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Abstract This study was conducted to investigate the effects of different concentrations of βmercaptoethanol on the development ability of oocytes in Dorper sheep at the age of 6 weeks. In this study， 6 Dorper sheep of 6 weeks old were chosen to induce oocyte collection in vivo by gonadotropin， and A and B grade cumulus oocyte complexes were obtained （COCs） with different concentrations of （0， 50， 70 and 100 μmol/L） of βmercaptoethanol in the mature liquid. The cleavage rate and blastocyst rate were counted after matured oocytes and capacitated sperm were coincubated and fertilized eggs were cultivated. The results showed that compared with the cleavage rate （59.13%） and the blastocyst rate （12.17%） of the control group， the cleavage rates （60.87%， 63.48% and 65.22%） and blastocyst rates （14.78%， 17.39% and 21.74%） of the test groups I， II and III were higher （P>0.05）， and the order was test group III>test group II>test group I>control group. The cleavage rate and blastocyst rate of the 100 μmol/L βmercaptoethanol group increased by 6.09 percentage points and 9.57 percentage points， respectively. It indicated that the addition of a certain amount of βmercaptoethanol in the mature liquid could improve the developmental ability of oocytes and the quality of fertilized eggs of the 6weekold Doper sheep. When the βmercaptoethanol concentration was within 100 μmol/L， the cleavage rate and the blastocyst rate of oocytes were on the rise. The optimal concentration of βmercaptoethanol in the mature liquid still needs further study.
Key words βmercaptoethanol; 6weekold Dorper sheep; Oocyte; Developmental ability
The invitro production technology of young sheep embryos began in the early 1970s. Worthington et al.[1]injected HCG into lambs of 5-6 weeks old and found that a large number of follicles in the ovary of the lamb began to develop. Trounson et al.[2]studied the oocytes of young lambs aged 10-16 weeks， and developed the oocytes in vitro to blastocysts. In the 21stcentury， scientists have conducted more indepth research on lamb follicular development and invitro fertilization techniques. Nowadays， a large number of oocytes of young sheep have been obtained by superovulation technology of young animals， which gave "test sheep" through a series of incubation and cultivation processes such as invitro maturation， fertilization and embryo development[3-12]. However， in the invitro production technology system of young sheep embryos， there have been problems such as poor oocyte development ability and insufficient oocyte cytoplasmic maturation[13-14]. To this end， the researchers mimicked the oocyte maturation conditions in vivo， and added fetal bovine serum （FBS）， glutathione （GSH）， follicle stimulating hormone （FSH）， 17βestradiol （17βE2）， luteinizing hormone （LH）， growth factors （IGF1， FGF， EGF） and energy substances to the mature liquid， with an attempt to improve the developmental ability of young oocytes in young sheep by improving oocyte maturation. Han et al.[15]studied the effects of glutathione on oocyte maturation and embryonic development. Cao et al.[16]studied the effects of vascular endothelial growth factor on the ultrastructural changes of mature oocytes of mature sheep in vitro. However， so far， there have been no reports on the effects of different concentrations of βmercaptoethanol in the mature liquid on the development of oocytes in young sheep. Therefore， in this study， the effects of different concentrations of βmercaptoethanol mature solutions on the developmental ability of oocytes in 6weekold Dorper sheep were investigated， aiming at providing reference for optimizing the invitro production technology system of young sheep embryos. Materials and Methods
Materials
Test time and location
The experiment was carried out in the Animal Genetic Engineering Laboratory of Institute of Animal Husbandry and Veterinary， Shanxi Academy of Agricultural Sciences from October 7 to 25， 2014.
Test sheep
Six 6weekold Dorper sheep were provided by the Jinzhong Linshan Farming Cooperative.
Frozen semen and estrus sheep serum （ESS）
Frozen semen and ESS of Dorper sheep were obtained in the laboratory.
