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Abstract The present study was conducted to explore ways of JIVET research on Texel sheep， and to provide technical support for the improvement of the JIVET technology system for research on Texel sheep. Superovulation was conducted on 4-8-week old Texel sheep， and in-vivo ovum collection， in-vitro oocyte maturation and fertilization， and fertilized-embryo culture and transplantation were adopted to perform JIVET research on Texel sheep. The results showed that 296 available oocytes were obtained at an average of 74 oocytes per sheep; 205 fertilized eggs were obtained at the 2-4 cell stage at an average of 51.25 eggs per sheep and a cleavage rate of 69.26%; and 66 of the fertilized eggs were transplanted to 11 receptor sheep. Six sheep were conceived at a conception rate of 54.55%， and 11 lambs were born. These results indicated that the present test could be used as a method for the study of JIVET technique on Texel sheep.
Key words Texel sheep; JIVET research; In-vitro fertilization; 2-4-cell fertilized ovum; Test-tube sheep
At present， there are 7 million to 8 million sheep in Shanxi Province， most of which are local sheep， with low production performance and poor economic benefits， which severely restrict the development of the sheep industry. In recent years， our province has introduced some Texel sheep to cross-improve local sheep， and the improvement effect is remarkable. However， the number of introduced high-quality sheep is far from meeting the needs of production. Although embryo transplantation technology is currently an effective technology that can realize the rapid breeding of Texel sheep and double the number of excellent breeds， the traditional multiple ovulation and embryo transfer （MOET） scheme is only used for adult sheep， the superovulation of which gives about 10 embryos each time， and the generation interval is at least 1.5 years old， which is long， resulting in a low reproduction speed.
The JIVET （juvenile in vitro embryo technology） researched and developed by the South Australian Government Reproductive and Development Research Center of Australia is an in-vitro production technology of juvenile embryos， as well as a new rapid reproduction technology system that integrates superovulation and egg collection of young animals， in-vitro maturation and fertilization of oocytes， and in-vitro culture and transplantation of fertilized eggs. The principle is that the number of antral follicles on the ovaries of lambs of 1 to 2 months old reaches a peak， gradually decreases with the arrival of sexual maturity， and remains constant until the age 33 of weeks. The ovaries of lambs aged 1 to 2 months are very sensitive to reproductive hormones， and follicular development rarely suffers from atresia. If induced by exogenous gonadotropins at this stage， an average of 80 to 160 oocytes can be obtained per lamb， which is about 10 times the number of eggs obtained by an adult ewe through superovulation， and the generation interval is only 1/3 of that of adult sheep， which greatly accelerates the reproduction speed. At present， scientists at broad have conducted in-depth research on the JIVET technology of superior sheep breeds and have achieved gratifying results[1-5]. Scientists at home also conducted JIVET technology research on Suffolk sheep， Kazakh sheep， Liaoning down-producing goat and small-tailed Han sheep raised in Xinjiang Autonomous Region， Liaoning Province， Shandong Province， Qinghai Province and Hebei Province[6-16]， but so far， there has been no report on the research of the JIVET technology of superior sheep breeds in Shanxi Province. For this reason， the Institute of Animal Husbandry and Veterinary Medicine， Shanxi Academy of Agricultural Sciences and China Agricultural University conducted research on the JIVET technology of Texel sheep， providing technical support for perfecting the JIVET technical system. Materials and Methods
Experimental materials
Experimental sheep
Texel sheep of 4-8 weeks old were provided by the sheep farm of the Institute of Animal Husbandry and Veterinary Medicine， Shanxi Academy of Agricultural Sciences， and the recipient sheep were provided by the sheep farm of Taiyuan Qinglian Breeding Co.， Ltd.
Frozen semen and estrus ewe serum
Texel sheep frozen pellet semen and estrus ewe serum （ESS） were made in laboratory.
Main hormones and reagents
Sheep CIDR， follicle stimulating hormone （FSH）， pregnant mare serum gonadotropin （PMSG）， 17β-estradiol （17β-E2） and luteinizing hormone （LH） were all purchased from Beijing Luxin Agriculture and Animal Husbandry Technology Co.， Ltd.
Culture media
Egg collection solution： M199+20 mmol/L Hepes+2% ESS+10 mg/ml heparin sodium; maturation solution： M199+20% ESS+10 μg/ml FSH+10 μg/ml LH+1 μg/ml 17β-E2; sperm capacitation fluid： synthetic fallopian tube fluid （SOF）+20 μg/ml heparin sodium; fertilization fluid： SOF+2% ESS; fertilized egg development fluid： SOF+8 mg/ml BSA+2% EAA+1% NEAA.
Experimental methods
ESS preparation
The blood of the estrus ewe was taken from the jugular vein， and the serum was prepared according to conventional method.
