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Abstract［Objectives］ This study was conducted to investigate the effects of lamb age and in vitro culture system of oocytes on the results of juvenile in vitro embryo transfer (JIVET).
［Methods］ Ten Dorper×smalltailed Han lambs aged 5 to 10 weeks were induced to superovulate via i.p. injection of pregnant mares serum gonadotropin (PMSG). The oocytes were matured in basal maturation solution or modified maturation solution, which was prepared by adding 200 μmol/L cysteine to the basal maturation solution. Then, the oocytes were fertilized in fertilization medium I containing 2% estrus sheep serum (ESS) or fertilization medium II containing 3 mg/ml bull serum albumin (BSA). Finally, the number of oocytes, oocyte maturation rate and cleavage rate of the lambs of different ages were determined.
［Results］ The average number of oocytes recovered per lamb was (111.00±16.97), (139.50±28.99), (108.50±17.68) and (42.00±11.31) for 5, 7, 8 and 10weekold Dorper×smalltailed Han lambs, respectively. The number of oocytes obtained from 5, 7 and 8weekold lambs was significantly higher than that from 10weekold lambs (P＜0.05), but there was no significant difference among 5, 7 and 8weekold lambs (P＞0.05). The maturation rate of oocytes cultured in modified maturation solution was 3.64% higher than that in basal maturation solution. The cleavage rate of oocytes in fertilization medium I was very significantly higher than that in fertilization medium II (P＜0.01).
［Conclusions］ The results of JIVET can be improved by harvesting oocytes from lambs aged 5-8 weeks, adding a certain amount of cysteine into oocyte maturation solution, and a certain amount of ESS into fertilization medium.
Key wordsJuvenile lambs; Age; Oocytes; In vitro culture system; JIVET; Maturation rate; Cleavage rate
Received: October 8, 2018Accepted: November 12, 2018
Supported by Special Fund for National Hair Sheep Industrial Technology System (CARS3924); Science and Technology Development Program of Shanxi Province ( 201203110241); Science and Technology Innovation Team Project of Shanxi Province (201705D13102820); Financial Support of Agriculture of Shanxi Province (NYGX201503); Talent Project for Science and Technology Development in Outlaying Poor Areas, Frontier Ethnic Minority Areas and Old Revolutionary Base Areas of Shanxi Province, China (2017Sy128).
Yangyi MAO (1960- ), male, P. R. China, professor, devoted to the research about sheep genetics and breeding, Email: mao7094728@126.com. * Corresponding author. Email: lhd2638@126.com.
Juvenile in vitro embryo transfer (JIVET) is a powerful technology to produce offspring and reduce the generation intervals of livestock via superovulation of young animals, in vitro maturation of oocytes and embryo transplantation. This method was first developed and reported by the South Australian Research & Development Institute (SARDI), and has now become a research topic in the fields of embryo biology and livestock reproduction［1-5］. JIVET technology has so far been studied experimentally in Suffolk sheep, Liaoning cashmere goat, Dorset Horn sheep and smalltailed Han sheep［6-12］. However, there are still some problems that need to be resolved in JIVET. For example, there is no standard procedure for superovulation of juvenile animals, the results of superovulation are quite variable among the lambs of different ages, some of the oocytes have poor ability to mature or to develop into blastocyst stage in vitro. Numerous studies have reported the effects of lamb age, hormone combinations, injection time and dose on superovulation during JIVET treatment［10, 13］. To simulate the in vivo conditions for oocyte maturation, fertilization and embryo development, gonadotropins, antioxidants, epidermal growth factor (EGF), and estrus sheep serum (ESS), bovine serum albumin (BSA), amino acids and other substances are added into in vitro oocyte culture medium, to improve the results of JIVET technology［14-15］. The present study was conducted to investigate the effects of lamb age, addition of cysteine into oocyte maturation solution, addition of ESS or BSA into fertilization medium on JIVET, and to provide a theoretical basis for optimizing JIVET system.
Materials and Methods
Animals
Dorper rams, Dorper×smalltailed Han lambs aged 10 weeks and recipient ewes were provided by the sheep farm of Jinzhong Linshan Cooperative. Dorper sheeps semen frozen in pellet and ESS were prepared by the Laboratory of Animal Genetic Engineering of Institute of Animal Husbandry and Veterinary, Shanxi Academy of Agricultural Sciences.
