RESUMO
A series of experiments were carried out to develop a new method to reduce pig polyspermic fertilization and produce more normal embryos, in vitro. Experiment 1 determined the effect of methyl-beta-cyclodextrin (MCD) treatment during cryopreservation on sperm acrosome reaction and sperm fertilization. Compared to the non-MCD-treated control, MCD treatment increased the percentage of acrosome-reacted spermatozoa at thawing and 2h after incubation in fertilization medium (P<0.01). Treatment with MCD also increased (P<0.05) sperm-penetration rate, number of spermatozoa in oocytes, and fertilization efficiency in the caffeine-free fertilization medium. Experiment 2 was designed to examine the effect of withdrawal of caffeine (caffeine-free) from fertilization medium on fertilization parameters and early embryo development. Using MCD-treated spermatozoa, there was no difference in sperm-penetration rate, oocyte cleavage rate, and blastocyst formation rate between the caffeine-free and caffeine-supplemented groups. However, polyspermic fertilization rate was lower, and fertilization efficiency and blastocyst cell number were higher in the caffeine-free group compared to the caffeine-supplemented group (P<0.05). Experiment 3 studied the effect of caffeine and different concentrations of spermatozoa on fertilization parameters. Sperm-penetration rate did not differ between the caffeine-free and the caffeine-supplemented groups at different sperm concentrations. Caffeine and sperm concentration had an effect on the number of spermatozoa in oocytes and on the polyspermic fertilization rate (P<0.002). Caffeine also affected fertilization efficiency (P<0.05). In conclusion, treating spermatozoa with MCD and withdrawing caffeine from fertilization medium may provide a new method to produce a large number of normal embryos, in vitro.
