ICSI Quarter Horse News – April 15, 2007

INFINITE POSSIBILITIES


Advances in technology make it possible to get an embryo from just one sperm and one egg.

Infinity. Eternity. To endure forever.

Generally, those words do not apply to living creatures – especially to horses, whose life expectancy is only about 30 years. However, for those on the cutting edge of technology in the equine industry, words like “living forever” could soon become a reality. That’s because new technology has been developed that could take equine breeding, which is already a high-tech endeavor, to an even higher level, perpetuating the genes of the best horses in the industry for years to come.

The technology is called Intracytoplasmic Sperm Injection – ICSI, pronounced “ick-see” – and involves taking just one sperm out of billions produced by a stallion and injecting it into one oocyte with the eventual result being an embryo, and hopefully, a foal.

In reality, “infinity” is an exaggeration. But certainly with the advent of advanced technologies such as cloning (producing another individual with the same genetics as the original) and ICSI (using only one sperm for fertilization of an egg), it is not far from the truth. The most likely scenario, however, is that ICSI could take the genetics of today’s top stallions so far into the future that stallions could possibly outlive
their usefulness and demand.

The idea of ICSI may be a new concept in equine reproduction, but it is nothing new for other species. In fact, humans have been using ICSI for in vitro fertilization for more than 25 years, and the method also has been successful in other species. So why has the use of this reproductive technology in horses lagged behind?

"We’ve done a lot of work with ICSI in the horse because traditional, standard in vitro fertilization does not work well in horses,” said Dr. Katrin Hinrichs, a professor in the department of veterinary physiology and pharmacology and holder of the Link Chair of Mare Reproductive Studies at Texas A&M University. “Standard IVF has been done in humans, sheep and goats, and all sorts of other species. With standard in vitro fertilization, you put the eggs and sperm into a dish, and the sperm do what they’re supposed to do – they penetrate the egg and fertilize it. But there hasn’t been any repeatable method to make that work in the horse."

So, when a stallion or mare suffers from decreased fertility, often due to age or illness, other methods become a necessary means of getting foals. When other techniques – such as artificial insemination, deep horn AI, video endoscope AI, embryo transfer or oocyte transfer – all fail, ICSI can be a viable, but expensive, option. It is also a valuable tool to a stallion owner whose horse dies suddenly, leaving little frozen semen behind for future use. And, while ICSI is often thought of as a helping hand for sub-fertile stallions, it can also be a blessing for fertility-challenged mares.

Researchers and leading equine reproductive veterinarians at both Texas A&M University and Colorado State University have been developing ICSI into a viable option for breeders. Though the technique is still in its infancy, there have already been foals successfully
born using the method.

THE PROCESS

“Intracytoplasmic Sperm Injection – ICSI – is very simple,” Hinrichs explained. “It is injecting one sperm into one egg.”

Of course, the process of selecting one sperm and actually injecting it into one oocyte is not quite as simple. The procedure requires quite a bit of set-up, as well as special microscopes to do the job.

“It’s an outgrowth of the oocyte transfer program,” said Dr. Edward Squires, a professor in the department of biomedical sciences animal reproduction and biotechnology laboratory at Colorado State University, and a leading expert on equine reproduction. “You’ve got to collect the eggs from the donor, which is an ultrasound-guided aspiration, so that part is pretty standard.”

The first step is obtaining an oocyte to inject. At Texas A&M, oocytes were obtained from mares at Texas slaughterhouses until a Texas court decision led to a cease in horse slaughter operations at those facilities earlier this year. Now, Hinrichs reports that ICSI research
has basically come to a grinding halt until other options become available. In the meantime, Texas A&M researchers have been collating their research data and publishing the results. At Colorado State, researchers don’t have access to slaughterhouse ovaries, but they’ve
been collecting oocytes from live mares on a routine basis for studies that have yielded positive results.

After eggs are collected from a donor mare, they are matured in an incubator to prepare them for fertilization. Meanwhile, the sperm is getting its own preparation.

“On a given day, for a research project we might have 20 oocytes that we want to inject, so we’d thaw one of the straws of semen and concentrate the sperm by centrifuging it, and then wash it to remove the sperm-freezing solution,” Hinrichs explained. “Then we would take a small portion of those sperm and put them in a little droplet on a microscope. We add a compound to make the fluid really viscous, which makes the sperm slow down so that you can actually pick them up with a tiny pipette. This is all done through the microscope, using a micro-manipulator.”

