Effect Of Resveratrol And Melatonin On The Quality And In Vitro Fertilization Ability Of Sex-sorted Holstein Bull Spermatozoa

It is a significant advantage for livestock production to maximize profit by controlling the gender of calves with the use of sex-sorted sperm by Flow cytometry.Meanwhile,sex-sorted frozen sperm plays an important role in the commercial production of sexed embryos.However,the sorting process will remove the sperm plasma that rich in antioxidant enzyme,and leads to the inadequacy of endogenous antioxidant enzyme.Meanwhile,the sperm suffer from oxidative stress and produces excessive reactive oxygen?ROS?,which will destroy the balance of anti-oxidative defense system in sperm,resulting in oxidative stress injury.As a powerful exogenous antioxidant,both melatonin?MT?and resveratrol?Res?can protect sperm availability and fertilization ability of non-sorted frozen sperm as a ROS scavenger.However,little information is still available about the effect of Res and MT on the sex-sorted bull sperm,less about the relative mechanism.Therefore,different concentrations of Res or MT was supplemented in the washing and fertilization medium,and sex-sorted sperm from three individual bulls were washed in washing medium and incubated in fertilization medium for 1.5 h.Then detected the effect of different concentrations of Res or MT to the quality and fertilization capacity of sex-sorted bull sperm,and the optimal concentration of these two kinds of antioxidants in the sex-sorted bull sperm was investigated.Meanwhile,the effect of melatonin receptor on capacity of bovine sex-sorted sperm was investigated by using melatonin receptor inhibitor.The results are as follo ws:1.Five different concentrations(0M,10^-3M,10^-4M,10^-5M,and 10^-6M)of resveratrol?Res?was supplemented in the washing and fertilization medium,and sex-sorted sperm from three individual bulls were washed twice in washing medium and then incubated in fertilization medium for 1.5 h.Then analysised the levels of reactive oxygen species?ROS?,phosphatidylserine?PS?externalisation,mitochondrial membrane potential???m?,ATP and malondialdehyde?MDA?,acrosomal integrity,blastocyst rate,and blastocyst quality after in vitro fertilisation?IVF?.The results for sex-sorted sperm from three bulls showed:?1?ROS and MDA levels in 10^-3 M and 10^-4 M Res groups were significantly lower than those of controls?P<0.05?;?2?the percentage of viable sperm,percentage of sperm with high??m,and the ATP content in 10^-33 M and 10^-4 M Res groups was significantly higher than those of controls?P<0.05?;?3?the percentage of viable sperm with acrosomal integrity,and the blastocyst percentage and quality of the 10^-44 M Res group were significantly higher than those of controls?P<0.05?.In conclusion,10^-4 M Res supplementation in washing and fertilisation medium of sex-sorted bull sperm significantly decreased ROS,PS externalisation,and MDA,and protected mitochondrial function and acrosomal integrity,thereby increasing blastocyst percentage and quality following IVF.2.Five different concentrations of melatonin(0M,10^-33 M,10^-5M,10^-7M and 10^-99 M)was supplemented in the washing and fertilization medium,and sex-sorted sperm from three individual bulls were washed twice in washing medium and then incubated in fertilization medium for 1.5 h.The n reactive oxygen species?ROS?,MDA level and endogenous antioxidants levels?glutathione peroxidase,GPx;superoxide dismutase,SOD;catalase,CAT?,apoptotic events?phosphatidylserine externalization,PS externalization;mitochondrial membrane potential,??m?,acrosomal integrity,capacitation events(calcium ion level,Ca²⁺level;cyclic adenosine monophosphate,cAMP level;capacitation level)and fertilization ability of these sperm were examined.The results for sex-sorted sperm from three bulls showed:10^-55 M MT can significantly decreased ROS level and increased GPx,SOD and CAT levels of sex-sorted bull sperm,which inhibited PS externalization and MDA levels,improved??m,acrosomal integrity,capacitation events and fertilization ability.3.The presence of melatonin type I receptor?MT1?and type I receptor?MT2?of bull sex-sorted sperm was determined by western blot.And according to the experiment,10^-5 M MT can effectively improve the fertilization ability.4-P-PDOT?selective MT2 antagonist?or Luzindole?nonselective MT1/MT2 antagonist?was supplemented in washing and fertilization medium with 10^-5 M melatonin to investigate the involvement of melatonin receptors on capacitation capacity of bovine sex-sorted sperm.The results showed:MT1 was proven to involve in the capacitation regulation of sperm by melatonin.In conclusion,this study found that both 10^-44 M Res or 10^-55 M MT can significantly improves the fertilisation capacity of bovine sex-sorted semen by remove ROS,inhibiting apoptosis and lipid peroxidation,protecting the structure and function of sperm,significantly improving the fertilization ability of the bull sex-sorted sperm.In addition,this study also shows that melatonin besides antioxidant effect,MT1 was also proven to involve in the capacitation regulation of sperm by melatonin through a series of signal transduction pathways.These findings contributed to improve the fertilization capacity of sex-sorted bull sperm and exploring the relative approachs.