Main hormones， reagents and consumable items
Luteinizing hormone （LH）， 17 βestradiol （17βE2）， follicle stimulating hormone （FSH） and pregnant horse serum （PMSG） were purchased from Beijing Luxin Agriculture and Animal Husbandry Technology Co.， Ltd. Heparin sodium and bovine serum albumin （BSA）， essential amino acids （EAAs） and nonessential amino acids （NEAAs） of Solarbio brand， M199， Hepes， βmercaptoethanol， NaCl， CaCl2·2H2O and NaHCO3 of SIGMA brand， round bottom glass test tubes， centrifuge tubes and petri dishes of Corning brand， and fourhole culture plates of Nunc brand， were all purchased from Shanxi Cell Biotech Co.， Ltd.
Main instruments
Ultraclean workbench （SWCJ2FD）， centrifuge （Sigma 316PK）， stereomicroscope （OLYMPUS SZX10）， researchgrade inverted microscopy system （OLYMPUS IX71） and carbon dioxide incubator （SANYO MCO5AC） were all provided by the Animal Genetic Engineering Laboratory of Institute of Animal Husbandry and Veterinary， Shanxi Academy of Agricultural Sciences.
Main surgical instruments and supplies
Scalpels， surgical scissors， hemostats， syringes， 75% alcohol and iodine were purchased from Shanxi Medical Equipment Company.
Culture media
Eggcollecting liquid： M199 （containing 20 mmol/LHepes， 2% ESS， 10 mg/ml heparin sodium）; mature liquid of the control group： M199 （containing 20% ESS， 10 μg/mlFSH， 10 μg/ml LH， 1 μg/ml 17βE2）; mature liquid of test group I， II， III： on the basis of the mature liquid in the control group， containing 50， 70 and 100 μmol/L βmercaptoethanol， respectively; capacitation liquid： synthetic oviduct fluid （SOF） （containing 20 μg/ml heparin sodium）; fertilizing liquid： SOF （containing 2% ESS）; development liquid of fertilized eggs： SOF （containing 8 mg/ml BSA， 2% EAA， 1% NEAA）.
Methods
Hormone induction and oocyte collection of 6weekold Dorper sheep
The lamb hormone induction method of Kelly et al.[3]was adopted， with a modification that the amount of PMSG injection was 300-350 IU per sheep. Oocytes were collected in vivo by the method of Bai[7]. ESS preparation
Blood was collected from the jugular vein of sheep in estrus for 1 d， and stood in a refrigerator for about 20 h at 4 ℃. The serum was obtained and inactivated in a water bath at 56 ℃ for 30 min， filtered through a 0.22 μm filter， and stored at -20 ℃.
Frozen semen preparation
The granule frozen semen was prepared by the conventional dry ice freezing method from the Dorper sheep semen with vitality of 0.8 or more.
Capacitation of sperm
Invitro capacitation was performed according to the method of Bai[7].
Oocyte maturation
The invitro maturation of oocytes was performed according to the method of Bai[7]with a modification that the A and B grade COCs of the 6 young Dorper sheep collected were randomly divided into 4 parts， which were then added into the mature liquid of the control group， the test group I， the test group II and the test group III in fourwell culture plates which had been equilibrated in a 5% carbon dioxide （38.5 °C， saturated humidity） incubator for 2 h， and then covered by mineral oil was covered. The liquids were then cultured for 24 h in a 5% carbon dioxide incubator.
Oocyte fertilization and fertilized egg culture
Invitro fertilization of oocytes and culture of fertilized eggs were performed according to the method of Bai[7]. The oocytes and sperms were coincubated for about 24 h， and the cleavage condition was observed under a microscope. The fertilized eggs were transferred to the fertilized egg development liquid for about 12 h to calculate the cleavage rate. The culture was continued for 4 to 5 d to observe the development of the fertilized eggs. And the morula rate or blastocyst rate were then calculated.
