63) Collection and cultivation of oocytes in vivo and in vitro

One way to obtain a large number of descendants of genetically valuable livestock females - oocytes fertilization and embryo culture outside the body (in vitro). Traditional methods (in vivo) reproduction of farm animals females produce significantly fewer gametes than males.

The problem of oocyte maturation and fertilization in vitro, followed by culturing embryos and their non-surgical transplant recipients and receiving numerous progeny definitively solved.

Fertilization in vivo matured oocytes in the ovary, when considered in isolation from the maturation of oocytes in vitro, is of limited use for the breeding of farm animals. In this case, as a result of induction of superovulation surgically or during slaughter cows can be obtained in vivo matured units full oocytes. Only a combination of fertilization of oocytes matured in vitro followed by transplantation of embryos can significantly increase the use of the genetic potential of female gametes.

So, if after induction poliovulyatsii per cow - get up to 10 donor tubalnyh, ie matured in vivo, and ovulated oocytes from the ovary is cow can be removed to 200 follicular oocytes. Oocyte maturation by culturing reached 80% or higher, and their fertility - 50-70%.

It is established that in vitro maturation of oocytes not equivalent natural that occurs in vivo in the oocyte prior to ovulation. As shown by numerous studies, the in vitro maturation of the kernel is without the full cytoplasmic maturation. Under the cytoplasmic maturation understand set of morphological, biochemical and physiological changes in the oocyte cytoplasm, resulting in the egg becomes fertilized and capable of further development.

By culturing the oocytes in vitro maturation of immature to understand the process of oocytes in artificial media in which immature oocytes undergo meiotic maturation to metaphase of the second division, that is, to the stage of readiness for fertilization.

To isolate the oocytes from follicles, as a rule, use the ovaries of slaughtered cows and less ovaries extracted operational putem.Posle oocyte retrieval of antral follicle premises in the culture medium is a first morphological changes associated with the nuclear maturation of oocytes, ie meiosis resumes. The first morphological sign of maturity - the destruction of the germinal vesicle - oocyte nuclei, ie prophase oocyte nucleus is converted to a meta-phase.

The first meiotic division, which is a pressure reducer, is replaced by the second equational division in which there is a division of chromosomes in mitosis and spindle formation of ahromatino of new yarns. At this stage of meiotic maturation division is blocked until the oocyte fertilization. On the completion of oocyte maturation in vivo or in vitro judged by the appearance of the polar body or towards the oocyte-mi metaphase of the second division sozrevaniya.Intensivny synthesis, carried out by the oocyte is associated with gene activity and an increase in the number of copies. Found that the first stage of embryonic development take place in an environment where the core does not produce RNA in the cytoplasm, and the process is controlled by RNA accumulated in oogenesis.

The obtained results show that changes in protein synthesis are not associated with nuclear and cytoplasmic maturation of crucial importance for normal fertilization and early embryo development up to the implantation of the embryo in the uterine wall of the recipient. In connection with this, methods for enhancing the synthesis of RNA and protein during maturation of oocytes in vitro. It is assumed that during the maturation of oocytes in vitro in the cytoplasm are not fully synthesized factor responsible for the formation of the male pronucleus.

65	)Methods of embryo transplantation

Embryo is now one of the most pressing issues in the field of animal farming. With the help of embryo transfer can dramatically increase the yield of the progeny of high-producing cows. Embryo or embriotechnology is to obtain one or more embryos from the uterus of breeding animals (donors) and transplant the uterus of cows (retsepient), where embryos develop to calving. This method, combined with superovulation in donors can get a great seed of high yielding animals. In this way the embryos can be implemented in either breed in other regions, using as retsepient beef cows. This method also facilitates the exchange of agricultural gene pool of animals between countries and continents. Embryo transfer can be used to produce offspring from valuable, but barren cows that have lost the ability to grow as a result of accident, illness or age.

When it was established that the rabbit has immunity against FMD, put forward the idea of ​​using the method for the recovery of transplant pups of foot and mouth disease of animals. Genital tract of the rabbit, which transplanted embryos can destroy FMD virus in embryos. Transplantation can be used for temporary storage of embryos. In the oviducts of rabbits can carry transcontinental transport of sheep embryos.

Removing the embryos until the 70s produced mainly by surgery, he was subsequently replaced by a less traumatic and time-consuming non-surgical, based on the introduction of a special probe into the uterus through the natural channel. The probe has three channels. One channel is for inflating the balloon that occludes the uterine horn, preventing leakage of fluid. According to another input channel saline at 25-30C, and the embryos are washed back with them through the third channel of the probe into the tube placed in a waterbath at 35C. From this liquid extracted embryos. On average superovulation of donor can be from 5 to 7 embryos.

Transplantation is carried out with the help of a special probe or a gun for insemination. Embryos were placed in the uterine horn. Pregnancy in females - retsepient checked on the level of progesterone in plasma on day 21.

Regulation of sex. In the practice of breeding animals is very important to learn to manage education in the offspring of male and female individuals. The method of separation of embryos by sex based on determining specific proteins males. This method is widely used in animal practice in many countries. In Canada since 1975 born calves, separated by sex on stage embryos. In the long term to get targeted individuals, male or female can be used microsurgical technique replace the X and Y chromosomes. Such manipulations are carried out on the plant cells and amphibian oocytes.

67 The method of in vitro fertilization. IVF is the basic assisted reproduction technique, in which fertilization occurs in vitro. The sperm and the egg are combined in a laboratory dish, and after fertilization, the resulting embryo is then transferred to the female's uterus. The first successful IVF offspring were rabbits born in 1959. The basic requirements for IVF are healthy ova, sperm that can fertilize, and a uterus that can maintain a pregnancy. Five general steps are followed for IVF : 1Superovulation: Normally, a female ovulates just one or more egg per cycle. With the use of fertility drugs, she may be able to produce many eggs, which can then be retrieved from the ovaries prior to ovulation to be used for artificial semination of another female unable to produce eggs or that quality of egg. Spontaneous ovulation during the cycle is typically prevented by the use of GnRH agonists or GnRH antagonists, which block the natural surge of luteinising hormone (LH). 2. Egg Retrieval: When follicular maturation is judged to be adequate, chorionic gonadotropin (hCG) is given. This agent would cause ovulation about 42 hours after injection. The eggs are retrieved using a transvaginal technique involving an ultrasound-guided needle piercing the vaginal wall to reach the ovaries. Through this needle follicles can be aspirated. It is common to remove between ten and thirty eggs.3. Fertilization: In the laboratory, the identified eggs are stripped of surrounding cells and prepared for fertilization. In the meantime, semen is prepared for fertilization by removing inactive cells and seminal fluid. The sperm and the egg are incubated together (at a ratio of about 75,000:1) in the culture media for about 18 hours. In most cases, the egg will be fertilized by that time and the fertilized egg will show two pronuclei. In certain situations, such as low sperm count or motility, a single sperm may be injected directly into the egg using intracytoplasmic sperm injection (ICSI). The fertilized egg is passed to a special growth medium and left for about 48 hours until the egg has reached the 6-8 cell stage. 4. Selection: Laboratories have developed grading methods to judge oocyte and embryo quality. Typically, embryos that have reached the 6-8 cell stage are transferred three days after retrieval. 5. Embryo Transfer: Embryos are graded by the embryologist based on the number of cells, evenness of growth and degree of fragmentation.The embryos judged to be the "best" are transferred to the patient's uterus through a thin, plastic catheter, which goes through her vagina and cervix. Several embryos may be passed into the uterus to improve chances of implantation and pregnancy.