Embryo Transfer in Small Ruminants:
- used a lot less than AI
What is it?
- Goal: to get more offspring out of elite females
- Elite females are used as egg “donors”
- Caused to super-ovulate through hormone therapy
O Females are allowed to ovulate and be bred
- Embryos develop to a determine stage and are then harvested
- Harvested embryos are put into “recipient” animals and are carried to term or frozen for future transfer
Why go to the trouble?
- More offspring out of elite females
- Number of breeding females can be reduced
- Increases rate of improvement
- More uniform set of offspring to market
- Storing of important genetic material
- International movement of important genetics
- Ability to breed out of season; frozen embryos
- Shorten lambing/kidding season
- Biosecurity
- Extend the productive life of a female who can no longer carry/raise offspring
Disadvantages:
- Cost
o Cost of AI and collection of embryos
- Risks to donors
o Scarring, infection, possibility of infertility
- Additional labor needed
- Keeping recipients on hand
- Genetic factor for super-ovulation
Initial Considerations:
- Is it economically feasible for your operation?
- Do you have the facilities to support a program?
- How many females will be in the program?
- When do you want the lamb/kid crop?
- How will you acquire/manage recipients?
- Are you willing to risk the reproductive soundness of valuable females?
- What sire will you use?
- Will artificial insemination be incorporated into the program?
Recipient Selection:
- GOOD embryos mean NOTHING without GOOD recipients
- Important factors for success…
o Appropriately sized
o Good milkers
o At least second parity females
o Calm disposition – good maternal characteristics
o Good body condition – a little on the thin side
o Consider using recipients that can be incorporated into your breeding program
o Get your recipients way ahead of time
Synchronization:
- Donors and recipients must be synchronized
o Start about 3 weeks before flush
o CIDR put in donors and recipients at the same time
o All ET technicians have different protocols
- Protocol changes based on season
- Different “drugs” can be used to achieve same result
Donor Synchronization:
- Estrus cycle of a goat is 21 days on avg
o Can be 18-23 days
o Ovulation is considered day 1 of cycle
o Estrus last from 24-48 hours
o Ovulation occurs 24-36 hours after onset of estrus
- Donors induced into heat and caused to super-ovulate
o CIDR is put in and left for 14 days
o Pull CIDR 8.5 days before flush
o FSH injections start around 3 days before CIDR pull
o 10cc of FSH is a standard total dose over 3 day period
o FSH dose usually decreases between first an last injection
o Donors will start coming into heat within 24 hours of CIDR pull
- Is it CRITICAL to correctly execute the protocol
o Familiarize yourself with the schedule ahead of time
o Use a checklist/dry erase board to record progress
o Colored chalk can help for marking donors after each injection
o A good chute aids the process
o Give shots as quick as possible from start to finish
o Look for pulled CIDRs
- Cut off end
- Heats and breeding
o If using AI – inseminate 48 hours after CIDR pull
o Use a teaser buck
o Check donors often
o Try to get 2-3 breedings on each donor
o Take the buck to the doe
o Record date and time of each breeding for each doe
o Re-CIDR donors 4.5 days after CIDR pull (108 hours)
- Simulates CL formation
Success Rate:
- Factors…
o General health of females (nutrition etc.)
