Effect of Antioxidants on Pig Semen Cryopreservation to Preserve Sperm Fertility after Thawing

Abstract

Boar semen cryopreservation has a high potential in the swine industry, allowing the large-scale use of genetically superior animals, improving efficiency, product quality, helping to reduce the risk of disease spread and gathering needs from the market. From a genetic point of view, semen freezing is desirable for genetic diversification, favouring a more efficient reproduction as well as the constitution of germplasm banks, including for repopulation in case of disease outbreak. However, freezing this semen for long periods for practical use is limited by the reduced viability and fertilization potential caused to sperm during the cryopreservation process and consequently low conception rates and smaller litters after artificial insemination. In part, the decrease in the fertilizing power of frozen spermatozoa may be associated with oxidative damage due to excessive formation of Reactive Oxygen Species (ROS), osmotic stress and cell damage due to ice formation during cryopreservation. To suppress the damage caused by ROS, the present study was conducted to determine the impact of supplementation with three antioxidants, these being ascorbic acid, a-tocopherol and reduced glutathione, evaluating the parameters of semen quality, viability, total and progressive motility, vigour and agglutination rate after thawing. For this purpose, semen was collected from five boars, each being collected three times, at weekly intervals, always at the same time. Immediately after harvesting, the macroscopic (colour, appearance, and volume) and microscopic evaluation of the semen (mass motility, concentration, progressive individual motility, spermatic vigour and spermatic morphology) were evaluated. Subsequently, the semen was placed at 15°C for two hours and centrifuged at 800 x g for 10 minutes also at 15°C, removing the supernatant. For the freezing medium, a base medium consisting of a commercial MR-A extender, supplemented with 3% v/v glycerol, 10% v/v egg yolk and 0.20% w/v Sodium Dodecyl Sulfate (SDS) was used. The nine treatments used in the study were, respectively, ascorbic acid at concentrations of 100, 200 and 400μL, a-Tocopherol at concentrations of 200, 400 and 800μM and reduced Glutathione at concentrations of 2.5, 5 and 10 mg/l and numbered as T1 to T9, respectively. In the control group, semen was frozen in a medium without adding any antioxidant. The semen belonging to the different treatments and to the control was placed in 0.25ml insemination French straws and incubated at 6°C for two hours. The subsequent freezing was carried out in nitrogen vapours (-120°C) for ten minutes and immersed in liquid nitrogen after this period. After 7 days, the semen was thawed in a water bath at 37°C for 20 seconds, the straws dried on paper, placed on a microscope slide heated to 37°C and evaluated according to the parameters described above. Regarding the comparison between the different treatments, it was observed that the sperm viability obtained in the treatments with ascorbic acid as well as glutathione reduced, was not statistically different from the control group. Higher values of ascorbic acid and reduced glutathione reduced sperm viability after thawing. As for the use of a-tocopherol at a concentration of 400μM, the best results of the entire study were obtained, with sperm viability of 31.52% (±1.50). Regarding sperm motility and agglutination rate, a-tocopherol also showed the best results at the concentration of 200μM, in which the mean sperm motility was 2.57 ± 0.15 and 2.07 ± 0.15, respectively. The results of the present study allow us to infer that the addition of 200μM or 400μM of a-tocopherol to the swine semen-freezing medium has a positive effect on sperm viability parameters after thawing.