Technology for producing identical twins

Obtainingidentical twins. The basic premise of the natural manifestations of multiple pregnancy in mammals is the simultaneous fertilization of at least two mature eggs by different sperm. Cattle is characterized by low twins frequency (an average of 0,025). Among calves- twins are sometimes found identical twins. The likelihood of such genetically identical twins is only 0.01%. Low rates of twins incidence and heritability do not allow to expect the high efficiency of selection. Therefore of great practical importance is the genotypes copy methods of highly productive animals on the basis of early embryo separation by microsurgery and micromanipulation techniques into two or more blastomeres capable to develop during the entire ontogeny, ie capable to express their totipotency. The possibility micromanipulation with individual embryos was proven in 1936 G Pincus. He introduced a single blastomeres of 2-cell rabbit embryo in oviduct of falsely pregnant rabbit. Embryos developed normally with differentiation of their cells to blastocyst stage (stages 2, 4 and 8 blastomeres, morula stage, blastocyst stage). Then offspring of rabbits and mice was received from two-cell embryos (stage 2 blastomeres) with the destruction of a single blastomere by puncturing it with a needle. These studies were the basis for the further improvement of micromanipulation technique with embryos of animals, and in particular to obtain identical twins. The latter is of great importance to intensification of livestock, since it facilitates the increase of output young animals from a single donor and allows to get genetically identical twins. SMWilladsen first reported in 1979 about getting of monozygotic twins in sheep by dividing 2-cell blastomere. He divided blastomeres into two separate cells, and impaired pellyutsida area (pellyutsida is formation preventing spillage of blastomeres, as well as contact with other embryos, foreign cells, white blood cells, sperm cells and facilitates the passage of the embryo through oviductcloged with agar. Enclosing separated blastomeres in agar, which is practically insoluble in the female genital tract, allowed them to survive and develop in vivo. For the cultivation of embryos enclosed in agar sheep was used as temporary recipients (sheep oviduct is the most suitable object for cows, horses and pigs embryo development up to blastocyst). Survival rate of "halves" of embryos at this stage after transplantation to recipients was about 50%.

In sheep, two-cell blastomeres can be obtained in a very short period of time. So, after 60 hours, most embryos are on the 4-cell stage of development. Due to significant fluctuations of intervals between injections of pregnant mare serum (PMS) and the beginning of hunting, between hunting season and the beginning of ovulation, it is almost impossible during the operation of sheep to find embryos, which are on the 2-cell stage. Later experiments showed that genetically identical twins can also be obtained from the 4 - and 8 - cell blastomeres by splitting them into two groups. These "half" were equally viable as a normal sheep embryos. It is established that embryos derived from 8-cell blastomeres have no vitality. It is believed that the sharp decrease in the number of cells of the embryo is a major factor reducing their ability to develop into viable blastocysts.

 Technique of enclosing in agar blastomeres of cattle embryos divided into parts to get identical twins was successfully used by SMWilladsen et al. (1981) in obtaining calves - identical twins. Investigations were carried out on 5-6-day-old embryos at morula stage, because in cows non-surgically method more appropriate to get embryos. Morula were divided in "half" or "quarters", enclosed in agar and transferred to the oviduct of anestralnoy sheep for 1-2 days. Then they were removed and surgically transplanted to recipients on the 6th and 7th day of the sexual cycle. Engraftment of "halves" was high (75%), while this figure in "quarters" was significantly lower (41%). The authors concluded that the ability of splitting cow embryo to regulate their development in the case of decreasing the number of cells is largely preserved even at morula stage. W.R. Allen and R.L. Pashen (1984) in experiments on horses proved the possibility of obtaining identical twins in the same manner using 2-8-cell blastomeres. G.Brem et al. (1983) have developed a technology for the production of identical twins using a 6-day-old embryos (morulae) received by non-surgical way. Embryos fixed with pipettes. With the help of micro knife pellyutsida zone was cut, and then incision was opened by glass needle. Micro knife or glass thread was introduced through a hole to cut morula into two halfs. One half of the embryo left in pellyutsida’s own zone, and the other was placed in a transparent area of unfertilized cattle oocytes.According to the authors, engraftment of separated embryos was 60%. From the all implanted embryos 50% of the pairs of twins were received, one out of three divided embryo gave a pair of twins. Subsequent experiments on the production of monozygotic twins have been focused on the use of late morulae and blastocysts, because at that stages of development protection from the pellyutsida zone is not insignificant. SMWilladsen and RAGodke (1984) carried out separation of sheep embryos at the late morula stage, at the stage of early, late and hatching blastocyst into two equal halves.

