论文部分内容阅读
Abstract [Objectives] This study was conducted to explore the effects of three different splitting fluids on the bisection effects of bovine morulae and blastocysts.
[Methods] With the help of a micromanipulator， the morulae and blastocysts that were routinely produced in the body from the 6th to the 8th d were divided into half-embryos in vitro to observe their development， and the half-embryos with a restored morphology and a blastocyst trophoblast were selected.
[Results] The success rates of bisection of morulae in PBS+0.2 mol/L sucrose and PBS+5% PVP were significantly higher than that in PBS （P<0.05）， and the values in the three liquids were 95.7%， 97.2% and 50%， respectively， while the blastocyst development rates and of half-embryos and the pregnancy rates of transplantation were not significantly different （P>0.05）. When blastocysts were bisected in PBS+0.2 mol / L sucrose and PBS+5% PVP， the success rates of bisection were significantly higher than that of PBS （P<0.05）， and the values in the three liquids were 96.8%， 95.5% and 50.0%， respectively， while the development rates of half-embryos and the pregnancy rates of transplantation were not significantly different （P>0.05）. The development rate of half-embryos bisected from blastocysts was significantly higher than that of morulae.
[Conclusions] This study improves the efficiency of bovine embryo bisection， which is beneficial to the application of embryo bisection technology in cattle breeding.
Key words Cattle; Embryo bisection; Blastocyst development rate
At present， embryo transfer is widely used as an effective means for livestock improvement and pure breeding， but the low pregnancy rate and high cost of embryo transfer are the main factors restricting its industrialization. Therefore， how can we reduce costs and increase the embryo pregnancy rate is one of urgent problems to be solved. Embryo bisection is the use of mechanical or chemical methods to artificially divide the pre-implantation embryos of mammals into two， four or even eighth embryos， cultivated in vivo or in vitro， and then transplanted into the recipients to obtain monozygotic twins or multifetal gestations. Since the research on embryo splitting started in 1942， different results have been achieved in mice， rabbits， cattle， sheep， horses， pigs， and even primates. Embryo bisection can provide a large number of genetically homogenous embryos， which requires an appropriate preservation condition to ensure that half-embryos are used for multiple studies. Therefore， in this study， cattle morulae and blastocysts were bisected in 3 different splitting fluids， aiming to obtain a dividing fluid that is more conducive to improving the success rate of dividing embryos of cattle and to exploring the effect of different embryo ages on embryo division. Materials and Methods
Source of embryos
Morulae and blastocysts （days 6-8， counted from day 0 on the day of insemination） were obtained from insemination in vitro. Embryos of grade A and B were selected for bisection.
Main drugs and equipment
Except for TCM-199 purchased from Gibco， most of the drugs were purchased from Sigma. The splitting liquid was PBS solution without Ca2+ and Mg2+， and 0.2 mol/L sucrose and 5% polyvinylpyrrolidone （PVP） were added to the PBS solution without Ca2+ and Mg2+ as the base solution. The instruments used included metal cutter， inverted microscope and disposable plastic dish.
Embryo bisection
Bisection of morulae
Several 70 μl of required splitting liquid droplets were made in a sterile plastic dish with a diameter of 100 mm. After washing the embryos twice with the splitting liquid without Ca2+ and Mg2+ for 2 min， the embryos were transferred to the droplets separately and placed under an inverted microscope （200×）. After adjusting the positions of the embryos， the embryos were cut with a metal cutter on the micromanipulator gradually from top to bottom， and each morula could be easily divided into two[1].
Bisection of blastocysts
The blastocyst bisection method was similar to that of morulae. It does not require pretreatment in PBS solution without Ca2+ and Mg2+， and can be directly bisected in the required splitting fluid. When dividing， we must pay attention to divide the inner cell mass of the embryo into two.
Judgment of half-embryo development ability
The divided half-embryos were cultured in the same way as whole embryos， and the pairs of half-embryos of each group were taken out from the splitting liquid， washed 2 to 3 times with a balanced culture medium， and then transferred to the culture medium， followed by 8-24 h of culture at 38.5 ℃ with 5% CO2 and saturated humidity. The number of half-embryos restored （number of successful divisions） was checked from 1 to 3 h， and the development rate of half-embryos was recorded at 24 h （the half-embryo developed to morula is an embryo with normal development ability）. After thawing the half-embryos， they were transferred to the monolayer of human granules and cultured for 24 h. Embryos that can reform a blastular cavity are viable embryos. The number of successful divisions and the number of blastocysts developed in each group were recorded.
Half-embryos were cultivated The developing half-embryos were cultivated. In this experiment， the success rate of embryo bisection， the development rate of half-embryo blastocysts were used as indexes to evaluate the effect of bisection.
