| The process of cryopreservation could cause high concentrations of reactive oxygen species(ROS) which is detrimental to spermatozoa. Sperm will be attacked by ROS, and lead to lipid peroxidation, which would do damaged to the membrane of sperm, resulted in decreasing the motility of sperm and the rate of fertilization, and those would reduce the delivery rate and limit the widely-used of artificial insemination(AI) finally. The objective of the present study was to determine whether addition of melatonin to freezing extender could protect rabbit spermatozoa against cryodamage. The freshly ejaculated semen was diluted with Tris-citrate-glucose extender supplemented with melatonin. The motility of post-thawed sperm was evaluated by visual estimation with an optical microscope, and kinds of sensitive fluorescent probes such as SYBR14/PI, FITC-PNA, JC-1,BODIPY 581/591C11 were used to asses of the membrane integrity, acrosome integrity, mitochondrial potentials and lipid peroxidation of the post-thawed sperm. Besides, we also evaluated the apoptosis of post-thawed sperm treated with different concentrations of melatonin. The results of this research were shown below:(1) Spermatozoa with motility, acrosome intact, membrane integrity and mitochondrial potentials in the treatments was significantly higher than that of the control. And lipid peroxidation of the post-thawed treated with 1mM melatonin was lower than of the other treatments. These observations suggest that addition of melatonin to the freezing extender leads to less lipid peroxidation and higher integrity of acrosome, plasma membrane and mitochondrial as well as higher motility. Supplementation of 0.1mM or1 mM melatonin with freezing extender may be recommended to facilitate the improvement of semen preservation technique for the rabbit breeding industry, however, the effect of melatonin treated with 5mM decreased.(2) The results of western blot showed that melatonin could decrease the apoptosis of post-thawed sperm; and also, the treatment with 1mM melatonin could relieve the apoptosis.The study suggested that the freezing extender supplied with 0.1mM melatonin may decreased the apoptosis of spermatozoa to improve the quality of sperm in rabbit. |
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