Frozen semen production
The semen of Texel sheep with a vitality of 0.8 or more was prepared by conventional dry ice freezing method to prepare frozen pellet semen.
Recipient sheep in estrus
The method of simultaneous estrus in recipient sheep adopted by Mao et al.[17] was applied with the difference that the recipient sheep was implanted with CIDR for 12-14 d， and the amount of PMSG injected intramuscularly was 320-360 U/sheep.
Superovulation in young sheep
The lamb superovulation method of Kelly et al.[1] was adopted. Four Texel young sheep with ear numbers T1104， T1110， T1112 and T1116 were superovulated. The difference was that the PMSG injection volume was 300-350 U/sheep.
Oocyte collection
After the donor lamb was anesthetized and fixed， and the surgical site was disinfected. Then， a scalpel was used to cut a small opening of 3-5 cm in the anterior abdomen of the breast slightly off the midline， and the ovary was gently pulled out from the abdominal cavity. The 18# needle on a 10 ml syringe with 2 ml of egg collection fluid was used to puncture the follicles above 2 mm in diameter on the ovary and suck out the cumulus-oocyte complexes （COCs）. After the egg collection was completed， the surgical opening was sutured and disinfected. The collected oocytes were placed in a 75 mm petri dish， and the well-formed A and B-grade COCs with more than 2 layers of cumulus cells and uniform cytoplasm were quickly picked out under a stereo microscope and placed in the maturation fluid.
In-vitro sperm capacitation
The in-vitro sperm capacitation method of Bai[6] was adopted.
In-vitro oocyte maturation and fertilization and fertilized egg culture
The collected A and B-grade COCs were put into the maturation fluid in a four-well culture plate， covered with mineral oil， and matured in a 5% CO2 incubator （38.5 ℃， saturated humidity） for about 24 h. The discharge of the first polar body from the oocytes was the criterion for the maturation of oocytes.
The mature oocytes were gently blown and beaten in a 0.2% hyaluronidase solution to remove part of the cumulus cells. They were then put into the fertilization fluid in the wells of the four-well culture plate， added with capacitated sperm （1×106-2×106 Pieces/ml）， and covered with mineral oil. The oocytes were fertilized and incubated in a 5% CO2 incubator for 24 h.
After removing the remaining sperm and cumulus cells attached to the fertilized eggs， they were put into the fertilized egg development fluid in a four-well culture plate and covered with mineral oil. Next， the four-well plate was put into an aluminum foil bag （filled with a mixed gas of 5% CO2-7% oxygen-88% nitrogen）， which was sealed. The fertilized eggs were incubated in a 5% CO2 incubator for about 24 h， and the cleavage condition was observed under a microscope， followed by counting the cleavage rate.
Fertilized egg transplanting
The operation method was the same as "Oocyte collection". The ovary and fallopian tube on the side with the ovulation point was gently pulled out of the abdominal cavity， and an embryo transfer device was used to suck 6 fertilized eggs at 2-4 cell stage and implanted into the ampulla of the fallopian tube.
Data processing method
The test data was processed in Excel.
Results and Analysis
The effect of superovulation
After superovulation， the Texel sheep of 4-8 weeks old showed enlarged swollen ovaries full of developing follicles. The COCs collected from Texel lambs T1104， T1110， T1112 and T1116 were 86， 72， 104， and 51， respectively， totaling 313， with an average of 78.25 COCs per animal. Among them， there were 296 A and B-grade COCs， with an average of 74.0 COCs/sheep. In-vitro oocyte development
After the 296 A and B-grade COCs were matured and cultured in vitro， 241 first polar bodies were discharged from the oocytes， that is， the maturation rate of oocytes was 81.41%. The mature oocytes and capacitated sperm were incubated together. After the fertilized eggs were cultured， 205 fertilized eggs split into 2-4 cells， that is， the cleavage rate was 69.26%， and the average was 51.25 eggs per sheep.
In-vitro transplantation effect of fertilized eggs at the 2-4 cell stage
Sixty six in-vitro fertilized eggs at the 2-4 cell stage were transplanted into the ampullae of 11 estrus recipient ewes. Six ewes were conceived and 11 lambs were born. Among the 6 ewes， 1 ewe gave birth to 3 lambs， 3 ewes gave birth to two lambs， and 2 lambs produced single lamb. The conception rate was 54.55%.