Statistical analysis
Test data was analyzed by analysis of variance to determine the significance of the difference.
Results and Analysis
According to the splitting fluid and embryo development stage， the divided half-embryos were divided into groups and cultured in vitro. It could be seen from Table 1 that the success rates of morula bisection in PBS+0.2 mol/L sucrose and PBS+5% PVP were significantly higher than that in PBS （P<0.05）， and the values in the three liquids were 95.7%， 97.2% and 50%， respectively， while the blastocyst development rates of embryos and the pregnancy rates of transplantation were not significantly different （P>0.05）. When blastocysts were bisected in PBS+0.2 mol/L sucrose and PBS+5% PVP， the success rates of bisection were significantly higher than that of PBS （P<0.05）， and the values in the three liquids 96.8%， 95.5% and 50.0%， respectively， while the development rates of blastocysts in half-embryo culture and the pregnancy rates of transplantation were not significantly different （P>0.05）. Adding 0.2 mol/L sucrose or 5% PVP to PBS was beneficial to improve the success rate of morula and blastocyst division.
Conclusions and Discussion
In order to improve the success rate of embryo bisection and avoid damage to embryo cells due to bisection， it is necessary to have a suitable solution as a carrier during embryo bisection， namely embryo splitting fluid[1]. Many researchers believe that adding or removing certain chemicals in the PBS solution as a splitting liquid can improve the success rate of embryo division. Studies have shown removing Ca2+ and Mg2+ in PBS can reduce the viscosity between embryonic cell clusters， relax the binding between cells， and facilitate bisection[2]. Nagashima et al.[3] reported that for mouse embryos， removing Ca2+ and Mg2+ in PBS can increase the success rate of bisection. Therefore， the PBS solution used in this study did not contain Ca2+ and Mg2+. It is also reported that the addition of a certain concentration of sucrose in PBS solution is beneficial to embryo division. Sucrose is a macromolecular substance. Due to the hyperosmotic effect， the cell mass shrinks and the liquid in the blastocyst cavity is reduced， which is conducive to symmetrical division and the improvement of the survival rate of embryos after division. In addition， the higher concentration of sucrose splitting liquid is helpful to prevent embryos from sliding during the splitting process， which is beneficial to increase the splitting speed and shorten the splitting time to reduce the damage to embryos[4-8]. The results of Suzuki et al.[9] pointed out that embryos in PBS solution had soft cells and were not easy to divide， and embryo cells were more damaged. However， in 12.5% sucrose solution， the split surfaces were easy to separate and damage to embryo cells was less. Zhang et al.[10] believed that in sucrose solution， the blastocysts showed a slight contraction state， and it was not easy to cause large mechanical damage during the division process. In the contraction state， the cell mass in the blastocyst was more obvious， which was convenient for symmetric division. TSuzuki et al.[11] compared the effects of thawing bovine embryos in sucrose solution and PBS， and believed that the sucrose splitting liquid could prevent the fusion of the two half-embryos left in the same zona pellucida， and could significantly improve the division effect of thawed embryos， which accords with the conclusion of study. However， Zhang et al.[12] believed that in sucrose， the blastocysts showed a slight contraction state， and the internal pressure of the blastocysts in the slightly contracted state was small， which was not easy to cause large mechanical damage during the segmentation process. And in the contracted state， the cell mass in the blastocysts was more obvious， which is convenient for symmetric division. In addition， higher concentrations of sucrose solution and imported splitting liquid are helpful to prevent the embryos from sliding during the splitting process， thereby increasing the splitting speed， shortening the splitting time， and reducing the degree of embryo damage. The results of this study showed that the presence of sucrose had no adverse effect on the blastocyst development rate of split embryos. It should be noted that when splitting in PBS+0.2 mol/L sucrose splitting liquid， embryos are more likely to adhere to the bottom of the dish after division. In order to reduce the degree of damage to embryos， 20% of calf serum must be added to the liquid after cutting， and then the half-embryos should be sucked out using a pipette， otherwise the embryos will adhere to the pipette wall and be lost easily. Recently， researchersalso reported that adding 50% polyvinylpyrrolidone （PVP） to PBS is beneficial to improve the success rate of embryo division. PVP is a high-molecular polymer， which can be quickly filled into the division gap after embryo division， and plays a role in protecting embryo cells. The division of cattle 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 improve the bisection success rate of bovine morulae and blastocysts. The experimental results of the influence of embryonic development stage on the development of bisected embryos prove that the embryos of different developmental stages have different development potentials after bisection. Blastocyst is superior to morula. Williams et al.[13]. found that half-embryos derived from blastocysts were transferred into the recipients during the embryo division test of cattle. Their pregnancy rate and fetal survival rate were higher than the half-embryos from morulae. It might be due to splitting. The cell connection of morulae is more likely to be broken than the connection between embryo cells in the blastocyst stage， which affects the further development of the split embryos. The movement of the embryos must be rapid and decisive to avoid damage to the embryos caused by back and forth or intermittent cutting. References
[1] 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.