Hormones and reagents
Folliclestimulating hormone (FSH), luteinizing hormone (LH), pregnant mares serum gonadotropin (PMSG), 17βestradiol (17βE2) and progesterone vaginal suppository (EaziBreed CIDR) were all purchased from Beijing Luxin Agriculture and Animal Husbandry Technology Co., Ltd.
Medium M199, cysteine, BSA, essential amino acids (EAAs), nonessential amino acids (NEAAs), and chemical reagents such as CaCl2?2H2O and NaHCO3 were purchased from Sigma. Instruments
The main instruments used in this study included Olympus Szx10 stereo microscope, Olympus IX71 inverted microscope and Sanyo MCO5AC CO2 incubator. Surgical supplies including scalpels, scissors and hemostats were all purchased from Shanxi Medical Equipment Company.
Culture media
Oocyte collection medium consisted of 20 mmol/L Hepes, 2% ESS and 10 mg/ml heparin sodium. Basal oocyte maturation medium contained 20% ESS, 10 μg/ml FSH, 10 μg/ml LH and 1 μg/ml 17βE2. Modified oocyte maturation medium was prepared by adding 200 μmol/L cysteine to the basic oocyte maturation medium. Sperm capacitating medium (CM) was synthetic oviduct fluid (SOF, containing 20 μg/ml heparin sodium). Fertilization medium I was SOF supplemented with 2% ESS. Fertilization medium II was SOF supplemented with 3 mg/ml BSA. Fertilized eggs were cultured in SOF supplemented with 8 mg/ml BSA, 2% EAA and 1% NEAA.
Time and location
In vitro maturation and fertilization of oocytes was carried out in the Laboratory of Animal Genetic Engineering of the Institute of Animal Husbandry and Veterinary Medicine, Shanxi Academy of Agricultural Sciences from May 5 to 23, 2014, and the fertilized eggs were transplanted into recipient ewes at the sheep farm of Jinzhong Linshan Cooperative.
Methods
Superovulation and oocyte collection
Ten Dorper×smalltailed Han lambs aged 5 to 10 weeks were induced to superovulate via i.p. injection of 300 to 350 IU pregnant mares serum gonadotropin (PMSG) per lamb as previously described［1, 3］. And oocytes were recovered from ovaries according to Bais method［15］.
Oocyte maturation and fertilization
According to Bai［15］, all GradeA and GradeB oocytes were randomly and equally divided into two copies, one were cultured for 24 h in basal oocyte maturation medium, and the other in modified oocyte maturation medium. The oocytes were considered to be mature when the cumulus cells spread out or the first polar body was released. Then, the matured oocytes cultured in each oocyte maturation medium were randomly and equally divided into two copies, one were fertilized in fertilization medium I, and the other in fertilization medium II. The fertilized eggs were further cultured in embryo culture medium for about 20 h, observed under a microscope to calculate cleavage rate. After another 4 to 5 d of culture, the blastocysts of in each dish were calculated to calculate blastocyst formation rate. Transplantation of embryos
The 2 to 8cell embryos were transplanted into recipient ewes according to Bais method［15］.
Data processing and analysis
The experimental data were sorted with Microsoft Excel, and analyzed with ttest and chisquare test for statistical significance.
Results and Analysis
Superovulation of 5to 10week Dorper×smalltailed Han sheep
The oocytes recovered from eight 5 to 10weekold lambs were shown in Table 1. The development and growth of their ovaries and follicles were shown in Fig. 1.
Table 1Oocytes recovered from each superovulated ewe lamb
Lamb No.Age∥weekTotal number of oocytesNumber of available oocytesAverage number of available oocytes
15123116111.00 a±16.97
259996
37119115139.50 a±28.99
47160149
58121114108.50 a±17.68
689694
710505042.00 b±11.31
8103434
Total802768
No. 1 and No. 2 are twin lambs; different lower case letters (a, b) within the column represent significant difference between means (P<0.05).
