In vitro penetration assay of boar sperm fertility: effect of various factors on the penetrability of immature pig oocytes

The present study was designed 1) to examine the influence of cumulus cells, ovary storage time and oocyte size on the penetrability of immature pig oocytes, and 2) to investigate the effect of 2 methods of treating the semen from different boars on the inter-assay variability of homologous in vitro penetration tests of boar sperm fertility. In Experiment 1, cumulus oocyte complexes, oocytes with spontaneous loss of the cumulus cells during collection, and oocytes mechanically stripped of cumulus cells were used. No differences were observed in oocyte penetrability among the 3 types of oocyte, although mechanical removal of the cumulus caused an increase (P < 0.005) in the degeneration rate compared with the other oocyte types. In Experiment 2, the oocytes were recovered from ovaries kept in PBS (30 degrees C) for 2, 4 or 6 h after slaughter of prepuberal gilts. Ovary storage did not modify the penetrability of oocytes but increased (P < 0.02) their degeneration rates. In Experiment 3, the diameters of fresh oocytes were determined after co-incubation with spermatozoa. They were classified into 4 groups according to diameter: A) < 105 microm, B) 105-115 microm, C) 116-120 microm and D) > 120 microm. Oocytes from Groups C and D exhibited higher (P < 0.05) penetrability than oocytes from the other groups. In Experiment 4, stored, diluted spermatozoa from 4 boars were pretreated by centrifugation at 50 x g for 3 min and subsequent concentration of the supernatants at 1,200 x g for 3 min. The pellets were treated (washed twice and preincubated for 40 minutes) before co-incubation with immature oocytes or used directly as untreated samples (unwashed and non-preincubated). A boar effect (P < 0.001) was evident for the parameters of in vitro penetration, independently of sperm treatment. When the oocytes were inseminated with untreated spermatozoa, the effects of the replicate and the boar-by-replicate interaction on the variability in oocyte penetrability were not significant. The results of this study indicate that the use of standardized immature pig oocytes and stored untreated, diluted spermatozoa can provide a useful method for optimizing the homologous in vitro penetration (hIVP) assay of boar fertility.