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Dr. Katrin Hinrichs--Setting the Standard in Assisted Equine Reproduction

Posted June 05, 2017


Dr. Hinrichs with staff and students in the reproduction lab

Dr. Katrin Hinrichs, professor and Patsy Link Chair of Mare Reproductive Studies at the Texas A&M College of Veterinary Medicine & Biomedical Sciences (CVM), grew up riding horses as a hobby. As an adult, she is internationally recognized for her research in equine reproductive physiology and for overseeing one of the few labs in the world capable of performing intracytoplasmic sperm injection (ICSI), a process that has now become the standard in assisted reproduction in horses.

A more complex and precise form of in vitro fertilization (IVF), ICSI is the only process that can efficiently produce a fertilized equine embryo outside of a mare’s body. Hinrichs’ research ultimately led to success and improved efficiency in ICSI, a goal that once seemed unreachable for equine reproduction researchers.

In the last year, Hinrichs was named a Texas A&M Regents Professor and awarded the third Simmet Prize in Assisted Reproduction by the International Congress on Animal Reproduction. Her other achievements include producing the first cloned horse in North America, named Paris Texas, as well as other cloned foals to aid in her research on the application of cloning equids. Although she faced a number of challenges throughout her journey, Hinrichs’ work paved the way for the clinical and research application of many forms of assisted reproduction in horses.

The Early Years

As a child, Hinrichs was infatuated with horses. When she was nine years old, her mother purchased the family’s first horse. Soon after, Hinrichs got a horse of her own and recalls working with horses ever since. She and her mother kept the horses in a nearby boarding stable until they moved from Orange County to northern California, where they were able to keep their horses in their backyard. “I owned a mixed-breed horse named Tico. When we moved up to northern California, we were right next to El Dorado National Forest,” Hinrichs recalled. “My mother and I used to go out and ride for miles in the forest. It was a great way to grow up.”

It was this passion for horses that motivated Hinrichs to become a veterinarian. “I’ve always been a horse fanatic. I always wanted to be a veterinarian growing up,” she said.

Hinrichs began her journey to veterinary school as a biochemistry major at the University of California (UC), Davis with an intention of attending veterinary school. Although biochemistry encompassed many of her interests, it did not seem to align with Hinrichs’ true passion.

“In my undergraduate studies, I kept telling people, ‘I want to know how muscles work,’” Hinrichs explained. “I started out in biology, switched to zoology and still did not learn about how muscles work. Then, I thought, ‘What I need is biochemistry,’ but that’s not what I needed either.”

After taking her first physiology class at the UC Davis School of Veterinary Medicine in 1974, Hinrichs realized that physiology was what she had wanted to learn about all along.  “What I wanted to learn was physiology, but I didn’t even know what the word ‘physiology’ meant at that time,” she said. “I kept trying to find it until I got into veterinary school.”

The Road to Research

Like many veterinary schools at the time, the students in the School of Veterinary Medicine at UC Davis were primarily male. “I specialized in equine medicine my last year, and out of the 13 people on the equine track, I was the only woman,” Hinrichs said. But, being the only woman in her field of study at UC Davis and one of few women in her graduating class did not deter her.

Hinrichs graduated from veterinary school in 1978 with the intention of becoming an equine practitioner; however, she was not the kind of applicant that practitioners with open equine positions were looking for. “The fact that I was a woman really got in the way because nobody wanted to hire a woman as an equine practitioner back then, so I got a job in a mixed-animal practice in northern California,” she said. “However, I was disappointed in the work; it was using only a small amount of what I had learned in veterinary school. I wanted to be a veterinarian all my life, and when I became one, I wasn’t happy.”

Things would soon change for Hinrichs. “I had a friend who went to the University of Pennsylvania as a visiting scholar in reproduction,” she said. “He wrote me and said, ‘I think you would like it here,’ so I applied for the large animal residency there. As soon as I started in academia, I knew that was exactly where I belonged because people were trying to be the best they could and learn the most they could.”

Hinrichs pursued a Ph.D. in comparative medical sciences at the University of Pennsylvania after she completed her residency studying the hormonal requirements for pregnancy in the horse and equine oocytes—unfertilized egg cells. After earning her Ph.D., she took a faculty position in reproduction at Tufts University in Massachusetts.

“My position at Tufts was an interesting change, because during my time at the University of Pennsylvania, I was in a strong and active section of equine reproduction. The section was probably one of the strongest in the world at that time,” Hinrichs explained. “Then, I went to Tufts, and I was the only equine reproduction faculty member. That was a very different environment. Luckily, I was able to get some money to do my research—and luckily there was tissue available.”

After moving to Massachusetts, Hinrichs married and had two daughters. At the urging of a colleague at Texas A&M, she applied for an open physiologist position at the CVM in 1998. She recalled her excitement over the seemingly endless possibilities in research Texas A&M offered. “I came to Texas to interview for the position, and it was amazing,” Hinrichs said. “Texas A&M helped me see the light. It was like being in a stall and then being released out into a pasture; there were so many more resources and opportunities here. I was really fortunate to get the position.”
Testing the Possibilities

When Hinrichs accepted the offer to teach physiology at the CVM she began researching cloning and reproduction. She brought along Dr. Young Ho Choi as a collaborator, who began as a post-doctoral trainee and is currently a senior research scientist. The two began exploring the possibility of producing a fertilized equine embryo in vitro, a basic research and clinical tool in other species that was not yet feasible in the horse.