As can be seen from Table 1, a total of 768 available oocytes of Grade A and Grade B were obtained from the eight superovulated ewe lambs. The number of available oocytes of 5, 7, 8weekold lambs was significantly higher than that of 10weekold lambs (P<0.05), while there was no significant difference in number of available oocytes among 5, 7 and 8weekold lambs (P>0.05).
Effects of culture medium on in vitro maturation of oocytes
In vitro maturation of the 768 GradeA and GradeB oocytes in different culture media were shown in Table 2. The maturation rate of oocytes in modified oocyte maturation medium was 3.64% higher than that in basal oocyte maturation medium, and X2 test showed that the difference between the two them was not significant (P>0.05).
Table 2In vitro maturation of oocytes (Grade A and Grade B)
MediumTotal number of oocytesNumber of matured oocytesMutation rate∥%
Modified oocyte maturation medium38429276.00 a
Basic oocyte maturation medium38427872.40 a
Total76857074.22
The same lower case letter within the column indicates insignificant difference between treatments (P>0.05).
Effects of culture medium on in vitro development of fertilized oocytes
The development of fertilized embryos in different media was shown in Table 3, Fig. 2 and Fig. 3. As shown in Table 3, fertilization rate and blastocyst formation rate of matured oocytes in fertilization medium I were both higher than in fertilization medium II. And X2 test revealed that there was an extremely significant difference in cleavage rate between fertilization media I and II, but there was no significant difference in blastocyst formation rate among the four different treatments (P>0.05). Table 3In vitro development of fertilized oocytes in different treatments
MediumFertilizationmediumNumber ofoocytesNumber ofcleaved embryosNumber ofblastocystsCleavagerate∥%Blastocystformation rate ∥%
Modified oocyte maturation mediumI146933163.70 Aa21.23
II146682646.58 Bc17.81
Basic oocyte maturation mediumI139832859.71 Ab20.14
II139602343.17 Bc16.55
Total570304108
The different upper case letters within column indicate extremely significant difference (P<0.01); different lower case letters indicate significant difference (P<0.05), and the same lower case letter indicate insignificant difference (P>0.05).
Agricultural Biotechnology2019
Transplantation of 2 to 8cell embryos
A total of 77 2 to 8cell embryos were produced, and nonsurgically transferred into the ampulla of the fallopian tube of 17 recipient ewes, acting as surrogate mothers. The results turned out that seven ewes got pregnant, and produced a total of 13 lambs. Among them, seven ewes gave birth to twins and one gave birth to singleton. The pregnancy rate of recipient ewes was 41.18%. on average, one JIVET lamb was produced using six 2 to 8cell embryos (Fig. 4).
Fig. 1Ovarian follicles of superovulated ewe lambs
Fig. 2In vitro cultured two to fourcell embryos
Fig. 3In vitro cultured blastocyst
Fig. 4Lambs produced via JIVET
Discussion
Effect of lamb age on JIVET technology
The young lambs were injected with gonadotropin to induce follicular development, and to produce a large number of oocytes, which is the basis for the establishment of JIVET system. It has been reported that lamb age is a critical factor influencing ovarian follicular development and JIVET results. In the study of Wang et al.［16］, gonadotropin was used to induce follicular development of Suffolk lambs of different ages, and the results showed that an average of 82.54 oocytes per lamb were produced by 5 to 6weekold lambs, and only 12.83 oocytes per lamb were obtained from 9weekold lambs. Our data showed that the number of available oocytes of 5, 7, 8weekold lambs was significantly higher than that of 10weekold lambs (P<0.05), which was basically consistent with previous studies［13, 16］. Generally, antral follicles first appear on fetal ovary at about 135 day of gestation. Then, this number continues to increase and reaches the maximum level 4 to 8 weeks after birth. The pituitary gland of lambs at this age is still incompletely developed, and the incidence of follicular atresia is limited［1］, so it is the best period to collect ovulated oocytes. Then, with the increase of lamb age and the development of the pituitary gland, lamb follicles gradually possess the ability to become dominant. Dominant follicles are more sensitive to folliclestimulating hormone (FSH), producing estradiol (E2) and inhibin, which inhibit the secretion of FSH from the pituitary gland. The nondominant follicles become atretic as FSH levels fall. Therefore, lambs aged 5-8 weeks are a preferred option of oocyte donors in JIVET system. Effect of in vitro oocyte mature solution on JIVET technology
Oocyte maturation solution is an important factor influencing the results of JIVET. Bai［5］ reported that the oocyte maturation rate of young Mongolian Sheep was increased to 80.60% by 100 μmol/L βmercaptoethanol to oocyte maturation solution. Our data showed that the oocyte maturation rate of 5 to 10weekold lambs was improved from 72.40% in basal oocyte maturation solution to 76.04% in modified oocyte maturation solution, which was prepared by adding 200 μmol/L cysteine to the basal oocyte maturation solution. Cysteine is a substrate for the synthesis of glutathione (GSH), which is a tripeptide containing with a γamide bond and a sulfhydryl group, composed of glutamic acid, cysteine and glycine. GSH, present in every body cell, has antioxidant and detoxifying properties. And it protects the functional proteins and enzymes required for cell development, amino acid transport, protein synthesis, and DNA replication from being oxidized during oocyte development.
