| The cryopreservation of semen originated in more than a hundred years ago, but so far semen freezing effect is not ideal. Oxidative damage is one of the reasons that cause the declined quality of frozen-thawed sperms. Melatonin has a strong antioxidant effect, but the impact of sperm motility and viability is controversial. The main component caffeine sodium benzoate is caffeine and sodium benzoate, caffeine has been applied to many species of semen cryopreservation process which can improve sperm motility. but caffeine sodium benzoate is little used in the research on frozen boar semen. Kunming white mice and Rongchang pigs were used as the objects in this study. Different concentrations of melatonin added into mouse sperm freezing extender R18S3 were 0 mg/mL (MO group),0.125 mg/mL (Ml group), 0.25 mg/mL (M2 group),0.5 mg/mL (M3 group), the aim of which was exploring it’s impact on the frozen-thawed sperm quality and reactive oxygen species(ROS). Add 5 mL of caffeine sodium benzoate into 100 mL thawing solution during boar sperm cryopreservation, the density, viability, acrosome integrity, membrane integrity and deformity rates of thawed sperm was detected.The results were as follows:(1) In the study of mouse sperm cryopreservation, the kinematic parameters in addition to velocity average path, linearity and straightness of sperm showed significant decrease when compared to that of the fresh group(P≤0.05). Compared with the group without adding melatonin frozen (M0), the total motility of sperm was significantly increased in M1 group and M2 group (P≤ 0.05),the velocity curved line of sperm in M1 group, M2 group, M3 group and the velocity straight line of sperm in M2 group and M3 group were all improved dramatically (P≤0.05). The sperm beat cross frequency of M2 group and the amplitude of lateral head displacement of M3 group were significantly higher than those in M0 group (P≤0.05), it is noteworthy that the progressive motility of sperm was significantly increased only in M1 group(P≤0.05). The percentage of viable sperms with ROS in M1,M2 and M3 group was less than MO group. Therefore, the M1 group of freezing extender was selected as the semen freezing protection liquid for subsequent experiments in mice.(2) The M1 group of freezing extender was used for mouse sperm cryopreservation, the pro-apoptotic gene box of sperm after thawing did not change significantly, but the anti-apoptotic gene bcl-xl mRNA expression was markedly improved (P≤0.05).(3) In the reserch of boar semen cryopreservation, compared with the fresh sperm, the density, motility, acrosome integrity rate, membrane integrity were decreased significantly (P≤0.05), and malformation rate of freezen-thawed sperm was increased markedly (P≤0.05).The thawing solution was added with caffeine sodium benzoate could significantly improve sperm motility after freezing (P≤0.05), the other quality indicators were also improved, but did not reach significant levels.Thus, adding 0.125 mg/mL melatonin into freezing extender during mouse sperm freezing process can inhibit oxidative damage, enhance the expression of anti-apoptotic genes, thereby improving the sperm quality after thawing. Caffeine sodium benzoate was added to improve the frozen-thawed quality of pig sperm, especially significantly improved the survival rate, can be considered as one of the additives in the future boar semen cryopreservation. |
PDF Full Text Request