Effect of hyaluronan on monospermic penetration of porcine oocytes fertilized in vitro.

An experimental series was carried out to examine the ability of exogenous hyaluronan (HA) to facilitate monospermic penetration of in vitro-matured (IVM) oocytes during conventional porcine in vitro fertilization (IVF). IVM oocytes were co-cultured in fertilization medium with frozen-thawed, ejaculated boar spermatozoa. Addition of 0.5 mg/mL HA to the fertilization medium increased the rates of monospermic penetration (p < 0.05) on cumulus-denuded oocytes, without detrimentally affecting penetration rates (rates of monospermy were 57% and 78% in 2 mM caffeine-containing fertilization medium without and with HA, respectively). Caffeine and cumulus cells affected the rate of monospermic fertilization (p < 0.02-0.03), and caffeine affected the rate at which oocytes were penetrated (p < 0.02). Moreover, exogenous HA (0.5 mg/mL) in the sperm pre-incubation medium promoted monospermic penetration rates without affecting overall penetration rates. The effects of exogenous HA on sperm penetration and monospermy were also compared among semen batches. After semen was pooled from three fertile boars and held in homologous seminal plasma (SP) for 3 (batch A), 10 (batch B) or 20 (batch C) h prior to being cooled, spermatozoa from each semen batch were co-cultured with IVM oocytes. Monospermy of cumulus-denuded oocytes was increased (p < 0.05) by the addition of exogenous HA within the same semen batch (batches B and C). Furthermore, co-culture with cumulus cells did not mask the effects of HA on batch-B semen. The concentration of exogenous HA (0, 0.05 and 0.5 mg/mL) during sperm-oocyte co-culture differentially affected the rates of penetration and monospermy of cumulus-intact oocytes, depending on the time spermatozoa were held in SP before being frozen. The highest penetration rate was obtained by exposing semen to SP for 20 h prior to being cooled (0.05 mg/mL HA). The results suggest that exogenous HA decreases polyspermy during conventional porcine IVF. Furthermore, it appears that the action of HA is dependent upon the priming action of seminal plasma.