Discussion
The effect of superovulation method on the JIVET technical system
The superovulation of young sheep with gonadotropins for developing follicles on the ovaries of young sheep and obtain a large number of oocytes is the basis of the JIVET technical system. In the research field of hormone-induced young sheep， Kelly et al.[1，3] studied the effects of follicular development on lambs of 1 to 2 months old injected with FSH 160 mg/sheep and PMSG 500 U/sheep， and obtained oocytes at 80.0 oocytes/sheep or above. Gou et al.[5] used FSH and PMSG to induce 1-month-old lambs， obtaining 79.1 oocytes per lamb. Guo et al.[8] used gonadotropins to induce Merino sheep and Kazakh Sheep of 4 to 8 weeks old to study the effect of ovarian follicle development， and obtained oocytes at 135.8 and 37.8 oocytes per sheep， respectively. Yu et al.[18] induced the follicular development in Tao Han hybrid sheep at the age of 1 and 3 months by injecting FSH 160 mg/sheep， 4 times in two days， and adding PMSG 500 U/sheep at the last injection of FSH. The 1-month-old lamb produced 79.0 oocytes per sheep， which was significantly higher than the 3-month-old lambs producing 18.5 oocytes per sheep. In this study， the effect of gonadotropins on the development of ovarian follicles in Texel sheep of 4 to 8 weeks old was studied， and 78.25 COCs were obtained per sheep. The number of oocytes obtained from young Texel sheep is higher than the results of Guo et al.[8] on young Kazakh sheep， but lower than the results of Guo et al.[8] on young Merino sheep， and basically the same as the results of Kelly et al.[8]， Gou et al.[5] and Yu et al.[18] on lambs of 1 to 2 months old. It shows that the method of using gonadotropins to induce the development of ovarian follicles in young Texel sheep is feasible. Effects of the methods of oocyte maturation and fertilization method and fertilized egg culture in vitro on the JIVET technical system
Methods of in-vitro maturation， fertilization and incubation of oocytes and fertilized egg culture are important parts of the JIVET technical system. The three constitute the JIVET technical culture system， and any problem in any culture link will affect the effect of JIVET technology. In the research of the JIVET technical culture system， Bai et al.[4] studied the effect of 600 μmol/L cysteine in the maturation fluid on the development of Mongolian lamb oocytes， and the fertilization rate was 86.9%. Wu et al.[19] studied the effect of in-vitro maturation time of Xinjiang fine-wool lamb oocytes on the effect of in-vitro fertilization， and the cleavage rate of oocytes matured for 24 h was 52.98%. Guo et al.[20] studied the effect of adding 100 μmol/L β-mercaptoethanol to the maturation solution on the oocyte development ability of sheep at the age of 4-8 weeks old， and the cleavage rate was 68.0%. In this study， the in-vitro oocyte culture system of Texel sheep at the age of 4-8 weeks was studied， and the cleavage rate was 69.26%. The cleavage rate of young Texel sheep is higher than that of Xinjiang fine wool lambs obtained by Wu et al.[19]， and basically consistent with the results of Bai et al.[4] and Guo et al.[20] on young sheep. It is indicated that the methods of oocyte maturation， fertilization and incubation and fertilized egg culture of young Texel sheep in vitro in this study are feasible.
Effect of transplantation method of fertilized eggs at 2-4 cell stage on the JIVET technical system
Sheep's egg and sperm combination is in the ampulla of the oviduct， and the development of the fertilized egg to the 16-cell stage is completed in the oviduct. Therefore， eggs fertilized in vitro should be transplanted into the fallopian tube at the 2-4 cell stage. Wang et al.[7]， Guo et al.[8]， Liu et al.[9]， Ma et al.[10] and Chen et al.[11] studied the JIVET techniques of Suffolk sheep， Kazakh sheep， Dorset sheep and small-tailed Han sheep， and the conception rates ranged from 16.6% to 53.6%. In this study， the eggs fertilized in vitro were transplanted at the 2-4 cell stage into the ampullae of the oviducts of the recipient ewes in estrus， and the conception rate was 54.55%， which is consistent with the results of the above studies. The results show that the transplantation method of fertilized eggs at the 2-4 cell stage used in this study is feasible. Conclusions
In this study， a total of 296 A and B-grade oocytes were obtained from 4 Texel sheep of 4-8 weeks old， at an average of 74.0 oocytes per sheep. After in-vitro maturation， fertilization， incubation and culture， 205 fertilized eggs at the 2-4 cell stage were obtained， at a cleavage rate of 69.26% and an average of 51.25 eggs per sheep. Sixty six fertilized eggs at the 2-4 cell stage were transplanted to 11 recipient sheep， among which 6 ewes were conceived， and gave birth to 11 "test tube sheep"， showing a conception rate of 54. 55%. The results show that the method of this study can be used as a method for the research on the JIVET technology of Texel sheep.
References
[1] KELLY JM， KLEEMANN DO， WALKER SK. Enhanced efficiency in the production of offspring from 4-to 8-week-old lambs[J]. Theriogenology， 2005， 63 （7）： 1876-1890.
