Abstract
The process of cryopreservation results in over-production of reactive oxygen species, which is extremely detrimental to spermatozoa. The aim of this study was to investigate whether addition of cysteine to freezing extender would facilitate the cryosurvival of rabbit spermatozoa, and if so, how cysteine protects spermatozoa from cryodamages. Freshly ejaculated semen was diluted with Tris-citrate-glucose extender supplemented with different concentrations of cysteine. The motility, intact acrosomes, membrane integrity, mitochondrial potentials, 8-hydroxyguanosine level and sperm-zona pellucida binding capacity were examined. Furthermore, glutathione peroxidase (GPx) activity, glutathione content (GSH), and level of reactive oxygen species (ROS) and hydrogen peroxide of spermatozoa were analyzed. The values of motility, intact acrosomes, membrane integrity, mitochondrial potentials and sperm-zona pellucida binding capacity of the frozen-thawed spermatozoa in the treatment of cysteine were significantly higher than those of the control. Addition of cysteine to extenders improved the GPx activity and GSH content of spermatozoa, while lowered the ROS, DNA oxidative alterations and lipid peroxidation level, which makes spermatozoa avoid ROS to attack DNA, the plasma membrane and mitochondria. In conclusion, cysteine protects spermatozoa against ROS-induced damages during cryopreservation and post-thaw incubation. Addition of cysteine is recommended to facilitate the improvement of semen preservation for the rabbit breeding industry.
Highlights
Cryopreservation of spermatozoa has long been an important strategy for preservation of male fertility
It was found that supplementation of 7.5 mM cysteine improved post-thaw motility, acrosome intactness, membrane integrity (p < 0.05; Fig 2A–2C), we found that addition of cysteine significantly increased the percentage of acrosome-intact viable sperm (S1 Fig), and addition of 7.5 mM cysteine showed the highest value (33.0±1.6%), compared to the control (19.7±0.6%)
At 0 h of incubation, addition of cysteine to either freezing extender or thawing solution led to an increase in GSH synthesis (p < 0.05)
Summary
Cryopreservation of spermatozoa has long been an important strategy for preservation of male fertility. The quality of the frozen-thawed spermatozoa is extremely affected during preservation [1]. Spermatozoa contain large amounts of unsaturated fatty acids in the plasma membrane, making them highly susceptible to reactive oxygen species (ROS) stress [2]. The process of cryopreservation lead to accumulation of ROS [3].
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