One sperm is picked up into the pipette, and with another holding pipette, an oocyte is held via vacuum. Then, an individual sperm is injected into an individual oocyte.

At Texas A&M, the ICSI procedure is done by Dr. Young Ho Choi, who developed a repeatable method of making ICSI work with horse oocytes. Dr. Choi works closely with Hinrichs and Dr. Dickson Varner in improving the procedure and making it a viable commercial option. At Colorado State, Squires and Dr. Elaine Carnevale, head of Assisted Reproduction Technology, have led the ICSI research. In the university setting, the initial process is the same, but there are differences between the procedures done at Texas A&M and those done at Colorado State after the individual sperm has been injected into an egg.

Hinrichs’ laboratory has developed a way to grow the sperm-injected eggs in an incubator for seven days, during which time they develop into a blastocyst that can be transferred into a mare via trans-cervical embryo transfer, which is a simple non-surgical procedure.

“We’ll do ICSI on those eggs and grow them to seven days in culture until they’re about the same stage as an embryo would be if you were using embryo transfer,” Hinrichs explained. “Then, we transfer them into the recipient mare through the cervix, non-surgically.

“It’s a lot less invasive, and in our environment, when we do research on mares, we can only do one surgery per mare – so if we wanted to have a recipient herd, every time we used one, we’d have to sell her and get a new mare. So that’s why we’ve had the real push here to do ICSI with early embryo development in the incubator in the lab, to get away from doing surgeries.”

Colorado State has a different approach. “With most of them, we do ICSI and then look at them the next day,” Squires said. “If they are two-cell embryos, and they look good, we’ll put them in the oviduct of the recipient. We put them in by flank incision. Under typical conditions with oocyte transfer, we’d put the egg back in the recipient by flank incision, and then we’d breed the recipient. So with ICSI, instead of breeding the recipient, we inject the egg [using ICSI], wait a day, and then put it in the oviduct.”

For researchers at Colorado State, the flank incision method has yielded positive results for the past five years, with pregnancies from 30 different mares in 2006. However, the researchers are looking to perfect a method for growing embryos in an incubator, and
have received a grant to research the use of a laparoscope in transferring the embryos into a recipient mare.

“I think there are some things that will change in the future,” Squires noted. “Really, the thing that needs to get more practical is how to put the egg back into the recipient mare once it’s been injected.”

GOING COMMERCIAL

Texas A&M and Colorado State have led the way in ICSI research, and as the technique is perfected, it is now going commercial. Colorado State offers it to commercial clients, and Joe Landers Inc., Weatherford, Texas, is offering the procedure as well.

Anne Buchanan, a graduate of Colorado State with a master’s degree in reproductive physiology, who spent four years working in a human IVF lab, does the ICSI procedure at Landers’ facility.

“The technique is very similar, so it was an easy crossover from humans to horses,” Buchanan said. “There’s a little bit of a difference in the equipment that we use because of the nature of the equine oocyte, but it’s pretty much the same procedure. I was in the equine and bovine lab at CSU, so my research was with horses and cattle, but again, it’s a very easy transition from one species to another in terms of reproduction.”

Buchanan spent most of 2006 doing research and perfecting the technique, and she said now they are ready to accept clients for the procedure on a limited basis.

“This year, we’re going to do some live mares,” she explained. “We did some transfers last year and had embryo development, so we’re pretty confident in our protocol and ready to start doing the procedure on some mares here. We have several stallions lined up and a lot of people who are interested in adding to that. This year, we’re going to keep kind of a limited roster so we don’t get ahead of ourselves.”

One of the stallions on the roster is Kay Floyd’s Freckles Playboy, the legendary cutting sire that died in 2003. Due to former AQHA regulations that prohibited the registration of foals conceived from frozen semen, very little of the stallion’s semen was frozen.

“When Joe mentioned freezing a little bit, I thought about my own self, that it would be fun to have a Playboy this year or next year,” Floyd
explained. “I wasn’t thinking about this.”

At first, Floyd was hesitant to use any of the precious little semen in an experimental procedure, but after seeing some successful results, she decided to go for it.

“We’re going to see what happens,” Floyd said with excitement in her voice. “At first when they started talking about this, I said no, we aren’t playing with Playboys! But, if the one sperm works, I thought well, why not? I’m excited about doing a little something with it.”

Much of the credit for the use of ICSI in a commercial setting belongs to Peter Stent, the owner of the late Docs Stylish Oak, which died of a heart attack in January 2005. Because of the unexpected death, Stent had relatively little frozen semen banked.

