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Abstract ［Objectives］ This study was conducted to explore the effects of embryos at different developmental stages on the bisection effect of embryos, improve the efficiency of bovine embryo bisection, and facilitate the application of embryo bisection technology in cattle breeding.
［Methods］The effects of two different bisection solutions on the bisection of morulae and blastocysts in vitro were explored. The morulae and blastocysts produced in vitro from cattle that developed to the 6th to 8th d were bisected by hands, and demi-embryos were cultured in vitro. Their development was observed.
［Results］ Morulae were bisected in PBS solution and PBS+0.2 mol/L sucrose, and the success rates of bisection were 50% and 95.2%, respectively. The success rate of bisecting morulae in PBS+0.2 mol/L sucrose was significantly higher than that in PBS (P<0.05), while the development rate of the bisected demi-embryos had no significant difference between the two (53.3%, 52.4%) (P>0.05). The success rates of blastocyst bisection in PBS solution and PBS+0.2 mol/L sucrose were 51.6% and 95.1%, respectively. The success rate of blastocyst bisection in PBS+0.2 mol/L sucrose was significantly higher than that in PBS (P<0.05), while the development rate of the bisected demi-embryos had no significant difference between the two (50.0%, 56.4%) (P>0.05).
［Conclusions］There were no significant differences between the success rates of bisecting bovine morulae and blastocysts in PBS+0.2 mol/L sucrose, which were both significantly better than those in pure PBS bisection solution, proving that PBS+0.2 mol/L sucrose bisection solution is suitable for bovine embryo bisection.
Key words Cattle; Embryo division; Success rate; Development rate; Morula; Blastocyst
Received: June 6, 2021 Accepted: August 9, 2021
Supported by Qiqihar Agricultural Research Project (NYGG-201904); China Agriculture Research System of MOF and MRRA.
Guizhi TONG (1967-), female, P. R. China, master, devoted to research about animal genetics, breeding and reproduction.
*Corresponding author.
At present, embryo transfer, as an effective means for livestock improvement and purebred breeding, is being widely used. However, the low pregnancy rate of embryo transplantation and high cost of embryo transfer are the main factors restricting its industrialization. Therefore, how to reduce the cost and increase the embryo pregnancy rate is one of the urgent problems to be solved. Embryo bisection is a biotechnology of dividing mammalian pre-implantation embryos into demi-, quarter- or even eighth-embryos by artificial mechanical or chemical methods, culturing them in vivo or in vitro, and then transplanting into recipients to obtain identical twins or multiple identical offspring［1-4］. Since the beginning of embryo bisection-related research in 1942, different degrees of results have been achieved in rats, rabbits, cattle, sheep, horses, pigs and other animals［5-7］. Embryo bisection can provide a large number of genetically homogenous embryos, which requires a proper storage condition to ensure that demi-embryos are used for multiple studies. Therefore, in this study, in-vitro bovine morulae and blastocysts were bisected in three different bisection solutions, aiming to obtain a bisection solution that is more conducive to improving the success rate of bovine embryo bisection, and to find out appropriate bisection solutions of embryos at different developmental stages. Materials and Methods
Embryo sources
Morulae and blastocysts (day 6-8) (the day of insemination was counted as day 0) obtained from in-vitro fertilization of cattle were collected, and A-level embryos were selected for experiment use.
Main agents and instruments
TCM-199, purchased from Gibco; metal cutting knife; inverted microscope; disposable petri dish; bisection solution D-PBS solution; 0.2 mol/L sucrose solution prepared with PBS solution the base solution.
Embryo bisection
Morula bisection
Free hand bisection method: Multiple 70 μl of bisection drops were made in the cover of a sterile petri dish with a diameter of 100 mm. The embryos were washed twice with a bisection solution, and then the embryos were moved into the bisection drops, 1 embryo per drop. Before use, the metal cutting knife should be sterilized by ultraviolet light for 15-20 min, and then placed in a sterilized petri dish for later use. Under a stereo microscope, a straight line was first scratched at the bottom of the bisection drops to fix the embryos, and the embryos were moved into the embryo bisection solution drops in the petri dish, and moved to the line mark. The petri dish was fixed with the left hand, and the dividing blade was clamped with hemostatic forceps in the right hand or directly by the right hand, moved to the top of the embryo to be bisected, aligned with the middle of the embryo’s inner cell mass, and moved down to press the middle of the embryo and divide it down. When dividing down, the action should be decisive and rapid.