In thiscase  part of the halves of embryos remained inside pellyutsida torn areas, while others were transplanted without it. Halves of embryos were transplanted to the same sheep from that they have been removed. 16 out of 18 sheep lambing, among them 7 heads gave birth to one lamb, 8 heads - to identical twins and one head gave birth to no monozygotic twins. Moreover, the value of zone pellyutsida in the development of separated blastocysts is not revealed. Engraftment of embryos’ halves on the stage of late morula and early blastocyst was almost two times lower (42%) than in the late stage or in the stage of hatched blastocysts (79%). Researchers have not received any single egg twins after embryo transfer, divided on morula stage, whereas one egg twins were obtained after transplantation of blastocysts, divided on all three stages of development. And there are reports about possibility of using embryos of cows and pigs in the later stages of development for obtaining of one egg twins (Willadsen, 1981; Zambeth et al., 1983; Baker et al., 1984; Rorie et al., 1985). Effective test for assessing survival of embryo halves is cultivating them within 2-4 hours between separation and the transplant. Culturing embryo halves for a night or more than 12 hours, was accompanied by a marked decline in their survival.



22. New approaches in improving the process of in vitro fertilization

Fertilizationin vitro. Oocyte fertilization "in vitro" is being made now in more than 20 species of animals. The first reports about the normal offspring obtained from mice was published in 1968, from the rats - in 1974 and from cattle pigs and sheep - in 1981-1984. Oocytes in vitro fertilization means that complex physiological processes in the body of a pregnant female, must take place in a relatively simple and static conditions. An important step in developing method of fertilization in vitro was the discovery of sperm capacitation phenomenon (Chang and Austin,1951). They established that fertilization occurs only if the sperms preliminary are located in female oviduct for several hours before ovulation.At this time they undergo certain physiological changes and become capable to fertilization. It is believed that sperm capacitation is first of all modification or removal of proteins and other macromolecular substances in the plasma membrane of the sperm. Across the plasma membrane proteolytic enzymes necessary for penetration of zone pellyutsida are washed. Duration of mouse and hamster’s sperm capacitation in uterus and in vitro is equal to 1-2 h, rats - 5-6 hours, whereas rabbits in uterus - about 6 hours, and in vitro - about 10 hours.

Chang (1959) for the first time received offspring after transplantation of oocytes fertilized in vitro by sperm which was capacitated in utero of rabbit. After working out of conditions for sperm capacitation in the female genital tract experiments were conducted on capacitation in vitro. For this purpose, initially oviduct and follicular fluid or blood serum was added to the medium. Later in mice capacitation and fertilization in vitro in a medium containing bovine serum albumin and sodium priuvat without additives of biological fluids were reached. The main medium for mice, rats and hamsters’ sperm capacitation and fertilization in vitro was Krebs-Ringer solution containing glucose, serum albumin, lactate and sodium pyruvate.

L.K.Ernst et al. (1983) performed in vitro fertilization without processing sperm. In these experiments, bovine semen was washed twice in media Brinstera or TC 199. The authors concluded that the key to a full fertilization is oocyte maturation. Percentage of dividing oocytes was the same when mature oocytes were inseminated with capacitated and non capacitated sperms. According to researchers, sperm capacitation is a natural process culminating during it passing through the cumulus cells at zone pellyutsida of oocyte. If oocyte is a full and surrounded by cumulus cells sperm capacitation is proceeding normally at the time of their contact with zone pellyutsida. Egg fertilization of cows with semen capacitated in vitro, was reached in the late 70's. For this purpose Bracett et al. (1978) used the physiological medium with high ionic strength. In these experiments, in contrast to previous experiments in which oocytes after maturation in vitro were used, eggs were extracted from pre-ovulatory follicles or oviduct shortly after ovulation. Later Iritani et al. (1984) showed that sperm can be capacitated not only in a high ionic strength, but also in an isotonic medium. Moreover, the authors came to the conclusion that capacitation can be made during the storage of sperm at 20 ° C. The effectiveness of in vitro fertilization is highly dependent on factors associated with eggs (Brackett et al., 1982). Thus, in a high ionic strength the penetration of capacitated sperm into oocytes was recorded in 40% of oocytes matured in the pre-ovulatory follicles or oviducts (called tubal oocytes), whereas among the oocytes, which are riped in vitro, fertilization is observed in only 10% of oocytes. Embryos in culture conditions in vitro developed to 4-8-cell stage, which did not allow experimenters to implement non-surgical transplantation (for non-surgical transplantation and the freezing of embryos is required to bring them to the stage of compaction).However, even if perfect technique in vitro fertilization of tubal oocytes will be designed huge oocyte genetic material containing in the follicles is remained unrealized in the reproduction and breeding. Only the development of a method of obtaining calves from in vitro fertilization of follicular oocytes (eggs matured in vitro) will create a real opportunity to significantly increase the effectiveness of transplantation and the creation of large banks of embryos from genetically valuable cows. Such a methodology will provide with zygotes and early embryos in sufficient quantity, required for nuclear transfer and recombinant DNA, as well as for the production of transgenic animals. The first calf from follicular oocytes riped "in vitro" after in vitro fertilization was born in 1983 (LK Ernst, et al.). Researchers used for this purpose oocytes matured in vitro, and noncapacitated fresh or frozen-thawed sperm. 1-7 embryos which were on 2-4-cell stage of crushing were transplanted by surgical methods into the egg wire of each heifer. Three embryos on four cell stage obtained from in vitro matured follicular oocytes collected from ovaries of two- and one-month heifers and from one mature cow were transplanted to one recipient. As a result of transplantation the alive calf was born. However, the surgical transplantation of embryo at their early stages of development into recipient egg wire greatly complicates biotechnology of transplantation. Therefore it is very important to obtain embryos at morula stage or blastocysts which are suitable for transplantation by non-surgical way.

