41 EFFECTS OF MATURATION PERIOD OF PORCINE OOCYTES ON DEVELOPMENT FOLLOWING SOMATIC CELL NUCLEAR TRANSFER

Abstract

Despite intensive research, porcine nuclear transfer is still characterized by low success rates. To determine the effect of maturation period of porcine oocytes on subsequent development following nuclear transfer, we investigated fusion rate, induction of activation and development to blastocyst stage of somatic cells. For this we used MII-oocytes after 38, 40, and 42 h of maturation culture as recipients. Oocytes surrounded by a compact cumulus mass were selected and placed into North Carolina State University (NCSU) 37 oocyte maturation medium supplemented with 0.1 mg mL⁻¹ cysteine, 10 ng mL⁻¹ epidermal growth factor, 10% porcine follicular fluid, 50 μm 2-mercaptoethanol, 0.5 mg mL⁻¹ cAMP, 10 IU each of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) for 22 h in humidified air with 5% CO₂ at 38.5°C. Subsequently the oocytes were moved to fresh NCSU37 maturation medium without cAMP, eCG and hCG and incubated for an additional 16, 18, and 20 h. In the first experiment, a total of 878 MII-arrested oocytes were enucleated, fused with pig fetal fibroblasts in calcium-free medium and activated approximately 3 h later with an electrical stimulus. This was followed by incubation in 6-dimethylaminopurine for 3 h and subsequent analysis of development in vitro. Maturation period had no effect on the frequencies of fusion (87% v. 75% v. 84%, respectively), and cleavage (82% v. 81% v. 87%, respectively), but when MII-oocytes recovered at 40 h of maturation were used as recipients, 41/279 (14,8%) the numbers of cloned embryos developing to the blastocyst stage on Day 7 of culture was significantly (ANOVA followed by multiple pairwise comparisons using Tukey test, 6 replicates, P < 0,05) higher than those of embryos reconstituted with oocytes collected at 38 h (27/285, 9.6%) and 42 h (16/314, 4.9%). In the second experiment, reconstructed embryos derived from oocytes matured for 40 h were surgically transferred to the oviducts of synchronized German Landrace gilts. Transfers were made on the first day of standing oestrus within 3 h of activation to assess their development in vivo. Synchronization was achieved by injections of 1500 IU eCG followed by 500 IU hCG 3 days later. Of 4 recipients receiving an average of 150 zygotes (range, 136 to 163), 2 became pregnant as determined by ultrasound between Days 25 and 35 of gestation. Of the two pregnant recipients, one subsequently farrowed 4 piglets on Day 115 of pregnancy. These results indicate that the maturation period is critical and affects development of porcine nuclear transfer embryos. This study was funded by the Deutsche Forschungsgemeinschaft (DFG; SFB265).