Data professing
Data processing was performed using Excel， and the χ2 test method was applied for significant analysis.
Results and Analysis
Inducing effect of young Doper sheep with gonadotropin
It can be seen from Table 1 that a total of 489.0 cumulusoocyte complexes （COCs） were collected from six 6weekold Doper sheep， with an average of 81.5 COCs per sheep. Among them， 460.0 were A and B COCs， with an average of 76.7 COCs per sheep. It can be seen from Fig. 1 that after gonadotropin induction， the ovary volume of young Doper sheep increased， in red color， and densely covered by follicles filled with follicular fluid.
Effects of different concentrations of βmercaptoethanol mature liquid on oocyte development 460.0 A and B oocytes were matured in the control group， test group I， test group II and test group III containing different concentrations （0， 50， 70 and 100 μmol/L） of βmercaptoethanol， respectively. After invitro fertilization incubation， fertilized egg development and other processes， oocyte development is shown in Table 2 and Fig. 2-Fig. 3.
It can be seen from Table 2 that the cleavage rate and blastocyst rate of the test groups I， II and III were higher than those of the control group （P>0.05）， and the order was test group III>test group II>test group I>control group. The cleavage rate and blastocyst rate of oocytes in the 100 mol/L βmercaptoethanol group increased by 6.09 percentage points and 9.57 percentage points， respectively， that is to say， the blastocyst rate increased more than the cleavage rate. It indicated that the addition of βmercaptoethanol to the oocyte mature liquid could increase the cleavage rate and blastocyst rate of young oocytes， especially the blastocyst rate. Within the βmercaptoethanol concentration of 100 μmol/L， the cleavage rate and blastocyst rate of oocytes increased.
Agricultural Biotechnology2019
Discussion
The invitro culture system of young animal embryos not only directly affects the degree of oocyte maturation and the ability to continue to develop， but also directly affects the efficiency of invitro production of young animal embryos. Bai[7]and Guo et al.[17]added 100 μmol/L βmercaptoethanol to the invitro mature liquid of Mongolian lamb and lamb oocytes from 4 to 8 weeks old. The results showed that the normal fertilization ratio of oocytes and the blastocyst rate increased significantly; the proportion of polyspermy decreased significantly; and the proportion of normal fertilization was not significantly different from that of adult sheep （P>0.05）. Quan et al.[18]found that the addition of βmercaptoethanol to the granulosa cell culture medium of bovine oocytes can increase the viable cell rate， and the viable cell rate increased with the increase of βmercaptoethanol concentration， and reached the highest value at the concentration of 25 μmol/L. Shu et al.[19]showed that the addition of 100 μmol/L βmercaptoethanol into bovine oocyte culture medium can significantly increase the blastocyst rate of bovine embryos and the number of cells in the cell mass of blastocysts after invitro fertilization. Lan et al.[20]found that the addition of βmercaptoethanolto the mature liquid of pig oocytes can increase the concentration of glutathione in the cytoplasm， which provides suitable intracellular conditions to the fertilized eggs， thereby preventing oxidative damage and helping the embryos develop to the blastocysts. In this study， the results showed that the addition of （50， 70 and 100 μmol/L） βmercaptoethanol to the oocyte mature liquid of 6weekold Doper sheep increased the cleavage rate and blastocyst rate， especially the blastocyst rate; and the cleavage rate （65.22%） and blastocyst rate （21.74%） of the test group III （containing 100 μmol/L βmercaptoethanol） were the highest. When the βmercaptoethanol concentration was within 100 μmol/L， the cleavage rate and the blastocyst rate of oocytes were on the increase， and the blastocyst rate increased more than the cleavage rate， indicating that βmercaptoethanol helps the oocytes to develop into the blastocysts. The results of this study are consistent with the results of Bai[7]， Guo et al.[17]， Quan et al.[18]， Shu et al.[19]and Lan et al.[20]. The reason might be that βmercaptoethanol is a low molecular weight mercaptoalcohol compound capable of reducing cystine to cysteine which is a substrate for the synthesis of glutathione （GSH）. GSH is present in every cell of organisms and has antioxidant and detoxifying effects. During oocyte development， it protects the functional proteins and enzymes required for cell development， amino acid transport， protein synthesis and DNA replication from oxidation through free mercapto groups （SH）， thereby eliminating the retarding or delaying effect of active oxygen free radicals （ROS） on further oocyte development， accelerating sperm depolymerization and pronuclei formation in fertilized eggs， reducing parthenogenetic activation and polyspermy， and increasing the proportion of normal fertilization， the developmental ability of fertilized eggs and blastocyst rate. Conclusions
The results of this study indicate that the addition of a certain amount of βmercaptoethanol to the mature liquid can improve the developmental ability of oocytes and the quality of fertilized egg of the 6weekold Doper sheep， and when the βmercaptoethanol concentration was within 100 μmol/L， the cleavage rate and blastocyst rate of oocytes showed an upward trend. The optimal concentration of βmercaptoethanol in the mature liquid still needs further study.
