Embryo Transfer Technology Flashcards
The 1st successful transfer of fertilized egg was reported in which animal and by whome?
Rabbit by walter and heape at cambridge 1891
1st success in which farm animals
Sheep and goat 1934
1st offspring after transfer of embryos on cattle and wine was in
1951
Enlist diff steps involved in embryo transfer
Selection of donors Synchronisation of oestrus Induction of superovulation Collection of embryo Evaluation of embryo Selection of recepient Transfer of embryos
Embryo transfer techniques have been used in which animals
Extensively in cattle and more sporadically on sheep goat and swine.
Cow is preferable than hiefer as embryo donor T or F
T. Embryo transfer programme is based on milk yield or genetic superiority
What is criteria for selection of donor
The age of donor should be between 2 to 5 years
Free from generic diseases and conformational abnormalities
Exhibit refular oestrus cycle
Superior production traits
Record of sound reproductive performance Including successive fertility and AI
Enlist harmones used for superovulation
PMSG pregnant mare serum gonatotrophins
FSH Follicle stimulating harmone.
HMG human menopausal gonadotrophins
Stage of collection of harmones
In the early follicular or luteal phase of the oestrus cycle
Most frequently used harmones
PMSG and FSH
Doses of PMSG and FSH
PMSG one single injection 2000 -3000 IU
FSH 35-50 mg twice daily for 4 days
___________ and _______ after beginning the gonadotrophin treatment _______ is administered to induce oestrus . Insemination is preferred at ____ days after oestrus
48 and 60 hours
Prostaglandin (PG)
2days later the oestrus
Embryos are recovered _____ days after insemination
8days
In case of catlle embryo recovery is done at
21 days 10th day Pmsg and fsh 11th day fsh 12th day pg and pg/fsh 14th day anti pmsg oestrus and inseminations
In bovine embryo is collected by which method
Non surgical method
Explain non surgical method in bovines
Special catheter ie foleys catheter are introduced in the cervix into uterine horn and embryos are flushed with 250-350 ml flushing medium
In other farm animals like sheep goat pig which method is used
Surgical method is used
Classify embryos according to there morphological appearance
- Excellent embryos app dev stage and perfect morphology
- Good embryos app stage and slight deviation in morphology eg minor damage of zona pellucida and excluded blastomeres
- degenerated or retarted embryo inapp stage and major morph deviation
- Unfertilised ova
In bovines _______ and ______ are best suited as reciepients
Heifers and young cows
Transfer of embryo
Non surgical methods in cattle
Transfer the embryo in to the tip of the uterine horn without damaging the endometrium
For successful embryo transfer the most imp is __________
Embryo control
It is imp to transfer the embryo _______ position
Ipsilateral means into the horn bearing corpus luteum
Cryopreservation preserving them for long distance shipments
Optimal method for bovine embryo includes one step addition of 1.4M glycerol as cryoprotectant ,a 20 minutes equilibration period and 0.25ml straws as embryo containers , slow cooling down at -35 Celsius and subsequent plunging to liquid nitrogen -196 celsius . Embryo are thawed by placing the straws directly into warm water .
Application of embryo transfer
Exploitation of femal repro system Significant facilitation of import and export for valuable genetic material Dev of new breeding concepts Gene conservation by freezing techniques Twin production Intro of. New genes into closed herds Manipulation of embryos Gene transfer
Enlist steps involved in invitro fertilisation
Oocyte recovery
Oocyte maturation
In vitro fertilisation of matured oocyte
IVF is quite successful resulting in about ________ of fertilized eggs
70%-80%
Which oocyte is selected for fertilization
2nd follicular phase of oestrous cycle …. Graffian follicles
Size of preovulatory follicles of diff animals lying against the ovary wall
Cattle 15mm
Sheep pig 8mm
_________ surgery can be used to recover oocytes from these follicles
Laparoscopy
Oocytes are obtained from
Oviducts , follicles , and surface of ovary after ovulation
Immature ovary collected from ovary
Culturing of oocytes
Cows are collected from the slaughter house and maintained in phosphate buffered saline at 30-32celsius for transportation
Oocytes are washed twice with
HEPES and once with BMOC-3 branchat and olivhant medium for oocyte collection
After washing 5-10 oocytes are transferred to 0.2ml of BMOC drops under parrafin oil at 37 celsius with 5% co2 in 95% air for 12-32hrs
_____ spermatazoa is essential for in vitro fertilisation
Capcitated
Capcitation of spermatazoa
Reexposure to of spermatozoa to seminal vesicle
In vitro fertilisation is done by
Oocytes are mixed with capcitated sperms in a petridish and incubated at 37 celsius with 5% CO2
In IVF which stage of fertilized zygote is added to recipient
Morula stage 16 celled
Sexing of sperm is done by
Flow cytometer attached with a sorter .
Explain flow cytometer Method
Sperm is stained with hoechst 33342 and sex specific sperms are separated by the use of flow cytometer
Method of flow cytometer has an accuracy of ________ of sexing sperms amd commercialy used in __________
90% cattle
Methods of embryo sexing
- Y specific DNA probes presence or absence of Y DNA chromosome
- PCR amplify y chromosome specific repeat sequence DYZI 500-8000 times on y chromosome
- PCR-RFLP PCR based genotyping a pseudoautosomal region present between Xand Y these regions are called ZFX and ZFY
Who described technique of micromanipulation
Lin injecting a mouse egg
Define micromanipulation
It is a technique whereby sperm egg and embryo can be handled on inverted microscope via joysticks that hydraullicaly operate glass microtools
1st to inject a sperm in oocyte by
Uehara and yanagimachi using hamster as a model
Monoclonal antibody was 1st showed by
Kholar and milstein hybrids between myeloma tumor cells and antigen stimulating b lymphocytes
Hybrid cells are formed by fusing the ________ cells and the __________ using a chemical __________
Myeloma cells
Antibody secreting B lymphocytes
PEG polyethylene glycol
Hybrid cells have character of both parents ________ of the cancerous myeloma cells and _______ propert of B lymphocytes
Infinite growth
Antibody secreting
Steps in production of monoclonal antibody production
Immunization Fusion Selection of hybrid cells Cloning of hybrid cells Large scale prod of ma cells
Animal used for inoculation is
BALB/mice
Cancerous myeloma cells which strain
Sp2/o
Fused cells are grown on ________ medium
HAT hypoxanthine aminopterin thymidine
The selection of hybrid cell formed is achieved by growing the cell on
HAT medium
Cloning of hybrid cell is done by
ELISA
For large scale production hybrido,as can be grown on tissue culture where they secrete upto ________ or in vivo as tumors in peritoneal cavity of balb mice where they produce upto _______
100ug 10-50ug/ml
40mg 2-20 mg/ml
Methods of dna fingerprinting
RFLP restriction fragment length polymorphism
PCR
AFLP amplified fragment length polymorphism
STR short tandem repeats
Dna fingerprinting syn
Dna profiling dna testing it is a process which uses fragments of dna to identify the unique genetic ,makeup of an individual
In 1984, ___________ at university of leicester in England was able to distinguish diff among individuals based solely on their dna composition
Sir Alec jeffreys
Read all methods from
Notes thanku dafffaaa ho ja amrinder
App 12 hour after the end of normal heat a single non fertilized ova is released T or F
T
