Embryo Transfer in the South American Camelids
by Paul Taylor
In the summer of 1998 a man named Bob Godke phoned our ranch in Montana. He said he had heard we knew how to do embryo transfer in the South American camelids. Godke, it turned out, was Dr. Robert Godke, Professor of Reproductive Physiology at LSU and world famous authority on advanced reproduction techniques. He was in Montana for a fly fishing holiday and decided to track down the rumor he heard at a meeting of the International Embryo Transfer Society (of which he is a past President).
Godke came to the Taylor Ranch for an afternoon of conversation which led to demonstrations and eventually to a full-blown cooperative project between LSU and the Taylors. He was quick to admit that he simply had not believed reports that an unpublished layman llama breeder, without any formal training in advanced reproduction, was doing embryo transfer in llamas with a high rate of success. Nobody else in the world had recorded more than a few successful embryo transfers in the lamas (less than 10 in total) and we were claiming more than one hundred live births.
The success of our llama embryo transfer program is due to a team effort. My wife, Sally, does most of the actual palpations, ultrasound scans and embryo flushes and transfers while I assist and take notes. Bob Godke realized the importance of this and began to refer to Sally as my "secret weapon". Teamwork, at least two people working closely together to manage the animals, do the technical work and keep accurate records, is essential for successful embryo transfer in camelids. Luckily for me, Sally enjoys this work and is very good at it.
When we began to investigate the state of advanced reproduction in camelids in 1992 we soon learned that embryo transfer in llamas and alpacas was considered to be nearly impossible and certainly impractical for any commercial purpose. There was nobody doing much research in this area. We had no intention of actually doing embryo transfer research ourselves, but we decided to investigate further. I subscribed to a scientific journal of animal reproduction, Theriogenology, which became my bible and my compass. I didn't realize it at the time, but Dr. Robert Godke was one of the editorial advisors for this publication.
We took a short-course in artificial insemination of cattle to learn the basics of rectal palpation. Rectal palpation, feeling of the uterus and ovaries through the elastic wall of the animal's rectum, is a very valuable tool for the study of reproduction. Rectal palpation is a window on the female reproductive organs and it is basic to the understanding of reproductive cycles and early pregnancy.
We set up a llama restraint chute and some tables in our garage and hired a local bovine ET technician, Jeanne Reyher, to show us how to do embryo flushes. In fact, this young woman was a big help to us, teaching us the basics of the technique as it is practiced in cattle and showing us where to get the supplies we needed, but after ten or twelve flushes she still had not found any llama embryos. It became obvious that we could not continue to pay the "reduced" fee of $100 per flush for a professional to make the 23 mile drive from Bozeman to our ranch two or three times a week. If we were to learn more about camelid reproduction and embryo transfer we would have to do most of the work ourselves.
We selected a group of ten normal female llamas to be held open for study. We began by palpating each of these girls every few days to learn how the ovaries changed during the natural cycle. At first we were very timid about palpating our llamas. After all, these animals were our friends, and almost all the female llamas we owned were quite valuable. We knew that rectal palpation had led to the rupture and subsequent death of several llamas, even when it was done by veterinarians experienced in palpating cattle, so we were very fearful that we would have the financial loss and guilt of killing one of our own females. In retrospect, after more than 10,000 rectal palpations of llamas, camels, guanacos, alpacas and even a vicuña without injury to any, this fear seems unwarranted. Still, we are as gentle and careful as possible in using this technique, and we know that our good record to date has required a little luck as well as skill and patience.
In the South American camelids, as almost any breeder/owner now knows, there is no true estrus or heat and normally no ovulation unless there is a breeding. Instead, these animals cycle continuously, starting with multiple small follicles developing on the ovaries. In llamas and alpacas these follicles grow at the rate of about one millimeter per day until the largest is about 7mm in diameter. At that point, all but the largest follicle are repressed and begin to shrink back to nothing. The dominant follicle continues to grow at the same rate until it reaches maximum normal size. In llamas this is usually about 12-14 millimeters in diameter. Alpaca follicles are slightly smaller, about 10-11 millimeters in diameter when fully developed.
The large dominant follicle persists at maximum size for several days before regressing (decreasing in size) at a rate of about 2-3 millimeters per day. Then the cycle begins all over again. Usually the new dominant follicle will develop on the opposite side from the previous dominant follicle. If it was on the left ovary in one cycle it will develop on the right ovary in the next cycle.
With rectal palpation we were able to feel all of this happening. It is possible to pick up the ovaries and turn them over in your fingers through the thin elastic wall of the rectum. With experience and practice we learned to judge the number and size of developing follicles quite accurately.