[2] SKRZYSZOWSKA M， SMORAG Z. Effect of splitting on cell losses and quality of bisected embryos[J]. Hheriogenology， 1987（27）： 276.
[3] NAGASHIMA H. Production of monozygotic mouse twins from microsurgically bisected morulae[J]. J Reprot Fer， 1984（70）： 357- 362.
[4] 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.
[5] CHAN AWS， DOMINKO T， LUETJENS CM， et al. Clonal propagation of primate offspring by embryo splitting[J]. Science， 2000（287）： 317-319.
[6] OPPENHEIM SM， MPYER AL， BONDURANT RH， et al. Sucessful pregnancy in goats carrying the irgenetically identical conceptus[J]. Theriogenology， 2000（541）： 629-639.
[7] 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.
[8] OZIL JP. Production of identical twins by bisection of blastocysts in the cow[J]. J Reprod Fertil， 1983（69）： 463-468.
[9] SUZUKI H， TOGASHI M， ADACHI J， et al. Developmental ability of zone-free mouse embryos is influenced by cell association at the 4-cell stage[J]. Biol Reprod， 1995， 53（1）： 78-83.
[10] ZHANG Y， QIE JM， WANG JC. Mouse embryo segmentation method and monozygotic twin test[J]. Journal of Northwest Agricultural University， 1987， 15（2）： 10-16. （in Chinese）
[11] TSUZUKI. Viability of frozem-thawed bovine embryos bisected in sucrose[J]. Theriogenology， 1986（26）： 333-339.
[12] ZHANG Y， WANG JC， QIAN JF. Segmentation test of goat frozen embryos[J]. Acta Veterinaria et Zootechnica Sinica， 1992， 23（3）： 193-197. （in Chinese）
[13] WILLIAMS TJ， ELSDEN RP， SEIDEL GE. Pregnancy rates with bisected bovine embryos[J]. Theriogenology， 1984， 22（5）： 521-531.
[Methods] With the help of a micromanipulator， the morulae and blastocysts that were routinely produced in the body from the 6th to the 8th d were divided into half-embryos in vitro to observe their development， and the half-embryos with a restored morphology and a blastocyst trophoblast were selected.
[Results] The success rates of bisection of morulae in PBS+0.2 mol/L sucrose and PBS+5% PVP were significantly higher than that in PBS （P<0.05）， and the values in the three liquids were 95.7%， 97.2% and 50%， respectively， while the blastocyst development rates and of half-embryos and the pregnancy rates of transplantation were not significantly different （P>0.05）. When blastocysts were bisected in PBS+0.2 mol / L sucrose and PBS+5% PVP， the success rates of bisection were significantly higher than that of PBS （P<0.05）， and the values in the three liquids were 96.8%， 95.5% and 50.0%， respectively， while the development rates of half-embryos and the pregnancy rates of transplantation were not significantly different （P>0.05）. The development rate of half-embryos bisected from blastocysts was significantly higher than that of morulae.
[Conclusions] This study improves the efficiency of bovine embryo bisection， which is beneficial to the application of embryo bisection technology in cattle breeding.
Key words Cattle; Embryo bisection; Blastocyst development rate
At present， embryo transfer is widely used as an effective means for livestock improvement and pure breeding， but the low pregnancy rate and high cost of embryo transfer are the main factors restricting its industrialization. Therefore， how can we reduce costs and increase the embryo pregnancy rate is one of urgent problems to be solved. Embryo bisection is the use of mechanical or chemical methods to artificially divide the pre-implantation embryos of mammals into two， four or even eighth embryos， cultivated in vivo or in vitro， and then transplanted into the recipients to obtain monozygotic twins or multifetal gestations. Since the research on embryo splitting started in 1942， different results have been achieved in mice， rabbits， cattle， sheep， horses， pigs， and even primates. Embryo bisection can provide a large number of genetically homogenous embryos， which requires an appropriate preservation condition to ensure that half-embryos are used for multiple studies. Therefore， in this study， cattle morulae and blastocysts were bisected in 3 different splitting fluids， aiming to obtain a dividing fluid that is more conducive to improving the success rate of dividing embryos of cattle and to exploring the effect of different embryo ages on embryo division. Materials and Methods
Source of embryos
Morulae and blastocysts （days 6-8， counted from day 0 on the day of insemination） were obtained from insemination in vitro. Embryos of grade A and B were selected for bisection.