o Body condition
o Breed
o Reproductive history
o Age
o Genetics
o Efficiency of semen (fresh/frozen)
o Stress
o Weather/season (males and females)
What to expect:
- 8 transferrable embryos per donor is good for mature females
- Some donors wont stimulate
- Does 2-5 years old average more embryos than maiden does or old does
- Infertile and degenerated embryos aren’t uncommon
- 70% conception on recipients is average
- Seasons make a big difference
Surgical vs. Non-surgical:
- Sheep and goats CAN NOT be flushed like cattle
o Can’t be palpated
o Cervix is smaller/tighter and not straight
o Post ovulation – cervix is particularly constricted
- Traditional procedure is surgical
o Incise the abdomen and bring uterus outside the body to flush
- There is a non-surgical procedure available in goats
o Flush through a catheter like in cattle
Surgical Procedure:
Pros-
- Faster better recovery rate (questionable)
- More technician available
- Uterus is accessible in ALL animals
Cons-
- More invasive
- Greater risk of infection
- Lower/NO success on regressed flushes
- Scarring
Non-surgical procedure:
Pros-
- Less invasive
- Much less risk of scarring/adhesions
- Less infection
- Much quicker recovery for donors (can flush more often)
- Greater success in regressed flushes
Cons-
- Limited technicians
- Only available in goats
- Takes more time
- In some instances, it is impossible to pass a catheter
Flush Procedure – Surgical
- If flushing and transferring on the same day
o 12 donors is maximum
- Sometimes it is beneficial to flush half and transfer those embryos
o Then flush remaining donors and transfer
- Females must be off feed and water the night before flush day
- A clean, dust free environment is needed
- Running water is ideal
- Embryos usually collected 6-7 days after breeding
o By this time, they have been fertilized
o Usually in morula or blastocyst stage
- Waiting 6-7 days gives time for embryos migrate to uterine horns
o This makes it easier to retrieve the embryos
- All donors must be fully anesthetized
- Surgical site clipped and sanitized
- Does placed on cradles belly up
- Cradle inverted
- Trocars inserted and laparoscope introduced to check ovaries
o Ovulations turn into CLs
- Each CL indicates an ovulation
- You can estimate number of embryos to be recovered this way
- If no/few ovulations, might not flush
- Some might choose to look at ovaries during surgery (not ideal)
- Ultrasound is also a possibility
- Female is laid belly up on cradle
- Surgical drape is placed over animal exposing only surgery site
- Incision is made on midline ( 2”- 4”)
o Skin and muscle are incised
- Female is inverted to bring uterus into view of laparoscope
- Forceps are used to “grab” the uterus and bring it outside the body
- Cradle is then returned to flat position
- It is important to keep uterus irrigated during entire procedure
- Each uterine horn is flushed individually
- Embryos are usually towards the end of the horns
- General technique is to flush from tip of horn to base
- Foley catheter is inserted at base of horn
- A small hole is made at the top of the uterine horn
o Small catheter is placed through this entrance
- Flush media is forced through syringe, through catheter, and through uterine horn
- Media flows out through the Foley catheter
- Assistant is holding a collection dish below the Foley catheter
- All media is caught in dish
- After a thorough flush the catheters are removed
- Second horn is flushed in the same manner
Flush Media:
- Flush Media is a commercially prepared product
- Designed to simulate composition of uterine environment
- It has nutrients (protein source) , antibiotics (gentamycin and kanamycin), etc.
- We used Vigro Complete Flush
o Made by Bioniche Animal Health
- Catheters are removed and uterus is returned to body cavity
- Incision is sutured
o First muscle then skin
o Some prefer to use staples
- GIVE SHOT OF LUTALYSE
o Lutalyse causes CL on ovaries to die and not produce progesterone and not produce pregnancy
Non-surgical Procedure:
- Uterus is not externalized
o Catheter is passed through cervix similar to cattle
o No ability to palpate
o Ovaries checked for CLs with laparoscope just as in surgical procedure
- Vaginal speculum is used visualize cervix
o “Grab” the os of cervix with Alice forceps
o Makes passing catheter easier
- We used primarily cut down IMV ET sheaths (about 2mm diameter)
- “Blind” process
- Some does are more difficult to pass a catheter in than others
- Some smaller catheters are needed
- Passing catheter can take from 1min to 1 hour
- Once the catheter is passed through cervix:
- Surgical tubing is attached to catheter
- It is split two ways
o One is Flush media flowing in