We began to try to pass a catheter through the cervix. This passing of the cervix is another key to embryo transfer. You must pass a Foley catheter, a rubber tube with an inflatable collar to hold the tip in position just inside the uterus, through the cervix in order to flush an embryo from the uterus of a donor female. If you recover an embryo you must pass the tip of an ET "gun", a long syringe containing the embryo, through the cervix of the recipient to deliver the embryo to its new home. We had read about all of this and we had practiced a bit on cattle during our AI course, but doing it in llamas was quite a different thing.
The cervix of a llama or alpaca is of small diameter and has internal bumps and deviations that make life very difficult for a novice trying to get into the uterus. Learning to thread the cervix consistently, using one hand in the rectum to massage and manipulate the cervix while the other hand guides the catheter or ET gun in the vagina, was the single most difficult and frustrating part of our education.
Initially, we wasted a lot of time trying to flush llama embryos at four, five or six days after conception as bovine practitioners do. We found nothing. Finally I decided to try some flushes ten or twelve days after conception. Still we failed to find anything that looked like the photos I had seen of early bovine embryos. Often with these later flushes we would find an elongated empty sac about 1mm in diameter and an inch or more in length that looked very much like a nylon stocking under the microscope. I assumed this was cellular debris from the wall of the uterus or maybe even from the lining of the oviduct.
Of course, this was an embryo, but we only realized that after several more months of flushing slightly earlier each time and finding, sometimes, a slightly smaller sac-like structure than the time before. When we flushed 8 days after the breeding we found that the empty sac-like structure was spherical, about 1mm in diameter. At 7 days the fluid-filled sphere of soft tissue was only about half a millimeter in diameter and there was a distinct dark spot evident on the surface.
The ova and early embryos of all mammalian species are enclosed in a capsule called the zona pellucida. Several days after fertilization the embryo breaks out of the zona and this process is referred to as hatching. Embryo transfer procedures in cattle (and in all other species I had read about) were done while the embryo is still contained in the zona. By this time I knew that we were recovering hatched embryos. The dark spot on the surface was called the inner cell mass and it was the part which ultimately developed into the new animal. All the rest, 99.9% of the elongated nylon stocking we saw in later-stage flushes, becomes the placenta.
Whenever we flushed earlier than 6.5 days after breeding we found nothing, and we recovered only hatched embryos when we flushed later than 6.5 days. It was evident that llama embryos, unlike those of other species, hatch in the oviduct and reached the uterus only after hatching. This was a stunning conclusion. Finally we had to accept that zona-intact embryos, at least in llamas, could not be recovered with non-surgical flushing of the uterus.
We were not really set up for surgical procedures and did not want to do this kind of surgery on our llamas in any case. Further, we were sensing a reaction against our ET research from other llama breeders who felt our work was stressful for the animals. Doing surgery to find early embryos was out of the question, so we decided to work with the hatched embryos we already knew how to retrieve non-surgically.
From that time onward things fell into place quickly. We were hooked on this line of research and made the decision to build a proper clinic and lab where we could do the work effectively. We bought a high quality stereomicroscope and began to look into the purchase of an ultrasound machine.
Dr. Walter Bravo, the well-known camelid researcher, came to Bozeman for a visit and he brought his own portable ultrasound machine for us to use for a couple of days. We had no problem learning how to use this machine because we already had the palpation skills and knowledge of the structures we were feeling (uterus, ovaries and follicles). All we had to do was point the ultrasound head at what we wanted to scan and see what it looked like on the screen. We placed an order for a new Aloka 500V ultrasound (about $20K at that time) while Dr. Bravo was still with us. When he departed he said, "Now I think you will have everything you need to do successful embryo transfer."
We began to synchronize our potential recipients with our donors by breeding them to a vasectomized male. The idea was to get the recipients to ovulate at the same time as the donors so they would be at the same stage of the cycle when the transfer was attempted. Soon we found we could accomplish the same thing with an injection of leutenizing hormone. This is the same hormone that all the camelids release naturally following a normal breeding and it causes the release of the ovum from a ready follicle at a predictable time after injection.
We did our first transfers in November and December of 1994 and soon saw the first evidence of success, an embryonic vesicle or fluid-filled space in the uterus of one of the recipients. This vesicle, which is the continuation of growth of the "nylon stocking" structure we had seen under the microscope after doing 10 and 12-day flushes, becomes visible on ultrasound about 14 days after conception. This was a momentous occasion for us, but we knew the real proof of a successful transfer would come when the fetal heartbeat started at about 30 days. When we finally saw a heartbeat in one of our transferred embryos Sally broke into tears. We had worked several hours almost every day for over two years and we had spent nearly $70,000 of our own money on the project without any guarantee we would ever succeed.