Main drugs and equipment
Except for TCM-199 purchased from Gibco， most of the drugs were purchased from Sigma. The splitting liquid was PBS solution without Ca2+ and Mg2+， and 0.2 mol/L sucrose and 5% polyvinylpyrrolidone （PVP） were added to the PBS solution without Ca2+ and Mg2+ as the base solution. The instruments used included metal cutter， inverted microscope and disposable plastic dish.
Embryo bisection
Bisection of morulae
Several 70 μl of required splitting liquid droplets were made in a sterile plastic dish with a diameter of 100 mm. After washing the embryos twice with the splitting liquid without Ca2+ and Mg2+ for 2 min， the embryos were transferred to the droplets separately and placed under an inverted microscope （200×）. After adjusting the positions of the embryos， the embryos were cut with a metal cutter on the micromanipulator gradually from top to bottom， and each morula could be easily divided into two[1].
Bisection of blastocysts
The blastocyst bisection method was similar to that of morulae. It does not require pretreatment in PBS solution without Ca2+ and Mg2+， and can be directly bisected in the required splitting fluid. When dividing， we must pay attention to divide the inner cell mass of the embryo into two.
Judgment of half-embryo development ability
The divided half-embryos were cultured in the same way as whole embryos， and the pairs of half-embryos of each group were taken out from the splitting liquid， washed 2 to 3 times with a balanced culture medium， and then transferred to the culture medium， followed by 8-24 h of culture at 38.5 ℃ with 5% CO2 and saturated humidity. The number of half-embryos restored （number of successful divisions） was checked from 1 to 3 h， and the development rate of half-embryos was recorded at 24 h （the half-embryo developed to morula is an embryo with normal development ability）. After thawing the half-embryos， they were transferred to the monolayer of human granules and cultured for 24 h. Embryos that can reform a blastular cavity are viable embryos. The number of successful divisions and the number of blastocysts developed in each group were recorded.
Half-embryos were cultivated The developing half-embryos were cultivated. In this experiment， the success rate of embryo bisection， the development rate of half-embryo blastocysts were used as indexes to evaluate the effect of bisection.
Statistical analysis
Test data was analyzed by analysis of variance to determine the significance of the difference.
Results and Analysis
According to the splitting fluid and embryo development stage， the divided half-embryos were divided into groups and cultured in vitro. It could be seen from Table 1 that the success rates of morula bisection in PBS+0.2 mol/L sucrose and PBS+5% PVP were significantly higher than that in PBS （P<0.05）， and the values in the three liquids were 95.7%， 97.2% and 50%， respectively， while the blastocyst development rates of embryos and the pregnancy rates of transplantation were not significantly different （P>0.05）. When blastocysts were bisected in PBS+0.2 mol/L sucrose and PBS+5% PVP， the success rates of bisection were significantly higher than that of PBS （P<0.05）， and the values in the three liquids 96.8%， 95.5% and 50.0%， respectively， while the development rates of blastocysts in half-embryo culture and the pregnancy rates of transplantation were not significantly different （P>0.05）. Adding 0.2 mol/L sucrose or 5% PVP to PBS was beneficial to improve the success rate of morula and blastocyst division.