Effect of in vitro fertilization medium on JIVET technology
As one of the main culture media in JIVET system, fertilization medium should contain the nutrients required for sperm fertilization, oocyte growth and embryo development, maintain cell pH and osmotic pressure, and prevent oocyte zona pellucida from hardening, so that sperm can penetrate the zona pellucida to get to the oocyte. Guo et al.［17］ reported that the oocyte cleavage rate of Kazakh lambs was improved to 69.00%, and blastocyst formation rate to 17.50% in SOF fertilization medium (containing 20% ESS, 10 μg/ml heparin sodium). According to Wu et al.［18］, the oocyte cleavage rate of Xinjiang finewool lambs was 52.88% in SOF (containing 2% ESS) fertilization medium. Our data showed that the oocyte cleavage rate of Dorper×smalltailed Han lambs aged 5 to 10 weeks in fertilization medium I (containing 2% ESS) was very significantly higher than in fertilization medium II (containing 3 mg/ml BSA). ESS contains cell growth factors, reproductive hormones and proteins. Reproductive hormones (progesterone, estrogen) can bind to the progesterone receptor and estrogen receptor on sperm plasma membrane, and activate Ca2+ channel on sperm membrane, induce intracellular Ca2+ release and Ca2+ influx, and finally activate the enzymes involved in sperm capacitation and acrosome reaction. FSH, LH, E2, cell growth factors and proteins can promote oocyte cytoplasmic maturation, prevent oocyte zona pellucida hardening, conducive to subsequent spermegg binding, fertilization and embryo development. BSA is only a macromolecular protein in serum, and it is unstable. Therefore, the addition of a certain amount of ESS into the fertilization solution I is beneficial to the in vitro fertilization of lamb oocytes. Conclusions
Our results showed that the number of oocytes obtained from 5 to 8weekold Dorper×smalltailed Han lambs was significantly higher than that from 10weekold lambs (P＜0.05). The cleavage rate of oocytes in fertilization medium I was very significantly higher than that in fertilization medium II (P＜0.01). The maturation rate of oocytes cultured in modified maturation solution was 3.64% higher than that in basal maturation solution. Our findings prove that it is possible to improve the results JIVET can be improved by recovering oocytes from lambs aged 5-8 weeks, adding a certain amount of cysteine into oocyte maturation solution, and a certain amount of ESS into fertilization medium.
References
［1］ KELLY JM, KLEEMANN DO, WALKER SK. Enhanced efficiency in the production of offspring from 4to 8 weekold lambs［J］. Theriogenology, 2005, 63(7): 1876-1890.
［2］ RANGELSANTOS R, MCDONALD M F, WICKHAM G A. Evaluation of the feasibility of a juvenile MOET scheme in sheep［J］. Proc New Zealand Society Anim Proc, 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］. Theriogenology, 2005, 63(7): 2020-2031.
［4］ 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.
［5］ 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.
［6］ 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): 1-4.
［7］ ZHANG XH, ZHANG SW, SONG XC, et al. Study on follicular development of female lamb and technology of oocyte maturation［J］. China Animal Husbandry & Veterinary Medicine, 2011, 38(11): 136-139.
