Optimization of the in vitro fertilization protocol for frozen epididymal sperm with low fertilization ability in Ban-A native Vietnamese pigs.

The aim of the present study was to improve the penetration during in vitro fertilization (IVF) of a frozen lot of epididymal sperm with a notoriously low fertilization ability of a Ban boar which is a native Vietnamese breed by optimizing different parameters of the IVF system. In Experiment 1, we determined that Pig-fertilization medium was superior medium to Tyrode's albumin lactate pyruvate-polyvinyl alcohol medium for IVF and defined the optimum the sperm concentration (1 × 106  sperm/ml). In Experiment 2, we clarified that partial removal of cumulus cells from cumulus-oocyte complexes by hyaluronidase treatment before IVF enhances sperm penetration, whereas complete cumulus removal reduces penetration. Finally, in Experiment 3 the elevation of concentration of caffeine in Pig-fertilization medium from 2 to 5 mmol/L and the prolongation of the co-culture of gametes from 3 to 5 hr significantly increased the total penetration rate from 15.2% to over 50%. In conclusion, the combination of partial oocyte denudation, an elevated caffeine concentration in Pig-fertilization medium and an extended interval of IVF with using an optimized sperm concentration was a potent way to improve the fertilization results for a frozen epididymal Ban sperm lot with low fertility.