References
[1] WORTHINQTON CA， KENNEDY JP. Ovarian response to exogenous hormones in sixweek old lambs[J]. Aust J Biol Sci， 1979， 32（7）： 91-95.
[2] TROUNSON AO， WILLADSEN SM， MOOR RM. Reproductive function in prepubertal lambs：ovulation， embryo development and ovarian steroidogenesis[J]. J Reprod Fertil， 1977， 49： 69-75.
[3] KELLY JM， KLEEMANND O， WALKER SK. Enhanced efficiency in the production of offspring from 4 to 8weekold lambs[J]. Theriogenology， 2005， 63（7）： 1876-1890.
[4] KELLY JM， KLEEMANN DO， WALKER SK. The effect of nutrition during pregnancy on the in vitro production of embryos from resulting lambs[J].Theriogenology， 2005， 63（7）： 2020-2031.
[5] GOU KM， GUAN H， BAI JH， et al. Field evaluation of juvenile in vitro embryo transfer （JIVET）in sheep[J].Animal Reproduction Science， 2009， 112（3/4）： 316-324.
[6] BAI JH， HOU J， GUAN H， et al. Effect of 2mercaptoethanol and cysteine supplementation during in vitro maturation on the developmental competence of oocytes from hormonestimulated lambs[J]. Theriogenology， 2008， 70（5）： 758-764.
[7] BAI JH. Premature sheep and cattle JIVET technology research[D]. Beijing： China Agricultural University， 2008. （in Chinese）
[8] GUO H， WANG PC， SHI WY， et al. Study on superovulation and embryo production technology of different sheep breeds[J]. Animal Husbandry and Veterinary Medicine， 2012， 44（10）： 20-24. （in Chinese）
[9] 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）
[10] LIU ZZ， LI HG， CHENG M， et al. Study on rapid propagation of suffolk sheep by using JIVET technology[J]. Animal Husbandry and Feed Science， 2016， 37（3）： 1-3. （in Chinese）
[11] 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） [12] 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）
[13] GUO H， WANG PC， DAI R， et al. Analysis of influencing factors on the oocyte in vitro embryo production in lambs[J]. Hubei Agricultural Sciences， 2013， 52（19）： 4726-4729. （in Chinese）
[14] LI YS， CAO HG， LIU Y， et al. Advance in goat and sheep juvenile in vitro embryo transfer[J]. Progress In Veterinary Medicine， 2011， 32（11）： 99-104. （in Chinese）
[15] HAN Y， YIN D， ZHANG T， et al. Research progress on effects of glutathione on maturation and embryo development of oocyte[J]. Jiangsu Agricultural Sciences， 2010（3）： 269-270. （in Chinese）
[16] CAO X， ZHAO YZ， LUO HL， et al. Effects of VEGF on the ultrastructure of ovine oocytes cultured in vitro[J]. Acta Veterinaria et Zootechnica Sinica， 2010， 41（2）： 167-173. （in Chinese）
[17] GUO H， WANG PC， SHI WY， et al. Effects of EGF and βmercaptoethanol on invitro fertilization and development of oocytes collected from hormonestimulated lambs[J]. Journal of Anhui Agricultural Sciences， 2012， 40（11）： 6537-6540. （in Chinese）
[18] QUAN FS， LIU Q， WANG L， et al. Effects of βmercaptoethanol and estrus sheep serum on bovine culumus cells in vitro culture[J]. Acta Agriculturae Borealioccidentalis Sinica， 2011， 20（12）： 22-25. （in Chinese）
