Reproductive Biotech

Superovulation treatment of the female with hormones so that more eggs are ovulated than normal

Superovulation alter normal level of FSH Follicle Stimulating Hormone Typical results are ovulation of 10 or more eggs

Superovulation recipient animals must be closely synchronized with the donor cow. Must be at the same stage in their heat cycle

EmbryoTransfer provides opportunity to introduce genetic material into populations of livestock while greatly reducing risk of transmission of diseases

ET Process super ovulate donor cow synchronize recipients breed donor cow (A.I.) when in heat

ET Process flush - remove fertilized eggs from donor before 7 days gestation plant embryo in the recipient animal

Transfer to recip. Non surgical common deposit embryo in uterus through cervix with AI straw and gun

non surgical usually 70-80% success rate

Surgical incision in recip through flank tip of uterine horn is exposed cut open and embryo is deposited

Surgical recip is sewn shut less than 60-65% success rate.

ET now allows for shipping of 500 registered holsteins in four sealed flasks in one seat on an airplane

Splitting Embryos remove embryo from cow 7 days after conception collect unfertilized egg and cut into zona pellucida

Splitting Embryos remove contents of unfertilized egg leaving the zona intact similarly, cut into zona around embryo

Splitting Embryos divide cells in embryo in half using a micro surgical blade remove 1/2 of the cells and transfer them into empty zona

Splitting Embryos seal zona with microjolt each 1/2 put into recipient animals

Cloning 6 days after conception, 32 cell embryo is removed from donor cow recipient egg cell is taken from a slaughterhouse

Cloning recipient cells DNA is removed one of the 32 cells is removed from the embryo

Cloning the single cell is inserted into recipient egg recipient cell is given a microjolt - fuses grafted DNA to plasma membrane

Cloning desired embryo will replace cells that are removed providing a continuing supply of DNA to be inserted into more recipient cells

Cloning resulting offspring are cloned identical siblings

Sexing Embryos usually done by determining if embryo is a male males have Y chromosome H-Y antigen can be detected in male cells

Detection of Y break open cells and stain chromosomes to identify presence of Y chromosome Karyotyping must examine at least 10 cells

Detection of Y 70% accurate use Y specific probe for ID Y specific probe attaches to Y chromosome

Detection of Y can be detected by radioactivity or special light 85% accurate

H-Y Antigen make an antibody by injecting H-Y antigen into mice attach dye onto the antibody for easy detection of male embryo

H-Y Antigen add antibody to embryo and if embryo is male it will glow if it is female it will remain dark

Advantages does not kill cell no damage to embryo fast - can be few hours or less than one hour old

Disadvantages need high skilled technician 80% accurate

Freezing Embryos cooling embryos -5C or 40 F can be stored several days freezing -196 C or -320 F must cool before freezing

Freezing Embryos slowly cooled to -25C at a rate of .5C per minute and then plunged into liquid Nitrogen

Freezing cryoprotectants are used to lower the freezing point so removal of water can be increased

Freezing Cryoprotectant used in cattle - glycerol also help prevent cell membrane from becoming brittle dilutes salt in cell

Thawing 500 C per minute complete is about 20 seconds glycerol is removed and embryo is placed in successive solutions

Thawing glycerol is removed and embryo is placed in successive solutions to re hydrate

Thawing if done too slowly embryo will burst because large ice crystals will form 70-80% success rate

Animals Genetic Transformation two tools embryo micromanipultion recombinant DNA technology

Genetic Trans. Occurs when new genetic info is introduced into animals genome

Methods ES - Embryonic Stem EC - Embryonic Carcinoma cells

ES or EC cells harboring the foreign gene are incorporated into blastocyst resulting in a mosaic animal

EC potential for tumor formation

Retrovirus infection retro. Carrying desired gene is used to transformhost close to 100% efficient

Microinjection of several hundred copies of DNA are injected into male pronucleus of one or two cell embryos

Microinjection inefficient mice - 10-15% are born only 25% express new trait

Screening is it transgenic? Incorporation of gene expression of gene germline incorporation

Agricultural Use rabbit sheep pig cow

The End !! :)