EMBRYO transferred to each foster mother so

EMBRYO TRANSFER IN CATTLE  Artificial Insemination has allowed genetic process to be achieved relatively, quickly through the widespread and efficient use of frozen semen for the past several decades.

Genetic programmes were limited to male side for genetic contribution because cows could relatively produce only one calf in one year. Today due to the advancement of embryo transfer technique cows can produce many offspring every year.Embryo transfer is a method of artificial breeding for the genetic improvement of cattle. Cows have a 21 day oestrus cycle with a day zero characterized by visual display of heat. Buffaloes are silent heaters. Approximately 12 hours after the end of heat a single non- fertilized egg (ova) is released from 1 or 2 ovaries.If the cow is bred at the end of heat the egg will be fertilized shortly after ovulation and develop into embryo. The process of embryo transfser is typically the same.

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But the cow is made to superovulate by hormone treatment so that at one single oestrus period instead of one usual ovum more ova are produced if the cow is bred at this stage more fertilized embryos may result.If these embryos are physically collected from that cow. Each of the fertilized embryo may be transferred to each foster mother so that, more calves can be produced simultaneously from each foster mother. One major advantage of embryo transfer is the cross breeding for genetic improvement can be achieved quickly.

The first successful transfer of fertilized rabbit eggs was reported in 1891 by Walter and Heape at Cambridge. The first successful embryo transfer in farm animals was done in 1934 in Sheep and Goats. The first offspring after transfer of embryos in cattle and swine was reported in 1951.

Embryo transfer includes a sequence of various steps which areSelection of donorsSynchronization of estrousInduction of superovulationEmbryo collectionEvaluation of embryosSelection of recipientsTransfer of embryosEmbryo transfer techniques have been extensively used in cattle and more sporadically in Sheep, Goats and Pigs. SELECTION OF DONORS Any regularly cyclic cow or heifer can successfully be termed as donor because technically it will respond to superovulatory treatment followed by embryo transfer.Usually there are many reasons that a cow is preferable than a heifer because embryo transfer programme is based on milk yield or genetic superiority. In addition the following are the criteria for selection of donor,Donor should be of age between three years and ten years.

Donor should be free from genetic diseases and conformational abnormalities.They should exhibit regular oestrus cycle.Superior production traits of economic importance.Previous record of sound reproductive performance including successive fertility and artificial insemination. INDUCTION OF SUPEROVULATION  Superovulation means the induction of multiple ovulations by application of exogenous hormones (PMSG – Pregnant Mare Serum gonadotrophins, FSH- Follicle Stimulating hormone, HMG – Human Menopausal Gonadotrophins) in the early follicular or in the luteal phase of the oestrus cycle in order to collect large number of fertilized eggs. Most frequenctly PMSG or FSH is used.

PMSG – one single injection (2000 to 3000 IU) as the substance has a very long half life time.FSH – Multiple injections of FSH (35-50 mg) twice daily for four days.48 and 60 hours after beginning the gondaotrophin treatment, Prostaglandins (PG) are administered to induce oestrus.

Inseminations are performed at oestrus two days later. Embryos are recovered 8 days after insemination. SCHEDULE FOR SUPEROVULATION IN CATTLE ·  Day of cycle PMSG (2000-3000 IU) FSH (35-50 mg) 0 OESTRUS (Spontaneous or after synchronization) 10 PMSG FSH/FSH 11 — FSH/FSH 12 PG/PG PG/PG/FSH/FSH 13 PG/PG/FSH/FSH 14 Anti PMSG Oestrus and inseminations) 15 16 17 18 19 20 21 Embryo recovery EMBRYO COLLECTION In the bovine, embryos are recovered by non surgical methods.

Special catheters(Foley’s catheters) are introduced via the cerevix into the uterine horn and embryos are flushed with 250 – 300ml flushing medium.Recovery rate is influenced byThe position of the embryos in the uterusThe flushing methodThe time of recoveryOvarian responseEmbryo viabilityIn other farm animals, Sheep, Pigs and Goats embryo recovery is performed by surgical methods. Disadvantage of the surgical method is the number of repetitions is limited by the occurrence of adhesions.

After recovering embryos from the flushing medium their further developmental capacity has to be evaluated. Embryo viability can be evaluated by various methods.Morphological evaluationMost frequently the morphological evaluation at various microscopical magnifications is used. Morphological criteria in embryo evaluation are shape, colour, number and compactness of cells, Size of the perivitelline space, number and size of vesicles and status of the zona pellucida. The ideal embryo is even, the perivitelline space is empty and of a regular diameter. According to their morphological appearance, embryos are classified into four groups.Excellent embryos: Embryos in the appropriate developmental stage with a perfect morphology.Good embryos: Embryos in the appropriate stage of development with slight morphological deviations.

Eg. Minor damage of the zona pellucida and excluded blastomeres or vesicles in the perivitelline space.Degenerated and / or retarded embryos: Embryos in the appropriate developmental stage with major morphological deviations (degenerated embryos)Unfertilized ova.Staining methodsEmbryo viability can be evaluated by various vital staining and fluorescence techniques. SELECTION OF RECIPIENTS  Recipients are selected based on theNormal physiological and health conditions.