[2] RANGEL-SANTOS R， MCDONALD MF， WICKHAM GA. Evaluation of the feasibility of a juvenile MOET scheme in sheep[J]. Proceedings of The New Zealand Society of Animal Production， 1991， 51 （1）： 139-142.
[3] KELLY JM， KLEEMANN DO， WALKER SK. The effect of nutrition during pregnancy on the in vitro production of embryos from resulting lambs[J]. Theriogenol， 2005， 63 （7）： 2020-2031.
[4] BAI JH， HOU J， GUAN H， et al. Effect of 2-mercaptoethanol and cysteine supplementation during in vitro maturation on the developmental competence of oocytes from hormone-stimulated lambs[J]. Theriogenol， 2008， 70（5）： 758-764.
[5] GOU KM， GUAN H， BAI JH， et al. Field evaluation of juvenile in vitro embryo transfer （ JIVET） in sheep[J]. Anim Reprod Sci， 2009， 112 （3/4）： 316-324.
[6] BAI JH. Study on JIVET technology of sheep and cattle before sexual maturity[D]. Beijing： China Agricultural University， 2008. （in Chinese）
[7] WANG LQ， HE ZL， LIN JP， et al. Study on the induced development effect of Suffolk lamb follicular[J]. Journal of China Agricultural University， 2015， 20 （4）： 141-146. （in Chinese）
[8] GUO H， WAN PC， SHI WY， et al. Study on in vitro production （ivp） of embryo utilizating superovulated oocytes from different breed lambs[J]. Animal Husbandry and Veterinary Medicine， 2012， 44 （10）： 20-24. （in Chinese）
[9] LIU ZZ， LI HG， CHEN M， et al. Study on rapid propagation of suffolk sheep by using JIVET technology[J]. Animal Husbandry and Feed Science， 2016， 37 （3）： 1-4. （in Chinese）
[10] MA SK， JIAO HS， MA LQ， et al. Application research on JIVET in lamb in Qinghai plateau area[J]. Chinese Qinghai Journal of Animal and Veterinary Sciences， 2015， 45 （2）： 13-14. （in Chinese） [11] CHEN XY， TIAN SJ， SANG RZ， et al. Inducement of lamb follicular development and embryo production in vitro[J]. Chinese Journal of Agricultural Biotechnology， 2008， 16 （3）： 456-460. （in Chinese）
[12] ZHANG XH， ZHANG SW， SONG XC， et al. Study on follicular development of female lamb and technology of oocyte maturation[J]. China Animal Husbandry and Veterinary Medicine， 2011， 38 （11）： 136-139. （in Chinese）
[13] WU HF， LI FD， CHANG WH， et al. The effect of different treatments on superovulation and in vitro fertilization （IVF） efficiency[J]. Journal of Gansu Agricultural University， 2011， 46 （6）： 1-5. （in Chinese）
[14] XU XS， LIU CJ， MAO XL. Detection of vitamin C content in 16 common fruits and vegetables in Zunyi City[J]. Vegetables， 2012（6）： 49-50. （in Chinese）
[15] YIN XJ， LIU L. Analysis on color reaction of amino acids with ninhydrin by RGB value method[J]. Guangzhou Chemical Industry， 2018， 46（07）： 64-66. （in Chinese）
[16] LI M， QI F， ZHOU Z， et al. Dynamics research on changes of salt and lactic acid content in pickling process of radish[J]. China Condiment， 2013， 38（9）： 31-36. （in Chinese）
[17] LIU W， ZHANG MM， SONG Y， et al. Non-enzymatic browning kinetics of pickled white radish with low salt[J]. Food Science， 2016， 37（13）： 65-70. （in Chinese）
[18] LIU LL， ZHANG H， LIANG CY. Effect of curing time on texture color of spicy cabbage flavored beef jerky[J]. Journal of Agricultural Science Yanbian University， 2019， 41（2）： 80-85. （in Chinese）
[19] XIAO LL， ZHONG K， FENG PH， et al. Optimization of green-protecting and brittleness-keeping method of green pepper and its curing process[J]. Sichuan Food and Fermentation， 2019， 55（5）： 40-44， 50. （in Chinese）
[20] ZHOU Q， LIU MJ， YOU HY. Study on the effect of fermentation conditions on the quality of pickled radish and its process optimization[J]. China Condiment， 2018， 43（2）： 12-16， 22. （in Chinese）
[21] YANG L， WU ZY， ZENG WW， et al. Quantitative systematic review of the growth and decline rule of Escherichia coli during the fermentation process of Sichuan pickle[J]. Food Safety and Quality Detection Technology， 2017， 8（12）： 4529-4533. （in Chinese）
[22] WANG F. Research of the change of nitrite content in the process of vegetables pickled[J]. Farm Products Processing， 2016（1）： 6-9， 11. （in Chinese）
[23] CHE ZQ， XIA YB， LEI C， et al. Study on the effect of pretreatment methods on the flavor and texture of radish before pickling[J]. Modern Food Science & Technology， 2017， 2017（1）： 197-205. （in Chinese）
Key words Texel sheep; JIVET research; In-vitro fertilization; 2-4-cell fertilized ovum; Test-tube sheep
At present， there are 7 million to 8 million sheep in Shanxi Province， most of which are local sheep， with low production performance and poor economic benefits， which severely restrict the development of the sheep industry. In recent years， our province has introduced some Texel sheep to cross-improve local sheep， and the improvement effect is remarkable. However， the number of introduced high-quality sheep is far from meeting the needs of production. Although embryo transplantation technology is currently an effective technology that can realize the rapid breeding of Texel sheep and double the number of excellent breeds， the traditional multiple ovulation and embryo transfer （MOET） scheme is only used for adult sheep， the superovulation of which gives about 10 embryos each time， and the generation interval is at least 1.5 years old， which is long， resulting in a low reproduction speed.
