Guide to Frozen-Thawed Embryo Transfer in Cows

Overview
This guide outlines the procedure for frozen-thawed embryo transfer in cattle, a widely used technique in commercial ET programs. It includes recipient preparation, thawing protocols, embryo handling, and transfer method to ensure high pregnancy rates.

Required Equipment (WTA Tech)
- ET Gun (Universal or 0.25 mL / 0.5 mL specific) WTA
- ET Sheaths (Smooth tip, compatible) WTA
- Sanitary Chemise (Disposable cover) WTA
- OB Sleeves (Gloves) WTA
- Waterproof Nylon Apron WTA
- Lubricant (Non-spermicidal)
- Straw Cutter WTA
- Embryo Thaw Unit or Water Bath (28–30°C or as per protocol)
- Paper Towels
- Sterile Forceps
- Embryo Handling Dish:
Option 1: Petri Dish 60 mm (standard)
Option 2: Five-Well Dish (for rinsing or observation)
- Embryo Transport Container: TSV Embryo and Oocyte Transporter (recommended for temperature stability during handling and transfer)
- Recipient Cow Identification Records

Step-by-Step Procedure

  1. Recipient Cow Preparation
    Restrain cow in chute with side access. Confirm recipient ID and ensure correct synchronization (Day 7 ideal). Confirm presence of corpus luteum (CL) on ovary (same side as transfer). Clean vulva and perineal region. Wear OB sleeve and lubricate rectal arm.

  2. Embryo Thawing
    For Direct Transfer (DT) Embryos:
    - Remove straw from nitrogen tank using pre-chilled forceps.
    - Immerse straw in 28–30°C water for 25–30 seconds (or per manufacturer's instructions).
    - Dry straw with sterile paper towel.
    - Gently shake straw to mix contents. 
    - Cut sealed end with straw cutter. Load into pre-warmed ET gun.
    For Conventional (non-DT) Embryos:
    - Thaw as above.
    - Expel embryo into Petri Dish 60 mm or Five-Well Dish containing holding medium.
    - Observe under microscope to confirm viability and orientation.
    - Use a sterile pipette to load embryo into ET gun.

  3. Transfer Technique
    - Insert gloved rectal arm and stabilize cervix.
    - Guide ET gun through the vagina and cervix.
    - Remove sanitary chemise.
    - Advance tip into the uterine horn on the side of the corpus luteum.
    - Slowly depress plunger over 5 seconds to deposit embryo.
    - Withdraw smoothly.
    - Record transfer details (recipient ID, embryo ID, side of CL, date).

  4. Post-Transfer Care
    - Avoid stress immediately after transfer.
    - Mark cow for pregnancy monitoring.
    - Schedule pregnancy diagnosis at 28–35 days post-transfer.

Best Practices

Embryo Handling: Keep embryos below −130°C until thawing. Minimize time from thaw to transfer.
Timing: Follow strict synchronization protocols.
Sanitation: Always use sterile equipment for each cow.
Thawing Accuracy: Precise time and temperature are critical to viability.
Transport: Use the TSV Embryo and Oocyte Transporter to maintain temperature stability if moving loaded guns or embryos between facilities.

Conclusion
Frozen embryo transfer enables flexibility in genetic programs and global transport. With precise thawing, careful handling, and rigorous recipient selection, pregnancy rates can approach fresh transfer success in well-managed herds.

Additional Notes and Best Practices
Synchronization Protocols
To ensure recipients are at Day 7 post-estrus, consider these widely used synchronization programs:

Ovsynch Protocol:
Day 0: GnRH injection
Day 7: PGF2α injection
Day 9: Second GnRH injection
Transfer 7 days after confirmed ovulation

Prostaglandin-Based Protocols:
Two PGF2α injections 11–14 days apart
Estrus expected 2–5 days after the second injection
Transfer 7 days after standing heat

CIDR-Based Protocols (Controlled Internal Drug Release):
Insert CIDR for 7 days
PGF2α given on removal
Estrus occurs within ~48–72 hours
Transfer 7 days post-estrus
Note: Synchronization choice depends on herd management, technician experience, and donor-recipient scheduling.

Recipient Selection Criteria
Ideal recipients should meet the following conditions:
- Age: 2–7 years old
- Body Condition Score (BCS): 2.75–3.5 on a 5-point scale
- Reproductive History: No history of metritis, dystocia, or retained placenta in the last calving
- Cyclicity: At least one confirmed natural estrus cycle before synchronization
- Health: No lameness, mastitis, or systemic illness
- Functional Corpus Luteum: Confirmed via palpation or ultrasound

Stress Management Recommendations
Reducing stress around handling and transfer significantly improves conception rates:
- Move cows calmly and quietly—avoid shouting or abrupt gestures
- Minimize the presence of dogs or unfamiliar people
- Use well-designed chutes and pens to avoid slipping or injury
- Schedule transfers during cooler parts of the day (morning)
- Allow recipients to rest undisturbed for at least 30–60 minutes after transfer

Managing Transfer Failure
If pregnancy is not achieved:

Review Records:
Was synchronization timing correct?
Were embryos properly handled (thawing temperature, timing)?
Were recipients healthy and cycling?

Resynchronize Recipients:
Repeat synchronization protocol after verifying reproductive status
Consider a different synchronization protocol if conception rates are consistently low

Evaluate Additional Factors:
Heat stress at transfer
Technician technique (depth of deposition, side of CL)
Embryo quality or source

Plan Next Steps:
Reschedule embryo transfer
If repeated failures occur, investigate herd nutrition, disease status, and handling practices

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