| Because of severe infectious diseases,crossbreeding of foreign pig breeds and high cost of seed preservation,the genetic resources of local pig breeds in China are facing an increasing risk of extinction,and semen cryopreservation is an effective measure to solve these difficulties.In this paper,first of all,liquid nitrogen injection method(large-scale industrialized semen freezing equipment,the cooling rate at 5-10℃is-15℃/min,the cooling rate at-10-140℃is-40℃/min),glycerin concentration of 3%,0.25ml tubules,sperm quality computer-aided analysis system and other technologies are used to 2-4 Hunan local pigs(Ningxiang pig,dawizi pig,Shaziling pig and Xiangxi black pig)were 1:1 diluted on site and transported to the laboratory at 17℃,microscopic examination,adding I solution,centrifugation,removing supernatant,adding II solution at 4℃for more than 1.5h,and0.25ml fine tube loading and freezing were completed within 15min.Secondly,in order to solve the problems such as the small number of boars and the difficulty of semen collection caused by the distance between pig resource fields,the simple and portable method of liquid nitrogen surface distance(simulating the freezing procedure of liquid nitrogen injection method)was studied.Then the freezing effect of liquid nitrogen injection method was compared with that of liquid nitrogen surface distance method.Finally,the technology of frozen semen thawing was improved.The results are as follows:1.Study on the freezing technology of pig semen by liquid nitrogen injectionThe frozen semen of four local pigs(Ningxiang pig,Shaziling pig,dawaizi pig and Tiegu pig)in Hunan Province were prepared by liquid nitrogen injection.The average activity of Tiegu pig before and after freezing was the highest,with an average of 72.2%,48.2%,which was not significantly different from that of Ningxiang pig before and after freezing(70.6%,46.4%)(P>0.05),compared with Shaziling pig(64.8%,40.9%)and dawaizi pig(62.5%,38.1%)There was significant difference(P<0.05);there was no significant difference(24.4%)(P>0.05)in the freeze-thaw vigor damage of four Hunan local breeds of pigs;among the four Hunan local breeds of pigs(Ningxiang pig,Shaziling pig,dawaizi pig,Tiegu pig),the higher the balance density(0.341,0.246,0.301,0.165),the higher the thaw density(0.327,0.222,0.245,0.158);the ejaculation of Shaziling pig The order of ejaculation was Shaziling pig(310ML)>Tiegu pig(263ml)>Ningxiang pig(246ml)and Weizi pig(242ml).The results showed that:1)the quality of individual primordial semen was significantly higher than that of frozen semen(P<0.05),there was a positive correlation between freezing activity and fresh semen activity,there was a positive correlation between balance density and thawing density,and there was a positive correlation between ejaculation amount and the number of tubules;2)pigs with high ejaculation amount did not have a good vitality.Therefore,it is possible to select lines with strong frost resistance to establish a cryopreservation library.2.Study on the cryopreservation of pig semen by liquid nitrogen surface distance methodOn the basis of freezing method,using liquid nitrogen surface distance method,foam box specification(long 35cm,wide 35cm,high 48CM,thick 5cm),liquid nitrogen surface one-step method,high 3cm,5cm,7cm frozen sperm motility(CM)had no significant difference(P>0.05),highly 3cm,5cm frozen sperm motility(exercise),local motion(activity),vitality(H)were significantly higher than those of)The activity of 3cm liquid nitrogen surface height was the best(52.17±6.12)%.There was no significant difference(P>0.05)in the m,cm and LM of spermatozoa when the thin tube was placed in the automatic lifting freezing device at different heights,among which the activity at the height of liquid nitrogen at 1cm under the iron grid was the best(51.6±8.1)%;the activity at 1cm and 2cm under the iron grid of the automatic lifting freezing device was V,and the fast motion(FM)was significantly higher than that of the iron grid of the automatic lifting freezing device Upper 1cm,upper 0cm and lower 3cm(P<0.05).Therefore,there is no significant difference between the activity after one-step unfreezing and that after one-step unfreezing,but the multi-step operation is more complicated.3.The comparison of liquid nitrogen injection method and liquid nitrogen surface distance method in pig semenThe activity of liquid nitrogen injection method was(63.4±15.3)%,while that of liquid nitrogen surface distance one-step method was(47.7±5.1)%,and that of liquid nitrogen surface distance step method was(45.8±10.3)%,which was significantly better than that of liquid nitrogen surface distance method(P<0.05).There was no significant difference between liquid nitrogen surface distance one-step method and liquid nitrogen surface step method(P>0.05).The disadvantage of liquid nitrogen injection method is that it takes a long time.If a small amount of tube is used,a large amount of liquid nitrogen will be wasted and the cost is high.Therefore,liquid nitrogen injection method should be used as much as possible when conditions permit,otherwise one-step method of liquid nitrogen surface distance can be used,and the step-by-step method of liquid nitrogen surface distance still needs to be improved.4.Study on thawing method of pig semenThe sperm motility(33.6±0.13)%,acrosome integrity rate(38.8±1.7)were significantly higher than those of 45℃,52℃and 60℃30s groups(P<0.05).There was no significant difference in acrosome integrity rate(P>0.05),deformity rate and 60℃30s group(P<0.05),but there was no significant difference between 45℃30s group and 52℃30s group(P>0.05).Therefore,38℃30s water bath is more suitable for 0.25ml of Hunan local pigs.To sum up,for the semen freezing of Hunan local pigs,glycerin concentration is 3%,sperm density is 2.0×10~9/ml,0.25ml thin tube,if possible,liquid nitrogen injection method should be used as far as possible,otherwise,one-step method of liquid nitrogen surface distance can be used,and 38℃30s water bath can be used for thawing of frozen semen to obtain better effect. |
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