| Background: Human reproduction insurance and assisted reproductivetechnology (ART) develop and complement each other. Sperm bank can provideinsurance to male reproduction .The tendency of future reproductive medicinemust be the establishment of various reproductive strong room. Freezing techis one of the key points. It is reported that there are 10 percent infertilecouples in the world. Basically ,40percent are caused by male factors..Recently, more infertile couples want to use ART to become pregnant .Spermfreezing is a powerful and important tech but it can make damage tospermatozoa. Cryopreservation protective media (CPM) is the crucial agent forthe success of sperm preservation in ultra low temperature. Many kinds of CPMhave been reported but the findings are going to be discussed. Single glycerinand GYC are most widely used at home and abroad, they both have two sides.Nowadays, freezing approaches include one step or step by step freezing,computer programmed cooling and so on. The basic methods are rapid freezingand slow freezing. Rapid freezing is based on ice crystal theory. Slow freezing isused to prevent cold shock and achieve cold balance. The mechanism ofcryodamage has not been clarified until now. Recent studies show that it may berelated to active oxygen and lipid peroxidation , antioxidation may have effecton sperm freezing. Findings are different and to be discussed. Thus it isimportant to establish proper cryopreservation method and find optimalprotectant for the study of cryodamage.Objective: Do research on the movement parameter, motility rate, normalmorphous ratio, acrosome integrity ,acrosomal enzyme activity, hypotensionswelling rate of sperm tail ,DNA maturation of sperm, function of mitochondria,and the ultrastructure of pre and post freezing sperms. Compare the function ofglycerin and GYC and the effect of rapid freezing and slow freezing on thesperm structure to provide experiment evidence for optimizing cryoprotectantand freezing method. We will further discuss influence of active oxygen andantioxygen on the pre and post freezing sperm and reveal the mechanism ofcryodamage.Methods: Semen analysis is based on the detection manual of humansemen and sperm-cervical mucus interaction(WHO,2001).Samples are healthymen with normal parameters ageing from 24 to 36 without external injury ,genetic disease and sexual disturbance. Medical examination doesn't finddysfunction of testis, epididymis and deferent duct . After sexual abstinence for3 to 7 days, obtain semen by ipsation , liquefy them in 37℃ water bath, freezethe sample whose activity ratio ≥50%,sperm count≥20×106/mL and theamount≥2mL.Every sample is divided into five parts. One is detectedimmediately, two are added GYC separately and the other two are added equaldose of 7.5% glycerin, then do rapid and slow freezing separately. Samplesare thawed after 3 to 6 months freeze. Detect the sperm movement parameter byWeili color sperm mass detection system. Use eosin –Y to detect the motilityrate and hypotension swelling rate. Do morphous analyzation by WLJY-9000morphous detection system and calculate the normal ratio. Apply Pap staining,then analyze the integrity of acrosome under microscope (100x). Detectacrosomal enzyme activity. Mitochondria function is examined by SDHreduction .The degree of DNA mature is detected by fluorescent test. Analyzeultra structure of sperm by ultrathin section under scanning electron microscope.Detect active oxygen content by luminal chemoluminescence.Results: ○1 In GYC and glycerin groups, the percentages of grade a,grade b, grade a+b sperm of rapid and slow freezing were all markedly lowerthan those of sperm before freezing(P<0.01).In GYC group, VCL,VSL,VAP,ALH,STR of sperm were all markedly lower than those of sperm beforefreezing in rapid and slow freezing method(P<0.01, P<0.05). In glycerin group,VCL,VSL,VAP,MAD,ALH,LIN,WOB,STR of sperm were all markedlylower than those of sperm before freezing in rapid and slow freezing method(P<0.05). The motility parameters of rapid freezing were no difference incomparison with slow freezing(P>0.05). In rapid freezing method, thepercentages of grade a, grade b, grade a+b sperm and VCL,VSL,VAP,STRwere all markedly lower than those of sperm before freezing(P<0.01, P<0.05).These parameters of GYC group were markedly higher than those of glyceringroup(P<0.01, P<0.05). In slow freezing method, the percentages of grade a,grade b, grade a+b sperm and VCL,VSL,VAP,MAD,ALH,LIN,WOB,STR were all markedly lower than those of sperm before freezing(P<0.01,P<0.05). The percentages of grade a, grade b, grade a+b sperm and VCL,VSL,VAP of GYC group were markedly higher than those of glycerin group(P<0.01,P<0.05).○2 The viability of sperm after thawing was markedly lower than thatof sperm before freezing(P<0.01). The viability of sperm in rapid freezing wasno difference in comparison with slow freezing(P>0.05). The viability of spermin GYC group was no difference in comparison with glycerin group(P>0.05).○3 In GYC and glycerin groups, the percentages of normalmorphology sperm were markedly lower than those of sperm before freezing(P<0.01). There would be no difference between rapid and slow freezing(P>0.05). There would be no difference between GYC and glycerin group(P>0.05).○4 Arcosomal integrity would be no difference between before andafter freezing(P>0.05). It is the same between rapid and slow freezing, betweenGYC and glycerin group(P>0.05). ○5 Acrosin activity of sperm after thawingwere all markedly lower than that of sperm before freezing(P<0.05). Therewould be no difference of sperm acrosin activity after 1 month, 3 mouths and 6mouths. It is the same between rapid and slow freezing(P>0.05). But, acrosinactivity of GYC group were markedly higher than that of glycerin group(P<0.05).○6 Hypotension swelling rate of sperm tail(Type B～G)afterfreezing were all markedly lower than that of sperm before freezing(P<0.05).There would be no difference between rapid and slow freezing(P>0.05). It is the same between GYC and glycerin group(P>0.05).○7 Themitochondria succinic dehydrogenase (SDH)activity after freezing was nodifference in comparison with before freezing(P>0.05).○8 The degree of DNAmature after freezing was no difference in comparison with before freezing(P>0.05).○9 In transmission electron microscope, the membrane of normalmorphology sperm was integrity. The inner and outer membranes of acrosomewere seen in focus. The karyotheca was integrity. The inner membranes ofacrosome was parallel with the karyotheca. The density of karyon washigher. The chromatin was uniformity. The morphology of mitochondria wasnormal. After freezing and thawing, The inner and outer membranes ofacrosome separated. The part of karyon was sunken. The morphology ofmitochondria was illegibility. 1○0 The ability to product active oxygen ofleukocytes and sperm was no difference in pre and post thawing in backgroundlevel(P>0.05).○11The motility parameters, viability, percentages of normalmorphology sperm, hypotension swelling rate of sperm tail(Type B～G)ofsperm in adding VE,VC,VE+VC was no difference in comparison with controlgroup(P>0.05). There would be no difference among VE,VC,VE+VC group(P>0.05)Conclusion: After freezing, all the parameter such as movementability ,motility rate ,eumorphism ratio, acrosomal enzymatic activity andhypertension swelling rate of sperm tail are lower than unfreezing status. Rapidand slow freezing methods both can be used in semen cryopreservtion withoutsignificant difference in parameters above. GYC is better than glycerin inprotecting the movement ability and the acrosomal enzymatic activity . Lipidperoxidation induced by active oxygen may be not the immediate cause forcryodamage.
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