Reproductive Biotech. Superovulation b treatment of the female with hormones so that more eggs are ovulated than normal.

Reproductive Reproductive BiotechBiotech

SuperovulationSuperovulation

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

SuperovulationSuperovulationalter normal level of FSHalter normal level of FSHFollicle Stimulating Follicle Stimulating HormoneHormone

Typical results are Typical results are ovulation of 10 or more ovulation of 10 or more eggseggs

SuperovulationSuperovulationrecipient animals must recipient animals must be closely synchronized be closely synchronized with the donor cow.with the donor cow.

Must be at the same Must be at the same stage in their heat cyclestage in their heat cycle

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

ET ProcessET Process

super ovulate donor super ovulate donor cowcow

synchronize recipientssynchronize recipientsbreed donor cow (A.I.) breed donor cow (A.I.) when in heatwhen in heat

ET ProcessET Processflush - remove fertilized flush - remove fertilized eggs from donor before eggs from donor before 7 days gestation7 days gestation

plant embryo in the plant embryo in the recipient animalrecipient animal

Transfer to recip.Transfer to recip.

Non surgicalNon surgicalcommoncommondeposit embryo in deposit embryo in uterus through cervix uterus through cervix with AI straw and gunwith AI straw and gun

non surgicalnon surgical

usually 70-80% usually 70-80% success ratesuccess rate

SurgicalSurgicalincision in recip through incision in recip through flankflank

tip of uterine horn is exposedtip of uterine horn is exposedcut open and embryo is cut open and embryo is depositeddeposited

SurgicalSurgical

recip is sewn shutrecip is sewn shutless than 60-65% less than 60-65% success rate.success rate.

ETET

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

Splitting EmbryosSplitting Embryosremove embryo from cow remove embryo from cow 7 days after conception7 days after conception

collect unfertilized egg collect unfertilized egg and cut into zona and cut into zona pellucidapellucida

Splitting EmbryosSplitting Embryos

remove contents of remove contents of unfertilized egg leaving unfertilized egg leaving the zona intactthe zona intact

similarly, cut into zona similarly, cut into zona around embryoaround embryo

Splitting EmbryosSplitting Embryosdivide cells in embryo in divide cells in embryo in half using a micro half using a micro surgical bladesurgical blade

remove 1/2 of the cells remove 1/2 of the cells and transfer them into and transfer them into empty zonaempty zona

Splitting EmbryosSplitting Embryos

seal zona with seal zona with microjoltmicrojolt

each 1/2 put into each 1/2 put into recipient animalsrecipient animals

CloningCloning6 days after conception, 6 days after conception, 32 cell embryo is removed 32 cell embryo is removed from donor cowfrom donor cow

recipient egg cell is taken recipient egg cell is taken from a slaughterhousefrom a slaughterhouse

CloningCloning

recipient cells DNA is recipient cells DNA is removedremoved

one of the 32 cells is one of the 32 cells is removed from the removed from the embryoembryo

CloningCloningthe single cell is inserted the single cell is inserted into recipient egginto recipient egg

recipient cell is given a recipient cell is given a microjolt - fuses grafted microjolt - fuses grafted DNA to plasma DNA to plasma membranemembrane

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

CloningCloning

resulting offspring are resulting offspring are cloned identical cloned identical siblingssiblings

Sexing EmbryosSexing Embryosusually done by usually done by determining if embryo is a determining if embryo is a malemale

males have Y chromosome males have Y chromosome H-Y antigen can be H-Y antigen can be detected in male cellsdetected in male cells

Detection of YDetection of Ybreak open cells and stain break open cells and stain chromosomes to identify chromosomes to identify presence of Y chromosomepresence of Y chromosome

KaryotypingKaryotypingmust examine at least 10 must examine at least 10 cellscells

Detection of YDetection of Y70% accurate70% accurateuse Y specific probe for use Y specific probe for IDID

Y specific probe attaches Y specific probe attaches to Y chromosometo Y chromosome

Detection of YDetection of Y

can be detected by can be detected by radioactivity or special radioactivity or special lightlight

85% accurate85% accurate

H-Y AntigenH-Y Antigenmake an antibody by make an antibody by injecting H-Y antigen into injecting H-Y antigen into micemice

attach dye onto the attach dye onto the antibody for easy antibody for easy detection of male embryodetection of male embryo

H-Y AntigenH-Y Antigen

add antibody to add antibody to embryo and if embryo embryo and if embryo is male it will glowis male it will glow

if it is female it will if it is female it will remain darkremain dark

AdvantagesAdvantages

does not kill celldoes not kill cellno damage to embryono damage to embryofast - can be few hours fast - can be few hours or less than one hour or less than one hour oldold

DisadvantagesDisadvantages

need high skilled need high skilled techniciantechnician

80% accurate80% accurate

Freezing EmbryosFreezing Embryoscooling embryos -5C or 40 Fcooling embryos -5C or 40 Fcan be stored several dayscan be stored several daysfreezing -196 C or -320 Ffreezing -196 C or -320 Fmust cool before freezingmust cool before freezing

Freezing EmbryosFreezing Embryos

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

FreezingFreezing

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

FreezingFreezingCryoprotectant used in Cryoprotectant used in cattle - glycerolcattle - glycerol

also help prevent cell also help prevent cell membrane from becoming membrane from becoming brittlebrittle

dilutes salt in celldilutes salt in cell

ThawingThawing500 C per minute500 C per minutecomplete is about 20 complete is about 20 secondsseconds

glycerol is removed and glycerol is removed and embryo is placed in embryo is placed in successive solutionssuccessive solutions

ThawingThawing

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

ThawingThawing

if done too slowly if done too slowly embryo will burst embryo will burst because large ice because large ice crystals will formcrystals will form

70-80% success rate70-80% success rate

AnimalsAnimalsGenetic TransformationGenetic Transformationtwo toolstwo toolsembryo micromanipultionembryo micromanipultionrecombinant DNA recombinant DNA technologytechnology

Genetic Trans.Genetic Trans.

Occurs when new Occurs when new genetic info is genetic info is introduced into animals introduced into animals genomegenome

MethodsMethods

ES - Embryonic Stem ES - Embryonic Stem EC - Embryonic EC - Embryonic Carcinoma cellsCarcinoma cells

ES or ECES or EC

cells harboring the cells harboring the foreign gene are foreign gene are incorporated into incorporated into blastocyst resulting in blastocyst resulting in a mosaic animala mosaic animal

ECEC

potential for tumor potential for tumor formationformation

Retrovirus Retrovirus infectioninfection

retro. Carrying desired retro. Carrying desired gene is used to gene is used to transformhosttransformhost

close to 100% efficientclose to 100% efficient

MicroinjectionMicroinjection

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

MicroinjectionMicroinjection

inefficientinefficientmice - 10-15% are bornmice - 10-15% are bornonly 25% express new only 25% express new traittrait

ScreeningScreening

is it transgenic?is it transgenic?Incorporation of geneIncorporation of geneexpression of geneexpression of genegermline incorporationgermline incorporation

Agricultural UseAgricultural Use

rabbitrabbitsheepsheeppigpigcowcow

The End !! :)The End !! :)