The JIVET （juvenile in vitro embryo technology） researched and developed by the South Australian Government Reproductive and Development Research Center of Australia is an in-vitro production technology of juvenile embryos， as well as a new rapid reproduction technology system that integrates superovulation and egg collection of young animals， in-vitro maturation and fertilization of oocytes， and in-vitro culture and transplantation of fertilized eggs. The principle is that the number of antral follicles on the ovaries of lambs of 1 to 2 months old reaches a peak， gradually decreases with the arrival of sexual maturity， and remains constant until the age 33 of weeks. The ovaries of lambs aged 1 to 2 months are very sensitive to reproductive hormones， and follicular development rarely suffers from atresia. If induced by exogenous gonadotropins at this stage， an average of 80 to 160 oocytes can be obtained per lamb， which is about 10 times the number of eggs obtained by an adult ewe through superovulation， and the generation interval is only 1/3 of that of adult sheep， which greatly accelerates the reproduction speed. At present， scientists at broad have conducted in-depth research on the JIVET technology of superior sheep breeds and have achieved gratifying results[1-5]. Scientists at home also conducted JIVET technology research on Suffolk sheep， Kazakh sheep， Liaoning down-producing goat and small-tailed Han sheep raised in Xinjiang Autonomous Region， Liaoning Province， Shandong Province， Qinghai Province and Hebei Province[6-16]， but so far， there has been no report on the research of the JIVET technology of superior sheep breeds in Shanxi Province. For this reason， the Institute of Animal Husbandry and Veterinary Medicine， Shanxi Academy of Agricultural Sciences and China Agricultural University conducted research on the JIVET technology of Texel sheep， providing technical support for perfecting the JIVET technical system. Materials and Methods
Experimental materials
Experimental sheep
Texel sheep of 4-8 weeks old were provided by the sheep farm of the Institute of Animal Husbandry and Veterinary Medicine， Shanxi Academy of Agricultural Sciences， and the recipient sheep were provided by the sheep farm of Taiyuan Qinglian Breeding Co.， Ltd.
Frozen semen and estrus ewe serum
Texel sheep frozen pellet semen and estrus ewe serum （ESS） were made in laboratory.
Main hormones and reagents
Sheep CIDR， follicle stimulating hormone （FSH）， pregnant mare serum gonadotropin （PMSG）， 17β-estradiol （17β-E2） and luteinizing hormone （LH） were all purchased from Beijing Luxin Agriculture and Animal Husbandry Technology Co.， Ltd.
Culture media
Egg collection solution： M199+20 mmol/L Hepes+2% ESS+10 mg/ml heparin sodium; maturation solution： M199+20% ESS+10 μg/ml FSH+10 μg/ml LH+1 μg/ml 17β-E2; sperm capacitation fluid： synthetic fallopian tube fluid （SOF）+20 μg/ml heparin sodium; fertilization fluid： SOF+2% ESS; fertilized egg development fluid： SOF+8 mg/ml BSA+2% EAA+1% NEAA.
Experimental methods
ESS preparation
The blood of the estrus ewe was taken from the jugular vein， and the serum was prepared according to conventional method.
Frozen semen production
The semen of Texel sheep with a vitality of 0.8 or more was prepared by conventional dry ice freezing method to prepare frozen pellet semen.
Recipient sheep in estrus
The method of simultaneous estrus in recipient sheep adopted by Mao et al.[17] was applied with the difference that the recipient sheep was implanted with CIDR for 12-14 d， and the amount of PMSG injected intramuscularly was 320-360 U/sheep.
