106 PREGNANCY RATE AFTER EMBRYO TRANSFER OF IN VIVO-PRODUCED OVINE EMBRYOS CRYOPRESERVED BY SLOW FREEZING OR VITRIFICATION

Abstract

Although slow freezing is the method of choice to cryopreserve in vivo-produced ovine embryos, vitrification has became an alternative procedure mostly developed for in vitro-produced bovine embryos. The aim of this work was to compare pregnancy rates after cryopreservation of in vivo-produced ovine embryos with slow freezing or open pulled straw (OPS) vitrification method. Ewes were synchronized using intravaginal sponges containing 60 mg of medroxyprogesterone acetate for 14 d. Superovulation was performed using a total dose of 176 IU of ovine FSH (Ovagen), in 6 decreasing doses (i.m.) from Day 12 to 14 of treatment (Day 0 = sponge placing). Ewes were hand mated with 2 rams of proven fertility. Embryos were recovered 6 days after estrous detection by surgical procedure, evaluated under stereomicroscope, and randomly assigned to the cryopreservation treatments. Slow freezing was performed in D-PBS supplemented with 1.78 M ethylene glycol, 0.1 M sucrose, 4 mg mL^-1 of BSA, and 20% serum. Embryos were loaded into 0.25-mL plastic straws and placed into a -7°C methanol bath chamber. After seeding embryos were cooled to -35°C at a rate of 0.5°C/min and then stored in liquid nitrogen. Thawing was performed by placing the straws in a 30°C water bath for 30 sec. Vitrification was performed by using the OPS method (Vajta et al. 1998) with minor modifications. Embryos were incubated in D-PBS supplemented with 1.78 M ethylene glycol, 1.3 M DMSO for 3 min and then transferred for 25 s in vitrification solution of D-PBS with 3.56 M ethylene glycol, 2.6 M DMSO, and 0.5 M sucrose, loaded in a 1 mL drop in the OPS, and immediately submerged into and stored in liquid nitrogen. Warming was performed in D-PBS plus 0.25 M sucrose for 5 min and then into D-PBS plus 0.15 M sucrose for another 5 min. Before embryo transfer, the presence of corpus luteum (CL) was detected by laparoscopic examination. One embryo per recipient was surgically transferred in the apical extreme of the uterine horn ipsilateral to the CL. Pregnancies were determined by ultrasonography 41 days after embryo transfer. Data were analyzed using the chi-square test. We found 47.8% pregnancy rate using slow freezing (11/23) and 43.5% pregnancy rate using OPS vitrification (10/23). Statistical differences were not detected (P = 0.09). We conclude that vitrification by OPS system, with minor modifications, is a suitable procedure for in vivo-produced ovine embryo cryopreservation.