| Cryopreservation of semen under ultra-low temperature has prominent application advantages in protecting animal germplasm resources.The technical solutions related to semen cryopreservation have been applied in humans and cattle,and has achieved satisfactory results.In this paper,Jiang Quhai pig semen was selected as an experimental sample to construct a technical solution for cryopreservation of pig semen,which lays a reliable technical support for the artificial insemination of pigs.The main results of this experiment are as follows:(1)Evaluation method and correlation study of pig semen qualityThe "hand grip method" was used to obtain the semen of the adult and healthy Jiang Quhai pig,and the phosphate buffer solution,the preservation solution and the frozen base liquid were used in order to dilute the semen.Stored the semen at 4? and then the sperm viability,the mitochondrial activity and the acrosome finishing rate were recorded.At the same time,oocytes were collected for external fertilization and embryo culture.The results showed that there was a significant positive correlation between external fertilization rate and sperm morphology.When the sperm viability increased,the cleavage rate increased relatively,and the fertilization rate of the oocytes was positively correlated with the sperm acrosome integrity;the higher the acrosome integrity,the more the ovum was more easily fertilized.and the fertilization rate of the oocytes was positively correlated with the mitochondrial activity.(2)Study on cryopreservation of pig semenIn this experiment,the semen of adult and healthy Jiang Quhai pig was included in the sample to analyze the different effects of cryoprotector and liquid nitrogen fumigation height on semen quality.The results showed that the optimal effect could be achieved by using glycol as cryoprotective agent after the defrosting of the semen.There were significant differences in motility,plasma membrane breakage rate,acrosome integrity rate and mitochondrial activity with dimethyl sulfoxide(DMSO)and propylene glycol groups(P<0.05),but no significant difference with glycerol group(P>0.05).Fumigation and freezing were carried out at different heights from the liquid nitrogen surface.The fumigation effect at a height of 9 cm from the liquid nitrogen surface was the worst,and there was a significant difference compared with the test indexes of 2 cm and 5 cm height fumigation(P<0.05).Compared with the fumigation effect of liquid nitrogen surface at the height of 2 cm and 5 cm,the acrosome integrity rate of the fumigation group was significantly higher than that of the 2 cm height nimigation group(P<0.05),and there were no significant differences in motility,plasma membrane damage rate and mitochondrial activity between the two groups(P>0.05).Sodium dodecyl sulfonate(SDS)and vitamin C(Vc)can be added to the frozen solution to optimize the semen freezing effect.However,the effect of SDS and Vc incorporation is the most ideal.Except for the Vc group,the exercise was lower than that of the other groups(P<0.05),and the other indicators were not significantly different(P>0.05).Glutathione(GSH)and hyaluronic acid(HA)were added to the frozen liquid,and the external fertilization cleavage rate did not change significantly(P>0.05).Viability,superoxide dismutase(SOD)activity and acrosome integrity rate were significantly increased(P<0.05).Adding GSH and HA together(5 mmol·GSH-1 and 900 ?g·mL-1)can achieve the best results.While enhancing the sperm motility and maintaining SOD activity,the external fertilization cleavage rate can be greatly improved.Different concentrations of Cuscuta extract(divided into low-dose group,medium-dose group and high-dose group)were added to pig semen freezing liquid to detect normal sperm morphology and SOD activity.The results showed that the normal rate of pig sperm morphology and plasma membrane integrity decreased significantly after freezing-thawing(P<0.05).The SOD activity of the sperm added to the dodder was higher than that of the frozen group,and the low dose group reached a significant level(P<0.05).The content of MDA in the middle dose group of Cuscuta was lower than other frozen groups,and there was significant difference between the low dose group and the Vc group(P<0.05).It can be seen that the appropriate dose of Cuscuta extract can inhibit the membrane damage induced by freezing,enhance the SOD activity of sperm and reduce the content of MDA. |
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