The Thai Journal of Veterinary Medicine


The aim of these experiments was to investigate the effects of sperm preparation on the success rate of in vitro fertilization in the pig. Three factors, the time period for capacitation, the type of sperm and the sperm motility-stimulating factors were studied by investigating their effect on the cleavage of pig follicular oocytes, after IVM-IVF-IVC. A total of 1,235 immature oocytes, collected from 3 to 6 mm follicles of gilt ovaries gathered at slaughterhouse were allocated to 3 experiments. Immature oocytes were cultivated in TCM199 NaHCO3 supplemented with 10 μg/ml of FSH/LH and 1 μg/ml of Estradiol-17ẞ for 44 hrs. After the end of culture, they were fertilized in vitro by freshly ejaculated sperm or by caudal epididymal sperm. The ejaculated and epididymal sperm were diluted in BTS commercial semen diluent at ratio of 1:1 and suspended in TALP medium containing PHE, Penicillamine+ Hypotaurine + Epinephine or 2.0 M caffeine +10 μg/ml heparin at pH 7.2 for 1 or 2 or 4 hrs. After the end of sperm incubation, 1x10 spz/ml were introduced to IVF TALP medium at pH 7.6 containing 20-30 matured oocytes and were incubated for 18 hrs. The oocytes were later cultivated in TCM199 NaHCO3 +10% FCS for 5 days. All in vitro processes were performed at 38.5°C in an atmosphere of 5% CO2 in air. In Experiment 1, the length of time for sperm capacitation was examined (1 vs 2 vs 4 hrs). A higher cleavage rate was found in 1 hr capacitation group (41.2%; 49/119) than the 2 hr or 4 hr groups (25.6%; 52/203 and 26.8%; 40/149 respectively) (P<0.01). In Experiment 2, a comparison was made between ejaculated and epididymal sperm. No difference in the cleaved embryo rates was found between the 2 types of sperm; 35.7% (25/70) vs 38.5% (55/143) respectively. In Experiment 3, sperm motility stimulating factors were examined, comparing PHE vs 2.0 M caffeine; sperm-oocyte incubation in a IVF media, supplemented with PHE, gave similar cleavage rates to that of caffeine (54.3%; 108/199 vs 53.9%; 89/165). The results indicates that the period of time of sperm capacation (1 hr) was an important factor in the success of IVF in the pig. Therefore, no difference whether ejaculated or epididymal sperm were used and there was no difference in the motility stimulating factors used (PHE or caffeine).


Faculty of Veterinary Science, Chulalongkorn University

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