The strong anti-hyaluronidase effect of ellagic acid markedly decreases polyspermy during in vitro fertilization, resulting in sustainment of the developmental potency in porcine oocytes.

The present study investigated the effects of ellagic acid, a type of polyphenol that does not have a glycan and is composed of four hydroxyl groups and two lactone functional groups, on porcine in vitro fertilization (IVF) by focusing on its anti-hyaluronidase activity. A comparative analysis of ellagic acid and apigenin, which is commonly used as a hyaluronidase inhibitor, was performed. It compared the effects of ellagic acid and apigenin on hyaluronidase activity at different concentrations. The results showed that 10, 20, and 40 µM ellagic acid strongly reduced hyaluronidase activity (P < 0.05). The addition of 20 µM ellagic acid, but not apigenin, to porcine IVF medium effectively reduced polyspermy without decreasing sperm penetration or the formation rates of male pronuclei in cumulus-free oocytes. However, neither ellagic acid nor apigenin affected the number of sperm that bound to zona pellucida (ZP) or the induction of zona hardening and protease resistance. The percentage of acrosome-reacting sperm that bound to the ZP was markedly lower in the presence of 20 µM ellagic acid than in the untreated and apigenin-treated groups, even though the antioxidant capacity of ellagic acid was weaker than that of apigenin. Furthermore, a markedly higher percentage of embryos developed to the blastocyst stage in the ellagic acid-treated group, and the apoptotic indexes of expanded blastocysts produced by the ellagic acid treatment during IVF were significantly low. Therefore, the anti-hyaluronidase effect of ellagic acid markedly suppressed the induction of the acrosome reaction in sperm that bound to the ZP, resulting in a marked decrease in polyspermy under conditions that maintained high sperm penetrability during IVF and sustainment of the developmental potency in porcine oocytes.

Addition of l-carnitine to the freezing extender improves post-thaw sperm quality of Okinawan native Agu pig.

The objective of the present study was to establish whether the addition of l-carnitine (LC), which exhibits antioxidant activity, to the freezing extender improves the quality of cryopreserved Okinawan native Agu pig sperm. Ejaculated sperm frozen in an extender supplemented with 0, 1, 2.5, or 5 mM LC was thawed, and the integrities of mitochondria and the plasmalemma and other sperm characteristics were evaluated. The treatment with different concentrations of LC effectively improved sperm motility, mitochondrial and plasmalemmal integrities, and the proteolytic activity of acrosomal contents after freeze-thawing (P < 0.05). The proportion of post-thaw sperm possessing intact mitochondria and plasmalemma and higher proteolytic activity of acrosomal contents was markedly higher among sperm frozen in the presence of 2.5 mM LC than among sperm frozen in the extender without LC (P < 0.05). Furthermore, although the addition of LC to the freezing extender had no effect on disturbance of DNA damage and caspase activity, sperm treated with 2.5 mM LC during freezing exhibited significantly higher penetrability into matured oocytes in vitro than untreated sperm. Collectively, these results indicate that the addition of LC to the freezing extender effectively improved the post-thaw quality of Agu pig sperm by preventing mitochondrial dysfunction caused by oxidative stress during cryopreservation.