The Modern Equine Vet
November 2023
Vol 13 Issue 11 2023
Click Here to View Full Issue

Cover Story | Reproduction

Vitrifying Larger Embryos Without Puncturing 

By Marie Rosenthal MS

Embryos that are between 300 µm and 480 µm may be vitrified without puncturing or aspirating them, and still achieve a fairly high pregnancy rate, according to Sandra Wilsher, PhD, who discussed the new technique at the British Equine Veterinary Association Annual Congress, recently held in Liverpool, England.

Embryos smaller than 300 µm tend to be easier to cryopreserve without additional manipulation, but the larger they become the more challenging it is to vitrify them, explained Dr. Wilsher, who practices reproductive medicine at Sharjah Equine Hospital in the United Arab Emirates.

“Equine embryos have always been a little bit challenging to vitrify, particularly if they’re over 300 microns,” Dr. Wilsher said. “This is chiefly due to the large blastocoele cavity filled with fluid, but also these embyros have a relatively small inner cell mass compared with their size.”

Also once they’ve started to blastulate, they are developing an endoderm layer and a capsule, which increases the number of layers through which any cryoprotectants must pass, she explained.

“However, we can get very good pregnancy rates when we puncture or aspirate this fluid. And what traditionally we’ve always thought is you must remove between 90% and 95% of this blastocoele fluid prior to vitrifying the embryo,” she said.

The amount of fluid as the embryo increases in size is the primary concern because it increases the risk for the development of ice crystals, which can damage the embryo. The most common technique is to puncture the embryo, remove some of the blastocoele fluid and run it through a vitrification protocol.

Many reproduction specialists tend to use a micro manipulator to hold the embryo during aspiration, and they do get excellent results with embryos between 300 µm and 560 µm. However, the manipulator can be expensive. Manual puncture uses fine needles which are easily damaged.

“However, once you get skilled at using the micromanipulator, you can get really good results vitrifying embryos up to about 800 microns,” she said.

Dr. Wilsher routinely uses the manual method for embryos less than 560 microns. In this case, one does not need to remove fluid, just make a hole, before vitrifying.

Both techniques require a steep learning curve, which can be challenging in a busy practice because not many clients “want you to practice kababbing their embryos,” she joked.

“However, you can get really good results with this if you get good at it, but you can only really do it on embryos that are up to about 560 microns. But you don’t need to aspirate the fluid, you just need to make the hole. And then we run through the vitrification protocols with them.”

Once the embryo is manipulated, Dr. Wilsher uses an equilibration medium, which has ethylene glycol (EG) and dimethyl sulfoxide (DMSO) in it, and the embryos are exposed to around 5 to 6 minutes. And then they move them into the actual vitrification solution, which has higher amounts of both EG and DMSO, for about 60 to 90 seconds before they are loaded onto the minimal vitrification device and plunged into liquid nitrogen.

SKIPPING STEPS
However, her group has come up with a method that appears successful in embryos up to about 480 µm, which enables them to skip the first step of puncturing and/or removing fluid.

They use commercial vitrification kits from Kitazato. The equilibration medium contains 7.5% DMSO and 7.5% EG; and the vitrification kit contains 15% DMSO and 15% EG.

“The only difference we have made in the protocol is we don’t puncture them, and we leave them in the equilibration solution for 15 minutes,” she said.

They drop the intact embryo into 2 separate 50 µL droplets for about 7.5 minutes each, although she said the total time, rather than the length in each droplet, is the most important aspect of the timing. Then, the embryo is moved into 2 separate droplets of the vitrification solution, which has the higher amounts of DMSO and EG for up to 90 seconds total time, which includes the time for loading onto the vitrification device, which in Dr. Wilsher’s facility, is Cryolock. Then it is capped quickly and plunged into the liquid nitrogen. She caps it before she puts it into the nitrogen.

“We normally average about 1 minute 20 seconds for the time before it’s dropped into the vitrification. But we have gone up to 1 minute 30. The thing to do is not to panic, which most people do. So, make sure it does stay in the vitrification solution for long enough. Don’t, because you’ve only got a minute and a half start to panic and load it too quickly. So, it’s only been in the solution for 1 minute or so. It’s better, if it has been [in the droplet] a little bit longer,” she advised.

THAWING EMBRYOS
They use the same protocol as they do for the aspirated or punctured embryos when thawing them. They warm the 1M sucrose solution and the microscope plate to 42° C uncapping the Cryolock and plunging it straight into the thawing media, for 1 minute.

Then they move it through the dilution solution that is at room temperature, which has a slightly lower level of sucrose to make sure it does not expand suddenly. Again, just as they did with the freezing protocol, they use 2 droplets and move the embryo from 1 to the other—leaving it in each droplet for about 2 minutes each, but a total of 4 minutes.

Then they move it to 2 droplets of the hold solution again for a total of 4 minutes at about 2 minutes a drop.

She admitted that most people do not warm the plate and solution up to 42° C. “The reason I started doing this is I read something about vitrification of some semen, and they were doing it at 42° C and got better results. We have compared doing it at 37° C versus 42° C, I haven’t really seen any big differences. So, it may be just that there isn’t really a benefit to doing it at this slightly higher temperature. I don’t have enough data to be able to say yay or nay,” Dr. Wilsher explained.

So, far—and she admitted it was early days—they have seen an 82% pregnancy rate with this new protocol, which was done in 11 embryos that were smaller than 300 µm.

“These have never posed a problem to us anyway—vitrifying them before without puncture. They’ve always been very successful,” she said.

When they used the protocol for larger embryos—300 µm to 500 µm—16 out of 20 resulted in pregnancies, so an 80% pregnancy rate without puncturing or aspirating the embryos.

However, the best results were among those that were no larger than 480 µm, she said. “The largest embryo that survived was 480 µm.

“If you are over 480 µm, basically you’re going to have to puncture it because It’s not possible to do it with this method at the moment,” she said, although they are still tinkering with the protocol in the hope that they can improve the odds for larger embryos. MeV

 

On left, 460 micron embryo prior to vitrification. The embryo after 4 minutes in equilibration solution.
Courtesy of Dr. Sandra Wilsher