Bovine IVF Experience Report
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Bovin IVF Experience Report |
7.8. Embryo Loading:
To load one embryo in straw, is needed around 300 µl of ET medium per embryo.
For 50 embryos, 15 ml of medium.
Embryos transport temperature is between 36°C 37°C.
7.9. Embryo Preservation:
Embryo preservation (cryopreservation) freezes excess or planned embryos in liquid nitrogen at -196 °C, usually via vitrification, allowing them to remain viable for over 10 year.
7.10. Embryos Vitrification:
Vitrification is an ultra-rapid cryopreservation technique in which embryos/oocytes are solidified into a glass-like state without ice crystal formation.
Procedure:
1. Prepare a culture dish with 50µl drops organized into rows for each embryo group, ensuring do not use a warming plate for the V1 and V2 steps.
2. Transfer the embryos from the IVC plate into the Wash drops at 36°C.
3. Move the embryos from the Wash drops into the V1 drops and start the timer immediately.
4. Transfer the embryos into the V2 drops after 8 minutes.
5. Ensuring the total time spent in V2 rows is exactly 20 seconds at room temperature (25°C).
6. Pick up the embryos and place them onto the straw.
7. Carefully remove any excess V2 medium from around the embryos on the straw.
8. Plunge the loaded vitrific straw directly into liquid nitrogen to freeze.
7.11. Embryos Devitrification:
Devitrification is the rapid warming and removal of cryoprotectants to restore normal cell function while avoiding ice crystal formation.
Procedure:1.
1.Place 500 μl of D1 medium into a dish and ensure it is warmed to 36°C - 37°C.
2.Remove the straw from the liquid nitrogen and locate which side the embryo is placed on.
3.Submerge the tip into the D1 medium and leave it for 1 minute, making sure not to press the embryo side against the bottom of the dish.
4. Prepare a dish with 50 μl droplets of D2 and D3 media, keeping them at room temperature (22°C - 25°C).
5. Transfer the embryos from the D1 medium into the D2 droplets and incubate for 3 minutes.
6. While incubating, prepare a new dish with 4 wash drops of ET medium (70 μl each) warmed to 36°C.
7. Once the 5 minutes in D3 are up, transfer the embryos through the 4 drops of ET medium to wash them thoroughly.
8. Place the washed embryos into the final central pool of ET medium at 36°C, where they are ready for culture or transfer.
8. EMBRYO TRANSFER:
Selected embryos are loaded into transfer straws.
Embryos are transferred into synchronized recipient animals.
Images may be recorded for documentation purposes.
During transport, embryos should be maintained in appropriate culture media with proper gas conditions.
Transfer should ideally be completed within 2 to 4 hour after preparation to ensure viability.
Embryo Transfer Procedure:
Embryo transfer procedure involves following step;
8.1. Synchronization Protocol:
| Day 0 | Day 7 | Day 9 | Day 17 |
|---|---|---|---|
| CIDR IN+EB+PG | CIDR OUT+PG+EcG | GnRH+EB | ET |
8.2. Selection of Recipient Animal (using ultrasound)
Selection is based on the quality and size of the Corpus Luteum (CL).
1. Grade 1 CL
Excellent
CL Size: ≥ 17 mm
Suitability: Highly suitable for embryo transfer
2. Grade 2 CL
Good
CL Size: 14 mm – 17 mm
Suitability: Suitable for embryo transfer
3. Grade 3 CL
Fair
CL Size: < 14 mm
Suitability: Less suitable but may be considered in some cases
Select animals with no large cavity or cyst in the CL.
If dominant follicle is present, administer GnRH injection to promote ovulation and luteinization.
8.3. ET Protocols:
1. Secure the recipient animal properly in a chute or crush to ensure minimal movement throughout the embryo transfer procedure.
2. Administer approximately 2 mL of lidocaine epidurally between the first and second coccygeal vertebrae to reduce straining and facilitate handling of the reproductive tract.
3. Wearing a lubricated rectal glove, gently enter the rectum, remove all fecal material.
4. Prepare a sterile embryo transfer rod by fitting the ET sheath and covering it with a sanitary protective sheath to maintain cleanliness during insertion.
5. Remove the embryo straw from the transporter and carefully load it into the transfer rod, ensuring that all components are correctly positioned and secured.
6. Insert one hand into the rectum to manipulate and align the cervix and uterus, providing a clear passage for the transfer rod.
7. Gently introduce the loaded ET rod through the vulva and vagina and guide it through the cervix under rectal control while avoiding unnecessary trauma.
8. Advance the rod into the uterine horn located on the same side as the functional CL and position the tip at the desired site for embryo deposition.
9. Slowly release the embryo, withdraw the transfer rod carefully, verify successful deposition, and record all relevant transfer details in the designated record sheet.
Post-Transfer Activities
1. Inspect the transfer equipment to ensure the embryo has been expelled successfully.
2. Remove all disposable materials according to biosecurity procedures.
3. Release the animal after confirming its normal condition.
Document the following information:
1. Recipient identification number
2. Embryo identification
3. Date of transfer
4. Technician performing the procedure
5. Side of CL (left or right)
8.4. Pregnancy Diagnosis:
Pregnancy diagnosis (P.D) is done by one method by rectal palpation (R.P) and done by Ultra sound machine.
P1 is done at 35 days after AI and ET by Ultra sound machine.
P2 is done at 90-96 days after A.I and ET by rectal palpation and Ultra sound.
9. In Vitro Embryo Production Trial and Results:
During my internship at FonGrow (Pvt.) Ltd., I participated in complete in vitro embryo production trials.
2.1. Trial 01:
The first trial was conducted on 09/04/2026. It was performed to gain practical experience in the in vitro fertilization (IVF) process. The procedure involved the collection and grading of oocytes, followed by in vitro maturation (IVM). Matured oocytes were fertilized using prepared semen samples and cultured under controlled laboratory conditions. Embryo development was monitored and recorded.
RESULTS:
Parameter Results
| Breed | Red Sindhi |
| No. of Oocytes | 30 |
| Viable Oocytes | 10 |
| Oocytes Selected for IVM | 30 |
| Semen Used | Red Sindhi 4611 |
| Cleaved Embryos | 13 |
| Embryos Produced | 07 |
| Vitrified Embryos | 02 |
First Trial Results
2.2. Trial 02:
The second trial was conducted on 17/05/2026. The second trial was performed following the same IVF protocol to further enhance practical skills and evaluate the consistency of laboratory procedures. Oocyte collection, maturation, fertilization, and embryo culture were carried out under standard conditions. Embryo development was observed at different stages, and the results were documented for comparison with the first trial.
RESULTS:
Parameter Results
| Breed | Red Sindhi |
| No. of Oocytes | 50 |
| Viable Oocytes | 20 |
| Oocytes Selected for IVM | 50 |
| Semen Used | Red Sindhi 4611 |
| Cleaved Embryos | 18 |
| Embryos Produced | 12 |
| Embryos transferred | 02 |
OBSERVATIONS:
Both trials provided hands-on experience in embryo production techniques and helped develop a better understanding of oocyte handling, fertilization procedures, embryo culture, and laboratory management practices used in reproductive biotechnology.