Study On The Key Technology Of Bovine Production In Vitro

In the current commercial frozen semen of cattle,there are many factors that can affect in vitro sperm capacitation,such as antifreeze.Before in vitro sperm capacitation,it is necessary to separate and purify thawed semen first,removing various additives,so as to provide the best environment for in vitro sperm capacitation.Therefore,it is of great significance to study the effects of different sperm purification method on in vitro fertilization,and to determine the optimal sperm purification method in order to improve in vitro embryos production efficiency.Synchronous estrus is a key part in the process of embryo transfer.The synchronization and the quality of corpus luteum of the recipient cows will directly affect the conception rate after embryo transfer.In the study,Holstein cows were used as the test subjects and two exogenous reproductive hormones,prostaglandin and progesterone were selected to study the effects of different synchronous programs on embryo transfer recipient cows in order to improve the utilization rate of recipient cows and conception rate after embryo transfer.Experiment 1:Effect of different sperm purification method on bovine embryo production in vitro.In this experiment,Percoll density gradient centrifugation and swim-up method were used to treat frozen semen,and then fertilized with oocytes after in vitro maturation,repeated 8 times,and counted sperm motility,sperm density,fertilization rate,cleavage rate,and blastocyst rate.The results of the test showed that there was no significant difference in sperm motility between Percoll treatment(0.65)and swim-up treatment(0.68)(p>0.05),but the sperm motility in both groups was significantly higher than untreated group(0.41)(p<0.05).The sperm density in Percoll group(9.08×10~6/m L)was significantly higher than that in swim-up(7.60×10~6/m L)(p<0.05).The fertilization rate of Percoll group(83.70%)was significantly higher than that of the swim-up(77.99%)(p<0.05).The cleavage rate of Percoll group(62.61%)was significantly higher than that of the swim-up(51.62%)(p<0.05).There was no significant difference in blastocyst rate between the Percoll group(31.18%)and the swim-up(30.94%)(p=0.06).Experiment 2:Effects of different synchronous estrus methods on embryo transfer of recipient bovine.Selected 90 healthy and moderately fat Holstein young cattle,randomly divided into two groups,according to the CIDR+PG and PG+PG methods for hormone injection,and 15young cattle waiting for natural estrus(control),after the first estrus on 7 d,the recipient bovine corpus luteal diameter(CLD)was measured by B-ultrasound,and embryo transfer was performed on eligible recipient cattle.The test results showed that the estrus rate was significantly higher in the CIDR+PG than the PG+PG group(91.11%vs 84.44%,p<0.05);and in the CIDR+PG group,85.37%(35/41)of estrus cattle were mainly in estrus within 2 d after the withdrawal of CIDR,while 73.68%(28/38)of estrus cattle in the PG+PG group were mainly in estrus within 2-3 d after the second injection of PG.The qualified rate of corpus luteum in the CIDR+PG(70.73%,29/41)was significantly higher than that in the PG+PG group(65.79%,25/38)(p<0.05).The CLD was divided into three groups:10 mm≤CLD≤15 mm,15 mm<CLD≤25 mm and CLD>25 mm,to study the relationship between CLD at 7 d and conception rate after different estrus treatment.There was no significant difference in conception rate between CIDR+PG groups(33.33%vs 35.71%vs 33.33%,p>0.05);there was also no significant difference in conception rate was observed between PG+PG groups(37.50%vs 36.36%vs33.33%,p>0.05).There was no significant difference in conception rate between CIDR+PG and PG+PG group(p>0.05).The research results show that Percoll density gradient centrifugation can improve sperm utilization,fertilization rate and embryo cleavage rate compared with swim-up method.Compared with the PG+PG method,CIDR+PG method had higher estrus rate,more concentrated estrus time,higher corpus luteum qualification rate and higher utilization rate of recipient cattle.There was no significant difference between CLD and conception rate in recipient cattle treated with different synchronous estrus methods.