| To improve the quality of frozen-thawing semen, the effects of preservation extenders, different treatments before cryopreservation, cryoprotections and frozen-thawing methods were studied on stallion semen.Experiment 1: The stallion semen preservation extenders and dosage forms in different temperatures were selected and compared the quality of different stallion semen in this experiment. The results showed that: (1)In room temperature, the sperm motility in 2.5%Gly extender, 7%Glu extender were higher significantly than that in 5%Gly extender, 0.9%Nacl extender(P<0.05), and the survival time was extreme significant(P<0.01). (2)In low- temperature, the sperm motility in 10%Milk powder extender, 5%Egg yolk extender were higher significantly than that in 0.8%Egg yolk extender(P<0.05) and extreme significantly than that in 7%Glu extender(P<0.01), and the survival time was longer extreme significant than that in 0.8%Egg yolk extender, 7%Glu extender(P<0.01). (3)In cryopreservation, the pellet frozen semen motility in 11%Lac extender, 10%Suc extender, 4%Fructose extender were extreme significantly than that in 0.1%EDTA extender, 3%Glu extender, 1.25%Tris extender(P<0.01). The straw semen motility were higher significantly than that in 3%Glu extender, 1.25%Tris extender(P<0.05) and 0.1%EDTA extender extreme significantly(P<0.01). Compared with the straw semen, the frozen-thawing motility and recovery of the pellet semen were significantly different(P<0.05). Acorosomal intrgrity, in 10%Suc extender, 4%Fructose extender were higher extreme significantly than that in 11%Lac extender(P<0.01), and higher significantly than that in 11%Lac in abnormality(P<0.05). There was no difference between frozen pellet semen and straw semen in frozen-thawing motility(P>0.05). (4)The pre-sperm motility and frozen-thawing motility of stallion was difference (P<0.05).Experiment 2: The effects of different treatments before cryopreservation were studied on the preservation of stallion semen in the experiment. The results showed that: (1) The frozen-thawed motility and recovery of stallion semen centrifugated at the same velocit for 7 min,10min were higher than those for 15min significantly(P<0.05). When the velocity of centrifugation was 2000r/min and 3000r/min, the motility and recovery of frozen-thawed were significantly worse than those centrifugated at 1000r/min and 1500r/min (P<0.05), respectively. There was no difference in motility and recovery of frozen-thawed semen when the centrifugation was 1000rpm for 7 min or 10min and 1500rpm for 7 min or 10min(P>0.05). (2) The motility and recovery of frozen-thawed semen at the ration of sperm to seminal plasma for 1/1 and 1/2 were significantly higher than those for 0/l and 1/4,respectively(P<0.05). (3) The motility and recovery of frozen-thawed semen at the ration of semen to extender for 1/2 and 1/3 were higher than those for 1/l significantly (P<0.05). (4) There was no difference in motility and recovery of frozen-thawed semen when the equilibrium time was 2h,3h and 4h(P>0.05), respectively. (5) Using copper screen and fluorous plate as frozen material, the motility and recovery of frozen-thawed semen were significantly higher than using steel plate(P<0.01). (6)The motility and recovery of frozen-thawed semen fumed over liquid N2 were better than that immerged into liquid N2. (7)The motility and recovery of frozen-thawed semen were not significant different(P>0.05) fumed over liquid N2 at -80℃,-110℃and -140℃, and the time were 5min,7min,10min and 15min, respectively.Experiment 3: The effects of different methods of cryoprotections and thawing were investigated on stallion semen cryopreservation in this experiment. The study indicated that: (1) Using Gly made in China, Gly made in Japan, Gly and DMSO as cryoprotective, the motility and recovery of frozen-thawed semen were better than using EG and DMSO significantly(P<0.05). (2)When the final glycerol concentration in the extender were 3.8%,5%,6%, the motility and recovery of frozen-thawed semen were better than that were 7% significantly(P<0.05)and 9% extreme significantly(P<0.01). (3)The motility and recovery of frozen-thawed semen in 0.1%EDTA thawing fluid were better than that in 6% Suc, 2.9%sodium citrate, 5% Glu thaw fluid significantly(P<0.05). The abnormality and acorosomal of frozen-thawed semen in 0.1%EDTA thaw fluid were worse than that in 6% Suc, 2.9% sodium citrate significantly(P<0.05), compared with 5% Glu, the difference were not significant(P>0.05). (4) The motility, recovery, abnormality and acorosomal of frozen-thawed semen using dry thawed method were not different significant from using wet thawed method(P>0.05). (5)The motility and recovery of frozen-thawed semen thawed in 58℃and 78℃were higher than semen thawed in 38℃significantly(P<0.05) and 4℃extreme significantly(P<0.01), but there were no difference in the abnormality and acorosomal of frozen-thawed semen (P>0.05).In a word, the extender of 7% glucose, 5% egg yolk and 4% fructose was suitable for the preservation of stallion semen at room temperature, low- temperature and cryopreservation, respectively. And the frozen-thawing motility and recovery of the pellet semen was better than the straw semen. The optimal methods and conditions estabolished for the preservation of stallion semen were that the collected semen was centrifugated at 1500rpm for 10min, the ration of sperm to seminal plasma was 1/2, 4% fructose(6% glycerol or 4.5% glycerol+1.5%DMSO) was cryopreservation extender, the ration of semen to extender was 1/2, equalized 3h in 4℃, copper screen was cryoprotective material, fumed over liquid N2, finally dry thawed or wet thawed in 58℃and 78℃was thawing method. |
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