Superovulation in young sheep
The lamb superovulation method of Kelly et al.[1] was adopted. Four Texel young sheep with ear numbers T1104， T1110， T1112 and T1116 were superovulated. The difference was that the PMSG injection volume was 300-350 U/sheep.
Oocyte collection
After the donor lamb was anesthetized and fixed， and the surgical site was disinfected. Then， a scalpel was used to cut a small opening of 3-5 cm in the anterior abdomen of the breast slightly off the midline， and the ovary was gently pulled out from the abdominal cavity. The 18# needle on a 10 ml syringe with 2 ml of egg collection fluid was used to puncture the follicles above 2 mm in diameter on the ovary and suck out the cumulus-oocyte complexes （COCs）. After the egg collection was completed， the surgical opening was sutured and disinfected. The collected oocytes were placed in a 75 mm petri dish， and the well-formed A and B-grade COCs with more than 2 layers of cumulus cells and uniform cytoplasm were quickly picked out under a stereo microscope and placed in the maturation fluid.
In-vitro sperm capacitation
The in-vitro sperm capacitation method of Bai[6] was adopted.
In-vitro oocyte maturation and fertilization and fertilized egg culture
The collected A and B-grade COCs were put into the maturation fluid in a four-well culture plate， covered with mineral oil， and matured in a 5% CO2 incubator （38.5 ℃， saturated humidity） for about 24 h. The discharge of the first polar body from the oocytes was the criterion for the maturation of oocytes.
The mature oocytes were gently blown and beaten in a 0.2% hyaluronidase solution to remove part of the cumulus cells. They were then put into the fertilization fluid in the wells of the four-well culture plate， added with capacitated sperm （1×106-2×106 Pieces/ml）， and covered with mineral oil. The oocytes were fertilized and incubated in a 5% CO2 incubator for 24 h.
After removing the remaining sperm and cumulus cells attached to the fertilized eggs， they were put into the fertilized egg development fluid in a four-well culture plate and covered with mineral oil. Next， the four-well plate was put into an aluminum foil bag （filled with a mixed gas of 5% CO2-7% oxygen-88% nitrogen）， which was sealed. The fertilized eggs were incubated in a 5% CO2 incubator for about 24 h， and the cleavage condition was observed under a microscope， followed by counting the cleavage rate.
Fertilized egg transplanting
The operation method was the same as "Oocyte collection". The ovary and fallopian tube on the side with the ovulation point was gently pulled out of the abdominal cavity， and an embryo transfer device was used to suck 6 fertilized eggs at 2-4 cell stage and implanted into the ampulla of the fallopian tube.
Data processing method
The test data was processed in Excel.
Results and Analysis
The effect of superovulation
After superovulation， the Texel sheep of 4-8 weeks old showed enlarged swollen ovaries full of developing follicles. The COCs collected from Texel lambs T1104， T1110， T1112 and T1116 were 86， 72， 104， and 51， respectively， totaling 313， with an average of 78.25 COCs per animal. Among them， there were 296 A and B-grade COCs， with an average of 74.0 COCs/sheep. In-vitro oocyte development
After the 296 A and B-grade COCs were matured and cultured in vitro， 241 first polar bodies were discharged from the oocytes， that is， the maturation rate of oocytes was 81.41%. The mature oocytes and capacitated sperm were incubated together. After the fertilized eggs were cultured， 205 fertilized eggs split into 2-4 cells， that is， the cleavage rate was 69.26%， and the average was 51.25 eggs per sheep.
In-vitro transplantation effect of fertilized eggs at the 2-4 cell stage
Sixty six in-vitro fertilized eggs at the 2-4 cell stage were transplanted into the ampullae of 11 estrus recipient ewes. Six ewes were conceived and 11 lambs were born. Among the 6 ewes， 1 ewe gave birth to 3 lambs， 3 ewes gave birth to two lambs， and 2 lambs produced single lamb. The conception rate was 54.55%.