[19] SHU JH， ZHANG ZP， LI XC， et al. Effect of βmercaptoethanol on the development of the bovine parthenogenetic embryos[J]. Journal of Northwest A & F University： Natural Science Edition， 2008， 36（12）： 24-28. （in Chinese）
[20] LAN ZB， HE RG， MO PF， et al. Effect of βmercaptoethanol on invitro fertilization and embryo development of porcine oocytes[J]. Guangxi Journal of Animal Husbandry & Veterinary Medicine 2008， 24（4）： 202-203. （in Chinese）
Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU
Key words βmercaptoethanol; 6weekold Dorper sheep; Oocyte; Developmental ability
The invitro production technology of young sheep embryos began in the early 1970s. Worthington et al.[1]injected HCG into lambs of 5-6 weeks old and found that a large number of follicles in the ovary of the lamb began to develop. Trounson et al.[2]studied the oocytes of young lambs aged 10-16 weeks， and developed the oocytes in vitro to blastocysts. In the 21stcentury， scientists have conducted more indepth research on lamb follicular development and invitro fertilization techniques. Nowadays， a large number of oocytes of young sheep have been obtained by superovulation technology of young animals， which gave "test sheep" through a series of incubation and cultivation processes such as invitro maturation， fertilization and embryo development[3-12]. However， in the invitro production technology system of young sheep embryos， there have been problems such as poor oocyte development ability and insufficient oocyte cytoplasmic maturation[13-14]. To this end， the researchers mimicked the oocyte maturation conditions in vivo， and added fetal bovine serum （FBS）， glutathione （GSH）， follicle stimulating hormone （FSH）， 17βestradiol （17βE2）， luteinizing hormone （LH）， growth factors （IGF1， FGF， EGF） and energy substances to the mature liquid， with an attempt to improve the developmental ability of young oocytes in young sheep by improving oocyte maturation. Han et al.[15]studied the effects of glutathione on oocyte maturation and embryonic development. Cao et al.[16]studied the effects of vascular endothelial growth factor on the ultrastructural changes of mature oocytes of mature sheep in vitro. However， so far， there have been no reports on the effects of different concentrations of βmercaptoethanol in the mature liquid on the development of oocytes in young sheep. Therefore， in this study， the effects of different concentrations of βmercaptoethanol mature solutions on the developmental ability of oocytes in 6weekold Dorper sheep were investigated， aiming at providing reference for optimizing the invitro production technology system of young sheep embryos. Materials and Methods
Materials
Test time and location
The experiment was carried out in the Animal Genetic Engineering Laboratory of Institute of Animal Husbandry and Veterinary， Shanxi Academy of Agricultural Sciences from October 7 to 25， 2014.
Test sheep
Six 6weekold Dorper sheep were provided by the Jinzhong Linshan Farming Cooperative.
Frozen semen and estrus sheep serum （ESS）
Frozen semen and ESS of Dorper sheep were obtained in the laboratory.