［8］ LI Y, HOU J, ZHENG YS, et al. Effects of heparin and sheep serum on in vitro capacitation of ram epididymal sperm［J］. Journal of Inner Mongolia Institute of Agriculture and Animal Husbandry, 1998, 19(4): 20-24.
［9］ MA SK, XIAO 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.
［10］ CHEN XY, TIAN SJ, SANG RZ, et al. Inducement of lamb follicular development and embryo production in vitro［J］. Journal of Agricultural Biotechnology, 2008, 16 (3): 456-460.
［11］ ZHANG SL, LIU DJ, WANG JG, et al. The study of shortening the breeding duration of cashmere goat through in vitro fertilization technology［J］. Journal of Inner Mongolia University, 1998, 29 (1):114-117.
［12］ LI YS, CAO HG, LIU Y, et al. Advances in research and application of lamb superovulation and in vitro embryo fertilization techniques［J］. Progress in Veterinary Medicine, 2011, 32 (11): 99-104.
［13］ YU Y, ZHONG X, WANG FL, et al. Comparison on follicle development of sheep lamb at different ages［J］. Chinese Journal of Animal Science, 2012, 48 (9): 17-21.
［Methods］ Ten Dorper×smalltailed Han lambs aged 5 to 10 weeks were induced to superovulate via i.p. injection of pregnant mares serum gonadotropin (PMSG). The oocytes were matured in basal maturation solution or modified maturation solution, which was prepared by adding 200 μmol/L cysteine to the basal maturation solution. Then, the oocytes were fertilized in fertilization medium I containing 2% estrus sheep serum (ESS) or fertilization medium II containing 3 mg/ml bull serum albumin (BSA). Finally, the number of oocytes, oocyte maturation rate and cleavage rate of the lambs of different ages were determined.
［Results］ The average number of oocytes recovered per lamb was (111.00±16.97), (139.50±28.99), (108.50±17.68) and (42.00±11.31) for 5, 7, 8 and 10weekold Dorper×smalltailed Han lambs, respectively. The number of oocytes obtained from 5, 7 and 8weekold lambs was significantly higher than that from 10weekold lambs (P＜0.05), but there was no significant difference among 5, 7 and 8weekold lambs (P＞0.05). The maturation rate of oocytes cultured in modified maturation solution was 3.64% higher than that in basal maturation solution. The cleavage rate of oocytes in fertilization medium I was very significantly higher than that in fertilization medium II (P＜0.01).
［Conclusions］ The results of JIVET can be improved by harvesting oocytes from lambs aged 5-8 weeks, adding a certain amount of cysteine into oocyte maturation solution, and a certain amount of ESS into fertilization medium.
Key wordsJuvenile lambs; Age; Oocytes; In vitro culture system; JIVET; Maturation rate; Cleavage rate
Received: October 8, 2018Accepted: November 12, 2018
Supported by Special Fund for National Hair Sheep Industrial Technology System (CARS3924); Science and Technology Development Program of Shanxi Province ( 201203110241); Science and Technology Innovation Team Project of Shanxi Province (201705D13102820); Financial Support of Agriculture of Shanxi Province (NYGX201503); Talent Project for Science and Technology Development in Outlaying Poor Areas, Frontier Ethnic Minority Areas and Old Revolutionary Base Areas of Shanxi Province, China (2017Sy128).
Yangyi MAO (1960- ), male, P. R. China, professor, devoted to the research about sheep genetics and breeding, Email: mao7094728@126.com. * Corresponding author. Email: lhd2638@126.com.
Juvenile in vitro embryo transfer (JIVET) is a powerful technology to produce offspring and reduce the generation intervals of livestock via superovulation of young animals, in vitro maturation of oocytes and embryo transplantation. This method was first developed and reported by the South Australian Research & Development Institute (SARDI), and has now become a research topic in the fields of embryo biology and livestock reproduction［1-5］. JIVET technology has so far been studied experimentally in Suffolk sheep, Liaoning cashmere goat, Dorset Horn sheep and smalltailed Han sheep［6-12］. However, there are still some problems that need to be resolved in JIVET. For example, there is no standard procedure for superovulation of juvenile animals, the results of superovulation are quite variable among the lambs of different ages, some of the oocytes have poor ability to mature or to develop into blastocyst stage in vitro. Numerous studies have reported the effects of lamb age, hormone combinations, injection time and dose on superovulation during JIVET treatment［10, 13］. To simulate the in vivo conditions for oocyte maturation, fertilization and embryo development, gonadotropins, antioxidants, epidermal growth factor (EGF), and estrus sheep serum (ESS), bovine serum albumin (BSA), amino acids and other substances are added into in vitro oocyte culture medium, to improve the results of JIVET technology［14-15］. The present study was conducted to investigate the effects of lamb age, addition of cysteine into oocyte maturation solution, addition of ESS or BSA into fertilization medium on JIVET, and to provide a theoretical basis for optimizing JIVET system.