L.K.Ernst et al. (1987) used the rabbit oviduct for the early stages of embryo development after fertilization of cows both in vitro and in the oviduct of rabbit (1987). They removed oocytes from follicles and cultured them in medium 199 with 20% fetal bovine serum adding estradiol, progesterone, testosterone and luteinizing hormone, and incubated overnight at 38 ° C in a humidified chamber containing 5% CO2, 5% O2 and 90% N2. Sperm capacitation was performed in a high ionic strength and in the oviduct of the estrous rabbit for 4-5 h before transplantation of oocytes. Via 19-20 h after connections of oocytes with sperm in rabbit oviduct or in vitro oocytes were transplanted into the oviduct of a false pregnancy rabbit for their further development for 4-5 days. Fertilization in this case was 22-25%. After 3 days the percentage of embryos that reached the morula stage after fertilization in the oviduct of rabbit was about 4 times higher than in fertilization in vitro.

By 4and 5 days, this difference is somewhat reduced, but still significant in favor of the oocytes fertilized in the oviduct rabbit. This indicates that in the rabbit oviduct there are more favorable conditions for sperm capacitation and fertilization of bovine oocytes than in culture medium. As a result of surgical and non-surgical embryo transfer at morula stage were obtained six pregnancies of cows, including one twins (5 of 6 pregnancies were given by oocytes taken from the ovaries of cows killed at the factory.) Two live calves were born from oocytes fertilized in the oviduct of rabbit or "in vitro". One calf was born as a result of non-surgical embryo obtained after fertilization in vitro. Engraftment of embryos derived from oocytes fertilized in rabbit oviduct was almost 6 times higher than in the case of fertilization "in vitro".Crister et al. (1986) used a ligature sheep oviducts for the development of bovine embryos up to morula stage and blastocyst. Much success at in vitro fertilization of oocytes also achieved by Irish researchers (Lu et al., 1987, 1988). Sheep oviduct was used by them as a temporary recipient of fertilized cells. 80% of oocytes divided up to 2-cell stage and higher, and almost half of them reached the stage of morula and blastocyst. Experiments on fertilization of sheep were conducted in two directions: "in vitro" and introduction of follicular oocytes in inseminated sheep oviduct. As in cows, the efficiency of fertilized oocytes was higher when follicular and ovulated cells were placed into oviduct with sperm than when using the system in vitro. Moreover, significant difference in the development of oocytes to blastocyst stage in cases of in vitro oocyte culture and in vivo (in the follicles) was not observed. Issues of fertilization "in vitro" in pigs are explored in lesser degree. Polge (1977) described the penetration of sperm into oocytes after in vitro maturation and their transplantation into the oviduct of estrous inseminated pigs. However, no egg is not developed to blastocyst stage.


23. Prospects for improving farm animal embryo culture in vitro

Cultivationin vitro embryosof farm animals. Species serum was the first medium which was tested for suitability for culture of cows’ and sheep’s embryos. In this medium development of fertilized cells limited to one division and stopped after 48 hours of cultivation.
The possibility of using follicular fluid for culture was described by Thibault in 1966. He observed cows fetal development from 1-4 cell blastomeres up to morula in this medium. Tervit et al. (1972) tested a synthetic follicular fluid. According to the authors, 9% of 8-cell sheep blastomeres developed to early stage of blastocyst after 6-day culture in synthetic follicular fluid at atmosphere of 5% CO2, 5% O2 and 90% N2. More than half of 8-cell embryos cultured in synthetic follicular fluid within 3 days after transplantation have gone ontogeny to the birth of offspring. However, the authors failed to get pregnancy after culturing unicellular embryos. Wright et al. (1976), observed the development of cows embryos from 1-2 cell blastomere up to hatched blastocysts in HAM F-10 medium. The following year similar results have been achievedby  Peters et al. by using Witten medium with 0.5% bovine serum albumin. They also noted that "in vitro" development of 1-4-cell embryos is limited in comparison with 8-cell embryos. This medium but with a high content of bovine serum albumin (15%) was the best for culturing pig embryos (Linder, Wright, 1978).