Main hormones， reagents and consumable items
Luteinizing hormone （LH）， 17 βestradiol （17βE2）， follicle stimulating hormone （FSH） and pregnant horse serum （PMSG） were purchased from Beijing Luxin Agriculture and Animal Husbandry Technology Co.， Ltd. Heparin sodium and bovine serum albumin （BSA）， essential amino acids （EAAs） and nonessential amino acids （NEAAs） of Solarbio brand， M199， Hepes， βmercaptoethanol， NaCl， CaCl2·2H2O and NaHCO3 of SIGMA brand， round bottom glass test tubes， centrifuge tubes and petri dishes of Corning brand， and fourhole culture plates of Nunc brand， were all purchased from Shanxi Cell Biotech Co.， Ltd.
Main instruments
Ultraclean workbench （SWCJ2FD）， centrifuge （Sigma 316PK）， stereomicroscope （OLYMPUS SZX10）， researchgrade inverted microscopy system （OLYMPUS IX71） and carbon dioxide incubator （SANYO MCO5AC） were all provided by the Animal Genetic Engineering Laboratory of Institute of Animal Husbandry and Veterinary， Shanxi Academy of Agricultural Sciences.
Main surgical instruments and supplies
Scalpels， surgical scissors， hemostats， syringes， 75% alcohol and iodine were purchased from Shanxi Medical Equipment Company.
Culture media
Eggcollecting liquid： M199 （containing 20 mmol/LHepes， 2% ESS， 10 mg/ml heparin sodium）; mature liquid of the control group： M199 （containing 20% ESS， 10 μg/mlFSH， 10 μg/ml LH， 1 μg/ml 17βE2）; mature liquid of test group I， II， III： on the basis of the mature liquid in the control group， containing 50， 70 and 100 μmol/L βmercaptoethanol， respectively; capacitation liquid： synthetic oviduct fluid （SOF） （containing 20 μg/ml heparin sodium）; fertilizing liquid： SOF （containing 2% ESS）; development liquid of fertilized eggs： SOF （containing 8 mg/ml BSA， 2% EAA， 1% NEAA）.
Methods
Hormone induction and oocyte collection of 6weekold Dorper sheep
The lamb hormone induction method of Kelly et al.[3]was adopted， with a modification that the amount of PMSG injection was 300-350 IU per sheep. Oocytes were collected in vivo by the method of Bai[7]. ESS preparation
Blood was collected from the jugular vein of sheep in estrus for 1 d， and stood in a refrigerator for about 20 h at 4 ℃. The serum was obtained and inactivated in a water bath at 56 ℃ for 30 min， filtered through a 0.22 μm filter， and stored at -20 ℃.
Frozen semen preparation
The granule frozen semen was prepared by the conventional dry ice freezing method from the Dorper sheep semen with vitality of 0.8 or more.
Capacitation of sperm
Invitro capacitation was performed according to the method of Bai[7].
Oocyte maturation
The invitro maturation of oocytes was performed according to the method of Bai[7]with a modification that the A and B grade COCs of the 6 young Dorper sheep collected were randomly divided into 4 parts， which were then added into the mature liquid of the control group， the test group I， the test group II and the test group III in fourwell culture plates which had been equilibrated in a 5% carbon dioxide （38.5 °C， saturated humidity） incubator for 2 h， and then covered by mineral oil was covered. The liquids were then cultured for 24 h in a 5% carbon dioxide incubator.
Oocyte fertilization and fertilized egg culture
Invitro fertilization of oocytes and culture of fertilized eggs were performed according to the method of Bai[7]. The oocytes and sperms were coincubated for about 24 h， and the cleavage condition was observed under a microscope. The fertilized eggs were transferred to the fertilized egg development liquid for about 12 h to calculate the cleavage rate. The culture was continued for 4 to 5 d to observe the development of the fertilized eggs. And the morula rate or blastocyst rate were then calculated.
Data professing
Data processing was performed using Excel， and the χ2 test method was applied for significant analysis.