Conclusions and Discussion
In order to improve the success rate of embryo bisection and avoid damage to embryo cells due to bisection， it is necessary to have a suitable solution as a carrier during embryo bisection， namely embryo splitting fluid[1]. Many researchers believe that adding or removing certain chemicals in the PBS solution as a splitting liquid can improve the success rate of embryo division. Studies have shown removing Ca2+ and Mg2+ in PBS can reduce the viscosity between embryonic cell clusters， relax the binding between cells， and facilitate bisection[2]. Nagashima et al.[3] reported that for mouse embryos， removing Ca2+ and Mg2+ in PBS can increase the success rate of bisection. Therefore， the PBS solution used in this study did not contain Ca2+ and Mg2+. It is also reported that the addition of a certain concentration of sucrose in PBS solution is beneficial to embryo division. Sucrose is a macromolecular substance. Due to the hyperosmotic effect， the cell mass shrinks and the liquid in the blastocyst cavity is reduced， which is conducive to symmetrical division and the improvement of the survival rate of embryos after division. In addition， the higher concentration of sucrose splitting liquid is helpful to prevent embryos from sliding during the splitting process， which is beneficial to increase the splitting speed and shorten the splitting time to reduce the damage to embryos[4-8]. The results of Suzuki et al.[9] pointed out that embryos in PBS solution had soft cells and were not easy to divide， and embryo cells were more damaged. However， in 12.5% sucrose solution， the split surfaces were easy to separate and damage to embryo cells was less. Zhang et al.[10] believed that in sucrose solution， the blastocysts showed a slight contraction state， and it was not easy to cause large mechanical damage during the division process. In the contraction state， the cell mass in the blastocyst was more obvious， which was convenient for symmetric division. TSuzuki et al.[11] compared the effects of thawing bovine embryos in sucrose solution and PBS， and believed that the sucrose splitting liquid could prevent the fusion of the two half-embryos left in the same zona pellucida， and could significantly improve the division effect of thawed embryos， which accords with the conclusion of study. However， Zhang et al.[12] believed that in sucrose， the blastocysts showed a slight contraction state， and the internal pressure of the blastocysts in the slightly contracted state was small， which was not easy to cause large mechanical damage during the segmentation process. And in the contracted state， the cell mass in the blastocysts was more obvious， which is convenient for symmetric division. In addition， higher concentrations of sucrose solution and imported splitting liquid are helpful to prevent the embryos from sliding during the splitting process， thereby increasing the splitting speed， shortening the splitting time， and reducing the degree of embryo damage. The results of this study showed that the presence of sucrose had no adverse effect on the blastocyst development rate of split embryos. It should be noted that when splitting in PBS+0.2 mol/L sucrose splitting liquid， embryos are more likely to adhere to the bottom of the dish after division. In order to reduce the degree of damage to embryos， 20% of calf serum must be added to the liquid after cutting， and then the half-embryos should be sucked out using a pipette， otherwise the embryos will adhere to the pipette wall and be lost easily. Recently， researchersalso reported that adding 50% polyvinylpyrrolidone （PVP） to PBS is beneficial to improve the success rate of embryo division. PVP is a high-molecular polymer， which can be quickly filled into the division gap after embryo division， and plays a role in protecting embryo cells. The division of cattle 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 improve the bisection success rate of bovine morulae and blastocysts. The experimental results of the influence of embryonic development stage on the development of bisected embryos prove that the embryos of different developmental stages have different development potentials after bisection. Blastocyst is superior to morula. Williams et al.[13]. found that half-embryos derived from blastocysts were transferred into the recipients during the embryo division test of cattle. Their pregnancy rate and fetal survival rate were higher than the half-embryos from morulae. It might be due to splitting. The cell connection of morulae is more likely to be broken than the connection between embryo cells in the blastocyst stage， which affects the further development of the split embryos. The movement of the embryos must be rapid and decisive to avoid damage to the embryos caused by back and forth or intermittent cutting. References
[1] 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.
[2] SKRZYSZOWSKA M， SMORAG Z. Effect of splitting on cell losses and quality of bisected embryos[J]. Hheriogenology， 1987（27）： 276.
[3] NAGASHIMA H. Production of monozygotic mouse twins from microsurgically bisected morulae[J]. J Reprot Fer， 1984（70）： 357- 362.
[4] 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.
[5] CHAN AWS， DOMINKO T， LUETJENS CM， et al. Clonal propagation of primate offspring by embryo splitting[J]. Science， 2000（287）： 317-319.
[6] OPPENHEIM SM， MPYER AL， BONDURANT RH， et al. Sucessful pregnancy in goats carrying the irgenetically identical conceptus[J]. Theriogenology， 2000（541）： 629-639.
[7] 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.
[8] OZIL JP. Production of identical twins by bisection of blastocysts in the cow[J]. J Reprod Fertil， 1983（69）： 463-468.
[9] SUZUKI H， TOGASHI M， ADACHI J， et al. Developmental ability of zone-free mouse embryos is influenced by cell association at the 4-cell stage[J]. Biol Reprod， 1995， 53（1）： 78-83.
[10] ZHANG Y， QIE JM， WANG JC. Mouse embryo segmentation method and monozygotic twin test[J]. Journal of Northwest Agricultural University， 1987， 15（2）： 10-16. （in Chinese）
[11] TSUZUKI. Viability of frozem-thawed bovine embryos bisected in sucrose[J]. Theriogenology， 1986（26）： 333-339.
[12] ZHANG Y， WANG JC， QIAN JF. Segmentation test of goat frozen embryos[J]. Acta Veterinaria et Zootechnica Sinica， 1992， 23（3）： 193-197. （in Chinese）
[13] WILLIAMS TJ， ELSDEN RP， SEIDEL GE. Pregnancy rates with bisected bovine embryos[J]. Theriogenology， 1984， 22（5）： 521-531.