| Porcine oocytes had been hard to be cryopreserved due to their high lipid content. The purpose of this study was to establish and optimize the procedures for the cryopreservation of porcine oocytes, which was significant either in theory or in practical use. In present study, porcine oocytes were collected from ovaries of slaughtered gilts in the slaughterhouse and used for cryopreservation and in vitro fertilization. Survival of oocytes was assessed by Trypan blue (TB) staining, Fluorescein diacetate (FDA) staining, maturation and fertilization in vitro and so forth. The effects of different freezing methods and different cryoprotectants on cryopreservation of porcine oocytes were investigated. Frozen or unfrozen oocytes and spermatozoa were respectively used to conduct in vitro fertilization and the impact of different culture systems on the developmental competence of porcine early stage embryos was also examined here. The results were shown as follows.1. All of three cryoprotectants, EG, DMSO and Gly were effective to cryopreserve pig oocytes in programmed freezing, resulting in post-thawing survival rate of 33.8%, 25.8% and 23.5%, respectively, which were higher than that of control significantly (2.5%, P<0.01). Among these three cryoprotectants, EG group was superior to other two groups in survival rate significantly (33.8% vs 25.8%, 23.5%, P<0.05).2. The self-made glass micropipettes (GMP) were adopted for oocyte freezing in pig and were confirmed to be a suitable and efficient method in vitrification of porcine oocytes. Compared with traditional programmed freezing method with plastic straws, GMP method could significantly improve the survival rate of porcine oocytes after freezing (34.5% vs 63.3%,P<0.05).3. The vitrification solution carrier would affect the survival rate of oocytes. When EFS40 was used as vitrification solution, different carrier, straw and GMP, resulted in different survival rate significantly (45.0% and 65.9%, P<0.05).4. Both programmed freezing method and vitrification method were available in cryopreservation of porcine GV stage oocytes. However, they yielded different survival rates (30.0% and 59.7%, P<0.05).5. Vitrified MII stage oocytes were fertilized with fresh spermatozoa, producing only 4.9% cleavage embryos in 48h after culture. The cleavage rate derived from vitrified oocytes was significantly lower than that from control (49.5%, p<0.01). The subsequent developmental rate to 4-cell stage from frozen oocytes was 1.7% and none developed to 8-cell stage.6. Matured oocytes in vitro were fertilized with frozen or fresh semen and then were cultured in vitro, resulting in different developmental percentage to 2-cell stage embryos (34.1% and 49.5%, P<0.05) and different developmental rate of morulae (3.2% and 13.3%, P<0.05).7. All of three culture systems, TCM199 medium (containing 15%FCS), TCM199 (containing 15%FCS) + cumulus cell layer and NCSU-23 medium, could support the development of pig embryos before 4-cell stage with much similar cleavage rate (44.7%, 41.9% and 49.1%, P>0.05). TCM199 medium could not support the development of porcine fertilized eggs to pass the 4-cell stage block. TCM199 + cumulus cells and NCSU-23 medium could enable porcine embryos develop to morulae, but these two groups could yield significantly different developmental rate of 8-cell stage and morulae (14.3% vs 24.2% and 3.3% vs 11.7%, P<0.05), suggesting that NCSU-23 was more suitable to be used for the cultivation of early stage embryos in pig. |
