The main aim of this work was to evaluate how supplementing freezing and thawing
media with reduced glutathione (GSH) and L-ascorbic acid (AA) affected the quality parameters
of frozen-thawed boar spermatozoa. With this purpose, semen samples of 12
ejaculates coming from 12 boars were used. Each ejaculate was split into seven aliquots to
which 5 mM of GSH and 100 mM of AA were added separately or together at two different
steps of freeze-thawing. Various sperm parameters (levels of free cysteine residues in
sperm nucleoproteins, sperm viability, acrosome membrane integrity, intracellular
peroxide and superoxide levels [ROS], and total and progressive motility) were evaluated
before freezing and at 30 and 240 minutes after thawing. Both GSH and AA significantly
improved boar sperm cryotolerance when they were separately added to freezing and
thawing media. However, the highest improvement was recorded when both freezing and
thawing media were supplemented with 5 mM of GSH plus 100 mM of AA. This
improvement was observed in sperm viability and acrosome integrity, sperm motility, and
nucleoprotein structure. Although ROS levels were not much increased by freeze-thawing
procedures, the addition of GSH and AA to both freezing and thawing extenders significantly
decreased intracellular peroxide levels and had no impact on superoxide levels.
According to our results, we can conclude that supplementation of freezing and thawing
media with both GSH and AA has a combined, beneficial effect on frozen-thawed boar
sperm, which is greater than that obtained with the separate addition of either GSH or AA.