The reproductive status.Lack of any reproductive disorders.Compatibility to the donor with respect to size of the foetus.Oestrus synchronization.In bovines, heifers and young cows are best suited as recipients. TRANSFER OF EMBRYOS Recently special catheters have been developed to perform non – surgical transfers in cattle.It is important to transfer the embryo in to tip of the uterine horn without damaging the endometrium.For a successful embryo transfer, the most important is embryo quality.

It is very important to transfer the embryo ipsilateral, that means into the horn bearing the corpus luteum. CRYOPRESERVATION OF EMBRYOS  Cryopreservation of embryos is an essential part of embryo transfer programme and it allows worldwide shipment of embryos.Reliable freezing methods have been developed for bovine and sheep embryos.An optimal method for bovine embryos includes a one step addition of 1.4M glycerol as cryoprotectant, a 20 minutes equilibration period and 0.25 ml straws as embryo containers, slow cooling down to -35°C and subsequent plunging to liquid nitrogen (-196°C).

Embryos are thawed by placing the straws directly into warm water. APPLICATIONS OF EMBRYO TRANSFER  Exploitation of female reproductive capacity (more offspring from valuable donors).Significant facilitation of import and export for valuable genetic material.Development of new breeding concepts.

Gene conservation by freezing techniques.Twin production (Embryo splitting).Introduction of new genes into closed herds.Manipulation of embryos.Gene transfer. MODULE-19: IN VITRO FERTILIZATION, SEXING, MICROMANIPULATION AND ANIMAL CLONING  Learning objectivesThe learner will learn aboutIn vitro fertilizationSteps in in vitro fertilizationSexing of EmbryosMicromanipulationAnimal Cloning  IN VITRO FERTILISATION (IVF)  Embryo transfer is not used widely because of its cost, technical difficulty and the limited supply of embryos available from super ovulated donors.

These limitations would be removed if we could fertilize in vitro, the thousands of oocytes that are present in a female’s ovaries.Breeding programmes would select for female genetics as easily as for male genetics. The genetic influence of a male would be increased further since the amount of semen required for fertilization in vitro is fraction of the amount needed for Artificial insemination.

Fertilization consists essentially the fusion of two cells, the oocytes from the female and spermatozoon from the male to form single cell, the Zygote.IVF is generally quite successful, resulting in about 70-80% of fertilized eggs. The practical difficulties arise in sourcing eggs for fertilization and in the development of fertilized zygotes to term.IVF involves three steps namelyOocyte recoveryOocyte maturation in vitroIn vitro fertilization of matured oocytes OOCYTE RECOVERY  In the second (follicular) phase of the oestrus cycle, a number of ovarian follicles (20 or so) grow and become filled with fluid. The fluid -filled space is called the antrum and such follicles are known as ‘antral follicles’ (or Graafian follicles).In the normal course of events just one of these matures and ruptures, releasing the egg for fertilization.

Super ovulation occurs when many more antral follicles are stimulated to mature by injection of gonadotrophins.Pre- ovulatory follicles lie against the surface of the ovary and quite large (about 15 mm in cattle, 8 mm in sheep and pigs).Laparoscopy surgery can be used to recover oocytes from these follicles, allowing them to be matured and fertilized in vitro.A number of eggs can be collected in this way from superovulated donors. The technical demands and cost of surgical procedure of oocyte recovery is high. IN VITRO CULTURE AND MATURATION OF OOCYTES Oocytes for in vitro fertilization are obtained from the oviducts, follicles and surface of ovary after ovulation either from live animals by flushing some medium or from slaughtered animals.

The immature oocyte is collected from the ovary and cultured in vitro to induce maturation.Cow ovaries are collected from the slaughter house and maintained in phosphate buffered saline at 30-32°C for transportation to the laboratory. Following aspiration of follicles, the oocytes are then observed under a dissecting microscope.The oocytes are graded according to their morphology, washed twice with HEPES buffer and once with BMOC-3 (Branchat and Olivhant medium for oocyte collection). After washing, 5-10 oocytes are transferred to 0.2 ml of BMOC-3 drops under paraffin oil at 37°C with 5% CO2 in 95% air for 12-32 hrs.Oocyte maturation in vitroLarge numbers of oocytes are allowed to mature under in vitro conditions.

PREPARATION OF SPERMATOZOA The capacitated spermatozoa is essential for in vitro fertilization. After capacitation ,the acrosome reaction in the head of the spermatozoa allows the release of enzymes for penetration into the zona pellucida of the oocytes.The capacitation process is completed durinng the movement of the spermatozoa from the oviduct to the uterus.Capacitation involves the removal, probably by the enzyme action, of macromolecular material located on the surface of the spermatozoa.

