506). Other studies report that, if a biological system is challenged by certain stresses, its ability to react and survive other stresses can be improved. The aim of our present study was to examine whether the survival rate of expanded mouse blastocysts could be improved by a certain pressure treatment before the freezing procedure. Morula stage mouse embryos were collected and cultured at 37°C with 5% CO₂ and maximal humidity in air in G 2.2 medium (Vitrolife, Göteborg, Sweden) to the expanded blastocyst stage. Embryos were randomly allocated to three groups. Embryos in Group I were equilibrated for 5 minutes in a solution containing 1.5 M ethylene glycol (EG) and 0.25 M sucrose in M2 (Sigma, St. Louis, MO, USA), supplemented with 10% FCS (Sigma), and then transferred into a vitrification solution (7 M EG, 0.5 M sucrose in M2 with 10% FCS) pre-loaded in a 0.25-ml plastic straw (7–9 embryos/straw). After 1-min exposure to the vitrification solution, the straw was slowly immersed in liquid nitrogen. Embryos in Group II were loaded into 0.08-mL straws (7–9 embryos/straw) with M2. Straws were placed into the chamber, filled with M2, of a special laboratory-made device that is capable of generating and precisely detecting hydrostatic pressure up to 150 MPa (1500 atm), and were exposed to 60 MPa pressure for 30 min. After the pressure treatment, embryos were frozen as described above. Straws were thawed by transfer into 30°C water for 30 s and then the embryos were recovered and placed in rehydration medium (0.5 M sucrose in M2 supplemented with 10% FCS) for 5 min. Embryos then were cultured in medium G2.2 as described above. A total of 27, 29 and 26 embryos were assigned to Group I, Group II and the untreated control group, respectively. Embryo viability and development were assessed at 6 and 20 h after culture as determined by morphological appearance and hatching. At 6 h, 16% (4/27) of the non-pressurized embryos were one-half expanded, at 20 hours 37% (10/27) were two-thirds and 30% (8/27) were one-half expanded; none of them were hatching. While at the pressure treated groups 89% (26/29) of the embryos were fully expanded at 6 hours, and 68% (20/29) were hatching at 20 h (untreated: 25/26 fully expanded at 6 h, 24/26 hatched at 20 h). Data were analyzed by chi-square test. We considered embryos which were at least two-thirds expanded. After 6 hours Group I differed from Group II and the control (P < 0.01). There was no significant difference between Group II and the control (P < 0.01). After 20 hours the same relations were seen. In the case of hatching, Group I differed from Group II and the control (P < 0.01). There was no significant difference between Group II and the control (P < 0.05). According to our results, the applied pressure treatment improved the in vitro development of the embryos after freezing. The re-expansion was faster and the survival rate was higher for those embryos that received pressure treatment before cryopreservation. Further experiments are needed to confirm and explore the in vitro and in vivo effects and benefits of pressure treatment before freezing.