197 The effects of pyridoxine supplementation during oocyte maturation on the in vitro production of pig embryos

Elevated levels of oxidative stress on both the sperm and pig oocytes reduces IVF success rates. Limiting the damage caused by oxidation, antioxidants, such as vitamin B₆ (pyridoxine) can be supplemented to the sperm and oocyte environment. The purpose of this study was to determine the effects of pyridoxine supplementation during oocyte maturation on reactive oxygen species (ROS) formation, catalase activity, IVF, and early embryonic development success rates. Oocytes were matured for 40–44 h supplemented with pyridoxine (0, 100, 250, 500 μM; Sigma-Aldrich Co.). After maturation, oocytes were denuded and either measured for ROS production (n = 40) by measuring the fluorescent intensity from the oxidation of 2′,7′-dichlorodihydrofluorescein diacetate and catalase activity (n = 659) by measuring the hydrogen peroxide decomposition rate, or fertilized (n = 839) using frozen-thawed boar semen (1.0 × 10⁵ sperm cells/mL) and co-incubated for 6–8 h followed by IVF analysis or embryo culture for 144 h. After IVF, a portion of the potential embryos (n = 240) were fixed, permeabilized, and stained with bisBenzimide H 33342 trihydrochloride and evaluated for penetration, polyspermy, and pronucleus formation rates while the remaining potential embryos (n = 599) were evaluated for cleavage and blastocyst formation at 48 h and 144 h post-IVF, respectively. Catalase activity and ROS fluorescent intensities of individual oocytes were analysed using a GLM and means were compared using l.s.d. after adjusting the value of the control group to 1. The IVF and embryo development data were reported as the percent observed/drop and mean percentages using a GLM were analysed with differences being compared using Tukey’s test. Supplementation of pyridoxine decreased (P < 0.05) ROS production compared with no pyridoxine supplementation (1.00 ± 0.11) and supplementation of 100 μM (0.62 ± 0.13) or 500 μM (0.69 ± 0.11) pyridoxine decreased ROS production compared with those oocytes supplemented with 250 μM (0.74 ± 0.10). Supplementation of pyridoxine had no effect on intracellular catalase activity at the end of maturation or sperm penetration rates, polyspermic oocytes, or oocytes with a male pronucleus. Pyridoxine supplementation during oocyte maturation decreased (P < 0.05) of cleavage formation by 48 h post-IVF compared with no pyridoxine supplementation (78.10 ± 0.03) but increased (P < 0.05) the percent of blastocysts observed by 144 h post-IVF compared with no pyridoxine supplementation (30.48 ± 0.03). Based on these results, supplementation pyridoxine during oocyte maturation reduces ROS production in the oocytes and increases blastocyst formation rates.