Re-exposure of the spermatozoa to seminal plasma leads to loss of capacitation and the process may well involve the restoration of the surface layer or ‘decapacitation factor’ which normally coats the spermatozoa as they pass through the male reproductive tract.Experimental procedures to induce the capacitation or acrosome reaction of the spermatozoa involve invitro incubation with follicular fluid and serum. IN VITRO FERTILIZATION IN FARM ANIMALS  The in vitro matured oocytes are mixed with the capacitated sperms in a petridish and incubated at 37ºC with 5 % CO2.After 24 hours incubation, the cumulus mass is removed manually and examined for fertilzation indicated by the presence of 2 pronuclei.

Further the fertilized oocytes were incubated for division.The morula stage embryos (16 cell stage) are transferred to recipient animals for further development of embryos.Among farm animals, invitro fertilization has been achieved in cattle, pig and goat; but the number of new born animals were limited. SEXING OF EMBRYOS ·        Embryo sexing in animals has the advantage to increase the milkyield or to increase meat production. The production of preferred sex is doneeither by sexing the sperm or by sexing the embryos. In case of sexing sperm,it could be used both in artificial insemination as well as in the in vitroproduction of embryos whereas the sexing of embryos could be done either invivo produced or in vitro produced embryos prior to their transfer to recipientanimals. METHODS Sexing the spermThis is done by using flow cytometer attached with a sorter. Inthis method, the sperm is stained with a stain – Hoechst 33342 and then sexspecific sperms are separated by using the flow cytometer’s sorter.

This methodhas an accuracy of 90 % sexing sperms. Commercially it is used in cattle.Embryo sexingY-specific DNA probesSexual differentiation of mammalian embryos is determined by the presence or absence of genetic elements located on the Y-chromosome. Molecular genetic techniques allow the detection of specific DNA sequences in interphase cells.Sex determination with Y-specific probes involves the identification and cloning of a probe (a DNA fragment with a nucleic acid sequence found only on the Y-chromosome) and the use of this probe to diagnose the presence or absence of Y-chromosomal DNA in a sample of embryonic cells.

PCRUsing polymerase chain reaction (PCR) to amplify a Y-chromosome-specific repeat sequence, DYZI, that is present 500-8000 times on the Y chromosome. The presence of this amplified target sequence indicates the presence of the Y-specific DNA sequence in the sample DNA and, hence, indicates that the animal is male.A disadvantage of this technique is that the absence of the amplified target sequence may be due either to the absence of the sequence in the sample DNA or to a failed reaction,PCR-RFLPSecond method involves PCR-based genotyping. In brief, a pseudo-autosomal region (common to both X and Y chromosomes) present in both X and Y chromosomes is amplified. These homologous regions are called ZFY (in the Y chromosome) and ZFX (in the X chromosome).The amplified product is then digested with restriction enzymes that take advantage of restriction enzyme fragment length polymorphisms between ZFY and ZFX. The digested DNA is separated via electrophoresis. The ZFX and the ZFY DNA are identified by their different digestion patterns.

MICROMANIPULATION In 1966, Lin described the technique of micormanipulating and injecting a mouse eggSubsequently, transgenic animals have been produced by introduction of foreign genes at the pronuclear stages of fertilized, one-cell zygotesMost of the successes have been with mouse and recently successful production of transgenic rabbit, pig, sheep and goat have been shownThis technique is a powerful tool for studying gene regulation and physiological functions of gene products in a normal host environmentMicromanipulation is the technique whereby sperm, eggs and embryos can be handled on an inverted microscope stage, performing minute procedures at the microscopic level via joysticks that hydraulically operate glass microtools.The injection of a single sperm into the cytoplasm of the oocyte, or intracytoplasmic sperm injection (ICSI), provided a satisfactory solution to the problems of the assisted fertilization techniques developed earlier.In this procedure, a single sperm is first immobilized by touching the sperm tail with an injection pipette (inner diameter 5–7 ?m). The injection pipette picks up the immobilized sperm, pierces the ZP and oolemma, and delivers the sperm inside the oocyte cytoplasm.In 1976 using hamsters as a model, Uehara and Yanagimachi were probably the first to report the injection of sperm into oocyte cytoplasm (ooplasm).It was later attempted on rabbit and human oocytes, although the first successful human pregnancy was not reported until 1992 by the Free University of Brussels’ group in Belgium.Micromanipulation technology has enabled the reproductive biologist to overcome inefficient steps in mammalian fertilization, the production of chimeric animals through blastocyst injection with embryonic stem (ES) cells and the introduction of specific genes into the genomes of domestic and laboratory animals.

This technology has also been used for the production of cloned animals and ES cell lineages from cloned embryos, using nuclear transfer. Moreover, micromanipulation is also used for microsurgical embryo biopsy to study the basic developmental biology of embryos during preimplantation development. ADVANTAGES AND DISADVANTAGES OF MICROINJETION Advantages The amount of DNA delivered per cell is not limited by the technique and can be  optimized. This improves the chance for integrative transformationThe delivery is precise, again increasing the chance of integrative transformation.The small structures can be injected containing only a few cells and with high regeneration potential.Since it is a direct physical approach, it is host-range­ independent.Disadvantages Injection can cause damage that affects embryonic survival and can result in quite high mortalities.Onlyone cell is targeted per injection

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