Materials and Methods
Animals
Dorper rams, Dorper×smalltailed Han lambs aged 10 weeks and recipient ewes were provided by the sheep farm of Jinzhong Linshan Cooperative. Dorper sheeps semen frozen in pellet and ESS were prepared by the Laboratory of Animal Genetic Engineering of Institute of Animal Husbandry and Veterinary, Shanxi Academy of Agricultural Sciences.
Hormones and reagents
Folliclestimulating hormone (FSH), luteinizing hormone (LH), pregnant mares serum gonadotropin (PMSG), 17βestradiol (17βE2) and progesterone vaginal suppository (EaziBreed CIDR) were all purchased from Beijing Luxin Agriculture and Animal Husbandry Technology Co., Ltd.
Medium M199, cysteine, BSA, essential amino acids (EAAs), nonessential amino acids (NEAAs), and chemical reagents such as CaCl2?2H2O and NaHCO3 were purchased from Sigma. Instruments
The main instruments used in this study included Olympus Szx10 stereo microscope, Olympus IX71 inverted microscope and Sanyo MCO5AC CO2 incubator. Surgical supplies including scalpels, scissors and hemostats were all purchased from Shanxi Medical Equipment Company.
Culture media
Oocyte collection medium consisted of 20 mmol/L Hepes, 2% ESS and 10 mg/ml heparin sodium. Basal oocyte maturation medium contained 20% ESS, 10 μg/ml FSH, 10 μg/ml LH and 1 μg/ml 17βE2. Modified oocyte maturation medium was prepared by adding 200 μmol/L cysteine to the basic oocyte maturation medium. Sperm capacitating medium (CM) was synthetic oviduct fluid (SOF, containing 20 μg/ml heparin sodium). Fertilization medium I was SOF supplemented with 2% ESS. Fertilization medium II was SOF supplemented with 3 mg/ml BSA. Fertilized eggs were cultured in SOF supplemented with 8 mg/ml BSA, 2% EAA and 1% NEAA.
Time and location
In vitro maturation and fertilization of oocytes was carried out in the Laboratory of Animal Genetic Engineering of the Institute of Animal Husbandry and Veterinary Medicine, Shanxi Academy of Agricultural Sciences from May 5 to 23, 2014, and the fertilized eggs were transplanted into recipient ewes at the sheep farm of Jinzhong Linshan Cooperative.
Methods
Superovulation and oocyte collection
Ten Dorper×smalltailed Han lambs aged 5 to 10 weeks were induced to superovulate via i.p. injection of 300 to 350 IU pregnant mares serum gonadotropin (PMSG) per lamb as previously described［1, 3］. And oocytes were recovered from ovaries according to Bais method［15］.
Oocyte maturation and fertilization
According to Bai［15］, all GradeA and GradeB oocytes were randomly and equally divided into two copies, one were cultured for 24 h in basal oocyte maturation medium, and the other in modified oocyte maturation medium. The oocytes were considered to be mature when the cumulus cells spread out or the first polar body was released. Then, the matured oocytes cultured in each oocyte maturation medium were randomly and equally divided into two copies, one were fertilized in fertilization medium I, and the other in fertilization medium II. The fertilized eggs were further cultured in embryo culture medium for about 20 h, observed under a microscope to calculate cleavage rate. After another 4 to 5 d of culture, the blastocysts of in each dish were calculated to calculate blastocyst formation rate. Transplantation of embryos
The 2 to 8cell embryos were transplanted into recipient ewes according to Bais method［15］.
Data processing and analysis
The experimental data were sorted with Microsoft Excel, and analyzed with ttest and chisquare test for statistical significance.
