144 Correlation between sperm characteristics and in vitro fertilization outcomes in frozen-thawed boar semen supplemented with glutathione and dithiothreitol

During the evaluation of fertilization, the presence of two pronuclei is the first sign of successful fertilization as observed during IVF. This study aimed to evaluate correlation for frozen-thawed boar sperm motility and velocity traits supplemented with glutathione (GSH) and dithiothreitol (DTT), and oocyte fertilization (pronucleus) rate. During semen cryopreservation, semen was supplemented with 4 different fraction B (control (no antioxidants), 5 mM DTT, 5 mM GSH, and 2.5 mM GSH + 2.5 mM DTT) extender and loaded into 0.25-mL freezing straws, then placed in vapour for 20 minutes, later plunged into a nitrogen tank (−196°C). Pig ovaries were collected from the local abattoir (Molare Meats), then transported to laboratory for further evaluation within an hour in 0.9% saline water in a Thermos flask at 38°C. A total of 440 oocytes were fertilized per treatment with frozen thawed semen supplemented with either 5 mM GSH, 5 mM DTT, 2.5 mM DTT + 2.5 mM GSH, and control. Prior to IVF, semen straws were thawed for 10 s in the air and 1 minute in warm water (37°C). Boar sperm motility was analysed using a computer-assisted sperm analysis. A drop of 50 μL of capacitated diluted sperm (~1 × 10⁶ sperm/mL) was used for IVF. Sperm and oocytes were co-incubated at 38.5°C in a moist atmosphere of 5% CO₂ for 18 h. Presumptive zygotes were assessed for fertilization status (O PN, 1 PN, 2 PN, >2 PN, and total fertilization). Data were analysed using GenStat^® statistical program with the GLM procedure. Treatment means were compared using Fisher’s protected t-test least significant difference at a 0.05 level of significance. Pearson correlation coefficients were calculated to test the relationships between sperm motility, velocity, and fertilization rate in the presence or absence of antioxidants. The results for frozen thawed sperm total motility (TM) were 32.0 ± 6.6, 28.3 ± 10.6, 26.0 ± 9.4, and 22.4 ± 11.1 for control, 5 mM DTT, 5 mM GSH, and 2.5 mM GSH + 2.5 mM DTT, respectively (P > 0.05). The coefficients were lower to higher with negative or positive correlation values ranging from r = −0.09 to 0.97 between sperm motility and fertilization rates (P > 0.05). There was a positive relationship (r = 0.28; P > 0.05) between sperm rapid motility and presumptive zygotes with <2 PN. Sperm TM was negatively correlated with fertilization rate (r = −0.06; P < 0.05) and presumptive zygotes with 1 PN (r = −0.09; P < 0.05). Moreover, there was a negative correlation (r = −0.23; P < 0.05) between sperm progressive motility and presumptive zygotes with 2 PN. In conclusion, boar sperm motility and velocity traits were negatively correlated with fertilization whereby presumptive zygotes with 2 PN were observed.