Reduced glutathione and procaine hydrochloride protect the nucleoprotein structure of boar spermatozoa during freeze–thawing by stabilising disulfide bonds

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Abstract
One important change the head of boar spermatozoa during freeze–thawing is the destabilisation of its nucleoprotein structure due to a disruption of disulfide bonds. With the aim of better understanding these changes in frozen–thawed spermatozoa, two agents, namely reduced glutathione (GSH) and procaine hydrochloride (ProHCl), were added at different concentrations to the freezing media at different concentrations and combinations over the range 1–2 mM. Then, 30 and 240 min after thawing, cysteine-free residue levels of boar sperm nucleoproteins, DNA fragmentation and other sperm functional parameters were evaluated. Both GSH and ProHCl, at final concentrations of 2 mM, induced a significant (P < 0.05) increase in the number of non-disrupted sperm head disulfide bonds 30 and 240 min after thawing compared with the frozen–thawed control. This effect was accompanied by a significant (P < 0.05) decrease in DNA fragmentation 240 min after thawing. Concomitantly, 1 and 2 mM GSH, but not ProHCl at any of the concentrations tested, partially counteracted the detrimental effects caused by freeze–thawing on sperm peroxide levels, motility patterns and plasma membrane integrity. In conclusion, the results show that both GSH and ProHCl have a stabilising effect on the nucleoprotein structure of frozen–thawed spermatozoa, although only GSH exerts an appreciable effect on sperm viability.

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