In rapid succession we passed several milestones in our research project. We tested many different formulas for culture medium until we were sure we had one that would sustain early llama embryos in the lab or anywhere else for at least two days. Two days is enough time for an embryo to be moved from almost anywhere in the world to almost anywhere else. We learned that early llama embryos are not sensitive to injury from chilling and can survive at any temperature between normal body temp and freezing.
We developed an effective protocol for superovulation of llamas, the production of multiple ova at the same time through the use of follicle stimulating hormone. We bred one of our superstimulated females to a male and produced multiple embryos at the same time. Embryos produced in this way are fraternal siblings. They have the same mother and father, but different combinations of their genes. Soon we took the next step, transferring these multiple embryos into multiple recipients, and produced the world's first llama triplets. Each of these babies was carried by a different surrogate mother, but they all resulted from multiple ova fertilized at a single natural breeding. We named them Kate, Duplicate and Triplicate, though they were quite different from each other in coat color and body style.
Up until this time we had used ordinary females as our donors for this work. Now we began to hold our best females open for a year or so at a time so we could get multiple babies from them in combination with the various great males we had at our disposal. If there was to be any compensation to us, any profit from this research, this was it. In this way we were able to evaluate several years worth of a promising female's production in one year. It was possible to see quickly which males made the best breeding combination with a given female and to learn of genetic defects or problem combinations as well.
We taught our embryo transfer technique to our partners in Chile and Argentina and started small pilot projects in both countries. Our Chilean partners, the von Baers of Temuco, built a small clinic on their ranch and their two daughters, both now veterinarians, received a grant from the government of Chile to do further research in camelid reproduction. In Argentina we learned how to do successful ET under field conditions, without shelter or electricity. In one three week period there I did twelve field transfers that resulted in ten babies
I was offered a seat on the prestigious Embryo Movement Subcommittee of the US Animal Health Association, where I rubbed elbows with and was adopted by the top people in the world of embryo transfer. These men and women helped us enormously with advice and encouragement. Dr. Tony Wrathall, Chairman of the Import/Export Committee of the International Embryo Transfer Society, helped us get a protocol for international movement of camelid embryos from the OIE, an international body that provides guidelines for animal health authorities in every country of the world.
It was through the USAHA connection that we were contacted by a young woman who was doing ET in racing camels for the Sheikh of Dubai. Her name was Lulu Skidmore, and she hoped to produce a cross between a camel and a llama. She was authorized by the Sheikh to invite us to come to Dubai, all expenses paid, to consult about our work in llamas and to assist in the capture and training of llamas running wild on the grounds of the Sheikh's desert palace. We didn't know what kind of conditions we would encounter, so we packed camping gear just in case. As it turned out, Lulu picked us up at the ultra-modern Dubai International Airport and whisked us off to the Dubai Hilton. The llamas turned out to be guanacos, but the hybrid project eventually succeeded (see Camel/Lama Cross).
One of the most gratifying uses of our knowledge of embryo transfer came when some friends of ours in the llama breeding business asked for help with an expensive female who was unable to carry a pregnancy to term because of a damaged cervix. We were able to help them get two babies by two different males transferred into two of their own recipient females. There have been many other instances when we could help other breeders with problem females, and we routinely offer advice and assistance to veterinarians across the country when llamas in their practices have reproductive problems. We always make the time to explain what we know about camelid reproduction to anyone who is interested because so little is published on this subject.
To lessen the worries of other llama breeders about the possibility that our work is stressful to the animals we invited all the breeders in our area, about forty in all, to come to our ranch for a barbeque and demonstration of an actual flush and transfer. Not every flush results in recovery of an embryo and not every transferred embryo results in a pregnancy, but it was more the handling and care of donors and recipients that we hoped to demonstrate. We were very pleasantly surprised when this first public embryo flush produced a beautiful, typical embryo, a miniature soccer ball, for all to view under the microscope. We went on to do the transfer, and everyone agreed that the work caused less stress to the animals than toenail trimming or grooming. This news passed around the llama community by word of mouth and the animal welfare concerns ended.
Luckily, this flush and transfer produced a pregnancy, but we could not have guessed the run of luck that would follow. The pregnancy went well and we invited the same breeders to return one year later to see the resulting baby. This embryo was from one of our best imported females bred to our best imported male, so we had reason to hope it would produce a better than average baby. When the baby was born we were relieved that it was quite handsome. It was a male, and we named him Demo.