o The other is media coming out of the uterus
o 3 way valve controls flow
o Flush media (in IV bag) is elevated on IV stand for gravity flow
- Once the catheter is passed through cervix:
- Technician should be able to feel bifurcation and flush each uterine horn individually
- Each horn is filled with media, massaged externally, and then media is allowed to flow out into embryo filter
- Increasing media is used for each flush
- Each horn flushed approx. 4-5 times
- Media in filter is periodically being checked for embryos
o If estimated number aren’t recovered…keep flushing
- Emcom Embryo Filter
o Filter screen is 75
o Embryos are about 100 microns
- If embryo recovery is slow/poor:
o With regressed CLs:
§ Embryos could already be expelled
- Embryo development could be delayed and embryos are still in the very tips of uterine horns
- Previous surgical flushes caused a disfigured, contorted uterus with adhesions
- There could be a blockage in the uterine horns preventing a good flush
- A laparoscopic camera can be used in conjunction with a steel flush catheter to look for blockages
o This is the future of non-surgical flushing
Embryology:
- After flushing the donor—embryology is the same for surgical and non-surgical flushing
o Process requires at least two people
§ One to flush and one to act as embryologist
o Media that has been flushed out of uterus must now be searched under microscope
§ Embryos are not visible to naked eye
o Scopes used are “stereo” microscopes
§ Large field of view and low magnification
- Flush media is emptied from filter into a “grid dish”
o Some surgical flush techs flush directly into a dish
o Filters must be emptied and the screen thoroughly washed
- Surgical flushes are “cleaner” and easier to search
- Non-surgical flushes contain lots of endometrial cells that must be sorted through
- When an embryo is found it is removed with a pipette and moved to another dish with “holding media”
- Embryos from each donor are placed in separately labeled dishes
- Holding media:
o Designed to simulate uterine environment
o Provides essential amino acids, growth factors, enzymes, energy substrates and antibiotics
o Just for holding embryos between flushing and freezing/transfer
o Not for embryo culture
o Sometimes embryo quality actually appears to improve during holding period
o We used Holding Plus from ViGro
- Most embryos will be 6-7 days old
o Usually early “morula” to late “blastocyst”
o Infertile and degenerate embryos can be sorted out
o Viable embryos are graded for stage and quality
o The only way to be proficient at grading:
§ Practice, Practice, Practice
- Embryos can be frozen or transferred to recipients
- Better quality embryos handle the freezing process better
- Zona must be intact for freezing
- If freezing and transferring:
o Freeze the better quality embryos
o Transfer the rest
- Freeze as quickly after flushing as is practical
Transfers:
- Recipients are synchronized at same stage as donors
o They are “ready” to accept embryos
- If recipients responded correctly, they will have ovulated and formed functional CLs
- Any recipient not seen in heat after CIDR pull should be rejected
- Any recipient appearing to be coming back into heat on transfer day should be rejected
- Each recipient (sheep and goats) usually receives two embryos……occasionally three
Transfer Procedure:
- Recipients are fully anesthetized
- Placed in cradles..belly up
- Surgical site is prepared
- Cradle is inverted
- One trocar is inserted precisely like before
- A small incision ( 1 cm ) is made on midline for forceps to go through
- Laparoscope is introduced to check for functional CLs
- If good CLs are located…the recipient is ready for a transfer
- Laparoscope and cannula are removed
- Small end of uterine horn is brought through the incision with forceps
- Whichever side had the CLs or better quality CLs is the side to make the transfer in
- About 1.5” of uterus is exposed with a diameter similar to a pencil
- A small hole is made in the uterus
- Embryos are loaded in a “tomcat” catheter with a small syringe attached
- Tip of catheter is inserted through the hole in the uterine horn
- Syringe is plunged and embryos are deposited into uterus
- Uterus is put back in body cavity
- One suture is required to close the incision
- RECORDS ARE IMPORTANT
o Each recipient should have an original ear-tag with donor information and estimated birth date of transferred embryos
§ About 143 days from transfer date
o All donors and recipients can be offered feed and water immediately after the procedure
o Some form of antibiotic injection is beneficial
o Wait at least two weeks before turning in a buck with recips
o Donors can be bred on next cycle