1. Cloning and splitting

2. Cloning (nuclear transfer):
Cloning is the production of a copy or copies of an individual and occurs in animals either naturally or artificially, when an embryo is split to produce identical twins. The word “clone” has also been used to describe animals produced by nuclear transfer for the production of an unlimited number of genetically identical offspring.

3. The first successes in cloning livestock were with sheep, by fusing a cell from a 16-cell embryo to an oocyte that had its chromosomes removed (enucleated oocyte). However, the biggest breakthrough in nuclear transfer came when it was demonstrated that a viable offspring could be produced by fusing cultured adult somatic cells with enucleated oocytes to produce Dolly the sheep in 1997.

4. Inications and applications
1-The main application of cloning to the livestock industry
is for expanding superior genetics. Animals of high genetic merit, male or female, can be selected for cloning based on any desirable trait, including growth, feed efficiency, and disease resistance. Production of numerous animals of exceptional genetic merit would allow for more rapid genetic progress and the associated economic benefits. The nutrition, reproduction, and health of these animals should be more easily managed because of animal uniformity.
2-Nuclear transplantation has also been useful for salvaging
old genetics and exotic breeds. Germ plasm from a breed of cattle on Enderby Island that had essentially gone extinct was preserved using somatic cell nuclear transfer from the only remaining cow.

5. 3-Additionally, this technology can be used to import and propagate new or exotic breeds of cattle that may offer other desirable genetic traits not currently available for use in cross
breeding schemes.
4-tissues can be harvested from recently deceased animals and propagated for use in cloning. This also has a tremendous advantage in the slaughterhouse, where superior carcasses can be selected and tissues taken for future cloning.

6. disadvantages 1-the current efficiency of equine cloning is very low.
1-the current efficiency of equine cloning is very low.
2-Consuming a very much genetic materials with very low percent of success.
3-Ethical determinations and a very low percent of social and religious acceptance especially in human cloning.

7. Methodology of cloning
1-Cloning first requires a supply of cells from the donor animal to be cloned. It is not yet clear which cells are the most effective for producing live cloned offspring.
2-Currently, biopsies are taken either from fetuses or adults, essentially from any tissue in the body, and then propagated in culture to generate fibroblasts.
3- It is critical for the practitioner to ensure that the area to be biopsied be thoroughly cleaned with disinfectant, and the tissue be collected using sterile technique in order to remove any chances of contamination, which could render the sample unusable.

8. 4-Tissue biopsies are transported to the lab, where they are minced and/or treated with enzymes and cultured to obtain cell lines. These cell lines can be expanded in large quantities and frozen in liquid nitrogen for future use. At the time of cloning, cells are removed from tissue culture plates by trypsin treatment and held in suspension until transfer.
5-Nuclear transfer involves removing the chromosomal DNA from mature oocytes and transferring the genetic material from a cell of the donor animal to be cloned through a process of fusion and reprogramming (Fig 1).

9. 6-Briefly,:
a- immature oocytes are collected either from slaughterhouse ovaries or by transvaginal oocyte retrieval (TVOR) and matured in vitro as described above.
b- After 18 to 22 hours, oocytes are stripped of their surrounding cumulus cells, and those with visible polar bodies (mature metaphase II oocytes) are selected for further manipulations.
c- Using micromanipulators, each oocyte is enucleated by piercing the zona pellucida with a glass needle and removing the polar body with a small amount of the adjacent cytoplasm, containing the oocyte’s chromosomal material, which is then discarded.
d- A donor cell from the animal to be cloned is then transferred through the same hole in the zona of each enucleated oocyte.

10. e-The donor cell is then fused to the enucleated oocyte by alignment in a fusion chamber and applying one or two DC pulses of 2.25KV/cm, 15ìsec each, although voltage and timing may vary. Fused couplets are activated by either chemical or electrical stimulation in order to mimic sperm penetration and finish the reprogramming and maturation process.
f-Reconstructed cloned embryos are then cultured in vitro for 6 to 9 days and evaluated for development.
g-Viable compact morula and blastocyst stage embryos are transferred to synchronized recipients as described above and carried to term in order to produce live cloned offspring.