Investigation of the Efficacy of Dithiothreitol and Glutathione on In Vitro Fertilization of Cryopreserved Large White Boar Semen

SIMPLE SUMMARY: Boar sperm has proven to be extremely susceptible to cold shock and sensitive to peroxidative damage due to the high membrane content of polyunsaturated fatty acids. As a result, free radicals and oxidative stress can have a significant impact on the outcomes of in vitro fertilization (IVF). The objectives of this study were to evaluate the properties of sperm motility, viability, and morphology under induced oxidative stress; compare the antioxidant capacity of dithiothreitol (DTT) and glutathione (GSH) following the cryopreservation of Large White boar semen; investigate the ability of the cryopreserved Large White boar semen to fertilize the matured gilts oocytes under the same conditions; and to compare the efficacy of DTT and GSH antioxidants in improving the oocyte fertilization by the cryopreserved Large White boar semen. In this study, hydrogen peroxide (H(2)O(2)) was used as a source of oxidative stress (control). The treatment of boar semen with H(2)O(2) reduced the percentage of sperm progressive motility (PM; 86.70 ± 10.24) after 3 h of incubation period (p < 0.05). The combination of GSH + DTT treatment reduced the percentage of sperm total motility (TM; 22.45 ± 11.14), PM (10.41 ± 4.59) and rapid motility (RAP; 9.20 ± 3.55) following thawing (p < 0.05). For IVF, fresh semen (11.84%) and a combination of DTT + GSH (14.10%) recorded a high percentage of zygotes with >2 pronucleus. However, the DTT treatment recorded a high percentage of zygotes with two pronuclei (19.76%). ABSTRACT: The objectives of this study were to evaluate the properties of sperm motility and morphology under induced oxidative stress, compare the antioxidant capacity of dithiothreitol (DTT) and glutathione (GSH) following the cryopreservation of Large White boar semen, investigate the ability of cryopreserved Large White boar semen to fertilize the matured gilts oocytes and compare the efficacy of DTT and GSH antioxidants in improving the oocyte fertilization by cryopreserved Large White boar semen. The semen was collected from three Large White boars (ten ejaculates per boar) and transported (37 °C) to the laboratory. Semen freezing extenders were supplemented with 5 mM DTT, 5 mM GSH and a combination of 2.5 mM DTT + 2.5 mM GSH. A liquid nitrogen vapor method was used to freeze boar semen. Gilts’ ovaries were collected from the local abattoir and transported (37 °C) to the laboratory. The slicing method was used to retrieve the oocytes from the ovaries. Fresh semen and frozen-thawed semen were used for in vitro fertilization (IVF). For frozen-thawed semen, four treatments (control, 5 mM DTT, 5 mM GSH, and a combination of 2.5 mM DTT + 2.5 mM GSH) were used during IVF in order to evaluate the fertilizing ability of the antioxidants. The supplementation of 5 µM DTT to H(2)O(2)-treated semen significantly improved progressive motility (PM) by 14.82%. A combination of 2.5 mM DTT + 2.5 mM GSH treatment reduced percentage of sperm total motility (TM) and rapid motility (RAP) following thawing (p < 0.05). Fresh semen and a combination of 2.5 mM DTT + 2.5 mM GSH treatment recorded a higher percentage of zygotes with polyspermy (p < 0.05). The control treatment numerically recorded a high percentage of zygotes with 1 PN, while the 5 mM DTT treatment recorded a high percentage of zygotes with 2 PN.