| Cryopreservation of sperm,in vitro maturation of oocytes and in vitro development of embryos are the technical basis for artificial breeding of animals.Improvement of sperm motility after freezing and improvement of oocyte maturation and embryo quality in vitro are of great significance for animal production.Melatonin?melatonin,MT?synthesized mainly by the pineal gland,and is closely related to reproductive behavior and endocrine regulation.Melatonin and its metabolites can directly scavenge reactive oxygen species?ROS?,increase glutathione?GSH?level and inhibit the activity of cell stimulating enzymes.Melatonin is one of the most effective substances to reduce cellular oxidative damage.In this paper,the effects of melatonin on sperm freezing,oocyte maturation in vitro and in vitro fertilization and embryo development were studied.The results are as follows:1.The sperm motility,plasma membrane integrity,mitochondrial membrane integrity,acrosomal integrity and ROS level produced during cryopreservation were studied by adding different concentrations of MT(0,10-3,10-44 and 10-5)to semen freezing dilution of three different Angus cattle.It was found that concentrations of MT at 10-3M and 10-4M significantly increased the vitality,plasma membrane integrity,and mitochondrial membrane integrity of Angus cattle sperm.Compared with the control group,the acrosomal integrity of the spermatozoa significantly increased when 10-3M MT was supplemented The addition of 10-3M MT reduced nearly 2 times ROS level in comparison to the control.Therefore,addition of 10-33 M MT is the best concentration in frozen semen.2.The effects of different concentrations of MT(0,10-7,10-8,10-9,10-10)on the maturation of oocytes and the effect of MT on the intracellular reactive oxygen species?ROS?level were investigated.The results showed that the addition of 10-77 M MT has a significant increase in the maturation rate of oocytes,and the ROS level reduced 2.4 times to the control.3.The effects of 10-77 M MT on embryonic cleavage,blastocyst formation,apoptosis and development-related gene expression were investigated.The results showed that the addition of MT at 10-77 M significantly increased the cleavage rate,blastocyst rate and blastocyst cell number The treatment of 10-77 M MT reduced the ROS level by 1.6-fold to the control at the 8–cell stage;and significantly increased the antioxidant and anti-oxidant activity at the 8-cell embryos.The expressions of the genes associated with cell apoptosis?CAT,BCL-2?and development-related genes?C23,MFN1?significantly increased,while the expressions of the pro-apoptotic genes?Caspase-3,BAX?significantly decreased.In summary,the effects of melatonin on Angus cattle semen cryopreservation,oocyte maturation,in vitro fertilization and embryo development were comprehensively evaluated,the melatonin have different actions on sperm freezing,oocyte maturation and fertilization,and embryo culture at different concentrations.Supplements of melatonin at 10-33 M concentration improved spermatozoa survival;addition of melatonin at 10-7 M concentration increased the maturation of oocytes and embryo development after in vitro fertilization. |
PDF Full Text Request