Results and Analysis
Inducing effect of young Doper sheep with gonadotropin
It can be seen from Table 1 that a total of 489.0 cumulusoocyte complexes （COCs） were collected from six 6weekold Doper sheep， with an average of 81.5 COCs per sheep. Among them， 460.0 were A and B COCs， with an average of 76.7 COCs per sheep. It can be seen from Fig. 1 that after gonadotropin induction， the ovary volume of young Doper sheep increased， in red color， and densely covered by follicles filled with follicular fluid.
Effects of different concentrations of βmercaptoethanol mature liquid on oocyte development 460.0 A and B oocytes were matured in the control group， test group I， test group II and test group III containing different concentrations （0， 50， 70 and 100 μmol/L） of βmercaptoethanol， respectively. After invitro fertilization incubation， fertilized egg development and other processes， oocyte development is shown in Table 2 and Fig. 2-Fig. 3.
It can be seen from Table 2 that the cleavage rate and blastocyst rate of the test groups I， II and III were higher than those of the control group （P>0.05）， and the order was test group III>test group II>test group I>control group. The cleavage rate and blastocyst rate of oocytes in the 100 mol/L βmercaptoethanol group increased by 6.09 percentage points and 9.57 percentage points， respectively， that is to say， the blastocyst rate increased more than the cleavage rate. It indicated that the addition of βmercaptoethanol to the oocyte mature liquid could increase the cleavage rate and blastocyst rate of young oocytes， especially the blastocyst rate. Within the βmercaptoethanol concentration of 100 μmol/L， the cleavage rate and blastocyst rate of oocytes increased.
Agricultural Biotechnology2019
Discussion
The invitro culture system of young animal embryos not only directly affects the degree of oocyte maturation and the ability to continue to develop， but also directly affects the efficiency of invitro production of young animal embryos. Bai[7]and Guo et al.[17]added 100 μmol/L βmercaptoethanol to the invitro mature liquid of Mongolian lamb and lamb oocytes from 4 to 8 weeks old. The results showed that the normal fertilization ratio of oocytes and the blastocyst rate increased significantly; the proportion of polyspermy decreased significantly; and the proportion of normal fertilization was not significantly different from that of adult sheep （P>0.05）. Quan et al.[18]found that the addition of βmercaptoethanol to the granulosa cell culture medium of bovine oocytes can increase the viable cell rate， and the viable cell rate increased with the increase of βmercaptoethanol concentration， and reached the highest value at the concentration of 25 μmol/L. Shu et al.[19]showed that the addition of 100 μmol/L βmercaptoethanol into bovine oocyte culture medium can significantly increase the blastocyst rate of bovine embryos and the number of cells in the cell mass of blastocysts after invitro fertilization. Lan et al.[20]found that the addition of βmercaptoethanolto the mature liquid of pig oocytes can increase the concentration of glutathione in the cytoplasm， which provides suitable intracellular conditions to the fertilized eggs， thereby preventing oxidative damage and helping the embryos develop to the blastocysts. In this study， the results showed that the addition of （50， 70 and 100 μmol/L） βmercaptoethanol to the oocyte mature liquid of 6weekold Doper sheep increased the cleavage rate and blastocyst rate， especially the blastocyst rate; and the cleavage rate （65.22%） and blastocyst rate （21.74%） of the test group III （containing 100 μmol/L βmercaptoethanol） were the highest. When the βmercaptoethanol concentration was within 100 μmol/L， the cleavage rate and the blastocyst rate of oocytes were on the increase， and the blastocyst rate increased more than the cleavage rate， indicating that βmercaptoethanol helps the oocytes to develop into the blastocysts. The results of this study are consistent with the results of Bai[7]， Guo et al.[17]， Quan et al.[18]， Shu et al.[19]and Lan et al.[20]. The reason might be that βmercaptoethanol is a low molecular weight mercaptoalcohol compound capable of reducing cystine to cysteine which is a substrate for the synthesis of glutathione （GSH）. GSH is present in every cell of organisms and has antioxidant and detoxifying effects. During oocyte development， it protects the functional proteins and enzymes required for cell development， amino acid transport， protein synthesis and DNA replication from oxidation through free mercapto groups （SH）， thereby eliminating the retarding or delaying effect of active oxygen free radicals （ROS） on further oocyte development， accelerating sperm depolymerization and pronuclei formation in fertilized eggs， reducing parthenogenetic activation and polyspermy， and increasing the proportion of normal fertilization， the developmental ability of fertilized eggs and blastocyst rate. Conclusions
The results of this study indicate that the addition of a certain amount of βmercaptoethanol to the mature liquid can improve the developmental ability of oocytes and the quality of fertilized egg of the 6weekold Doper sheep， and when the βmercaptoethanol concentration was within 100 μmol/L， the cleavage rate and blastocyst rate of oocytes showed an upward trend. The optimal concentration of βmercaptoethanol in the mature liquid still needs further study.