Results and Analysis
Superovulation of 5to 10week Dorper×smalltailed Han sheep
The oocytes recovered from eight 5 to 10weekold lambs were shown in Table 1. The development and growth of their ovaries and follicles were shown in Fig. 1.
Table 1Oocytes recovered from each superovulated ewe lamb
Lamb No.Age∥weekTotal number of oocytesNumber of available oocytesAverage number of available oocytes
15123116111.00 a±16.97
259996
37119115139.50 a±28.99
47160149
58121114108.50 a±17.68
689694
710505042.00 b±11.31
8103434
Total802768
No. 1 and No. 2 are twin lambs; different lower case letters (a, b) within the column represent significant difference between means (P<0.05).
As can be seen from Table 1, a total of 768 available oocytes of Grade A and Grade B were obtained from the eight superovulated ewe lambs. The number of available oocytes of 5, 7, 8weekold lambs was significantly higher than that of 10weekold lambs (P<0.05), while there was no significant difference in number of available oocytes among 5, 7 and 8weekold lambs (P>0.05).
Effects of culture medium on in vitro maturation of oocytes
In vitro maturation of the 768 GradeA and GradeB oocytes in different culture media were shown in Table 2. The maturation rate of oocytes in modified oocyte maturation medium was 3.64% higher than that in basal oocyte maturation medium, and X2 test showed that the difference between the two them was not significant (P>0.05).
Table 2In vitro maturation of oocytes (Grade A and Grade B)
MediumTotal number of oocytesNumber of matured oocytesMutation rate∥%
Modified oocyte maturation medium38429276.00 a
Basic oocyte maturation medium38427872.40 a
Total76857074.22
The same lower case letter within the column indicates insignificant difference between treatments (P>0.05).
Effects of culture medium on in vitro development of fertilized oocytes
The development of fertilized embryos in different media was shown in Table 3, Fig. 2 and Fig. 3. As shown in Table 3, fertilization rate and blastocyst formation rate of matured oocytes in fertilization medium I were both higher than in fertilization medium II. And X2 test revealed that there was an extremely significant difference in cleavage rate between fertilization media I and II, but there was no significant difference in blastocyst formation rate among the four different treatments (P>0.05). Table 3In vitro development of fertilized oocytes in different treatments
MediumFertilizationmediumNumber ofoocytesNumber ofcleaved embryosNumber ofblastocystsCleavagerate∥%Blastocystformation rate ∥%
Modified oocyte maturation mediumI146933163.70 Aa21.23
II146682646.58 Bc17.81
Basic oocyte maturation mediumI139832859.71 Ab20.14
II139602343.17 Bc16.55
Total570304108
The different upper case letters within column indicate extremely significant difference (P<0.01); different lower case letters indicate significant difference (P<0.05), and the same lower case letter indicate insignificant difference (P>0.05).
Agricultural Biotechnology2019
Transplantation of 2 to 8cell embryos
A total of 77 2 to 8cell embryos were produced, and nonsurgically transferred into the ampulla of the fallopian tube of 17 recipient ewes, acting as surrogate mothers. The results turned out that seven ewes got pregnant, and produced a total of 13 lambs. Among them, seven ewes gave birth to twins and one gave birth to singleton. The pregnancy rate of recipient ewes was 41.18%. on average, one JIVET lamb was produced using six 2 to 8cell embryos (Fig. 4).