Currently, scientific research in the field of agricultural animals fertilization "in vitro" is being further developed. They are aimed at the exclusion of temporary recipient (oviducts of rabbit, sheep, etc.) from the technology of early embryo development. Suitable replacement for temporary recipient may be monolayer of oviduct and granular cells.

24. Biotechnology to create chimeric animals (genetic mosaics)

Chimericanimals. The concept of a chimera means a compound animal. In the modern concept of the term chimera is mainly used for obtaining composite organisms that have genetically different cell populations originate more than one zygote or more than one embryo. Obtaining genetic chimeras or mosaics is currently one of the promising areas of biotechnology. The essence of this biotechnological method, based on the achievements of cell engineering and micromanipulation on early embryo consists in artificial combining of cell embryos from two or more animals, relating not only to one breed, but also to the different breeds and even species. Chimeric animals are signs of different genotypes. This is achieved by integrating blastomeres from two or more embryos or by injection of cells of one embryo into the cavity of the blastocyst of another embryo. All previous known mammalian chimeras were created by methods of aggregation of two (or more) of genotypically heterogeneous embryos or by microinjection of donors’ blastocyst intracellular cell mass in blastocoel of recipient. The first method is called aggregation, and the second is known as injection method Complex chimeric sheep embryos by integrating 2 -, 4 - and 8-cell blastomeres were obtained byFehilly et al. (1984). Each of these embryos consisted of an equal number of blastomeres of embryos from 2-8 parents. Results of the survey of 48 lambs in 2 months of age showed that 36 heads were chimeric by blood tests, by external signs or in those and other indicators. A year later, Butter et al. got chimeric lambs by injecting inner cell mass isolated from donor embryos into embryos’ blastocyst of recipients. From these 15 lambs 5 heads were identified as chimeras on blood groups and 1 head appears by its external signs. Chimeras in cattle were obtained by Brem et al. (1985) combining halves of 5-6-day-old embryos. 2 of 7 calves had evidence of chimerism. 1 calf was a chimera on suit of brown schwyz breed and Holstein-Friesian, although blood group it inherited from their Holstein-Friesian breed parents. Another calf was uncertain chimera. Chimeric calves by merging morulae without zone of pellyutsida were obtained by Church et al. (1985). They found that the transfer of each parent type to chimera is random, ie, offspring can develop from cells derived from any embryo, or from a combination of embryos.
Obtaining chimeric by cell fusion of embryos from different species is of great practical importance. It is known that no sheep or goat nurture hybrid offspring before birth. Typically, the embryos of an experimental hybrid pregnancy in sheep and goats at the end of the 2nd month are killed. The immediate cause of abortion in interspecific pregnancy is the strengthening of maternal immune responses to antigens of the fetus, leading to dysfunction of the placenta.Fehilly et al. (1984) showed that the blastomeres of sheep and goats that are enclosed in agar and placed for 4-5 days into oviduct of sheep can form a combined blastocysts that are viable and can develop to birth normal offspring. The merger of one blastomere from 4-cell embryos of sheep and goats 17 blastocysts were received, transplantation of which was ended with the birth of 7 lambs. They all looked like mostly in lambs, but 3 of them wool had that had transverse ridges and patches of hair sharply contrasting with the tight curly hair. By combining 8-cell embryos of sheep and goats researchers got five offspring similar to lamb, but 2 of them had similar deviations on coat, two offspring were like goat kids with some deviations into the coat. Animals with the outward signs of chimeras had blood sheep, except for one young animal, who had the blood group of parents of both species.The above experimental results indicate the feasibility of transplantation of chimeric embryos between closely related species of animals. Interspecies transplantation could be invaluable in preserving endangered species from extinction because usual embryo transfer may provide a small benefit, as the female recipient may not always be enough. Technique of obtaining chimeras can be used in breeding animals with desirable economic characteristics, as well as resistant to certain diseases.
Chimeric animals do not transmit to offspring their inherent genetic mosaicism. Like heterozygous or hybrid animals there is a splitting in the offspring, resulting in broken of valuable genetic combinations. Although chimeric animals support economic important signs only for a single generation they can be of great practical interest in the breeding of cattle. For example, you can create chimeric animals that combine features such as milk and meat productivity, which are antagonistic and incompatible in a single body. Creation of chimeras by injecting of certain embryo cell lines will improve the immune system and increase resistance to a range of diseases.


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