Effect of reduced glutathione (GSH) in canine sperm cryopreservation: In vitro and in vivo evaluation

Abstract

The aim of this study was to compare the in vitro and in vivo efficiency of different concentrations (0, 10 and 20 mM) of reduced glutathione supplemented to the extender for canine semen cryopreservation. Six normospermic dogs were used and each ejaculate was divided in 3 experimental groups, according to GSH concentration (GSH-0, GSH-10 and GSH-20 Groups). After thawing, samples were evaluated by sperm motility by computer-assisted sperm analysis (CASA), flow cytometric evaluation of plasma and acrosome membrane integrity, mitochondrial membrane potential and activity, chromatin susceptibility to acid-induced denaturation, and measurement of spontaneous and induced production of thiobarbituric acid reactive substances (TBARS). In vivo tests were carried out with GSH-0 and GSH-10 groups, for which six bitches were inseminated with semen cryopreserved in extender without GSH or containing 10 mM GSH. Intrauterine insemination was performed by cervical catheterization on the 5th and 6th days after the LH surge, detected by serum progesterone and LH assays. In the CASA evaluation, GSH-20 group had the lowest total and progressive motility and lower percentage of sperm with rapid and slow speed. Groups treated with glutathione showed lower percentage of acrosome damage, but higher percentage of plasma membrane injury. GSH-20 group had higher percentage of sperm with low mitochondrial activity and higher concentration of induced TBARS. Both groups (GSH-0 and GSH-10) had positive pregnancies. In conclusion, 20 mM GSH supplementation to canine cryopreservation extender promoted sperm damage, especially to mitochondrial activity. However, addition of 10 mM GSH resulted in acrosome protection, preserving fertility rate.