Minimum Volume Vitrification of Immature Feline Oocytes

This protocol is useful for the long-term conservation of the feline oocytes through a cryopreservation technique known as with vitrification, which is crucial for fertility and biodiversity preservation in animal species. The main advantages of vitrification are its speed since it can be completed in less than 17 minutes and its feasibility in field conditions if liquid nitrogen is available. The domestic cat is a viable model for wild endangered felines.

The development and optimization of cryopreservation protocols would allow establishing gamete biobanks for feline conservation programs. Begin by preparing a Repro vitrification plate with three conical wells in one row. Add 20 microliters of equilibration solution to the first well and 300 microliters of vitrification solution to the second and third wells.

To equilibrate the cumulus oocyte complexes, or COCs, use a small-bore pipette to transfer one or more complexes to the bottom of the first well. Slowly add 20 microliters of equilibration solution to the border of the drop and wait for three minutes, then add another 240 microliters of equilibration solution and wait for nine minutes. While waiting, prepare a box with liquid nitrogen and label a vitrification support with the experiment or cat identification code.

Put the box and support near the stereo microscope. After equilibrating the COCs, perform vitrification in less than 90 seconds. To vitrify the COCs, fill the small-bore pipette with the vitrification solution from the second well.

Take the COCs from the bottom of the first well and move them to the surface of the second well. Wash the pipette with the vitrification solution, then move the COCs to another area of the well and mix the surrounding solution. Fill the pipette with solution from another area of the well.

Move the COCs and mix the medium surrounding them with the pipette. Repeat this process one more time. Next, wash the pipette with the vitrification solution from the third well and use it to move the COCs to the bottom of the third well.

Again, mix the surrounding solution. After filling the pipette with solution from another area of the well, move the COCs and mix the solution. Repeat this process.

Fill the pipette tip with the vitrification solution from another area of the well and use it to load the COCs on the strip of the vitrification support close to the tip, then aspirate the excess medium. Immediately plunge the vitrification support in liquid nitrogen and use clamps to close it, making sure it remains immersed. Store the loaded vitrification supports in a goblet and keep them in a storage liquid nitrogen tank until warming.

Place the heating stage close to the stereo microscope and turn it on to 38 degrees Celsius. Warm the lid of a Petri dish on top of the stage, then transfer the vitrification supports from the storage tank to a box with liquid nitrogen. Prepare one row of a Repro plate for each vitrification support to be warmed.

Add 300 microliters of dilution solution to the first well and 300 microliters of washing solution to the second and third wells. Take the thawing solution out of the incubator and drop 100 microliters on the lid of the Petri dish. Use the clamps to open the vitrification support inside the liquid nitrogen.

Place the Petri dish lid under the stereo microscope, then quickly pick up the vitrification support and immerse its strip in the drop, moving it until all COCs detach. Remove the support from the drop as soon as it is empty, but leave the COCs in the thawing solution for one minute. Fill a pipette with dilution solution and use it to move the COCs from the thawing solution drop to the bottom of the first well with dilution solution.

Leave the COCs there for three minutes. Wash the pipette with the wash solution from the second well, then take the COCs and move them to the bottom of the second well. Wait for five minutes.

Wash the pipette with a solution from the third well and use it to move the COCs from the second well to the surface of the third well. Wait for the COCs to touch the bottom of the third well. Then repeat the process with another well area.

When finished, wash the pipette with culture medium and move the oocytes to a culture dish. The vast majority of gametes survive after oocyte vitrification and warming according to this protocol. A representative picture of a viable vitrified warmed cat cumulus oocyte complex stained with fluorescein diacetate and propidium iodide is shown here.

This table shows the percentage of survival after vitrification, which depicts post-warming data of oocytes intended for in vitro maturation. Out of 435 oocytes, 395 survived, scoring an overall 90.8%post-warming viability. However, some morphological changes can be noticed after warming.

The most frequent morphological abnormalities are changes in ooplasm shape and granulation, partial or sometimes total loss of cumulus cells, and zona pellucida fractures. When attempting this procedure, remember to prepare media in advance so that they are the right temperature before use and to follow protocol temperatures and timings to avoid cell toxicity. The protocol could also be applied to oocytes of other species.

If successful, this could also contribute to germ plasm banking for biodiversity preservation in endangered felines.