ART - Bovine ET Flashcards
Define embryo transfer.
Embryo transfer is a set of procedures where embryos are produced and collected from a donor and transferred into recipients which will cary the pregnancy to term
What are the advantages to embryo transfer?
Increase number of offspring from superior females
Faster genetic improvement
Obtain offspring from old or injured animals incapable of breeding or calving naturally
Increase farm income through embryo sales
Disease control
Import and export embryos easier than live animals
Conservation of genetic material
What are the steps involved in embryo transfer?
- Donor selection
- Recipient selection
- Estrus synchronization of donor and recipient
- Superovulation of donor
- Artificial Insemination of Donor
- Embryo Recovery
- Evaluation of Embryos
- Transfer of embryos/Cryopreservation of embryo
- Micromanipulation
What is the basic principle of superovulation of the donor cow?
To stimulate extensive follicular development through the use of gonadotropic hormones which is given intramuscularly to induce ovulation of multiple oocytes instead of one
What is the only FDA approved product in this country for superovulation?
Folltropin (follicle stimulating hormone - procine origin)
When is the best time for superovualtion?
For optimum response, gonadotropin treatment is initiated during mid-luteal phase (days 9-12) of a normal estrous cycle
How often can donor cows be superovulated?
In 6-8 week intervals
Why is it important to superovulate around day 9-12 of the estrous cycle?
Because there will be a new wave forming - we want to catch it at the beginning of the follicular wave so we can initiate superovulation
What should be done prior to giving an epidural?
the area should be clipped and scrubbed using an alcohol scrub until clean
How do you prep the donor for AI of the donor?
Wipe the perineal region clean with water or a wet paper towel and secure the tail off to the side
What should you avoid when clenaing the perineal region of the donor?
Avoid using disinfectants, soaps, or harsh chemicals to prevent contamination (spermicidal and embryocidal)
What equipment is used for embryo recovery?
Catheter and stylet, flushing media, Y-junction tubung, and filter
What are the two methods that an embryo can be collected by?
Closed system or gravity flow method and open system or syringe method
What are the two catheter placements that an embryo can be collected by?
Horn flush and body flush
What is the first step for all embryo recoveries?
A metal sylet is inserted into the catheter and the catheter and stylet are advanced through the cervix into the uterus
Describe the closed system or gravity flow method of embryo recovery.
A foley catheter is placed in the uterine horn or body. The cuff is located about 2 inches from the tip of the catheter is inflated to prevent media from escaping the cervix. The stylet is then removed from the catheter. The flushing media bag is placed higher than the cow to allow for gravity flow. The Y-junction tubing is connected to the catheter, media bag, and filter. The clamp is opened to let flushing media to flow in. When the uterus feels full, the clam can be closed (IN) and outflow (OUT) clamp can be opened. During this process, the uterus is massaged to remove all media containing embryos. The process is repeated 5-7 times per horn.
Describe the open flush or syringe flush method of embryo recovery.
60 mL catheter tip syringes with flushing media is connected to the catheter already in place. Media is flushed through the catheter into the uterine horn and the syring is pulled back. The uterine horn is massaged to recover all media. Media from the syring is then filtered.
How is a horn flush done?
A foley catheter is placed a couple of inches passing the external bifurcation. The cuff is inflated in the horn to secure the catheter in place and to prevent media from escaping.
What are the advantages of the horn flush?
Easier to recover the fluid. Less change of leaving embryos behind. Uses less flushing media
What are the disadvantages of the horn flush?
Slower procedure
How is a body flush done?
Foley catheter tip is placed a couple of inches past the cervix. The cuff is inflated in the body. Both sides should fill with flushing media.
What are the advantages to the body flush?
It is a faster procedure
What are the disadvantages of the body flush?
Fluid goes from one side to the other. It is difficult to empty the uterus. You may leave embryos behind
How is the filter prepped?
The filter is opened and rinsed into a gridded dish using flushing media to remove any embryo that might be adhered to the screen
What type of microscope is used for evaluating embryos?
stereomicroscope at a minimum of 50x with 10x on the eye piece
How is embryo searching done?
A gridded searching dish is used to search for embryos in a pattern and as they are found, they are transferred into a 4 well dish containing holding media
Describe the characteristics of a Day 7 bovine embryo.
It measures approximately 140-150 micrometers in diameter and are still within the zona pellucida
What does a code 3 stage of development mean?
Early morula
What does a code 4 stage of development mean?
morula
What does a code 5 stage of development mean?
early blastocyst
What does a code 6 stage of development mean?
blastocyst
What does a code 7 stage of development mean?
expanded blastocyst
Code 1 quality indicates an exellent or good quality embryo. Describe an embryo of this code.
Irregularities relatively minor. At least 85% of the cellular material should be an intact viable embryo mass.
Code 2 quality indicates a fair quality embryo. Describe an embryo of this code.
Moderate irregularities in overall shape of the embryo mass or size. At least 50% of the cellular material should be intact, viable embryo mass.
Code 3 quality indicates a poor embryo. Describe an embryo of this code.
Major irregulatirites in shape of embryo mass or size, color, and density. At least 25% of the cellular material should be intact.
Code 4 quality indicatese a dead or degenerating embryo. Describe an embryo of this code.
Degenerated embryos, oocytes or 1 cell embryo, non-viable.
What stage and quality of embryos are considered of transferrable quality?
Stages 4-7 and quality 1-2
What is used to cryopreserve embryos?
Cryoprotectants like ethylene glycol, glycerol, or DMSO
What are the advantages to cryopreservation?
Long term storage, eliminates the need for multiple recipients, world wide distribution, and easy export and import
How are embryos stored for cryopreservation?
Embryos are put into a cryoprotectant solution and loaded into a straw. Cotton end - ethylene glycol - air bubble - embryo - air bubble - ethylene glycol - plastic plug
What information needs to be on each straw of cryopreserved embryos?
Dam registration number, sire registration number, dams name sire name, date of flush, breed of animal, practitioner freezing number, and type of transfer
How do you prep for an embryo transfer?
Straws are thawed before transfer of embryo to the recipeitns. 5 seconds in the air and 20 seconds in a water bath at 80-85 F. The straw is dried off, plug is removed, and straw is loaded into the ET gun with cotton plug towards the bottom. The sheath and sheath protector are placed over the gun.
How is embryo transfer done?
Ovaries of the recipient are palpated for a CL and if one is present an epidural anesthetic is given. Transfer gun is carefully passed through the cervix, into the horn on the same side of the ovary with a CL and the embryo deposited. Damage to the uterus or too much manipulation should be prevented to avoid inflammatio and prostaglandin release.
What is embryo splitting?
The creation of identical twins by splitting an embryo
True or False: An embryo is no longer viable after you biopsy it.
False - you are only taking a few cells - it can continue to grow
What method can be used to determine gender?
PCR
What is the difference between IVF and embryo transfer?
IVF is collecting the oocytes to fertilize and ET is collecting already fertilized embryos
How is ovum pick up done?
An ultrasound guided needle is inserted into the ovary to connect oocytes
What is the application of IVF?
To obtain potential offspring in problem breeders, infertile cows, pregnant cows, young heifers that are not of breeding age, and terminally ill/injured animals
Is ET or IVF more efficient in utilizing sexed semen?
IVF
What conception rates are associated with IVF?
40-50% conception rates
What is cloning?
Cloning is the process of producing individuals with identical or virtually identicle DNA, either naturally or artificially
What are the applications for cloning?
Researching and treating human diseases and genetically altering animals for the production of transplant organs