Blastocyst bisection
The blastocyst bisection method was similar to that of morulae. There was no need to perform pretreatment in PBS without Ca2+ and Mg2+, and blastocysts could be bisected directly in the required bisection fluid. When dividing, attention should be paid to dividing the inner cell mass of embryos into two.
Evaluation of demi-embryo development ability
The divided demi-embryos were gently removed from the bisection drops, and washed with a well-balanced culture medium for 2 to 3 times, and the paired demi-embryos were then transferred into the culture medium and cultured under conditions of 8.5 ℃, 5% CO2, and saturated humidity for 8-24 h. The number of successful bisection and the number of developed embryos in each group were recorded.
Culture of bisected bovine embryos
The developed demi-embryos and trophoblast cells were co-cultured. In this experiment, the bisection success rate of in-vitro fertilized embryos and the demi-embryo development rate were used as indicators to evaluate the effect of bisection. Statistical analysis
Analysis of variance was performed on the test data to determine the significance of differences.
Results and Analysis
Effects of different bisection solutions on the bisection effects of morulae and blastocysts
According to the bisection solutions and embryo development stages, the bisected demi-embryos were divided into groups for in vitro culture. It can be seen from Table 1 that the success rates of bisecting morulae in PBS solution and PBS+0.2 mol/L sucrose were 50% and 95.2%, respectively. The success rate of bisecting morulae in PBS+0.2 mol/L sucrose was significantly higher than that in PBS (P<0.05), while the development rate of the bisected demi-embryos had no significant difference between the two (53.3%, 52.4%) (P>0.05). The success rates of bisecting blastocysts in PBS solution and PBS+0.2 mol/L sucrose were 51.6% and 95.1%, respectively. The success rate of bisecting blastocysts in PBS+0.2 mol/L sucrose was significantly higher than that in PBS (P<0.05), while the development rate of the bisected demi-embryos had no significant difference between the two (50.0%, 56.4%) (P>0.05). It indicated that PBS+0.2 mol/L sucrose was suitable for bovine embryo bisection, and was beneficial to improving the bisection success rates of bovine morulae and blastocysts.
Conclusions and Discussion
In order to improve the success rate of embryo bisection and avoid damage to embryonic cells due to division, a suitable solution is needed as a carrier for embryo bisection, namely embryo bisection solution. Many researchers believe that adding or removing certain chemicals in PBS solution as a bisection solution can improve the success rate of embryo bisection. Research results show that removing Ca2+ and Mg2+ in PBS solutions can reduce the viscosity between embryonic cell masses, relax the bond between cells, and facilitate bisection［8］. Nagashima et al.［6］ reported that for mouse embryos, removing Ca2+ and Mg2+ in PBS solution could improve the success rate of bisection. Therefore, the PBS solution used in this study did not contain Ca2+ and Mg2+. It has also been reported that adding a certain concentration of sucrose to PBS solutions is conducive to embryo bisection. Sucrose is a kind of macromolecular substance, and due to its hypertonic effect, it compresses cell masses and reduces fluid in the blastocyst cavity, which is conducive to symmetrical bisection and the improvement of the survival rate of embryos after bisection. In addition, a higher concentration of sucrose bisection solution helps prevent embryos from sliding during the bisection process, thereby helping to increase the bisection speed, shorten the bisection time, and reduce the degree of damage to embryos［3,9-11］. The results of Suzuki et al. (1995) pointed out that embryos in PBS solution were soft and difficult to be divided, and the embryonic cells were more damaged. However, when the embryos were divided in a 12.5% sucrose solution, the dividing surface was easy to separate and the embryonic cells were less damaged. Zhang et al.［2］ believed that in sucrose solutions, blastocysts showed a slight contraction state, which was not easy to cause major mechanical damage during the bisection process, and in the contraction state, cell masses in blastocysts were more obvious, which was convenient for symmetrical bisection. Suzuki et al.