Discussion
The effect of superovulation method on the JIVET technical system
The superovulation of young sheep with gonadotropins for developing follicles on the ovaries of young sheep and obtain a large number of oocytes is the basis of the JIVET technical system. In the research field of hormone-induced young sheep， Kelly et al.[1，3] studied the effects of follicular development on lambs of 1 to 2 months old injected with FSH 160 mg/sheep and PMSG 500 U/sheep， and obtained oocytes at 80.0 oocytes/sheep or above. Gou et al.[5] used FSH and PMSG to induce 1-month-old lambs， obtaining 79.1 oocytes per lamb. Guo et al.[8] used gonadotropins to induce Merino sheep and Kazakh Sheep of 4 to 8 weeks old to study the effect of ovarian follicle development， and obtained oocytes at 135.8 and 37.8 oocytes per sheep， respectively. Yu et al.[18] induced the follicular development in Tao Han hybrid sheep at the age of 1 and 3 months by injecting FSH 160 mg/sheep， 4 times in two days， and adding PMSG 500 U/sheep at the last injection of FSH. The 1-month-old lamb produced 79.0 oocytes per sheep， which was significantly higher than the 3-month-old lambs producing 18.5 oocytes per sheep. In this study， the effect of gonadotropins on the development of ovarian follicles in Texel sheep of 4 to 8 weeks old was studied， and 78.25 COCs were obtained per sheep. The number of oocytes obtained from young Texel sheep is higher than the results of Guo et al.[8] on young Kazakh sheep， but lower than the results of Guo et al.[8] on young Merino sheep， and basically the same as the results of Kelly et al.[8]， Gou et al.[5] and Yu et al.[18] on lambs of 1 to 2 months old. It shows that the method of using gonadotropins to induce the development of ovarian follicles in young Texel sheep is feasible. Effects of the methods of oocyte maturation and fertilization method and fertilized egg culture in vitro on the JIVET technical system
Methods of in-vitro maturation， fertilization and incubation of oocytes and fertilized egg culture are important parts of the JIVET technical system. The three constitute the JIVET technical culture system， and any problem in any culture link will affect the effect of JIVET technology. In the research of the JIVET technical culture system， Bai et al.[4] studied the effect of 600 μmol/L cysteine in the maturation fluid on the development of Mongolian lamb oocytes， and the fertilization rate was 86.9%. Wu et al.[19] studied the effect of in-vitro maturation time of Xinjiang fine-wool lamb oocytes on the effect of in-vitro fertilization， and the cleavage rate of oocytes matured for 24 h was 52.98%. Guo et al.[20] studied the effect of adding 100 μmol/L β-mercaptoethanol to the maturation solution on the oocyte development ability of sheep at the age of 4-8 weeks old， and the cleavage rate was 68.0%. In this study， the in-vitro oocyte culture system of Texel sheep at the age of 4-8 weeks was studied， and the cleavage rate was 69.26%. The cleavage rate of young Texel sheep is higher than that of Xinjiang fine wool lambs obtained by Wu et al.[19]， and basically consistent with the results of Bai et al.[4] and Guo et al.[20] on young sheep. It is indicated that the methods of oocyte maturation， fertilization and incubation and fertilized egg culture of young Texel sheep in vitro in this study are feasible.
Effect of transplantation method of fertilized eggs at 2-4 cell stage on the JIVET technical system
Sheep's egg and sperm combination is in the ampulla of the oviduct， and the development of the fertilized egg to the 16-cell stage is completed in the oviduct. Therefore， eggs fertilized in vitro should be transplanted into the fallopian tube at the 2-4 cell stage. Wang et al.[7]， Guo et al.[8]， Liu et al.[9]， Ma et al.[10] and Chen et al.[11] studied the JIVET techniques of Suffolk sheep， Kazakh sheep， Dorset sheep and small-tailed Han sheep， and the conception rates ranged from 16.6% to 53.6%. In this study， the eggs fertilized in vitro were transplanted at the 2-4 cell stage into the ampullae of the oviducts of the recipient ewes in estrus， and the conception rate was 54.55%， which is consistent with the results of the above studies. The results show that the transplantation method of fertilized eggs at the 2-4 cell stage used in this study is feasible. Conclusions
In this study， a total of 296 A and B-grade oocytes were obtained from 4 Texel sheep of 4-8 weeks old， at an average of 74.0 oocytes per sheep. After in-vitro maturation， fertilization， incubation and culture， 205 fertilized eggs at the 2-4 cell stage were obtained， at a cleavage rate of 69.26% and an average of 51.25 eggs per sheep. Sixty six fertilized eggs at the 2-4 cell stage were transplanted to 11 recipient sheep， among which 6 ewes were conceived， and gave birth to 11 "test tube sheep"， showing a conception rate of 54. 55%. The results show that the method of this study can be used as a method for the research on the JIVET technology of Texel sheep.
References
[1] KELLY JM， KLEEMANN DO， WALKER SK. Enhanced efficiency in the production of offspring from 4-to 8-week-old lambs[J]. Theriogenology， 2005， 63 （7）： 1876-1890.
[2] RANGEL-SANTOS R， MCDONALD MF， WICKHAM GA. Evaluation of the feasibility of a juvenile MOET scheme in sheep[J]. Proceedings of The New Zealand Society of Animal Production， 1991， 51 （1）： 139-142.
