Fertilization outside the body
With the development of technology nonsurgical embryo extraction in cows it became possible to receive multiple embryos without surgical intervention or slaughter. However, the effectiveness of this embryo extraction method is relatively low. Moreover this method allows to extract embryo which is in the uterus at morula and blastocyst stages. For genetic and cell engineering embryos at zygote stage (one-celled embryo) are necessary, which can be extracted from the oviduct only by operational way. Existing methods of hormonal control of ovulation in domestic animals do not possess of precision, so there is great difficulty in obtaining zygotes required for microsurgery by natural fertilization. In this regard, development of techniques of fertilization outside the body is of great importance in solving scientific problems and practical issues to improve the efficiency of breeding animals.With the development of in vitro techniques of fertilization it became possible to develop research on the cellular and genetic engineering in mammals and especially in domestic animals. What practical problems can be solved by using the technique of fertilization outside the body of the animal? First of all, the problem of obtaining the required number of embryos for transplantation remains unsolved, although embryo transfer being carried out quite successfully. Females donor are not predictable in the system of embryo transfer, because of the high variability of the number of ovulating animals and the number of ovulation. Secondly, this method would allow to obtain considerably more embryos for transplantation from animals with high genetic potential. It would be possible to create a bank of genetic material by collecting the ovaries in females of high value during slaughter that would significantly accelerate breeding of animals. Third, the method of in vitro fertilization is much faster and requires a much smaller sperm, and can be used for objective evaluation of fertilizing capacity of sperm and egg cells. Fourth, the method helps breeders to overcome infertility due to dysfunction of uterus and oviducts. Induction of superovulation, according to some researchers, is the cause of increased fetal mortality due to change of concentration and ratio of sex hormones. And, finally, discusses the methodology allows to obtain any number of identical twins, more efficient use of semen from bulls of high value.
Maturation of oocytes outside the body. In vitro fertilization of oocytes preceded by cultivation in vitro, in which maturation of oocytes occurs to metaphase 2. Spontaneous renewal of meiosis of oocytes isolated from rabbit follicles and cultivated in nutrient medium was discovered in 1935 by Pincus and Enzmanom. Moreover, the oocytes passed consistently all stages of maturation before metaphase 2, that is up to the stage of fertilization without any hormonal influences. Later this phenomenon was found in oocytes of other species of animals. By the time of its birth heifers oocytes has already reached diplotene stages of prophase of meiosis (a specific type of cell division), which is typical only for germ cells. Thus formed germ cells contain a single (haploid) set of chromosomes. In diplotene stage homologous chromosomes are separated, and among them a longitudinal slit is formed. At this stage of meiosis further maturation of oocytes is suspended until physiological maturity of the individual. Granulosa cells and some components of follicular fluid play an important role in the suppression of meiosis. After raising the level of luteinizing hormone in vivo or retrieval oocyte out of follicles inhibitory effect ceases and meiosis resumes. In addition, as noted above, the process of oocyte maturation up to metaphase 2 continues , ie oocytes ripen to a stage suitable for fertilization. It should be noted that this process is not the same maturity, which oocyte passes in vivo prior to ovulation. In cultivation of oocytes "in vitro" maturation of nucleus without cytoplasm is occured . As a result, in vitro matured oocytes are incapable of further development after fertilization, because in this process an important role play cytoplasmic factors responsible for the formation of the structure of proteins. Motlik and Fulka (1976) showed that after fertilization during the normal oocyte development an important role play substances released from the germinal vesicle to the cytoplasm. Thibault et al. (1976) found that steroids are required to achieve full physiological maturation of oocytes. Moor and Trounson (1977) point out that the cultivation of sheep oocytes in medium 199 with the addition of follicle-stimulating hormone (FCH) and luteinizing hormone (LH), as compared with the medium without hormones provides large numbers of oocytes to achieve metaphase Similar results were obtained on cows by Fukui et al. (1982) using estradiol, progesterone, luteinizing hormone and chorionic. A noticeable influence of sex steroid hormones (estradiol, progesterone, testosterone) on the efficiency of maturing oocytesb were observed, while the influence of gonadotropins (FSH, LH) was negligible.
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Staigmiller and Moor (1984) demonstrated that for the development of fertilized oocytes a certain critical number of follicular cells are required, while their presence for the manifestation of meiosis is not mandatory. Somatic cells are not only energy substrate for the oocyte, but also participate in the transfer of some precursors of amino acids, nucleotides and phospholipids in the oocyte. Follicular cells generate guidance signals that affect nucleus and synthesis of certain structural proteins. These signals are very important for the maturation of oocytes in the first 6-8 hours after the initiation of meiosis. Signal changes leads to violations in the ontogeny of the embryo. It is no coincidence that isolated follicle culture method is recognized by many researchers as the most modern method used for the preparation of oocytes for fertilization in vitro.. This method makes it possible to preserve the natural connection between the oocyte and somatic elements of the follicle. In this case, the follicle serves as a biofactory of steroid hormones. The method is as follows. Is isolated from ovarian The follicles is isolated during the slaughtering and transported to the laboratory in TC-199 medium. The ovaries are washed with sterile saline solution containing antibiotics, and then cut with a scalpel at the occurrence of blood vessels in the ovary. The follicles are clearly visible on the cut surface. Using tweezers follicles are separated from the connective tissue and cultured in CO2 - incubator in mentioned medium with addition 20% of fetal bovine serum and fetal hormones PMS (pregnant mare serum), FSH, LH, estradiol, and insulin for 48 hours. In such conditions, about 70% of oocytes mature up to the metaphase stage 2.
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Fertilization in 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.
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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 4 and 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.
Cultivation in vitro embryos of 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 achieved by 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.
Test questions: 1.Whatn is the practical significance of technique for fertilization outside the body? 2. Tell us about the results of research in the sphere of improving process of eggs’ maturation outside the body 3. Principles and approaches which are using in fertilization outside the body.
Lecture №8
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