［12］ compared the bisection effects of thawed bovine embryos in sucrose solutions and PBS. They believed that the bisection in sucrose bisection solutions could prevent the fusion of two demi-embryos remaining in the same zona pellucida, and could significantly improve the effect of frozen embryo bisection. The conclusion of this study is similar. However, Szell believed that the addition of sucrose had an adverse effect on the transplantation pregnancy rate of bisected sheep embryos, while the results of this study showed that the presence of sucrose had no adverse effect on the blastocyst development rate of bisected embryos. It should be noted that embryos are more likely to adhere to the bottom of plates after being cut when bisection is performed in the bisection solution of PBS+0.2 mol/L sucrose. In order to reduce the degree of damage to embryos, 20% calf serum must be added to the solution after cutting, and demi-embryos must be sucked out with a straw, otherwise embryos will stick to the wall of the straw and be lost easily. Recently, researchers have also reported that adding 5% vinylpyrrolidone (PVP) to PBS solution is beneficial to improving the success rate of embryo bisection. PVP is a kind of high molecular polymer, which can quickly fill into bisection gaps after embryos are divided, playing a role of protecting the embryo cells. The bisection of bovine morulae and blastocysts also confirmed this statement. In the bisection of bovine embryos, the addition of 0.2 mol/L sucrose and 5% PVP in PBS is beneficial to improving the bisection success rate of bovine morulae and blastocysts. The effect of embryonic development stages on the development of bisected embryos proved that embryos at different developmental stages had different developmental potentials after bisection. Blastocysts were better than morulae. Williams et al. (1984) found that after demi-embryos from blastocysts were transferred into recipients during the embryo bisection test of cattle, their pregnancy rate and fetal survival rate were higher than demi-embryos from morulae. It might be due to the fact that the cell connections of morula embryos are more likely to be destroyed than the connections between embryonic cells at the blastocyst stage during bisection, which affects the further development of bisected embryos. When bisecting embryos, the action must be rapid and decisive to avoid damage to embryos caused by back and forth pulling or intermittent cutting. This study showed that the PBS solution added with 0.2 mol/L sucrose could be used as a bovine embryo bisection solution. Although when bisecting embryos in PBS+0.2 mol/L sucrose, demi-embryos stuck to the bottom of the dish or in the straw very easily, PBS containing 20% FCS could be added after bisection. For different developmental stages of embryos, blastocysts were more suitable for embryo bisection than morulae. A simple and effective embryo bisection process was developed. The process industrializes the free-hand bisection method, is convenient for operators to control the accuracy of bisection, and minimize the damaging effect of the operation on embryos. It is conducive to the industrialization of the embryo bisection technology.
Agricultural Biotechnology2021
References
［1］ LIU YH, ZHU HB, HAO HS, et al. Research progress of mammalian embryo bisection technology［J］. Chinese animal husbandry and veterinary medicine, 2007, 34(3): 52-54. (in Chinese)
［2］ ZHANG Y, QIAN JM, WANG JC. Mouse embryo bisection method and identical twin test［J］. Acta Agriculturae Boreali-occidentalis Sinica, 1987, 15(2): 10-16. (in Chinese)
［3］ CHAN AWS, DOMINKO T, LUETJENS CM, et al. Clonal propagation of primate offspring by embryo splitting［J］. Science, 2000(287): 317-319.
［4］ LAMBETH VA, LOONEY GR, VOELKEL SA, et al. Microsurgery on bovine embryos at the morula stage to produce monozygotic twin calves［J］. Theriogenology, 1983(20): 85-95.
［5］ MATSUMOTO K. MIYAKE M, UTSUMIK, et al. Bisection of rat, goat and cattle blastocysts by metal blade［J］. Jpn J Anim Reprod, 1987(33): 1-5.
［6］ NAGASHIMA H. Production of monozygotic mouse twins from micro-surgically bisected morulae［J］. J Reprot Fer, 1984(70): 357-362.
［7］ NIBART M, SFIPONGPUN S, CEDDEN F, et al. Histological study of bovine intact and demi-embryos［J］. Theriogenology, 1988, 29(1): 283.
［8］ SKRZYSZOWSKA M, SMORAG Z. Effect of splitting on cell losses and quality of bisected embryos［J］. Hheriogenology, 1987(27): 276.
［9］ RHO GJ, JOHNSON WH, BETTERIDGE KJ. Cellular composition and viability of demi-and quarter-embryos made from bisected bovine morulae and blastocysts produced in vitro［J］. Theriogenology, 1998(50): 885-895.
［10］ OPPENHEIM SM, MPYER AL, BONDURANT RH, et al. Sucessful pregnancy in goats carrying the irgenetically identieal conceptus［J］. Theriogenology, 2000(541): 629-639.
［11］ REICHELT B, NIEMANN H. Generation of identieal twin piglets for lowing bisection of embroyos at the morula and blastoeyst stage［J］. Reprod Fertil, 1994(100): 163-172.
