The use of animals for this experiment was approved by the University of Illinois's Institutional Animal Care and Use Committee. Thus, the aim of the present study was to investigate how supplementing a commercial freezing medium with the specific antioxidants GSH, BHT, or the combination affects in vitro fertilizing ability of sperm. Reports on a variety of antioxidants have been shown to be beneficial, have no effect, or be detrimental in sperm cryosurvival. GSH is known to decrease sperm cryodamage and increase the reproductive performance of FTS. The antioxidant GSH is the most abundant thiol in cells and, among other functions, is vital for the maintenance of the intracellular redox balance by the reduction of peroxides and free radicals. īutylated hydroxytoluene is a synthetic analog of vitamin E that keeps the autooxidation reaction under control by converting peroxy radicals to hydroxyperoxides. However, it is unclear whether their combined use would have beneficial or detrimental effects, even though one (GSH) is a cofactor for the GPx antioxidant enzyme, and the other (BHT) protects sperm membranes against ROS-induced damage. Improved semen quality and post-thaw fertility have been reported with the antioxidants reduced glutathione (GSH), , ] and butylated hydroxytoluene (BHT). The damage can be reduced by the use of antioxidants in the freezing medium to mitigate oxidative stress. Lipid peroxidation, caused by ROS, has been reported as one of the main causes of sperm damage during freezing and thawing processes. The sperm damage that occurs during the cryopreservation is related to the cell's susceptibility to reactive oxygen species (ROS) and reactive nitrogen species (RSN). Sperm cryodamage results in reduced fertility when used for artificial insemination compared to liquid-stored semen, ,, , ]. ĭespite the noted advantages and reports of improvement in fertility success over the years, the use of FTS remains limited due to the damage cryopreservation causes to the sperm plasma membrane, acrosome and sperm motility. Although sperm cryopreservation is the best method to store spermatozoa for long periods, it is only used in specific cases, such as preservation of valuable genetic material (germplasm banks), safety strategies in case of natural disasters, long-distance transport of sperm, and in combination with sex-sorting. This is important for a country like Brazil which depends on imported genetics and is at the moment free of diseases like porcine reproductive respiratory syndrome (PRRS) and African swine fever (ASF). FTS can provide increased flexibility for on-farm use and allow additional time for disease tests of semen. Therefore, we suggest the supplementation with 1 mM of BHT in the formula of commercial freezing media used in the present experiment.įrozen-thawed boar semen (FTS) accounts for <1% of commercial inseminations performed in the swine industry but is used more often for international exchange among nucleus farms to help maintain genetic diversity. On the other hand, media supplemented with BHT improved the in vitro fertilizing ability of the frozen-thawed sperm cells. These results indicate that commercial media supplemented with GSH increased motility but impaired in vitro fertilization rate. However, for the blastocysts rate, there were more (+22.75%) blastocysts (P ≤ 0.05) for BHT compared to Control. For IVF, semen cryopreserved with both antioxidants (BHT + GSH) had a negative (P < 0.05) impact on fertilization rate (−54.11%) compared to Control. In contrast, there was a higher (P ≤ 0.05, +21.18%) superoxide anion production in the Control than in the BHT + GSH. No (P > 0.05) differences between Control and other treatment groups were observed in viability, or acrosomal and mitochondrial membrane integrity however, the proportion of capacitated spermatozoa were reduced (by −21.17%) in semen treated with BHT + GSH compared to Control (P ≤ 0.05). At all-time points evaluated, total and progressive motility were greater (P ≤ 0.05) in semen cryopreserved with GSH than in Control. Flow cytometry assessments were performed at 60 min after thawing. Semen was evaluated for motility kinetics at 30, 120, and 240 min after thawing. On arrival, samples were split into the treatments with the following additions before cryopreservation 1) semen without additional antioxidants (Control), 2) semen with 1 mM BHT, 3) semen with 2 mM GSH, and 4) semen with 1 mM BHT+2 mM GSH. Samples were diluted 1:1 in an extender and cooled before overnight shipping at 17 ☌ to the freezing lab. One ejaculate was collected from 30 high-fertility boars in a weekly collection rotation. This work aims to evaluate how supplementing a commercial freezing media with butylated hydroxytoluene (BHT), or reduced glutathione (GSH), or their combination affected in-vitro measures of boar sperm after cryopreservation.
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