Fundamentals of Biotechnology

1. Fundamentals of Biotechnology Animal Biotechnology

2. Manipulation of reproduction and Transgenic animal • Valuable products from cell culture: • Animals are a source of high protein, food and leather which men have been using since the time immemorial. • The most common farm hoofed animals are cattle, sheep, goats and pigs but unlike plant their management is expensive. • In spite these animals have been our companion since the dawn of civilization. • Improvement can be carried out in reproductive system of animals,their milk protein and milk products and several novel products which normal lack in animals via biotechnological methods.

3. Manipulation of reproduction in animals

4. In animal sexual reproduction -----fertilization ----fusion of 2 nuclei coming from two gametes. • This sexual reproduction maintains the genetic trait of organism. • Naturally female mammals produce one egg in a month. Ruminants can have only one offspring per year. • Due to increase in population there is needed to increase products and number of animals too —possible by biotechnology.

5. Artificial insemination: • Artificial insemination, or AI, is a fertilization procedure in which sperm is artificially placed into a woman’s cervix (intracervical insemination) or uterus (intrauterine insemination). • The woman’s menstrual cycle is closely monitored. • The semen to be implanted is “washed” in a laboratory, which increases the chances of fertilization while removing unnecessary, potentially harmful chemicals. • The semen is inserted into the woman. • Artificial insemination is widely used for livestock breeding, especially for dairy cattle and pigs. Techniques developed for livestock have been adapted for use in humans.

6. Semen and its Storage: • The motility and the number of the sperms are examined under microscope. • About 0.2 ml bull semen contains about 10 million motile sperm. • The ejaculate can be stored via cryopreservation at -196 in liquid nitrogen for long time. • One ejaculated semen is sufficient to inseminate about 500cows.

7. Ovulation Control: • In many animals it is difficult to find out oestrous b.c it persists for short time or occurs a night. • In herd it would be economical to get synchronised oestrous. • Possible by using hormones e.g. progestron or prostaglindine. • These hormones regulate ovulation cycle of female and results in total synchrony of oestrous.

8. Sperm Sexing: • Sperms are produce in the tests of males and ova in female’s ovaries. • Each contains half of the chromosomes as compared to somatic cells. • One ejaculate conations half X & half Y chromosomes. • In dairy industry demand of female is more than male. Live stock industry prefers animals of one sex. • Therefore via AI technology X and Y chromosomes can be detected and sex of progenies determined accordingly.

9. Embryo Transfer: • The multicellular organism that develops in the uterus from the fertilised egg, from the time of first cell division when it becomes a foetus (until birth). • Embryo transfer methods cannot be used widely because of its high cost, technical difficulties and limited supply of embryo from superovulated donor. • The foster mother simply acts as incubator and does not make any genetic contribution to offspring. • In ruminant females there is chance to increase the number of egg production.

10. Multiple Ovulation (Superovulation): • Ovulation differs in different animals e.g. 21 days in cows and horse, 16 days in sheep and goats, etc. • Ovulation occurs as result of gonadotropic hormones circulation. • Increase in hormones egg production can be increase e.g. in cattle 8-10 eggs are superovulated, Which may go to 60. • However it depends on health, nutrition, breed of animals and environment in which they are living. • Female to be superovulated are frequently injected with prostaglandin F2a, to synchronise oestrous • After 10 days of oestrous they are injected with hormones FSH up to 4 days followed by PGF2a treatment. FSH induced super ovulation.

11. Multiple Ovulation transfer (MOET): • After 6-8 days of fertilization the embryo will be recovered. In cattle via non surgical methods while in sheep via surgical methods (using catheter in both cases) • In general one superovulated female results into 5-6 progenies. In cattle 50-60 % of progenies can be achieved from embryo transfer method.

12. Embryo splitting (Demi embryo): • The embryo of blastocyst stage can be split into equal two halves and transferred in to female to produce identical twins. • This technology (embryo splinting technology) has increased the rate of pregnancy. • Embryo Sexing: • Very important to know sex of the embryo b.f transfer. • PCR etc techniques should be used to know the sex.

13. In Vitro Fertilization (IVF) Technology: • The term in vitro means in glass or in artificial condition, • IVF refers to the fact that fertilization of egg by sperm had occurred not in uterus but oust side the uterus at artificially maintained optimum condition. • Revolutionized the field of animal Biotechnology. • e.g. an animal produces about 4-5 offspring in her life through normal reproduction, • whereas through IVF technology the same can produce 50-80 offspring in her life.

14. It is very useful technology. It involves the procedure: • Taking out the egg from the female donor • In vitro maturation of egg couture by keeping in an incubator • Fertilization of the egg in test tubes by semen obtained from superior male • Implantation of the 7 day old embryo in to surrogate mother

15. Embryo Cloning: • A clone is population of cells or organism derived asexually from a single ancestor. • They are genetically identical to each other to their common ancestor. • Cloning means the production of exact genetic replica of copies of an individual. • Quardriparantal Hybrids: • During 1960s B. Mintz at Cancer research institute, Philadelphia (USA), demonstrate this experiment. • Fusion of embryos of two different species of mouse (four parents) • Joshi used the experiment and produce a hybrid of goat and sheep named geep in 1998.

16. Nuclear transfer: • Involves the removal of a single blastomere from a cleavage stage embryo with fine micropipette of glass, and placing it under the outer membrane of an unfertilized mature enucleated oocytes. • First time by R.Briggs and T. Night at cancer research institute Philadelphia (USA), in 1955. • In 1960 Gurdon at Oxford University, UK referred differentiated intestinal nuclease of frog in to the nuclease free unfertilized egg of different amphibian spices –mature to frog. • Similarly Dolly the world first mammalian clone has been created via this technique.

17. Embryonic Stem (ES) Cells: • Cloning of mice could not be done as such as sheep via nuclear transplantation. • b.cbefore first embryonic division the cell has started its process of differentiation. • Therefore the use of ES cells has been made an alternative approach. • ES cells are the pluripotent cells isolated from inner cell mass of early embryo. These ES can be induced to generate many different types of cells • e.g. Muscle cells, nerves cells, liver cells, pancreatic cells etc. • These have great interest for production of transgenic animals because these are plentiful and maintained in culture, can be transformed very easily. • Their genome can be modified.

18. Via IVF fertilize mouse egg. The embryo cells undergo several step of cleavage. • And the dividing cells will be accumulating at one corner of embryo. • These accumulated cells are called inner cell mass (ICM). • ES can be used for cloning in two ways • Creation of chimera • Nuclear transplantation • Production of chimera: • ES cell from black donor of trophoblast stage injected into blastocoels space of albino embryo. • Implantation in surrogate mother resultant progeny will be chimric mouse.