Anxious to carry on his great stallion’s legacy, he began to research ICSI and had the procedure done with some of the great horse’s frozen semen in 2006 at Colorado State, with successful results.

“What we’ve been doing is trying to work in a collaborative effort with Colorado State and with Joe in pushing the technology forward to make it more commercially viable,” Stent explained. “We’ve only done a few inseminations with Stylish, but I’m hoping to do more when we get the process to the point where we won’t be wasting semen.”

Buchanan noted that Stent’s efforts have made a great impact on her work at Landers.

“He’s been really instrumental in getting his technique out of the universities and into the commercial world,” she said. “A lot of the mares that we’re going to use this year belong to him and will be bred to Docs Stylish Oak. We are the only operation, outside the university setting, in the U.S. that is doing this commercially, and we’re really excited about it.”

Still, the procedure is very new, and Stent is adamant that at least a 50 percent success rate is achieved before moving forward with breeding more mares.

“We’re going cautiously here, and we feel like we’re making great progress,” Stent said. “We have one baby that was born at CSU. It’s very exciting. It’s good for us, but I think it’s good for the industry, too, to advance the use of ICSI.”

WHAT THE FUTURE HOLDS

For stallion owners like Floyd and Stent, the advent of ICSI makes the emotional loss of a valuable stallion easier to bear, knowing they can still get foals despite a horse’s death. However, the procedure is just as important for mares that have died unexpectedly. Though the science is progressing, the conception rate has been low for researchers at Texas A&M using the post-mortem ovaries.

“Right now what we’ve been doing is ICSI on oocytes that have been recovered from mares postmortem,” Hinrichs said. “If a mares dies, and the owners really, really want to see if they can get a foal from her, they’ll send ovaries to us. We recover the eggs, mature them in an incubator, and then we get a semen sample from the stallion they want to breed the mare to, and then do ICSI.”

Unfortunately, the procedure using client horses hasn’t been very successful for a variety of suspected reasons.

“We have not had a good success rate with the post-mortem ovaries from client horses,” Hinrichs said. “So far, we’ve transferred six embryos for five pregnancies, only one of which has gone on. We don’t know what to attribute this low rate to, because our
normal work is also with post-mortem ovaries, but those are from the slaughterhouse. We get ovaries from mares post-mortem at the slaughterhouse, bring them to our lab and then do exactly the same thing with them as we do with our client horse ovaries. With these ovaries, we get about a 30 percent blastocyst rate and 70 percent of those develop normally after transfer.”

Hinrichs sees three major differences in the slaughterhouse ovaries versus those of valuable client mares, the first of which being the time it takes to get the ovaries to her lab.

“In most of our cases, it has been over 16 hours [to get ovaries] from clients,” she said. “They’ve arrived the next morning, so that’s really detrimental to the ovary’s health.”

Mare health can also become an issue in recovering enough healthy oocytes for ISCI. “A lot of them were old and sick. A typical history
is that she’s a 21-year-old mare that’s had laminitis for three years and been down three months,” Hinrichs stated. “So it could just be the quality of the eggs is just not high anymore.”

Last, but not least, is the method of death. While slaughterhouse horses are euthanized via captive bolt, most client horses are euthanized with barbiturates, which the researchers at Texas A&M suspect also could have an adverse effect on the ovaries. The one pregnancy that did work from a client horse had none of the three variables (old age/poor health, delayed ovary delivery and drug-induced euthanasia) present.

“Interestingly, the one pregnancy that has gone on was from a mare that wasn’t euthanized,” Hinrichs noted. “She died on the table during surgery, and they got the ovaries to us within about six hours. She was a relatively young mare that had acute colic, so all three of those things didn’t apply to the one [pregnancy] that went on.”

Still, the overall success rate was very low.

“We had 22 oocytes altogether from this mare’s ovaries. Eleven of them matured and we did ICSI on them,” Hinrichs said. “We got two embryos from that for transfer, and one pregnancy. Even when using oocyte transfer with oocytes collected post-mortem, I think they’ve got about 17 percent efficiency. With post-mortem ovaries, you get oocytes from all the follicles you can see. These oocytes do as well as could be expected since you are using any size follicle, which could have been growing, on its way up, or on its way out – degenerating.”

Despite the low numbers from deceased client horses, Hinrichs is confident that it would be much higher using a live client horse.

