Preservation Technology And Cryodamage Mechanism Of Sperm In Equine

Horse is one of the main livestock,has been closely related to human life,the weakening of horse working value with the rise of industrial machinery,and gradually fade out of people’s lives.In recent years,with the improvement of people’s living standards,the modern horse industry,take culture and entertainment as a sign,quietly rising in our country.The development of the modern horse industry urgently needs to breed horse by artificial insemination,however,preservation of semen in vitro is a technical bottleneck in the application of artificial insemination of horses in China.In odor to solve this problem,we have carried out a series of experiments and obtain the following results:（1）Semen were place in INRA96? or INRA82 extender,which p H was adjusted to 6.5,6.7,6.9,7.1,7.3 and 7.5,evaluate the quality of semen storage 4℃ after 1,24,48 and 72 h.The Results show that post storage 48 h the total mobility in INRA96?,which p H were 7.1 and 7.3,were 81.33 ± 1.15 and 80.33 ± 4.04 significantly higher than p H 6.5（72.67 ± 3.81）and 7.5（73.00 ± 4.58）group（p <0.05）,and no significant difference with other group;the total mobility in INRA82,which p H were 6.9,were 74.75 ± 3.51 significantly higher than p H 6.5（67.75 ± 3.14）and 7.5（52.50 ± 1.53）group（p <0.05）,and no significant difference with other group.Our data suggestion that the most suitable p H is between 6.9-7.3 for cooled storage hose semen,When the p H is lower than 6.7 or higher than 7.3 will be detrimental to the preservation of sperm.（2）To further optimize the horse sperm freezing methods,we investigated the impact of the equilibrium time,freezing and thawing method for sperm motility,membrane integrity and mitochondrial membrane potential after freeze-thawing.The results show that thawing sperm motility and membrane integrity of the equilibrium 120,180 and 240 min group was significantly higher than the equilibrium 0,45,90 min and 8 h group;the liquid nitrogen steam freezing method,which the distance of semen from liquid nitrogen surface 2cm and 4cm obtained similar results with program freezing method;high temperature rapid thawing method（75 ℃,7s and 46 ℃,20s）gained higher post-thaw motility than the conventional thaw method（37 ℃,30s）.In summary we believe that combination of equilibrium 120²40 min,liquid nitrogen steam frozen of 2cm and 4cm surface from liquid nitrogen and high-temperature rapid thawing method（75 ℃,7s and 46 ℃,20s）can get better sperm frozen effect in horse sperm freezing process.（3）In order to clarification the impact of seminal plasma concentrations on the quality of cooled stored and frozen-thawed equine semen.We select 4 4-12 years old thoroughbred stallions,collect semen and place in extender containing different concentrations of seminal plasma to store or freeze,and then evaluate the quality of semen after storage 1,24,48,72 and 96 h or thawing.The results showed that TM,PM,RAP and VAP of addition of 30% and 40% seminal plasma were significantly lower than 0%,10% and 20% group P <0.05),TM of addition of 10% seminal plasma was significantly higher than 0% and 20% group after cooled-stored 24 h,while the control group（0%）was significantly higher than 10 % after cooled-stored 96 h.The effect of addition of seminal plasma on cryopreservation showed dose-dependence,Accompanied by increasing concentration of seminal plasma post-thaw TM,PM,RAP and viability gradually decreasing.（4）We investigated the effects of five cryoprotectants（CPAs）and cryoprotectant combinations on the post-thaw motility,progressive motility,viability,mitochondrial membrane potential and defective acrosomes in stallion sperm.In experiment one,our objective was to compare the impact of different concentrations（2.5,3.5 and 5%）of a single CPA,including glycerol（Gly）,ethylene glycol（EG）,dimethyl sulp Hoxide（DMSO）,methyl formamide（MF）,and dimethylformamide（DMF）for horse sperm cryopreservation.In experiment two,to determine whether the use of two or more CPAs can improve post-thaw sperm quality,Gly,MF and DMF,which showed good results in experiment 1,were used to prepare seven combinations of freezing liquid with different mixtures of cryoprotectant.The 3.5% Gly,MF and DMF were used as a control group.The results show that post-thaw motility,progressive motility,viability,and mitochondrial membrane potential for all concentrations of EG and DMSO were lower than the 3.5 and 5% Gly and MF and 2.5 and 3.5% DMF（P<0.05）.The 3.5% concentration achieves a higher post-thaw motility and progressive motility than the 2.5% and 5% concentrations for all CPAs.The results of the combination cryoprotectant experiments show differences in progressive motility and viability.The viability in the Gly（2/3）+MF（1/3）was 44.65% and was higher than the Gly（1/3）+MF（1/3）+DMF（1/3）（30.96%）,MF（2/3）+DMF（1/3）（35.05%）,Gly（32.21%）,and MF（33.76%）（P<0.05）.The progressive motility in the MF（2/3）+ Gly（1/3）was 36.00% and was higher than in the DMF（25.00%）and MF（2/3）+DMF（1/3）（22.67%）（P<0.05）.These results suggest that using the appropriate cryoprotectant combination instead of a single cryoprotectant improves horse sperm cryopreservation.