| Tan sheep is a special local breed in Ningxia Autonomous Region and its surrounding provinces.Tan sheep fur is white and beautiful,meat is delicious and has no taint.Therefore,the Tan sheep industry has a broad market prospect.However,Tan sheep has shortcomings such as small size,slow growth and development,and low reproductive performance,which affects the economic benefits of Tan sheep breeding farms(households)and the development of Tan sheep industry.Improving the reproductive efficiency of Tan sheep and cultivating a new strain of Tan sheep with the characteristics of large size,fast growth and high reproductive performance are the key to promoting the development of Tan sheep industry.MSTN(myostatin)is a regulatory factor that hinders muscle growth and development,and its expression in skeletal muscle inhibits muscle growth and development.FecB(fecundity booroola)gene is a major gene for lambing traits in sheep,the mutation of its key site can increase the number of ovulation and lambing.The author’s team has obtained MSTN gene knockout Tan sheep rams and ewes by using gene editing technology(CRISPR-Cas9),identified and screened FecB gene mutation rams and ewes in the natural population of Tan sheep by using fluorescent quantitative PCR technology.In order to rapidly expand the individual numbers of MSTN gene knockout Tan sheep and FecB gene mutation Tan sheep,and to accumulate more breeding materials for breeding a new Tan sheep strain with high reproductive performance and fast growth.This experiment used MSTN gene knockout Tan sheep rams and MSTN gene knockout Tan sheep ewes or normal ewes,as well as FecB gene mutation Tan sheep rams and FecB gene mutation Tan sheep ewes or normal ewes as the subjects,the technique of artificial insemination of natural estrus or synchronous estrus ewes was used to expand the population of MSTN gene knockout and FecB gene mutation Tan sheep.At the age of 15 days after birth,lambs were cultivated with pellet feed,weighed and measured regularly,and gene detection was carried out on all surviving lambs.The test contents and results are as follows:(1)4 MSTN knockout Tan sheep rams and 330 normal Tan sheep ewes were used as the experiment objects,the rams were used to test the ewes respectively in the morning and evening each day.318 ewes in estrus were artificially inseminated by vaginal insemination at 12h and 24h after estrus,and artificial insemination was performed on returning estrus ewes for a total of 3 estrus periods.The number of pregnant ewes was counted as the number of lambing ewes.A total of 220 ewes were pregnant and 233 lambs were born,the pregnancy rate of ewes was69.18%,and the lambing rate was 110.95%.Blood was collected from the jugular vein of 205 lambs surviving at 15 days of age,sanger sequencing technology was used to detect MSTN gene sequence of offspring lambs.The sequencing results showed that 72 of the offspring were MSTN gene knockout-positive individuals,and the positive rate of offspring was 35.12%;195lambs were survived at 60 days,and the survival rate was 83.69%;the results of body weight monitoring within one year of age showed that the weight of all individuals increased steadily,and the weight of the offspring of MSTN gene knockout was significantly higher than that of the negative individuals at each growth stage(p<0.05).(2)4 MSTN gene knockout Tan sheep rams,12 MSTN gene knockout Tan sheep ewes,5FecB gene mutation rams,52 FecB gene mutation ewes and 526 normal Tan sheep ewes were used as the experiment objects,and the following 4 propagation combinations were used:(1)3MSTN knockout rams and 12 MSTN knockout ewes,(2)4 MSTN knockout rams and 268 ordinary ewes,(3)5 FecB gene mutation rams and 52 FecB gene mutation ewes,(4)5 FecB gene mutation rams and 258 ordinary ewes,to start an expansion experiment.twice PG method and NRID+PMSG+PG method were used for estrus synchronization of ewes,or natural estrus ewes were selected through estrus test of rams.Artificial insemination was carried out at the bottom of vagina 12h and 24h after estrus of ewes respectively,and artificial insemination was carried out for returning estrus ewes again,with a total of 3 estrus periods.The number of pregnant ewes was counted as the number of lambing ewes.A total of 410 ewes were pregnant,456 lambs were born,the pregnancy rate of ewes was 69.97%,and the lambing rate was 111.22%.351 lambs survived at 60-day-old,and the survival rate of 60-day-old lambs was 76.97%.After collecting blood from jugular vein of surviving lambs,Sanger sequencing technology was used to detect MSTN gene sequence of offspring lambs,and the FecB gene sequence was detected by fluorescence quantitative PCR.In the first group(11 MSTN gene knockout ewes were in estrus and bred)8ewes were pregnant and 11 lambs were born,the percentage of lambing ewes was 72.72%,and the lambing rate was 137.50%,5 lambs survived at 60 days of age,of which 3 were FecB gene mutation and MSTN gene knockout lambs(genotype: MB),2 lambs were MSTN knockout heterozygotes(genotype: M-).In the second group(267 ordinary ewes were in estrus and bred)187 ewes were pregnant and 201 lambs were born,the percentage of lambing ewes was 70.04%,and the lambing rate was 107.49%,163 lambs survived at 60 days old,of which 12 lambs were FecB gene mutation and MSTN gene knockout individuals(genotype: MB),70 lambs were MSTN gene knockout heterozygotes(genotype: M-),15 lambs were FecB gene mutation heterozygotes(genotype: B-),66 lambs were ordinary sheep(genotype:--).In the third group(51 FecB gene mutation ewes were in estrus and bred)36 ewes were pregnant and 54 lambs were born,the percentage of lambing ewes was 70.59%,and the lambing rate was 150.00%,44 lambs survived at 60 days of age,22 of which were FecB gene mutation homozygous(genotype: BB),22 lambs were FecB gene mutation heterozygous.In the fourth group(257 ordinary ewes were in estrus and bred)179 ewes were pregnant and 190 lambs were born,the percentage of lambing ewes was 69.65%,and the lambing rate was 106.14%,139 lambs survived at 60 days old,of which 125 lambs were FecB gene mutation heterozygous,and 14 lambs were ordinary sheep.According to the genotypes of the offspring,the offspring were divided into M-(72),MB(15),--(80),B-(162),and BB(22)groups.The results of weight monitoring within one year of age showed,the weight of the offspring of each group increased steadily,the weight of the individuals in the M-group was significantly higher than that of the--,B-,and BB groups at each growth stage,and the weight of the individuals in the BB group was significantly lower than the other groups at each stage(p<0.05).A total of 553 living offspring of Tan sheep were obtained through the experiments,including 139 Tan sheep with MSTN gene knockout,184 Tan sheep with FecB gene mutation,and 15 Tan sheep with MSTN gene knockout as well as FecB gene mutation,which provided breeding materials for the aggregation breeding of a new strain of Tan sheep with high reproductive performance and faster growth characteristics.The results of the experiments showed that MSTN gene knockout rams breeding with MSTN gene knockout ewes or normal ewes would obtain MSTN gene knockout offspring with faster weight gain.Compared with the normal ewes,FecB Gene Mutation ewes have higher reproductive efficiency,the offspring of FecB gene Mutation homozygotes have relatively small birth weight and weight gain.When the population of gene editing Tan sheep reaches a certain scale,conventional breeding technology can be used to expand the population of gene editing Tan sheep. |
