Bovine IVF Experience Report
6. OPU (OVUM PICK UP)
This procedure involves the collection of oocytes from donor animal. It is a minimally invasive, ultrasound-guided procedure used to retrieve immature oocytes from a cow's ovaries, for in vitro fertilization (IVF).
Steps of OPU Procedure:
This procedure involves following step;
1. Prepration
The cow is placed in a crush with a head gate and given caudal epidural to prevent movement and minimize discomfort.
2. Imaging & Aspiration
A transvaginal ultrasound transducer is inserted, and a 18 to 20-gauge needle is passed through the vaginal wall to aspire ovarian follicles (typically 5-8 mm).
3. Vacuum System
A suction pump (usually 50-60 mm Hg) creates a vacuum to pull the follicle contents through silicone tubing into a collection tube maintained at 37°C.
4. Collection
Follicular fluid is collected, and sent to a lab for maturation and fertilization.
7. IN-VITRO EMBRYO PRODUCTION (IVEP)
In-Vitro Embryo Production (IVEP) is an assisted reproductive technology in which maturation, fertilization occurs outside the body under controlled laboratory conditions. The procedure involves oocyte retrieval, maturation, sperm preparation, fertilization, and embryo culture.
PROCEDURE:
7.1:FILTERATION & SEARCHING OF OOCYTES
After Ovum Pick Up (OPU), the aspirated follicular fluid is received in sterile falcon tubes in the IVF Laboratory at 37 oC.
7.1.1 Filteration Procedure
1. The follicular tubes are labeled properly with breed name, animal tag, aspiration time, number of follicles aspirated and OPU technician.
2. The follicular fluid is poured through a sterile 100 µm cell strainer.
3. The filter traps the cumulus-oocyte complexes (COCs) while allowing blood and debris to pass through.
4. The filter is gently washed with wash media to recover any attached oocytes to the filter.
5. The retained material is transferred into a clean dish for further examination/searching.
Purpose of Filtration
Removes blood clots and debris.
Prevents loss of oocytes.
Makes searching easier and clearer.
7.1.2:Searching of Oocytes
After filtration, the dish is placed under a stereo microscope.
Steps
We systematically scans the entire dish.
Cumulus-oocyte complexes appear as round structures surrounded by cumulus cells.
Each identified Oocyte is picked using a 10 µl pipette.
7.2.WASHING & GRADING OF OOCYTES:
7.2.1.WASHING:
After retrieval and searching, the collected cumulus-oocyte complexes (COCs) are subjected to washing with holding/wash media to remove follicular debris, and contaminants.
Procedure:
1. Pre-warmed washing media (37°C) is prepared in sterile culture dishes.
2. Each oocyte is transferred sequentially into 2-3 droplets of media using a pipette.
3. Gentle pipetting is done to remove attached debris and excess follicular fluid.
4. Care is taken to avoid mechanical stress or damage to the oocyte.
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| Washing of oocytes during IVEP |
7.2.2. GRADING
Before maturation, the oocytes are graded based on the number of cumulus cell layers and the appearance of the cytoplasm. Oocytes with dense cumulus cells and clear cytoplasm are considered healthy and have a higher chance of maturing and fertilizing. Those with fewer cumulus cells or dark, irregular cytoplasm are considered lower quality. This grading helps us to select the best oocytes for further procedures like IVM.
1. Grade A: Multiple layers of COCs, Uniform Cytoplasm
2. Grade B: Partial Expansion of cumulus cells, Slightly less compact layer, Normal Cytoplasm
3. Grade C: Few cumulus cell layers, Partially denuded areas, Slightly cytoplasmic irregularity
4. Naked Oocytes:
These oocytes have zona pellucida and lacks cumulus cells, resulting in very low viability.
5. Degenerated Oocytes:
Exhibit abnormalities.
They are not viable for fertilization
Degenerate Ooplasm
7.3 IVM Media Prepration
The IVM media is prepared under sterile conditions inside a laminar airflow cabinet 12 hours prior to use to allow proper equilibration.
A sterile 3 ml glass vial is taken and 500 µl of IVM media is added into it. The media is then carefully overlaid with 250 µl of sterile mineral oil to prevent evaporation and maintain pH stability. After preparation, the vial is placed in a CO₂ incubator at 38.5°C with 5-6% CO for 12 hours.
Immature oocytes are then transferred into the IVM media and cultured in a CO₂ incubator. A maximum of 40-45 oocytes are cultured per vial. The oocytes are incubated for 24 hours to allow maturation.
7.3.1. IN-VITRO MATURATION (IVM)
After grading of the cumulus-oocyte complexes (COCs), immature oocytes categorized as suitable for further development are selected for in-vitro maturation. IVM is the process in which immature oocytes are cultured in specialized maturation media to allow progression to the MII stage outside the body for 24h.
7.4:IN-VITRO FERTILIZATION:
Fertilization of oocytes occurs in two steps. First, bull semen is prepared using semen preparation methods and washed in IVF medium. Second, matured cumulus-oocyte complexes are inseminated with the prepared sperm in IVF medium.
7.4.2. IVF Media Prepration:
IVF media is prepared 12 hours before the fertilization procedure.
The prepared media is placed in a CO₂ incubator at 38.5°C for equilibration.
The concentration of IVF media depends upon the no of oocytes and no of semen doses used.
For the preparation of 1 mL IVF medium:
945 µL IVF medium is taken.
11 µL H media is added.
