No abstract

Sperm cryopreservation is the best technology for long-term storage of the semen. However, the damage of boar spermatozoa by cryopreservation is more severe than in other animal species and a standardized freezing protocol for efficient cryopreservation has not been established yet. Semen quality and freezability vary greatly between breeds as well as between individual boars and even the season. Boar spermatozoa are sensitive to low temperatures; they sustain damage and a high rate of mortality and freezing/thawing the boar semen may strongly impair the sperm function and decrease the semen quality. The freezability of boar semen can be influenced by a cryopreservation procedure, and also by using various additives to freezing and thawing extenders such as antioxidants. In order to obtain acceptable results after thawing the boar semen, it is necessary to combine an optimal amount of additives (glycerol, egg yolk, sugars, antioxidants), cooling and warming velocities.

Aminoguanidine is a selective inhibitor of the inducible nitric oxide synthase (iNOS) and a scavenger of reactive oxygen species (ROS). Numerous studies have shown the antioxidant properties of aminoguanidine in several cell lines, but the in vitro effects of this compound on spermatozoa under oxidative stress are unknown. In this study, we tested the hypothesis that aminoguanidine may protect against the detrimental effects of oxidative stress in boar spermatozoa. For this purpose, sperm samples were incubated with a ROS generating system (Fe2+/ascorbate) with or without aminoguanidine supplementation (10, 1, and 0.1 mM). Our results show that aminoguanidine has powerful antioxidant capacity and protects boar spermatozoa against the deleterious effects of oxidative stress. After 2 h and 3.5 h of sperm incubation, the samples treated with aminoguanidine showed a significant increase in sperm velocity, plasma membrane and acrosome integrity together with a reduced lipid peroxidation in comparison with control samples (p < 0.001). Interestingly, except for the levels of malondialdehyde, the samples treated with 1 mM aminoguanidine did not differ or showed better performance than control samples without Fe2+/ascorbate. The results from this study provide new insights into the application of aminoguanidine as an in vitro therapeutic agent against the detrimental effects of oxidative stress in semen samples.

Hydrogen sulphide (H 2 S) is involved in the physiology and pathophysiology of different cell types, but little is known about its role in sperm cells. Because of its reducing properties, we hypothesise that H 2 S protects spermatozoa against the deleterious effects of oxidative stress, a condition that is common to several male fertility disorders. This study aimed i) to determine the total antioxidant capacities of Na 2 S and GYY4137, which are fast-and slow-releasing H 2 S donors, respectively, and ii) to test whether H 2 S donors are able to protect spermatozoa against oxidative stress. We found that Na 2 S and GYY4137 show different antioxidant properties, with the total antioxidant capacity of Na 2 S being mostly unstable and even undetectable at 150 µM. Moreover, both H 2 S donors preserve sperm motility and reduce acrosome loss, although the effects were both dose and donor dependent. Within the range of concentrations tested (3-300 µM), GYY4137 showed positive effects on sperm motility, whereas Na 2 S was beneficial at the lowest concentration but detrimental at the highest. Our findings show that Na 2 S and GYY4137 have different antioxidant properties and suggest that both H 2 S donors might be used as in vitro therapeutic agents against oxidative stress in sperm cells, although the optimal therapeutic range differs between the compounds. Hydrogen sulphide (H 2 S) is the most recently discovered gaseous molecule that participates in a variety of biological functions, as do nitric oxide (NO) and carbon monoxide (CO). In mammals, H 2 S can be synthesised by enzymatic or non-enzymatic pathways 1. Overall, it seems likely that most of the H 2 S produced within an organism is generated by the H 2 S-synthesising enzymes: cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulphurtransferase (3-MST), with the latter coupled with cysteine aminotransferase (CAT) 2. In the male reproductive system, the expression of H 2 S-generating enzymes has been reported in the testis 3,4 , epididymis 5 , penile corpus cavernosum 6 , and spermatozoa 7,8 , which strongly suggests that this gasotransmitter is involved in sperm physiology to some extent. In a recent study, Wang et al. found that asthenospermic men show reduced levels of H 2 S in their seminal plasma and that exogenous H 2 S supplementation improves their sperm motility 8. In contrast, in boar spermatozoa, H 2 S exerts no or negative effects on sperm motility, viability, and mitochondrial membrane potential 9. With both positive and negative effects documented, there is still controversy concerning the role of H 2 S in sperm cells. This apparent discrepancy might, at least partly, be a result of H 2 S dose-and donor-dependent effects 10. According to their chemical structure and source, H 2 S donors include inorganic salts and derivatives of phosphorodithioate, garlic extracts, thioaminoacids, and anti-inflammatory drugs 11. On the basis of their release mechanism, H 2 S donors can be classified in two categories: slow-and fast-re...

Nitric oxide (NO) is a major gasotransmitter involved in several physiological processes of male reproduction. There is, nevertheless, little information concerning the role of NO during semen storage. The aim of this study was to evaluate the effect of NO on boar semen stored at 17 o C for 72 h. For this purporse, sperm samples were treated with 0.625, 1.25, 2.5, 5, and 10 mM aminoguanidine (AG) or Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), a selective and non-selective NO synthase (NOS) inhibitor, respectively. Moreover, sodium nitroprusside (SNP), a NO donor, was used at the dose of 18.75, 37.5, 75, and 150 μM. Sperm motility, membrane integrity, and acrosomal status were evaluated at 0, 4, 24, 48, and 72 h of semen storage. A significant increase of the amplitude of lateral sperm head displacement (ALH), and both curvilinear and straight-line velocity (VCL and VSL, respectively) was observed at 72 h of semen storage in samples treated with 0.625 mM AG, probably because of the antioxidant properties of this NOS inhibitor. Contrarily, 0.625 mM L-NAME showed no effect on boar sperm parameters during the entire period of semen storage. Moreover, AG and L-NAME at 10 mM negatively affected sperm kinetics and acrosome integrity, which may provide further support to the notion that low NO levels are necessary for a normal sperm function. The concentrations of SNP used in this study had mostly no or negative effects on boar sperm parameters during semen storage. In conclusion, the results from this study increase the understanding of the role of NO on boar sperm physiology.