Bovine IVM

Summary

This video provides a detailed, step-by-step process of collecting and processing oocytes from bovine ovaries for in vitro maturation. It covers the initial steps of obtaining ovaries from the abattoir, washing, and disinfecting them to prevent bacterial contamination. The script explains the technique for aspirating follicles, collecting oocytes, and washing them multiple times to ensure purity. The video then demonstrates the process of incubating the oocytes for 22 hours, observing the expansion of the cumulus mass as a marker of successful maturation. This comprehensive process is crucial for effective in vitro reproduction techniques.

Takeaways

• 😀 The process begins with collecting ovaries from the abattoir in the morning and preparing them for further processing.
• 😀 Ovaries are washed in a physiological saline solution to prevent bacterial contamination from the gastrointestinal tract.
• 😀 The mesovarium (connective tissue) is removed from the ovaries using scissors before washing them.
• 😀 Alcohol is used to disinfect the surface of the ovaries, eliminating remaining bacteria before they are moved to a second beaker for further washing.
• 😀 Follicle aspiration is performed using a needle to gently extract oocytes from follicles ranging from 2 to 8 millimeters in diameter.
• 😀 The oocytes are stored in tubes in an incubator for at least 30 minutes, allowing them to settle at the bottom of the tube.
• 😀 Supernatant (fluid above the oocytes) is removed, and the oocytes are resuspended in wash solution before being transferred to a Petri dish.
• 😀 A stereo microscope is used to select the best oocytes with intact cumulus masses, ensuring high-quality samples for maturation.
• 😀 The selected oocytes are grouped in sets of 60 and placed in maturation media for in vitro maturation (IVM) in a CO2 incubator.
• 😀 After 22 hours in the incubator at 39°C with 5% CO2, the expansion of the cumulus mass is observed as a sign of successful oocyte maturation.

Q & A

What is the main purpose of the procedure described in the script?

The procedure is designed to collect bovine oocytes (egg cells) from ovaries, followed by in vitro maturation to prepare them for reproductive purposes.

Why is physiological saline used during the collection process?

Physiological saline is used to wash the ovaries and prevent contamination from bacteria that might be present in the gastrointestinal tract, which is close to the reproductive tract.

What is the role of the mesovarium during the ovarian collection process?

The mesovarium is the connective tissue that holds the ovaries in place. It is removed using scissors to ensure that only the ovaries are collected for the procedure.

How is bacterial contamination minimized during the collection process?

The ovaries are dipped in alcohol after the initial wash to eliminate any remaining surface bacteria before transferring them into the second beaker for further processing.

What is the significance of maintaining physiological temperature during the procedure?

Maintaining physiological temperature (around 37°C) is crucial to ensure that the biological processes, such as oocyte aspiration and maturation, proceed effectively and accurately, mimicking the natural conditions of the body.

What is the purpose of the aspiration step in oocyte collection?

The aspiration step involves extracting the fluid from the follicles to collect the oocytes. By moving the needle in a specific manner, the procedure ensures the maximum yield of oocytes from follicles ranging from 2 to 8 millimeters in diameter.

Why is the syringe emptied into a tube and stored in an incubator for 30 minutes after aspiration?

The syringes are emptied into tubes to allow the oocytes to settle at the bottom of the tube. The 30-minute incubation period helps to facilitate this settling process and ensures that the oocytes can be effectively separated from the surrounding fluid.

What is the purpose of adding wash solution during the post-aspiration steps?

The wash solution is added to resuspend the oocyte pellet, helping to separate the oocytes from the remaining follicular fluid and ensuring they are properly prepared for further processing.

How are the oocytes prepared for in vitro maturation?

The oocytes are carefully transferred to a Petri dish under a stereo microscope, where they are selected based on their integrity (complete compact cumulus mass) and then washed before being placed in a medium designed for in vitro maturation.

What does the expansion of the cumulus mass indicate during in vitro maturation?

The expansion of the cumulus mass, which occurs after 22 hours in the incubator, is a morphological indicator that the oocytes have successfully completed the in vitro maturation process.