Page 170 - January 2018

ICSI fertilization takes place outside the mare, using a special injection microscope.
embryos to get a 45-day pregnancy. After 45 days, most of those mares stay pregnant just like embryo transfer mares or mares that are naturally bred. The loss rate after 45 days is not any greater in embryo transfer mares or in ICSI recipient mares. Getting to 45 days is the tricky part,” he says.
“At of the end of August 2017 we’d done 308 aspirations and averaged 7.5 eggs per aspi- ration, so we averaged about one embryo per aspiration. There is a lot of variation in mares in terms of how many eggs you get and how fertile they are and how well they are going to do. In general, we have to aspirate a mare twice to get a 45 day pregnancy,” says Beck.
“If we average 7.5 eggs per aspiration and do that twice, we end up with about 15 eggs. By the time you go through all the losses that occur every step of the way, we’ll probably get a pregnancy at 45 days because we’ve transferred two embryos—but only half of them will make it all the way. Those are the ballpark numbers and most people don’t understand the losses. When I call someone up and tell them we got 12 eggs, they are already deciding what they are going to do with 12 foals, but it doesn’t work that way. That’s the big picture in terms of expectations, with ICSI,” he says.
ICSI is different from simple embryo transfer. With embryo transfer it is unusual to get more than one pregnancy. “I’ve been doing embryo transfer for 25 years and there are only a few you get two from. We often get more than one with the ICSI procedure, especially
if we are doing it from immature eggs because there are so many eggs to work with. If you are working with mature, pre-ovulatory oocytes, you will only have one,” Beck explains. He recently did the procedure on a mare with a Walk Thru Fire ICSI breeding that produced five embryos to freeze from one aspiration of immature oocytes. One of the advantages of ICSI is being able to take ovaries out of dead mares and take the eggs from the ovaries and make babies. “It works best if we can get those ovaries within six hours or less after the mare dies. A mare at Louisiana State University at Baton Rouge died recently on the operating table during a surgery. By coincidence, the surgeon’s wife worked in a human ICSI lab. So, she took the eggs out of the ovaries of that dead mare and sent us the eggs and we got a pregnancy,” says Beck. “Usually people just send us the ovaries and we take the eggs out and inject them. Our percentages are less,
and get progressively less, the longer the time between death of the mare and us receiving the ovaries, but that’s another nice aspect of ICSI. I’ve been trying to inform the insurance com- panies because they are paying off on some of these very valuable mares. I tell them that if they can get the ovaries here, we will partner on the
world of reproduction because they frequently have more than one big follicle. If you are col- lecting mature oocytes you can sometimes get two or three from a warmblood because they often ovulate more than one. They also have
a lot of follicles, including many small ones. Warmblood mares tend to be very fertile and do well with ICSI in our experience. The mare we got nine embryos from was a warmblood and the owners were ecstatic. With Quarter Horses there’s usually only one mature follicle, sometimes two. When you aspirate a big follicle you only get one egg, and only get it about 75 or 80% of the time.”
If you miss it, you start over and have
to recycle the mare. The advantage of this method, however, is that those eggs are very fertile. Mother Nature has matured them and they are more fertile than the oocytes from immature follicles.
“The immature follicles are small and there are usually lots of them. The actual number will vary with the season and with the age of the mare, but we have aspirated as many as 33 eggs from one mare at one time,” he said. “You aspirate all the little follicles that you can go into, and you can get lots of eggs. An old mare may have only two or three follicles, but we can usually get at least one or two. We can always get eggs, but we still don’t know why the ICSI procedure doesn’t work on some mares. We’re still trying to figure this out.”
The immature eggs are not as fertile as those harvested from mature follicles. “We have to put them in maturation media and mature them. When they become as mature as the mature eggs that were ready to ovulate, we inject sperm into them,” Beck explains. “It takes 30 hours to mature these eggs and only about 60 to 70% of them respond. If you have 10 eggs you will lose three or four that didn’t mature,” says Beck.
“They keep dropping off. If we started with 10 and end up with seven, we inject all of those with sperm (one sperm for each egg). We put them in the incubator and only about half of them will start dividing (cleavage). Now, we are down to three or four eggs. It’s survival of the fittest.” he explains.
Those fertilized eggs have to keep dividing for the next seven days and become an embryo. “Out of those three or four, hopefully one or maybe two will make it. Once we have the embryo, we transfer it into a recipient mare.” he says. “Not every transfer results in pregnancy. We have about 80 to 85% pregnancy rate on transfers we do.”
ICSI embryos also have a very high rate of early embryonic loss—about three times higher than in embryos you flush out of the mare. This is somewhat related to the age of the donor mare. “About 20% of ICSI pregnancies terminate with early embryonic loss sometime before 45 days,” says Beck.
These are some of the things people don’t realize until they are trying to do it. “We gener- ally tell people that it takes seven immature eggs to result in an embryo, and it takes two
Fertilized eggs have to keep dividing for 7 days to become an embryo.
168 SPEEDHORSE, January 2018
equine health