［12］ SUZUKI H, TOGASHI M, ADACHI J, et al. Developmental ability of zona-free mouse embryos is influenced by cell association at the 4-cell stage［J］. Biol Reprod, 1995, 53(1): 78-83.
［Methods］The effects of two different bisection solutions on the bisection of morulae and blastocysts in vitro were explored. The morulae and blastocysts produced in vitro from cattle that developed to the 6th to 8th d were bisected by hands, and demi-embryos were cultured in vitro. Their development was observed.
［Results］ Morulae were bisected in PBS solution and PBS+0.2 mol/L sucrose, and the success rates of bisection were 50% and 95.2%, respectively. The success rate of bisecting morulae in PBS+0.2 mol/L sucrose was significantly higher than that in PBS (P<0.05), while the development rate of the bisected demi-embryos had no significant difference between the two (53.3%, 52.4%) (P>0.05). The success rates of blastocyst bisection in PBS solution and PBS+0.2 mol/L sucrose were 51.6% and 95.1%, respectively. The success rate of blastocyst bisection in PBS+0.2 mol/L sucrose was significantly higher than that in PBS (P<0.05), while the development rate of the bisected demi-embryos had no significant difference between the two (50.0%, 56.4%) (P>0.05).
［Conclusions］There were no significant differences between the success rates of bisecting bovine morulae and blastocysts in PBS+0.2 mol/L sucrose, which were both significantly better than those in pure PBS bisection solution, proving that PBS+0.2 mol/L sucrose bisection solution is suitable for bovine embryo bisection.
Key words Cattle; Embryo division; Success rate; Development rate; Morula; Blastocyst
Received: June 6, 2021 Accepted: August 9, 2021
Supported by Qiqihar Agricultural Research Project (NYGG-201904); China Agriculture Research System of MOF and MRRA.
Guizhi TONG (1967-), female, P. R. China, master, devoted to research about animal genetics, breeding and reproduction.
*Corresponding author.
At present, embryo transfer, as an effective means for livestock improvement and purebred breeding, is being widely used. However, the low pregnancy rate of embryo transplantation and high cost of embryo transfer are the main factors restricting its industrialization. Therefore, how to reduce the cost and increase the embryo pregnancy rate is one of the urgent problems to be solved. Embryo bisection is a biotechnology of dividing mammalian pre-implantation embryos into demi-, quarter- or even eighth-embryos by artificial mechanical or chemical methods, culturing them in vivo or in vitro, and then transplanting into recipients to obtain identical twins or multiple identical offspring［1-4］. Since the beginning of embryo bisection-related research in 1942, different degrees of results have been achieved in rats, rabbits, cattle, sheep, horses, pigs and other animals［5-7］. Embryo bisection can provide a large number of genetically homogenous embryos, which requires a proper storage condition to ensure that demi-embryos are used for multiple studies. Therefore, in this study, in-vitro bovine morulae and blastocysts were bisected in three different bisection solutions, aiming to obtain a bisection solution that is more conducive to improving the success rate of bovine embryo bisection, and to find out appropriate bisection solutions of embryos at different developmental stages. Materials and Methods
Embryo sources
Morulae and blastocysts (day 6-8) (the day of insemination was counted as day 0) obtained from in-vitro fertilization of cattle were collected, and A-level embryos were selected for experiment use.
Main agents and instruments
TCM-199, purchased from Gibco; metal cutting knife; inverted microscope; disposable petri dish; bisection solution D-PBS solution; 0.2 mol/L sucrose solution prepared with PBS solution the base solution.
Embryo bisection
Morula bisection
Free hand bisection method: Multiple 70 μl of bisection drops were made in the cover of a sterile petri dish with a diameter of 100 mm. The embryos were washed twice with a bisection solution, and then the embryos were moved into the bisection drops, 1 embryo per drop. Before use, the metal cutting knife should be sterilized by ultraviolet light for 15-20 min, and then placed in a sterilized petri dish for later use. Under a stereo microscope, a straight line was first scratched at the bottom of the bisection drops to fix the embryos, and the embryos were moved into the embryo bisection solution drops in the petri dish, and moved to the line mark. The petri dish was fixed with the left hand, and the dividing blade was clamped with hemostatic forceps in the right hand or directly by the right hand, moved to the top of the embryo to be bisected, aligned with the middle of the embryo’s inner cell mass, and moved down to press the middle of the embryo and divide it down. When dividing down, the action should be decisive and rapid.