“Now, if you were using one egg that a valuable mare was just about ready to ovulate, and you collected it out of her pre-ovulatory follicle, that should be a different story because that egg is perfect,” Hinrichs explained. “That egg was from a follicle that grew up, had all the stimulation it needed, and the follicle was going to ovulate. In other species anyway, the blastocyst development from those eggs, from pre-ovulatory follicles right before ovulation, is 70 to 80 percent. We haven’t done enough work in this area, and that’s something we’re going to concentrate on this year. We would like to do more ICSI with what we call invivo-matured oocytes, which are oocytes taken from a live mare right before they were going to ovulate. It should give us a lot higher blastocyst development.”

At Colorado State, researchers have been receiving client ovaries from deceased mares for the past five years with some encouraging results. According to Squires, one out of four shipped ovaries has resulted in a live foal.

Dr. Dickson Varner holds the Pin Oak Stud Chair for Stallion Reproductive Studies and is a professor in large animal clinical sciences at Texas A&M, and, like his colleague Hinrichs, is encouraged by reproductive advances made in equine studies during the past few years. His focus on sperm function, sperm preservation techniques and testicular health are particularly useful in the development of ICSI.

Varner helped to quantify what the ICSI technique could mean for sub-fertile or deceased stallions with little frozen sperm.

“Depending on the stallion, there could be anywhere from a billion to 20 billion-plus sperm in an ejaculate in a normal stallion. A sub-fertile stallion will produce anywhere from 100 million to 2 billion [per ejaculate], with a day or two of sexual rest between collections,” Varner explained. “The more you collect semen from them, the less sperm you will get. When these stallions start producing only a billion or even 500 million sperm on a daily basis, even though that sounds like a lot of sperm, those horses are generally sub-fertile. The average stallion will produce five to six billion sperm a day, which is 60,000 to 70,000 sperm per second.”

By using ICSI, a sub-fertile stallion could have enough sperm to inseminate several hundred mares or more with one ejaculate of semen. Even using deephorn or video endoscope insemination, often referred to as “low-dose insemination,” requires five million to 100 million sperm, making ICSI a much more viable option when sperm supply is limited by sub-fertility or death. “Most of the stallions you are concerned about are the stallions that are older, with reduced fertility and reduced sperm numbers,” Varner said. “In those stallions, we’re still looking at up to 1 [billion] to 2 billion sperm produced in an ejaculate."

“But the fascinating thing is, you take the sperm in one ejaculate from one of those stallions and conceivably, theoretically, hypothetically,
have enough sperm in that one ejaculate for maybe 100 million inseminations.”

– Dr. Dickson Varner

Looking at the numbers, it becomes easy to imagine a stallion’s genetic material being available for as long as frozen semen can be stored – up to thousands of years – much longer than any one human will live to see.

Varner’s research also has yielded positive results in using twice-frozen and diluted sperm with ICSI.

“We did an experiment where Dr. Varner collected a stallion, froze the semen by standard techniques, so there’s about 100 million sperm in each straw, and then thawed that, diluted it 100 fold, so made 100 straws out of one and froze it again. So, in one straw, we had a million sperm,” Hinrichs noted. “We thawed the straw, concentrated the sperm and then used that for the ICSI. We found that there was no difference in embryo development between that twice-frozen, diluted sperm and the original frozen sperm.

“That was a really important finding because one of the reasons to use ICSI is if someone only has a couple straws left of a really important stallion. That might give you maybe one chance at one pregnancy if you use it for standard insemination, but with ICSI, one straw could give you, theoretically, a thousand or more tries with ICSI.”

Additionally, the experiments of Hinrichs and Varner have proven that in some cases, even nonmotile sperm can be utilized, although with lower success rates.

“We used motile sperm that had been frozen twice, and we also used sperm that weren’t motile.” Hinrichs said. “The motile sperm gave us a normal blastocyst development rate, which in our lab is about 25 to 35 percent, and the non-motile sperm still gave us 13 percent blastocysts. So that was really exciting. That’s something that really needs to be looked at more, but certainly, some of these non-motile sperm from the twice-frozen semen could give us embryos.”

Hinrichs said it is difficult to freeze just one sperm, and while ultimately only one sperm is injected into an oocyte, many more are needed in order to select the most viable one for the job.

“You’d have to adjust the dose of ICSI to find the quality of sperm you need, with normal shape and hopefully with motility, so you’d probably require at least a couple thousand [sperm] in the straw,” Hinrichs explained. “You really couldn’t easily freeze just one sperm.”