[3] KELLY JM， KLEEMANN DO， WALKER SK. The effect of nutrition during pregnancy on the in vitro production of embryos from resulting lambs[J]. Theriogenol， 2005， 63 （7）： 2020-2031.
[4] BAI JH， HOU J， GUAN H， et al. Effect of 2-mercaptoethanol and cysteine supplementation during in vitro maturation on the developmental competence of oocytes from hormone-stimulated lambs[J]. Theriogenol， 2008， 70（5）： 758-764.
[5] GOU KM， GUAN H， BAI JH， et al. Field evaluation of juvenile in vitro embryo transfer （ JIVET） in sheep[J]. Anim Reprod Sci， 2009， 112 （3/4）： 316-324.
[6] BAI JH. Study on JIVET technology of sheep and cattle before sexual maturity[D]. Beijing： China Agricultural University， 2008. （in Chinese）
[7] WANG LQ， HE ZL， LIN JP， et al. Study on the induced development effect of Suffolk lamb follicular[J]. Journal of China Agricultural University， 2015， 20 （4）： 141-146. （in Chinese）
[8] GUO H， WAN PC， SHI WY， et al. Study on in vitro production （ivp） of embryo utilizating superovulated oocytes from different breed lambs[J]. Animal Husbandry and Veterinary Medicine， 2012， 44 （10）： 20-24. （in Chinese）
[9] LIU ZZ， LI HG， CHEN M， et al. Study on rapid propagation of suffolk sheep by using JIVET technology[J]. Animal Husbandry and Feed Science， 2016， 37 （3）： 1-4. （in Chinese）
[10] MA SK， JIAO HS， MA LQ， et al. Application research on JIVET in lamb in Qinghai plateau area[J]. Chinese Qinghai Journal of Animal and Veterinary Sciences， 2015， 45 （2）： 13-14. （in Chinese） [11] CHEN XY， TIAN SJ， SANG RZ， et al. Inducement of lamb follicular development and embryo production in vitro[J]. Chinese Journal of Agricultural Biotechnology， 2008， 16 （3）： 456-460. （in Chinese）
[12] ZHANG XH， ZHANG SW， SONG XC， et al. Study on follicular development of female lamb and technology of oocyte maturation[J]. China Animal Husbandry and Veterinary Medicine， 2011， 38 （11）： 136-139. （in Chinese）
[13] WU HF， LI FD， CHANG WH， et al. The effect of different treatments on superovulation and in vitro fertilization （IVF） efficiency[J]. Journal of Gansu Agricultural University， 2011， 46 （6）： 1-5. （in Chinese）
[14] XU XS， LIU CJ， MAO XL. Detection of vitamin C content in 16 common fruits and vegetables in Zunyi City[J]. Vegetables， 2012（6）： 49-50. （in Chinese）
[15] YIN XJ， LIU L. Analysis on color reaction of amino acids with ninhydrin by RGB value method[J]. Guangzhou Chemical Industry， 2018， 46（07）： 64-66. （in Chinese）
[16] LI M， QI F， ZHOU Z， et al. Dynamics research on changes of salt and lactic acid content in pickling process of radish[J]. China Condiment， 2013， 38（9）： 31-36. （in Chinese）
[17] LIU W， ZHANG MM， SONG Y， et al. Non-enzymatic browning kinetics of pickled white radish with low salt[J]. Food Science， 2016， 37（13）： 65-70. （in Chinese）
[18] LIU LL， ZHANG H， LIANG CY. Effect of curing time on texture color of spicy cabbage flavored beef jerky[J]. Journal of Agricultural Science Yanbian University， 2019， 41（2）： 80-85. （in Chinese）
[19] XIAO LL， ZHONG K， FENG PH， et al. Optimization of green-protecting and brittleness-keeping method of green pepper and its curing process[J]. Sichuan Food and Fermentation， 2019， 55（5）： 40-44， 50. （in Chinese）
[20] ZHOU Q， LIU MJ， YOU HY. Study on the effect of fermentation conditions on the quality of pickled radish and its process optimization[J]. China Condiment， 2018， 43（2）： 12-16， 22. （in Chinese）
[21] YANG L， WU ZY， ZENG WW， et al. Quantitative systematic review of the growth and decline rule of Escherichia coli during the fermentation process of Sichuan pickle[J]. Food Safety and Quality Detection Technology， 2017， 8（12）： 4529-4533. （in Chinese）
[22] WANG F. Research of the change of nitrite content in the process of vegetables pickled[J]. Farm Products Processing， 2016（1）： 6-9， 11. （in Chinese）
[23] CHE ZQ， XIA YB， LEI C， et al. Study on the effect of pretreatment methods on the flavor and texture of radish before pickling[J]. Modern Food Science & Technology， 2017， 2017（1）： 197-205. （in Chinese）