Blastocyst bisection
The blastocyst bisection method was similar to that of morulae. There was no need to perform pretreatment in PBS without Ca2+ and Mg2+, and blastocysts could be bisected directly in the required bisection fluid. When dividing, attention should be paid to dividing the inner cell mass of embryos into two.
Evaluation of demi-embryo development ability
The divided demi-embryos were gently removed from the bisection drops, and washed with a well-balanced culture medium for 2 to 3 times, and the paired demi-embryos were then transferred into the culture medium and cultured under conditions of 8.5 ℃, 5% CO2, and saturated humidity for 8-24 h. The number of successful bisection and the number of developed embryos in each group were recorded.
Culture of bisected bovine embryos
The developed demi-embryos and trophoblast cells were co-cultured. In this experiment, the bisection success rate of in-vitro fertilized embryos and the demi-embryo development rate were used as indicators to evaluate the effect of bisection. Statistical analysis
Analysis of variance was performed on the test data to determine the significance of differences.
Results and Analysis
Effects of different bisection solutions on the bisection effects of morulae and blastocysts
According to the bisection solutions and embryo development stages, the bisected demi-embryos were divided into groups for in vitro culture. It can be seen from Table 1 that the success rates of bisecting morulae in PBS solution and PBS+0.2 mol/L sucrose were 50% and 95.2%, respectively. The success rate of bisecting morulae in PBS+0.2 mol/L sucrose was significantly higher than that in PBS (P<0.05), while the development rate of the bisected demi-embryos had no significant difference between the two (53.3%, 52.4%) (P>0.05). The success rates of bisecting blastocysts in PBS solution and PBS+0.2 mol/L sucrose were 51.6% and 95.1%, respectively. The success rate of bisecting blastocysts in PBS+0.2 mol/L sucrose was significantly higher than that in PBS (P<0.05), while the development rate of the bisected demi-embryos had no significant difference between the two (50.0%, 56.4%) (P>0.05). It indicated that PBS+0.2 mol/L sucrose was suitable for bovine embryo bisection, and was beneficial to improving the bisection success rates of bovine morulae and blastocysts.
Conclusions and Discussion
In order to improve the success rate of embryo bisection and avoid damage to embryonic cells due to division, a suitable solution is needed as a carrier for embryo bisection, namely embryo bisection solution. Many researchers believe that adding or removing certain chemicals in PBS solution as a bisection solution can improve the success rate of embryo bisection. Research results show that removing Ca2+ and Mg2+ in PBS solutions can reduce the viscosity between embryonic cell masses, relax the bond between cells, and facilitate bisection［8］. Nagashima et al.［6］ reported that for mouse embryos, removing Ca2+ and Mg2+ in PBS solution could improve the success rate of bisection. Therefore, the PBS solution used in this study did not contain Ca2+ and Mg2+. It has also been reported that adding a certain concentration of sucrose to PBS solutions is conducive to embryo bisection. Sucrose is a kind of macromolecular substance, and due to its hypertonic effect, it compresses cell masses and reduces fluid in the blastocyst cavity, which is conducive to symmetrical bisection and the improvement of the survival rate of embryos after bisection. In addition, a higher concentration of sucrose bisection solution helps prevent embryos from sliding during the bisection process, thereby helping to increase the bisection speed, shorten the bisection time, and reduce the degree of damage to embryos［3,9-11］. The results of Suzuki et al. (1995) pointed out that embryos in PBS solution were soft and difficult to be divided, and the embryonic cells were more damaged. However, when the embryos were divided in a 12.5% sucrose solution, the dividing surface was easy to separate and the embryonic cells were less damaged. Zhang et al.［2］ believed that in sucrose solutions, blastocysts showed a slight contraction state, which was not easy to cause major mechanical damage during the bisection process, and in the contraction state, cell masses in blastocysts were more obvious, which was convenient for symmetrical bisection. Suzuki et al.