Buchanan estimates that straws could be as small as 200,000 sperm per dosage, while A&M’s experiments, in order to be controlled, have used one million sperm per “ICSI dose.” Still, if a straw of a million sperm could be used versus a straw of 30 million or more sperm used in the video endoscope method, that is 30 more possibilities for a foal.

“If you only needed a million in a straw, you’d still end up with a lot of straws,” Varner said. “Semen is not going to be the limiting factor. You’re going to have enough semen to last a long time.”

Choi, Hinrichs and Varner have also produced an ICSI-derived foal from sperm that were freeze-dried, rather than stored in liquid nitrogen, so alternatives to conventional freezing may be on the forefront.

SUCCESS RAISES QUESTIONS

As with any new technology, as success becomes apparent, even more questions and potential problems can arise. For instance, sometimes fertility issues can simply be nature’s way of natural selection, with only the strongest surviving.

“A concern we also have with intracytoplasmic sperm injection and sub-fertile stallions is that they are sub-fertile for a reason,” Varner explained. “The sperm is built to deliver a package of genes to the oocyte, and it could be that there’s some disruption in the genes themselves in these sub-fertile stallions such that sperm can establish fertilization, but the genetic codes are wrong and the embryo could die early. Studies in humans, for instance, have revealed that damaged DNA in the sperm can result in reduced pregnancy rates, as well as early embryonic loss and problems in young children, such as susceptibility to disease. As we advance with these new techniques, we’re creating potentially new problems that need to be sorted out.”

Cost also can be a mitigating factor in deciding to use ICSI. Squires puts the cost for a pregnant recipient to be between $6,000 and $8,000 – making the procedure an option only for a select number of stallions and mares.

“I think it’s a technique that has a place in the industry for stallions that have died and people want to continue to use them,” Squires stated. “But, it will be a small number of foals that are born because it’s so expensive. You’re going to have to have the right mare and the right stallion to justify it.”

And one thing that everyone – Hinrichs, Varner, Squires and Buchanan – all agree on is that with ICSI, the supply of semen will most definitely outlast the demand for that stallion.

“With ICSI, we’re still talking about a finite supply of sperm, but realistically, it would last longer than anyone would want foals from that stallion,” Hinrichs said. “Hopefully, genetics are going to improve from generation to generation, so sooner or later, that stallion’s foals won’t be competitive with the new generation stallion’s foals.”

Varner compared today’s top stallions with those of the past.

“Theoretically, if Man O’ War were still available today, would people still breed to Man O’ War?” Varner asked. “It’s unlikely they would, just because of the progress that has been made in genetic lines. And, I would suspect when Smart Little Lena is the same age as Man O’ War [1917-1947], people will look back, smile and realize that he was very good in his day, but one would expect improvements in genetic lines to make him a caveman of sorts.”

Squires compared Smart Little Lena to current leading cutting sire High Brow Cat.

“I certainly agree that we should be making better horses instead of using the same ones forever,” Squires noted. “Look at High Brow Cat. He’s come along, and even though Smart Little Lena is still an extremely valuable horse, in some ways, High Brow Cat may have surpassed him in popularity.”

Buchanan, who is a cutter, noted that some of yesterday’s popular stallions might not be able to pack a powerful punch in today’s competitive world of showing.

“The only barrier is the stallion’s relevance in whatever sport they’re in,” Buchanan said. “Some older horses may not be relevant to the sport today because they’re not subscribed to all the incentives, and their sons of sons of sons are out breeding today as well. If someone is considering do this, they need to consider if the cross is relevant today.”

For Peter Stent, who still has mare owners calling toget a breeding to the late Docs Stylish Oak, the answer to the question of relevance is obvious. His stallion’s offspring are still on the winners’ charts and in the headlines. For others, however, it could be a tougher decision. When taking all the factors into consideration – time, money, mare care and the possibility of failure – the researchers predict that the use of ICSI will cater to a small number of breeders whose outcome will outweigh the expense.

“Embryo transfer is always the first line,” Hinrichs said. “If a mare is capable of becoming pregnant, and a stallion has enough good-quality sperm to where you can breed the mare, inseminate her and she’ll conceive, then do embryo transfer. It would only be in those cases in which the mare can’t produce an embryo, or you’re dealing with a stallion that only has a couple straws of semen left, or he’s had an injury and is not producing adequate sperm numbers, that you’d use ICSI.”

And, as with most new technologies that become available in the equine industry, only time will tell what impact intracytoplasmic sperm injection will have on the equine industry.