References
[1] WORTHINQTON CA， KENNEDY JP. Ovarian response to exogenous hormones in sixweek old lambs[J]. Aust J Biol Sci， 1979， 32（7）： 91-95.
[2] TROUNSON AO， WILLADSEN SM， MOOR RM. Reproductive function in prepubertal lambs：ovulation， embryo development and ovarian steroidogenesis[J]. J Reprod Fertil， 1977， 49： 69-75.
[3] KELLY JM， KLEEMANND O， WALKER SK. Enhanced efficiency in the production of offspring from 4 to 8weekold lambs[J]. Theriogenology， 2005， 63（7）： 1876-1890.
[4] KELLY JM， KLEEMANN DO， WALKER SK. The effect of nutrition during pregnancy on the in vitro production of embryos from resulting lambs[J].Theriogenology， 2005， 63（7）： 2020-2031.
[5] GOU KM， GUAN H， BAI JH， et al. Field evaluation of juvenile in vitro embryo transfer （JIVET）in sheep[J].Animal Reproduction Science， 2009， 112（3/4）： 316-324.
[6] BAI JH， HOU J， GUAN H， et al. Effect of 2mercaptoethanol and cysteine supplementation during in vitro maturation on the developmental competence of oocytes from hormonestimulated lambs[J]. Theriogenology， 2008， 70（5）： 758-764.
[7] BAI JH. Premature sheep and cattle JIVET technology research[D]. Beijing： China Agricultural University， 2008. （in Chinese）
[8] GUO H， WANG PC， SHI WY， et al. Study on superovulation and embryo production technology of different sheep breeds[J]. Animal Husbandry and Veterinary Medicine， 2012， 44（10）： 20-24. （in Chinese）
[9] 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）
[10] LIU ZZ， LI HG， CHENG M， et al. Study on rapid propagation of suffolk sheep by using JIVET technology[J]. Animal Husbandry and Feed Science， 2016， 37（3）： 1-3. （in Chinese）
[11] 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） [12] 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）
[13] GUO H， WANG PC， DAI R， et al. Analysis of influencing factors on the oocyte in vitro embryo production in lambs[J]. Hubei Agricultural Sciences， 2013， 52（19）： 4726-4729. （in Chinese）
[14] LI YS， CAO HG， LIU Y， et al. Advance in goat and sheep juvenile in vitro embryo transfer[J]. Progress In Veterinary Medicine， 2011， 32（11）： 99-104. （in Chinese）
[15] HAN Y， YIN D， ZHANG T， et al. Research progress on effects of glutathione on maturation and embryo development of oocyte[J]. Jiangsu Agricultural Sciences， 2010（3）： 269-270. （in Chinese）
[16] CAO X， ZHAO YZ， LUO HL， et al. Effects of VEGF on the ultrastructure of ovine oocytes cultured in vitro[J]. Acta Veterinaria et Zootechnica Sinica， 2010， 41（2）： 167-173. （in Chinese）
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Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU