Effects Of Long-term Low Temperature Storage Of Sheep Semen On Sperm Genetic Substance

This study based on the development trend of sheep semen diluent with super long time low temperature preservation sheep semen influence on sperm genetic material,selection of soybean lecithin diluent(group1),patent yolk diluent(group 2)and glucose-citric acid diluent(control group)was carried out on the acquisition of fresh sheep semen long time kept at low temperature,and at different time points on sperm DNA,RNA integrity test and application effect of study,in order to reveal the sheep with sperm genetic material to save the time and the changes of genetic material change on sperm quality and the influence of auxiliary reproductive outcomes,This study provide a theoretical and experimental basis for the production and application of ultra-long time low temperature preservation sheep semen and the formulation of standards.1.To determine the effect of low temperature preservation for a long time on DNA integrity of sheep sperm.In this study,fresh sheep semen was collected by false vagina method.The sheep semen was stored at 0h,24 h,72h,144 h,216h and 288 h respectively,and DNA integrity was determined by TUNEL method and SCGE method.The results showed as follows: with the prolongation of storage time,the DNA damage of sheep sperm in the three groups was gradually increased,and from the 24 h,the glucose-citric acid dilution was significantly higher than that in the soybean lecithin dilution and yolk dilution(P<0.01).However,there was no significant difference between soybean lecithin diluent and yolk diluent at different time points,and the DNA damage of sperm increased significantly when the two diluent preserved the semen to 216 h.The results showed that the sperm viability was negatively correlated with the sperm DNA damage rate.Both SCGE and TUNEL assay could be used as effective methods to evaluate the quality of sheep sperm.2.In order to establish an effective method for RNA extraction from sheep sperm and detect the changes in RNA integrity of sperm with ultra-long time low temperature preservation.The sperm RNA was extracted from fresh sheep semen by kit method,and the quality of reverse transcripted c DNA and sperm RNA was verified.After that,sheep semen from the three groups of diluent were storage at 0h,24 h,72h,144 h,216h and 288 h respectively for RNA extraction and RNA integrity detection.Results: The established method of RNA extraction from sheep sperm could stably extract RNA from sheep sperm.The obtained RNA was of high purity and good quality,without contamination from other non-sperm cells.The RNA content of sheep single sperm cell was about 40.54 fg for the first time.After low temperature preservation of sheep spermatozoa with three groups of diluent,there was no significant difference in sperm RNA integrity between glucose-citric acid diluent and the other two groups at 0-24 hours,but sperm RNA could not be obtained after 72 hours.When sheep semen were low temperature storage with soybean lecithin diluent and patented yolk diluent,the RNA content(36.2～45.7 fg)and RNA integrity of single sperm did not decrease with the extension of preservation time.3.To verify whether sheep semen can produce healthy offspring after being low temperature storage for a long time.Fresh sheep semen was low temperature storage with soybean lecithin diluent and patented yolk diluent,respectively.Sperm viability,acrosomal integrity,plasma membrane integrity and acrosomal protease activity were detected at different time points of low temperature preservation.Three hundred and twenty-nine ewe in estrus were artificially inseminated with sheep semen preserved in two diluent for 216 h and fresh sperm diluted in glucose-citric acid diluent diluent.After insemination,the conception rate,lambing rate,female lambing rate and birth weight of male and female lambs were detected.The results showed that the sperm viability,acrosomal integrity,plasma membrane integrity and acrosomal proteinase activity of sheep semen were gradually decreased when the soybean lecithin diluent and yolk diluent were kept at low temperature for a long time.There was no significant difference in the pregnancy rate(72.4%vs73.2%vs78.5%),lambing rate(126%vs125%vs130%),female lambing rate(47.88%vs46.47%vs51.22%)and the average birth of male and female lambs after semen artificial insemination with soybean lecithin diluent and yolk diluent stored at low temperature for 216 h,compared with that of fresh semen diluted with glucose-citric acid diluent.In conclusion,the DNA integrity of sperm decreased gradually with the extension of preservation time and significantly decreased at 216 h after low temperature preservation of sheep semen with soybean lecithin dilution and yolk dilution.The integrity of sperm RNA did not significantly decrease with the extension of preservation time within 288 h.In addition,sheep semen can be preserved at low temperature for a long time and artificial insemination can still achieve good effect of conception and produce healthy offspring.This study revealed the changes of genetic material of sheep sperm with ultra-long time low temperature preservation and its effect on offspring,and was helpful for the production and application of ultra-long time low temperature preservation of sheep semen and the formulation of standards.