（5）The effect of different yolk components（yolk plasma proteins,low-density lipoprotein and yolk soluble protein）,soy lecithin and cholesterol and β-cyclodextrin on Eqine sperm cryopreservation.The results showed that yolk plasma proteins,low-density lipoprotein and Livetin have a significant cryopreservative function,but no significant difference be observe in different components of the yolk and whole egg yolk;Addition of 1% soy lecithin obtained similar cryopreservation result with the control group（4% egg yolk）（P> 0.05）,but Post-thaw TM,PM and RAP supplemented with 2% and 4% soy lecithin group were significantly lower than the control group（P <0.05）;there is no significant difference in different concentrations of β-cyclodextrin-cholesterol group and the control group thawed sperm Motility（P>0.05）（6）Five kinds of sugars（glucose,fructose,lactose,trehalose and raffinose）supplementation in INRA82 extenders on the quality of cooled stored and frozen-thawed equine semen.Semen samples were collected from 4 stallions,and diluted into extender contented different sugar to cold store or cryopreservation.Evaluated total motility and progressive motility after cold-storage 1,24,48,72 and 96h;thawing TM,PM,viability and mitochondrial membrane potential of semen were evaluated.The results showed that the semen TM and PM of supplementation of lactose and raffinose respectively was significantly lower than supplementation of glucose,fructose and trehalose group（P <0.05）after cold storage 24 h,there were no significant difference among glucose,fructose and trehalose group.No significant in difference sugar combinations after cold storage 1,24,48,72 and 96 h.The sperm TM and PM supplementation of raffinose was significant lower than glucose,fructose,or trehalose group after freeze-thawing,but the viability and mitochondrial membrane potential were significant higher than other group;no significant difference in TM PM and mitochondrial membrane potential among supplementation of glucose,fructose,lactose,trehalose group;the thaw viability of addition fructose group was significant lower than supplementation glucose group;the thaw sperm viability of supplementation of lactose+glucose +trehalose group was significantly higher than（P<0.05）Lactose + fructose,lactose + trehalose,lactose + glucose + fructose,and lactosse+fructose + trehalose groups（P <0.05）,the thaw sperm viability of supplementation of fructose+ lactose was significantly lower than other group except lactose + trehalose（P <0.05）.the thaw sperm mitochondrial membrane potential of supplementation of Lactose + glucose + fructose was significantly higher than fructose+lactose group（P <0.05）.（7）To asses whether the skim milk or native non-soluble milk fraction of routinely extenders can be replaced by solubility sodium caseinate（SC）and whey protein（WP）.INRA96 was as the standard reference extender,moreover,milk-free INRA82（salts citrate）and milk-free INRA96（Hank‘s）were as base extenders.The results show（Ⅰ）addition of WP and SC alone can play a positive role for sperm cool storage,but the data was variation when using different base extender and with or without SP;simultaneously adding SC and WP in base extender could gain a stable preservation effect;（Ⅱ）salts citrate was found more suitable for combined with SC+WP than Hank’s;（Ⅲ）the optimal concentration supplementation with SC and WP was 40g/L and 10g/L;（Ⅳ）post-thawing VAP,VSL and BCF values of semen frozen with treatment extender significantly higher than commercially extender.（8）Evaluate the effects of 8 anti-oxidants,namely,glutamine（5m M）,glycine（10m M）,L-cysteine（2.5m M）,taurine（25m M）,Vc（0.4mg/m L）,Ve（0.5mg/m L）,melatonin（0.001mg/m L）and methionine（2.5m M）in INRA82 on equine sperm quality after chill or freeze-thaw.The extender supplemented with 25 m M taurine and 0.5mg/m L Ve led to higher TM（78.00±3.00%）and PM（38.33±6.81%）,when compared to the control group（P < 0.05）after storage 5℃ for 48 h.However,the MT in INRA82 with 2.5m M L-cysteine is lower than control after storage 5℃ for 48 h（P < 0.05）.No significant differences were observed following the supplementation of the extender with 5m M glutamine,10 m M glycine,0.4mg/m L Vc,0.001mg/m L melatonin or 2.5m M methionine.In experiment two,The freeze extender supplemented with 0.5mg/m L Ve or 2.5m M methionine significantly increasing the mitochondrial membrane potential compare to the control（P<0.05）.No significant differences were observed for motion patterns,PMI and mitochondrial membrane potential of frozen–thawed equine spermatozoa in extender containing other antioxidants.（9）Evaluate insect AFP at 0（control）,0.1,1,10 and 100 μg /m L for its effect on post thaw sperm quality.The results showed that in the 1μg/m L Mp AFP treatment group the post-thaw RAP,PM and PMI are 58,42,and 44.65% for adding 1μg/m L AFP respectively,significantly higher than control group 49.33,37.33 and 32.21% in the control.However,treatment with 100μg /m L Mp AFP showed lower TM,PT,RAP,PMI and mitochondrial membrane potential than the control.Our study firstly provides the evidence that insect antifreeze protein Mp AFP698 supplemented with semen extender can improve post-thaw sperm quality in equine at low concentrations.（10）In order to further determine the effect of the extender,animal experiments were conducted.The results show that 601 mares the cycle pregnancy rate of cooled storage semen using our exrtender are 63.39%,similar with INRA96?,and the cycle pregnancy rate of thawing semen reached 50%（15/30）.（11）Using iTRAQ technique to detect the protein expression difference between fresh and thawing semen,and the differential protein was analyzed by bioinformatics.Each group of samples was set up two duplicate samples,and the data obtained from the mass spectrometry analysis were searched by Mascot 2.2 software in the Uniprot_equuscaballus₂8004 database.A total of 2073 proteins were identified.Meet the difference times greater than ± 1.5,P<0.01 conditions,differentially expressed proteins number of 3 groups were 112（thaw VS.fresh）,90（half-life thaw VS.thaw）,128（half-life thaw VS.fresh）respectively.