Study On Parameters Optimization And Application Of Cryopreservation Of Jiangquhai Boar Semen

The long-term conservation of mammalian semen is paid more and moreattention in animal resource protection,good male animal utilization,farm biosecurity and cost saving,etc.Currently,a breakthrough has been made in the protocols for semen freezing of many animals.And most of them were successfully used in human,canine and cattle.However,the cryopreservation of boar semen still have some problems.Therefore,it is necessary to establish an effective cryopreservation protocols for boar semen.In this study,Jiangquhai boar semen was cryopreserved with 0.25 ml straw,aiming at studying the effects of freezing and thawing on survival and developmental competence.Based on the formed freezing protocol,the enzymatic activity,the ultrastructure and the differential proteins were researched in order to clarify the cryo-damage mechanisms.At the same time,freezing protocol was be used in artificial insemination.This study contained the following 5 parts.1.Effects of cryopreservation on the quality of boar semenThe purpose of the experiment was to optimize the method to cryopreserve boar semen.Firstly,to explore the correlation of evaluation methods on boar semen quality,the motility,PI staining,hypotonic swelling test,Rh123 staining and FITC-PNA staining of boar sperm were checked,The results showed that various indicators had strong linear correlation with each other,and the correlation degree between acrosome integrity and IVF rate was the highest.Secondly,the propose was to observe the effect of cryopreservation on the Jiangquhai boar semen treated with different cryoprotanctant,0.3%dimethylsulfoxide(DMSO),propanediol(PDO),ethylene glycol(EG)and glycerine were added into the diluent,respectively.The results showed that glycerine gave the best results,and there were significant difference between the glycerine group and other two groups(DMSO group and PDO group)not only in post-thaw motility rate,but also in low membrane damage rate,acrosome intact rate and activity of mitochondria(P<0.05).But there were no statistical significant between the glycerine group and EG group(P>0.05).Thirdly,the aim was to probe the effect of cryopreservation on the Jiangquhai boar semen treated with different rang-height from liquid nitrogen(LN2),semen was cryopreserved.When rang-height from LN2 was 9 cm,there were worse freezing-thawing results than 2 cm and 5 cm rang-height(P<0.05).Compared rang-height 2 cm with 5 cm,there were no significant difference in motility,membrane damage rate and mitochondrial activity between them(P>0.05),besides acrosome intact rate.Forthly,the aim was to explore the effect of cryopreservation on the boar semen treated with different thawing methods,boar semen was thawed with 37 ? water for 30 s Vs room temperature for 20s then 37 ? water for 10 s.The results showed that the former was better than the latter in the motility,plasmalemma intact rate,acrosome intact rate and in vitro fertilization embryonic development.At last,to investigate the effect of supplementing extended boar semen with different amounts of reduced glutathione(GSH,1,3,5,7 and 9mmol/L),hyaluronan(HA,300,600,900 and 1200 ?g/mL)and combination of GSH and HA prior to freezing on post-thaw sperm characteristics(motility,intact acrosome rate,cleavage rate of in vitro fertilization embryo,SOD activity and MDA content).The results showed that 5mmol/L group has the best effects among GSH groups.There were distinct differences between 5mmol/L GSH group and free GSH group(P<0.05)in motility,intact acrosome rate,SOD activity and MDA content except for the cleavage rate.900?g/mL HA group had better effects than other HA groups.Except cleavage rate had no statistical difference,there was significant difference(P>0.05)between 900 ?g/mL HA group and free HA groups in motility,intact acrosome rate,SOD activity and MDA content.The effect of associated addition of 5mmol/L GSH and 900?g/mL HA was better than other combination of GSH and HA groups.And there were significant difference between it and without GSH and HA group about all the indexes.We suggest that combination of 5 mmol/L GSH and 900 ?g/mL HA can effectively improve the quality of frozen-thawed boar semen.2.Effects of cryopreservation on the enzymatic activity of boar semenIn order to clarify the changes of the enzymatic activity,fresh and frozen-thawed boar semen were taken as an object of study.The activity of the antioxidases(including superoxide dismutase SOD,glutathione reductase GR,catalase