Demo was one of the first ET llama babies accepted for registration by ILR under the intentionally burdensome rules then in effect. He was accepted into the '99 Spring Celebration Llama Sale and became the first ET llama ever sold at auction when he was 18 months old. His selling price was not very impressive, but the new owners loved him and thought he showed some promise. They began showing him in the Midwest under his new name, Toconao's Tocayo, and he eventually won a championship at an ALSA show. That made him eligible to compete against more than ninety other Champions in the ALSA National Finals, where, as a two year old, he was chosen 1999 National Grand Champion Heavy Wool Male!
Along the way we have studied semen collection, evaluation and freezing. We did some work with AI, artificial insemination, and produced two pregnancies using this technique. On several occasions we sent chilled llama semen to Lulu in Dubai for use in insemination of camels. It didn't work. AI in the camelids remains a low-percentage procedure despite years of work by many researchers, especially in South America.
Our work on selection and preparation of recipient animals is probably our most significant contribution to the science of embryo transfer. Some female llamas will carry almost every embryo transferred into them and others reject every transferred embryo. Learning how to identify the good recips and how to prepare them before the transfer was the real secret of our success to date. We now have a few veteran females who have carried an ET pregnancy to term for each of five successive years. Incidentally, the relationship between these surrogate mothers and their ET babies is indistiguishable from any normal mother/baby relationship. Neither the mothers nor the babies know or care that this is not a genetic bond.
Once we flushed an important embryo that was damaged, probably by the turbulence of the flushing liquid. It had a rupture in the surface of the sphere and we knew from past experience that it would certainly die unless the hole could be repaired. I was able to close this opening under the microscope using a scalpel to press and seal the open edges together. We transferred the repaired embryo and it developed into a normal healthy baby.
During the past four years I have spent hundreds of hours trying to develop a practical freezing technique for camelid embryos. These hatched embryos we work with are very sensitive to damage by the cryoprotectant solutions that are used effectively in freezing unhatched embryos of other species. Through our connection with LSU we are now collaborating with Dr. Stanley Leibo, the leading authority in the world on the freezing of embryos. With help from Dr. Leibo and Dr. Godke we expect to find a practical method for freezing camelid embryos in the near future. This will allow longterm storage of embryos and permit, finally, international trade in camelid embryos.
Throughout our journey into the science of embryo transfer and llama reproduction we have received visits from interested researchers from many countries. A team of scientists from the University of Saskatchewan came twice and stayed with us for several days each time while we provided them with oocytes, embryos and semen for study. Two important researchers from Argentina visited last summer to discuss how our techniques could be used to increase the population of vicuñas in their country. In these cases we have said that we were not interested in publishing in scientific journals and that our visitors were free to publish anything they learned without mention of our assistance. Recently, Dr. Godke convinced me that we should publish our basic embryo transfer protocol just to establish our credibility within the scientific community. In the January, 2000, edition of Theriogenology we have the first-ever scientific paper under our own names.
Lately we have taken an interest in cross-species transfer among the camelids. As evidence built that all the camelids are very closely related it seemed logical that they should be able to carry each other's transferred embryos. A friend loaned us a pair of alpacas and we quickly produced two alpaca pregnancies in llama surrogate mothers. Both of these pregnancies are proceeding normally and the babies, the first resulting from cross-species embryo transfer in the camelids, will be born in the summer of 2000. We expect them to be larger than normal at birth because of the extra room and nourishment available in the llama uterus, and they should have all the milk they can drink to help them get the most from their normal alpaca genetics. We have no intention to continue to produce alpacas babies carried by llamas, but we think the implications of this demonstration may lead to practical applications in some future scenarios.
Let me say that production of camelid babies through embryo transfer is expensive, difficult and time consuming. ET is useful and worthwhile to increase the production of a few special females and to obtain crucial breeding information about these females earlier than would otherwise be possible. It is not a commercially viable way to mass produce camelid babies. There is just too much hard work involved.
I now believe that international trade in camelid embryos can have a very positive effect on US breeders, not only by protecting them from market damage due to importation, but also by allowing them to market their own genetics, arguably the best in the world, to serious breeders in other countries.
I believe registry policies can and should be used to guide the use of ET and other advanced reproductive techniques in any species, but this can only work in the longterm if these policies are reasonable. Typically, in other species, embryo transfer has at first been rejected, then gradually accepted as a useful tool without any significant implications for the general market or the genepool. While we wait for this evolution of registry policies in the camelid species we continue, with great satisfaction, to learn more