321 PREGNANCIES ESTABLISHED FOLLOWING TRANSFER OF IN VITRO-PRODUCED FRESH AND FROZEN–THAWED BUFFALO (BUBALUS BUBALIS) EMBRYOS

The objective of this study was to evaluate the feasibility of obtaining pregnancies in the buffalo (Bubalus bubalis) species, following transfer of fresh and frozen–thawed in vitro-produced embryos during the months of April and May in southern China. Embryos produced from oocytes recovered by ovum pickup (OPU), from Murrah and Nili-Ravi buffaloes, were transferred into synchronized local F1 swamp � Murrah/Nili-Ravi heifers to establish pregnancies. In vitro maturation (IVM) was performed in 50-�L droplets of TCM-199 supplemented with 10% FBS, 0.5 �g mL-1 of LH, 5 �g mL-1 of FSH, and 1 �g mL-1 of estradiol in a humidified gas atmosphere of 5% CO2 in air at 39�C for 22 to 24 h. A concentration of 2 million spermatozoa was prepared in TALP supplemented with 0.6% BSA fraction V, 60 �g mL-1 of heparin, 0.2 mM penicillamine, and 0.1 mM hypotaurine, and co-incubated in 50-�L droplets up to 24 h. Co-culture on granulosa cell monolayer in TCM-199 supplemented with 10% FBS (GIBCO, Grand Island, NY), 0.25 mM Na pyruvate, and 50 �g mL-1 of kanamicin was allowed up to 8 days. A total of 923 antral follicles were punctured and 647 cumulus–oocyte complexes (COCs) were recovered (70%) over 3 replicates on 40 buffalo donors in twice-weekly collections. A total of 462 COCs were selected based on the presence of at least one layer of granulosa cells and were used for IVM and IVF, with a resulting cleavage of 257 (55.6%), leading to 22 morula (4.7%) and 93 blastocysts (20.1%). Embryo development was evaluated from Days 6 to 8, and grades 1 and 2 blastocysts to expanding blastocysts were used for transfer as fresh or following freezing and thawing. Recipients were synchronized by the Ovsynch protocol, and to match the recipient estrous cycle to the stage of in vitro embryo development, a second gonadotropin-releasing hormone was administered to recipients at the time of OPU on donors. For both fresh and frozen–thawed embryos, each recipient received 2 embryos into the uterine horn ipsilateral to the ovary bearing the corpus luteum. Following the Ovsynch protocol, a total of 26 out of 47 recipients (57.4%) were found to be suitable as recipients by palpation per rectum, receiving 52 fresh embryos and resulting in 7 pregnancies (26.9%). Thirty embryos were cryopreserved by conventional freezing using 1.8 M ethylene glycol and a freezing protocol, from seeding to plunging into liquid nitrogen, of -0.3�C min. Subsequently, embryos were transferred following thawing into 15 out of 20 recipients originally subjected to the Ovsynch protocol. Only 2 recipients were diagnosed as pregnant (13.3%). No statistical difference in pregnancy rate was found following the transfer of fresh vs. frozen–thawed embryos (P > 0.05). Further improvements are needed to optimize the establishment of pregnancies. A closer monitoring by ultrasound of recipients subjected to synchronization protocols will favor a better selection of more suitable animals.