［12］ compared the bisection effects of thawed bovine embryos in sucrose solutions and PBS. They believed that the bisection in sucrose bisection solutions could prevent the fusion of two demi-embryos remaining in the same zona pellucida, and could significantly improve the effect of frozen embryo bisection. The conclusion of this study is similar. However, Szell believed that the addition of sucrose had an adverse effect on the transplantation pregnancy rate of bisected sheep embryos, while the results of this study showed that the presence of sucrose had no adverse effect on the blastocyst development rate of bisected embryos. It should be noted that embryos are more likely to adhere to the bottom of plates after being cut when bisection is performed in the bisection solution of PBS+0.2 mol/L sucrose. In order to reduce the degree of damage to embryos, 20% calf serum must be added to the solution after cutting, and demi-embryos must be sucked out with a straw, otherwise embryos will stick to the wall of the straw and be lost easily. Recently, researchers have also reported that adding 5% vinylpyrrolidone (PVP) to PBS solution is beneficial to improving the success rate of embryo bisection. PVP is a kind of high molecular polymer, which can quickly fill into bisection gaps after embryos are divided, playing a role of protecting the embryo cells. The bisection of bovine morulae and blastocysts also confirmed this statement. In the bisection of bovine embryos, the addition of 0.2 mol/L sucrose and 5% PVP in PBS is beneficial to improving the bisection success rate of bovine morulae and blastocysts. The effect of embryonic development stages on the development of bisected embryos proved that embryos at different developmental stages had different developmental potentials after bisection. Blastocysts were better than morulae. Williams et al. (1984) found that after demi-embryos from blastocysts were transferred into recipients during the embryo bisection test of cattle, their pregnancy rate and fetal survival rate were higher than demi-embryos from morulae. It might be due to the fact that the cell connections of morula embryos are more likely to be destroyed than the connections between embryonic cells at the blastocyst stage during bisection, which affects the further development of bisected embryos. When bisecting embryos, the action must be rapid and decisive to avoid damage to embryos caused by back and forth pulling or intermittent cutting. This study showed that the PBS solution added with 0.2 mol/L sucrose could be used as a bovine embryo bisection solution. Although when bisecting embryos in PBS+0.2 mol/L sucrose, demi-embryos stuck to the bottom of the dish or in the straw very easily, PBS containing 20% FCS could be added after bisection. For different developmental stages of embryos, blastocysts were more suitable for embryo bisection than morulae. A simple and effective embryo bisection process was developed. The process industrializes the free-hand bisection method, is convenient for operators to control the accuracy of bisection, and minimize the damaging effect of the operation on embryos. It is conducive to the industrialization of the embryo bisection technology.
Agricultural Biotechnology2021
References
［1］ LIU YH, ZHU HB, HAO HS, et al. Research progress of mammalian embryo bisection technology［J］. Chinese animal husbandry and veterinary medicine, 2007, 34(3): 52-54. (in Chinese)
［2］ ZHANG Y, QIAN JM, WANG JC. Mouse embryo bisection method and identical twin test［J］. Acta Agriculturae Boreali-occidentalis Sinica, 1987, 15(2): 10-16. (in Chinese)
［3］ CHAN AWS, DOMINKO T, LUETJENS CM, et al. Clonal propagation of primate offspring by embryo splitting［J］. Science, 2000(287): 317-319.
［4］ LAMBETH VA, LOONEY GR, VOELKEL SA, et al. Microsurgery on bovine embryos at the morula stage to produce monozygotic twin calves［J］. Theriogenology, 1983(20): 85-95.
［5］ MATSUMOTO K. MIYAKE M, UTSUMIK, et al. Bisection of rat, goat and cattle blastocysts by metal blade［J］. Jpn J Anim Reprod, 1987(33): 1-5.
［6］ NAGASHIMA H. Production of monozygotic mouse twins from micro-surgically bisected morulae［J］. J Reprot Fer, 1984(70): 357-362.
［7］ NIBART M, SFIPONGPUN S, CEDDEN F, et al. Histological study of bovine intact and demi-embryos［J］. Theriogenology, 1988, 29(1): 283.
［8］ SKRZYSZOWSKA M, SMORAG Z. Effect of splitting on cell losses and quality of bisected embryos［J］. Hheriogenology, 1987(27): 276.
［9］ RHO GJ, JOHNSON WH, BETTERIDGE KJ. Cellular composition and viability of demi-and quarter-embryos made from bisected bovine morulae and blastocysts produced in vitro［J］. Theriogenology, 1998(50): 885-895.
［10］ OPPENHEIM SM, MPYER AL, BONDURANT RH, et al. Sucessful pregnancy in goats carrying the irgenetically identieal conceptus［J］. Theriogenology, 2000(541): 629-639.
［11］ REICHELT B, NIEMANN H. Generation of identieal twin piglets for lowing bisection of embroyos at the morula and blastoeyst stage［J］. Reprod Fertil, 1994(100): 163-172.
［12］ SUZUKI H, TOGASHI M, ADACHI J, et al. Developmental ability of zona-free mouse embryos is influenced by cell association at the 4-cell stage［J］. Biol Reprod, 1995, 53(1): 78-83.