| Cryopreservation of pig semen has great potential for long-term preservation,long-distance transportation,breed improvement,etc.It is also an important tool for assisted reproductive technology and germplasm storage.The application in cattle and other species has gradually matured,and plays an important role in daily production and breed improvement.While in pigs,only 1% of all artificial inseminations conducted worldwide are made using frozen–thawed boar sperm.Based on the current research,it is believed that freeze-thawing procedures may strongly impair the sperm function and survival and thus decrease the reproductive performance.Such cryodamage is largely due to the high sensitivity of boar sperm to cold shock and peroxidative damage because of the composition of their plasma membrane,which contents high levels of unsaturated phospholipids and low cholesterol levels.In response to this phenomenon,some methods have been made to improve cryopreservation efficiency.However,Its display different efficiency among various species.There are still many methods that are applicable to semen of other species such human mouse sheep cattle,etc.while not suitable for pig semen cryopreservation.To better improve the efficiency of semen cryopreservation,the species specificity should be considered.Based on the study of other species,it is also necessary to deeply analyze the specific freezability mechanism in pigs.In this study,we conducted an in-depth study on the mechanism of pig sperm cryopreservation injury,using sperm plasma membrane lipidomics analysis and sperm/seminal plasma metabolomics methods to explore the mechanism of pig sperm freezability and identification biomarkers related to freezability.In this study,we analyzed the semen from common pigs such as Yorkshire and Landrace,and10 semen with good and poor freezability were screened according to the quality of sperm after thawed.Through quality analysis of thawed sperm,it was found that compared with fresh sperm,the sperm motility,mitochondrial activity,and plasma membrane integrity after thaw decreased significantly,and the level of ROS increased significantly.At the same time,compared with the good freezability sperm,the ROS level in the sperm with poor freezability group increased significantly,and the integrity of the plasma membrane decreased significantly.In order to further analyze the detailed mechanism of this difference in freezability,this study this shtudy is based on the combination of ultra high-performance liquid tandem chromatography quadrupole time of flight mass spectrometry,technology(UHPLC-QTOFMS),combined with metabolites analysis methods in sperm with different freezability.According to the identification,a total of 442 metabolites were detected in sperm.We analysis these metabolites with OPLS-DA model,it was determined that the contents of oleic acid,2,4-dinitrotoluene,4-hydroxyl-6-methylpyran-2-one,aurapten,N8-acetylspermidine,oleamide these metabolites have significant differences between sperm with good and poor freezability.According the KEGG analysis found that these metabolites are mainly involved in fatty acid biosynthesis and biosynthesis of saturated fatty acids,present studies has shown that oleic acid can interact with lipids in the plasma membrane to regulate the fluidity and permeability of the plasma membrane,and at the same time can be used as an energy substrate to regulate sperm motility.It is inferred from this rusult that these metabolites will directly or indirectly affect the freezability of sperm,and the plasma membrane may play an important role.In order to explore the relationship between sperm plasma membrane composition and sperm freezability,this shtudy is based on the UHPLC-QTOFMS technology,combined with lipidomics analysis methods,to analysis fresh sperm,and sperm with good and poor freezability.The composition of the plasma membrane of sperm is analyzed.Through mass spectrometry data collection,a total of 225 types of sperm membrane quality were identified.Results showed that compared with fresh sperm,in the frozen sperm plasma membrane,the content of cholesterol,phosphatidylcholine,Lysophosphatidylethanolamines and Lysophosphatidylcholines increased significantly,and the content of sphingomyelins decreased.After thawed,the content rate of glycerophospholipid in the sperm plasma membrane increased from 77.6 to 91.2%,in which the content rate of phosphatidylcholine increased from 52.35% to 74.21%,and the content rate of sphingolipid component(including ceramide and sphingomyelin)decreased from 20.9% to 3.7%.After thawed,the plasma membrane composition of the sperm tends to become more simplification.It can be inferred that the cryopreservation process causes the sperm plasma membrane composition to tend to be simplification,which may be affected a potential factor of sperm quality after thawed.In addition,by comparing the plasma membrane components of sperm withgood and poor freezability,it was found that dihydroceramides(18: 0/18: 0),4 kinds of hexosylceramides(18:1/20:1,18:0/22: 1,18:1/16:0,18:1/18:0),lactosylceramides(18:1/16:0),2 types of lysophosphatidylethanolamines(18:2,20:2),5 kinds of phosphatidylcholines(16:1/18:2,18:2/16:1,14:0/20:4,16:0/18:3,18:1/20:2).Two kinds of phosphatidylethanolamines(14:0/20:4,18:1/18:3)have significant differences between sperm withgood and poor freezability,interestingly except(140/20:4)similar the fatty acid structure of phosphatidylcholine and phosphatidylethanolamine were higher in the low freezability group,the other lipid content was positively correlated with freezing resistance.In order to further judge the state of the sperm plasma membrane,this study uses scanning electron microscopy,transmission electron microscopy techniques to observe the morphology of the plasma membrane from the sperm head,middle and end piece.The results show that compared with fresh sperm,the plasma membrane in the end piece of frozen spermatozoa has a higher proportion of abnormalities,and plasma membrane of each piece of the high freezability sperm is better than that of the low freezability sperm.Among them,the plasma membrane of the head and the middle piece of the low freezability sperm is more damaged.Interesting,Comprehensive metabolome analysis,lipidome analysis and electron microscopy observation can infer that the difference in the metabolism level of the spermatozoa with different freezability is likely to affect the composition and physical and chemical properties of the plasma membrane,which in turn affects the freezability of the sperm.In addition to the level of sperm metabolism,the composition of seminal plasma also has an important effect on the freezability of sperm.Seminal plasma is a mixture mainly derived from the secretion of the testis,epididymis and accessory glands.On the one hand,its components can reflect the physiological state of the individual,and on the other hand,as the external environment of the sperm,it participates in the transportation of sperm and affects the sperm quality and freezability.This study started with seminal plasma metabolites and used metabolomics to screen the differences in seminal plasma composition ofgood and poor freezability semen.Using UHPLC-QTOFMS technology,a total of 953 metabolites were identified with seminal plasma in this experiment.Through OPLS-DA modeling and screening,the content of 50 metabolites are significant differences betweengood and poor freezability groups was finally determined.These candidate biomarkers are mostly involved in the biosynthetic metabolic pathways to amino acids such as alanine,aspartic acid,glutamic acid,arginine,proline,cysteine,and methionine,which implies that individual amino acids The level of metabolism may affect the freezing tolerance of sperm.In order to further to confirm and screen the freezing tolerance biomarkers in seminal plasma,we select 12 candidate biomarkers related to amino acid metabolism for verification by targeted metabolomics,and finally determine D-aspartic acid,N-Acetyl-L-glutamate,inosine content showed significant differences between thegood and poor freezability groups,and their content of seminal plasma was negatively correlated with freezing tolerance.In this study,through routine sperm quality testing combined with analysis methods such as metabolomics,lipidomics,and electron microscopy,it was finally determined that the level of lipid metabolism,plasma membrane composition and seminal plasma in the semen ofgood and poor freezability.There are significant differences in some metabolites related to amino acid metabolism,which provide a new theoretical basis and research ideas for studying the mechanism of sperm freezing tolerance,improving the efficiency of cryopreservation of pig semen,and screening freezing tolerance biomarkers. |
PDF Full Text Request