CAT),functional enzymes(Hyaluronidase(HYD),acrosome enzyme(ACE)and metabolic enzymes(acid phosphatase(ACP),ATPase creatine kinase(CK))were detected.The results showed that the activity of other enzymes all decreased obviously(P<0.05),besides the activity of GR enzyme increased significantly(P<0.05).The experiment indicates the cryopreservation destroys the enzyme system of the Jiangquhai boar semen,and lowered enzyme activity may be one of the reasons why the quality of the boar semen thawed reduces.3.Effects of cry opreservation on the ultrastructure of boar semenTo investigate the ultrastructural changes of ciyopreserved Jiangquhai boar sperm.Based on fresh and cryopreserved sperm,samples of SEM and TEM were prepared respectively.The results showed that the length of the sperm in Jiangquhai boar was about 54.4?m.It is composed of 4 parts,including head,neck,body and tail.The normal sperm is with intact membrane,acrosome and mitochondria,smooth acrosome,aligned mitochondria,homogeneous chromatin.There were different degrees of damage in thawed sperm:neck ruptured or separated,acrosome lost,membrane expanded or lost,and mitochondria dislocated.In conclusion,cryodamage mainly appears in the member,acrosome and mitochondria.4.Effect of cryopreservation on differential proteins of boar spermIn order to reveal the cryodamage mechanism of Jiangquhai boar sperm,differential expression proteins between fresh and frozen-thawed sperm were studied by using an approach of proteomic methods.Fresh and frozen-thawed sperm were collected.The expressed proteins were detected using two dimensional gel electrophoresis(2-DE)technology.All expressed proteins of before and after frozen-thawed sperm were analyzed by MALDI Tof/Tof mass spectrometry.The mass spectrometry data was analyzed by Mascot and subjected to biological information analysis by blast2go.A total of 42 differentially expressed proteins were revealed by spectrum analysis as well as MASCOT online retrieval.These proteins are involved in 7 biological functions including binding,catalytic activity,structural molecule activity,molecular function regulator,antioxidant activity,nucleic acid binding transcription factor and transporter activity.According to the result of GO and KEGG database,15 proteins were identified as meaningful differentilally expressed proteins.5 differential zymoproteins were searched,including like 14 kDa phosphohistidine phosphatase-like(like-PHP14),enolase(ENO),dihydrolipoyl dehydrogenase(PDC3),glutathion peroxidase(GSH-Px).and proteasome subunit beta type-5,and they were all reduced in boar sperm after being frozen-thawed;6 differential fertilization-related proteins were searched,inculding acrosin-binding protein,carbohydrate-binding protein AQN-1 precursor,voltage-dependent anion-selective channel protein 2(VDAC2),zinc finger protein(ZFP),lactadherin precursor,phosphatidylethanolamine-binding protein 4,and the others all reduced expect arbohydrate-binding protein AQN-1 precursor;4 differential cytoskeleton-related proteins were searched,including F-actin-capping protein subunit beta variant ?,cytoplasmic dynein 2 heavy chain 1-like,Beta-tubulin and outer dense fiber protein 2,and they were all reduced expect F-actin-capping protein subunit beta variant ?.The results indicate there lie differential proteins between fresh Jiangquhai boar sperm and frozen-thawed Jiangquhai boar sperm,which are classified as 3 kinds of proteins,zymoproteins,function-related proteins and cytoskeleton-related proteins.In brief,freezing and thawing have impacts on energy metabolism,cytoskeleton,fertilization and oxidation resistance of Jiangquhai boar sperm.The research provide the foundation for researching proteomics about boar sperm and reveal the cryodamage mechanisms of boar semen.But it need more research to explore the effects of differential expression proteins on sperm function and embryo development..5.Application on artifical insemination of frozen semen in Jiangquhai boar0.25 mL straws frozen Jiangquhai boar semen were produced to observe the result of artificial insemination(AI).The results showed that fresh semen had obviously better effective than frozen semen(P<0.05).For fresh semen part,there were no marked difference of between conventional AI(CAI)and intrauterine insemination(IUI)in pregnacy rate(PR),farrowing rate(FR)and total number of piglets per litter(TNB),P>0.05.For frozen semen part,there were statistical difference between CAI and IUI in PR and FR(P>0.05).But there were significant meaning between them in TNB(P<0.05).