44 µL PHE solution is added.
The final volume is adjusted to 1ml.
The H and PHE media is added for enhancing the sperm motility and life.
7.4.3. IVF Drops Preparation:
1. Fertilization drops are prepared in 35mm plates using IVF media.
2. The volume of each drop is 70μL.
3. Drops are covered with 400μL mineral oil to prevent evaporation and maintain culture conditions.
4. Prepared drops are incubated at 38.5°C in a CO2 incubator until insemination.
7.4.1.Semen Prepration Methods:
Bovine IVF semen preparation selects high-quality sperm by separating motile cells from debris, dead cells, and extenders using swim-up (for high motility)density gradient centrifugation (DGC, using Percoll for high recovery of motile sperm). In FonGrow IVF lab we use the density gradient method.
Density Gradient Centrifugation:
Involves layering semen over a colloidal suspension. Centrifugation separates cells by density, with high-quality, motile spermatozoa forming a pellet at the bottom while dead sperm and bacteria are trapped in the gradient layers.
Semen Prepration Steps:
1. Thaw/Collect Semen
Bull semen is collected or thawed if frozen.
Frozen bull semen straw is thawed in a water bath at 37°C for 30 seconds. The sample is allowed to liquefy at room temperature.
2. Motility Assessment
After thawing, a small drop of semen is placed on a warm slide.
Sperm motility is evaluated under a microscope.
Only samples showing satisfactory progressive motility are processed further.
3. Prepare Percoll Gradient
Percoll is prepared & incubated 30 minutes before the procedure to ensure proper layering.
In a centrifuge tube, 200 µL of Percoll & 200µL of sperm is added at the bottom and mix them slightly. 400 µL of Percoll is gently layered on bottom to form a gradient.
4. Layer Semen:
Thawed semen is carefully layered on top of the gradient without disturbing the layers.
5. Centrifugation:
The tube is centrifuged at 4700 rpm for 6 minutes for conventional and for sexed at 7100 rpm for 6 minutes.
Healthy motile sperm settle at the bottom, while debris and dead sperm remain in the upper layers.
6. Collect Motile Sperm:
The sperm pellet at the bottom is carefully collected and transferred to a new tube.
7. Wash Sperm:
The collected sperm is washed with 1000µL IVF medium and centrifuged again to remove Percoll.
The tube is centrifuged at 1700 rpm for 3 minutes for conventional and for sexed at 3500 rpm for 3 minutes. Prepared sperm is now ready for insemination of oocytes.
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| Percoll Density Gradient and Centrifugation during Semen Preparation |
7.4.4. Oocyte Fertilization :
1. Selection of Mature Oocytes:
After in vitro maturation (IVM), cumulus-oocyte complexes (COCs) are examined.
Mature oocytes with expanded cumulus cells are selected for fertilization.
2. Washing of Oocytes:
The selected oocytes are washed in drops of IVF medium to remove maturation media.
3. Transfer to Fertilization Drops:
Oocytes are placed into fertilization drops containing IVF medium under mineral oil.
In one drop we can put upto 20-25 oocytes.
4.Insemination:
The prepared sperm suspension is added to the fertilization drops at the required concentration.
5. Incubation:
Oocytes and sperm are incubated at 38.5°C in a CO2 incubator with 5-6% CO2 gas concentrations for 18-22 hours.
7.5.EMBRYO CULTURE PROCESS:
Approximately 18-22 hours after insemination
6.5.1. IVC Media Prepration:
In vitro culture (IVC) media is prepared 12 hours before use.
Media volume is adjusted according to the number of oocytes to be cultured.
Media drops are placed in culture dishes and covered with mineral oil to prevent evaporation and maintain stable conditions.
500μL IVC media in 1 well covered with 300μL mineral oil.
It can reside about 45 oocytes.
And for washing we will prepare IVC media according to no of drops.Each drop will be of 70μL.
7.5.2. PROCEDURE:
IVC Procedure involves following steps;
1.Denudation:
Denudation is the process of removing the cumulus cells surrounding the oocyte after fertilization.
This is done by gentle pipetting. Pipetting will be done at 50μL.
2. Washing:
Denuded zygotes are carefully washed in IVC wash media to remove residual cumulus cells, sperm, and debris.
3. Transfer to Culture Drops:
Clean zygotes are transferred to in vitro culture (IVC) drops prepared with IVC media.
Drops are covered with mineral oil to prevent evaporation.
4. Incubation:
Zygotes are incubated at 38.5°C in a mixed gas incubator. The concentration of gases will be 6% CO2, 6% O2 and 88% N2.
7.6.Embryo Assessment:
After embryo culture, embryos are evaluated to determine their quality, development, and suitability for transfer or cryopreservation.
The Assessment typically includes:
Day 3 Assessment (Cleavage Stage): Evaluates the cell division visually under stereo microscope
Day 5\6 Assessment (Blastocyst Stage): Evaluates the development stage (expansion), the inner cell mass, and the trophectoderm.
7.6.1. Other Observations:
Arrested Development: Embryos that fail to divide or degenerate are discarded.
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| Morphological appearance of Cultured Embryos |
7.7. Embryo Grading
Blastocyst Stage
Grade | Inner Cell Mass | Trophectoderm (TE) | Quality
A | Many tightly packed cells | Many Cohesive Cells | Excellent
B | Severly loosely packed cells | Few Cells | Good
C | Free Cells,Poorly Organized | Few Scattered Cells | Poor