Fig. 1Ovarian follicles of superovulated ewe lambs
Fig. 2In vitro cultured two to fourcell embryos
Fig. 3In vitro cultured blastocyst
Fig. 4Lambs produced via JIVET
Discussion
Effect of lamb age on JIVET technology
The young lambs were injected with gonadotropin to induce follicular development, and to produce a large number of oocytes, which is the basis for the establishment of JIVET system. It has been reported that lamb age is a critical factor influencing ovarian follicular development and JIVET results. In the study of Wang et al.［16］, gonadotropin was used to induce follicular development of Suffolk lambs of different ages, and the results showed that an average of 82.54 oocytes per lamb were produced by 5 to 6weekold lambs, and only 12.83 oocytes per lamb were obtained from 9weekold lambs. Our data showed that the number of available oocytes of 5, 7, 8weekold lambs was significantly higher than that of 10weekold lambs (P<0.05), which was basically consistent with previous studies［13, 16］. Generally, antral follicles first appear on fetal ovary at about 135 day of gestation. Then, this number continues to increase and reaches the maximum level 4 to 8 weeks after birth. The pituitary gland of lambs at this age is still incompletely developed, and the incidence of follicular atresia is limited［1］, so it is the best period to collect ovulated oocytes. Then, with the increase of lamb age and the development of the pituitary gland, lamb follicles gradually possess the ability to become dominant. Dominant follicles are more sensitive to folliclestimulating hormone (FSH), producing estradiol (E2) and inhibin, which inhibit the secretion of FSH from the pituitary gland. The nondominant follicles become atretic as FSH levels fall. Therefore, lambs aged 5-8 weeks are a preferred option of oocyte donors in JIVET system. Effect of in vitro oocyte mature solution on JIVET technology
Oocyte maturation solution is an important factor influencing the results of JIVET. Bai［5］ reported that the oocyte maturation rate of young Mongolian Sheep was increased to 80.60% by 100 μmol/L βmercaptoethanol to oocyte maturation solution. Our data showed that the oocyte maturation rate of 5 to 10weekold lambs was improved from 72.40% in basal oocyte maturation solution to 76.04% in modified oocyte maturation solution, which was prepared by adding 200 μmol/L cysteine to the basal oocyte maturation solution. Cysteine is a substrate for the synthesis of glutathione (GSH), which is a tripeptide containing with a γamide bond and a sulfhydryl group, composed of glutamic acid, cysteine and glycine. GSH, present in every body cell, has antioxidant and detoxifying properties. And it protects the functional proteins and enzymes required for cell development, amino acid transport, protein synthesis, and DNA replication from being oxidized during oocyte development.
Effect of in vitro fertilization medium on JIVET technology
As one of the main culture media in JIVET system, fertilization medium should contain the nutrients required for sperm fertilization, oocyte growth and embryo development, maintain cell pH and osmotic pressure, and prevent oocyte zona pellucida from hardening, so that sperm can penetrate the zona pellucida to get to the oocyte. Guo et al.［17］ reported that the oocyte cleavage rate of Kazakh lambs was improved to 69.00%, and blastocyst formation rate to 17.50% in SOF fertilization medium (containing 20% ESS, 10 μg/ml heparin sodium). According to Wu et al.［18］, the oocyte cleavage rate of Xinjiang finewool lambs was 52.88% in SOF (containing 2% ESS) fertilization medium. Our data showed that the oocyte cleavage rate of Dorper×smalltailed Han lambs aged 5 to 10 weeks in fertilization medium I (containing 2% ESS) was very significantly higher than in fertilization medium II (containing 3 mg/ml BSA). ESS contains cell growth factors, reproductive hormones and proteins. Reproductive hormones (progesterone, estrogen) can bind to the progesterone receptor and estrogen receptor on sperm plasma membrane, and activate Ca2+ channel on sperm membrane, induce intracellular Ca2+ release and Ca2+ influx, and finally activate the enzymes involved in sperm capacitation and acrosome reaction. FSH, LH, E2, cell growth factors and proteins can promote oocyte cytoplasmic maturation, prevent oocyte zona pellucida hardening, conducive to subsequent spermegg binding, fertilization and embryo development. BSA is only a macromolecular protein in serum, and it is unstable. Therefore, the addition of a certain amount of ESS into the fertilization solution I is beneficial to the in vitro fertilization of lamb oocytes. Conclusions
Our results showed that the number of oocytes obtained from 5 to 8weekold Dorper×smalltailed Han lambs was significantly higher than that from 10weekold lambs (P＜0.05). The cleavage rate of oocytes in fertilization medium I was very significantly higher than that in fertilization medium II (P＜0.01). The maturation rate of oocytes cultured in modified maturation solution was 3.64% higher than that in basal maturation solution. Our findings prove that it is possible to improve the results JIVET can be improved by recovering oocytes from lambs aged 5-8 weeks, adding a certain amount of cysteine into oocyte maturation solution, and a certain amount of ESS into fertilization medium.
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