| Boar semen cryopreservation technology is the key auxiliary technology to improve the output value of pig breeding industry and reduce the risk of breeding.However,due to the gap in breeding data such as average litter size between frozen semen and fresh semen,the actual production and application of frozen semen in artificial insemination is greatly limited.The key problem is that boar sperm is sensitive to low temperature damage,and the mechanism of boar sperm damage caused by frozen resuscitation is not perfect,so it is difficult to optimize it specifically.At the same time,vitrification of semen is becoming another hot research direction in semen freezing technology.At present,the vitrification freezing procedure of human sperm has been gradually improved,and the freezing technology is becoming mature.The vitrification procedure of sperm from dogs,horses,donkeys,sheep and other animals has also achieved good results.However,so far,there is no perfect scheme for the vitrification and freezing of boar sperm.Only sporadic studies have shown that the chromatin integrity of boar sperm after vitrification is not significantly different from that of fresh sperm,which can better preserve the genetic information of boar sperm.Therefore,this study using the metabolome and proteomics analysis and molecular experiments,to explore the mechanism of frozen recovery to boar sperm injury,and optimize the frozen system,evaluate the influence of frozen on boar sperm,and compared with the influence of slow freezing,the main test results are as follows:1.Slow thawing damage boar sperm mitochondria cause ROS leakage,the free energy driven ROS to eliminate is suppressed is the key to sperm deathUsing Hoechst33342 with propidium iodide(propidium iodide,PI),DCFH-DA staining and the change of sperm quality before and after slow freezing by JC-1 staining,we found that the sperm plasma membrane was damaged after freezing and thawing,and the mitochondrial membrane potential level decreased with the increase of reactive oxygen species.The addition of iron chelator mesylate decreased(P<0.05)and ROS decreased(P<0.05),but failed to prevent sperm death;the addition of GPX4 analogue EBsen decreased(P<0.05),but could not prevent sperm death.Later,sperm from high and low vitality groups after freezing and thawing were selected as the research objects for metabolome sequencing,and the selected differential metabolites were enriched and analyzed.The results showed that in the process of freezing and thawing,the change of sperm motility was reflected in the elimination driven by free energy in mitochondria.To test this point,Of boar sperm ATP levels before and after freezing and thawing,We found that the ATP level of sperm decreased significantly after freezing and thawing(P<0.05);At the same time,the amount and ratio of NADP~+and NADPH in the four groups of fresh concentrate group,high vitality group after freezing and thawing group,and low vitality group after freezing and thawing group were tested,Found that in the low vitality group compared to the fresh concentrate group,The ratio of NADP~+and NADPH increased significantly(P<0.05),Significant decrease in NADPH content(P<0.05),This was further exacerbated by the addition of Ebselen treatment in the low vitality group,Indicating that the free energy driving force required for ROS elimination at this time,Making it difficult to convert NADP~+to NADPH,Validated the results of the sequencing analysis,That is,the suppression of free energy-driven ROS elimination is critical for sperm death.2.The method of metal surface was explored and the corresponding freezing scheme was optimizedIn the optimization of boar sperm vitrification freezing system,initially using sphere method,film method and metal surface method as glass freezing boar sperm method,95glass freezing scheme designed based on the three methods,the results show that only one based on metal surface method vitrification freezing boar sperm success in surviving sperm.Then on the basis of the successful scheme,the vitrification cryodilution,freezing before incubation time,resuscitation procedures and frozen storage time optimization,the final vitrification freezing scheme can make the recovery after sperm activity rate up to 9.00±0.61%,compared with the current published research scheme,the construction of boar sperm vitrification freezing system has a certain perfect.3.Vitrification can avoid the metabolic disorder of boar sperm caused by freezing and thawing to some extentTo assess the effect of vitrifreezing on boar sperm,DCFH-DA staining and JC-1staining showed that the ROS level was significantly lower than the slow freezing group(P<0.05),the mitochondrial membrane potential was significantly higher than the slow freezing group(P<0.05),and the ROS and mitochondrial membrane potential levels of boar sperm after MSV freezing did not change significantly compared with fresh boar sperm.Metabolomic analysis of the vitrification group and the slow freeze-thaw group and the low motility group found that the metabolites in the vitrizing group were more similar to the slow freeze-thaw group.When the differential metabolites from the glass-frozen group and the slow freeze-thaw boar sperm were selected for analysis,the function of the enriched metabolites in the sperm was related with free energy-driven ROS elimination,which was further clarified by the analysis of proteomological differential metabolites.Then,the amount and ratio of NADP~+and NADPH in the fresh sperm group,vitrification group and slow frozen sperm group were detected respectively,and the ratio of NADP~+,NADP~+and NADPH also increased significantly(P<0.05),but the difference was that the difference of NADPH content in the vitrification frozen group was not significant compared with the fresh concentrate group,indicating that the free energy driving force required for the elimination of reactive oxygen species was maintained and NADP~+was normally transformed to NADPH.In conclusion,this study found that the cryogenic injury caused by slow freezing and thawing may be related to mitochondria-induced reactive oxygen species leakage,especially related to the decreased efficacy of mitochondria-derived free-energy driven ROS elimination process.However,after the metal surface-based vitrification scheme optimized in this study,the survival rate of pig sperm after resuscitation reached 9.00±0.61%,which showed some improvement compared with the previous vitrification scheme of pig sperm.At the same time,compared with slow freezing and thawing,it can avoid mitochondrial damage to a certain extent,and maintain the free energy driving force required for reactive oxygen species elimination,and avoid the in-vivo imbalance of ROS in sperm.The healthy metabolic pattern of pig sperm after vitrification and thawing makes the vitrification and freezing of pig sperm have some potential application prospects. |
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