In conventional IVF, sperm are placed in a dish with a mature oocyte, and one sperm penetrates the oocyte to fertilize it. Given ideal laboratory conditions, the egg will develop into an early embryo, which can be transferred to a recipient female for further development. However, traditional IVF had yet to be successfully achieved in horses. Unlike other livestock species, such as cattle, horse eggs and sperm do not seem to respond to traditional IVF methods. Therefore, Hinrichs and Choi took a different approach by looking into a more complex assisted reproduction method: ICSI.

In ICSI, a single sperm is manually injected into the cytoplasm, the fluid that fills the cell, of the mature oocyte. It is then placed in an incubator in hopes that fertilization occurs and an early embryo can develop. The embryo can then be transferred into a recipient mare’s uterus for gestation. Because so few sperm are needed, in theory, a single straw of frozen semen from a valuable stallion can produce thousands of offspring. This means that deceased stallions can continue to reproduce so long as they have provided frozen sperm. Mares who are no longer able to reproduce naturally, but still produce healthy oocytes, can also continue to produce offspring through ICSI.

Overcoming Challenges and Moving Forward

To see if the ICSI process could even be performed in their lab, Choi began working with a micromanipulator and a powerful microscope that allowed for manipulation of the horse oocyte. Using the micromanipulator, Choi was able to hold the oocyte in place and inject a single sperm into the egg through a pipette. This marked the beginning of a journey toward successful assisted reproduction for horses in vitro; but the rest of the journey would not be easy.


Dr. Hinrichs with a student in the lab

After Hinrichs and Choi discovered that the ICSI process could be successfully performed to fertilize a horse oocyte in the lab, the next challenge was to provide the ideal conditions for an early embryo to develop—a goal that would take over two years to reach. “We could put the sperm into the oocyte, but we did not have the right environment for it to develop in vitro,” Hinrichs explained. “IVF had never worked in horses, so nobody had produced early equine embryos in the laboratory, so no one had done any studies on how you culture an equine embryo to get it to develop.”

Promising results led to research support in the form of grants, which were instrumental in the success of Hinrichs’ ICSI program. More funding led to more research, and Choi and Hinrichs discovered that an equine embryo needed a complex medium to grow for seven to 10 days until it could be placed in a recipient mare. After more successful attempts at producing early equine embryos in vitro, Hinrichs and Choi were able to move on to perform the process clinically.

“It took a couple of years for us to develop a method where an embryo could develop in vitro to the point where we could transfer it to the uterus of a recipient mare to make a pregnancy,” Hinrichs said. “It turns out the developing equine embryo needs a lot to survive; it needs a complete cell culture medium. Luckily, you can buy a cell culture medium at the cell culture store. It’s got everything in it a cell would ever need.”

The clinical ICSI program quickly became successful. In 2015 Hinrichs and Choi performed over 450 procedures on oocytes from valuable client-owned mares. A large part of this demand is due to low semen supplies of stallions who are deceased or too old to reproduce any longer. In comparison to other forms of assisted reproduction, such as artificial insemination, ICSI is more efficient in these cases. For example, artificial insemination of a mare with frozen semen could potentially take several straws of sperm to produce a pregnancy, while for ICSI, one straw of frozen sperm can be thawed and diluted so that it yields enough doses to perform hundreds of ICSI procedures.

In addition to external grants, Hinrichs credits The Patsy Link Equine Research Endowment Fund as playing a major role in the success of her and Choi. “The Patsy Link endowment was what funded us to keep researching ICSI so that we could get the process to work,” Hinrichs said. “This helped fund Dr. Choi’s salary and our supplies in the laboratory. Recently, the clinical income has started to replace the Link funding, freeing up money to support other equine research programs at Texas A&M.”

Increasing the efficiency of ICSI solved many challenges associated with assisted reproduction in horses. The research and time Hinrichs and Choi devoted to successfully performing ICSI also aided advancements in another aspect of assisted equine reproduction: cloning.

Cloning: The Next Step

Cloning, a process that has been successful in many species, such as cats, cattle, and deer, was also a goal of Choi and Hinrichs’, and the findings from performing ICSI helped to advance their work on cloning. In reproductive cloning, researchers recover unfertilized eggs from mares, and eliminate all DNA from the eggs. They then collect a tissue sample, usually skin, from the valuable donor horse, and culture cells from the sample. The DNA from the donor cell is then transferred through a needle into an egg that had its own DNA removed. Given the right laboratory conditions, an early-stage embryo can develop and will be placed into a mare’s uterus for further development. The resulting foal has the same genetics as the donor horse—an “identical twin” born years later.

Hinrichs funded her research in cloning through research agreements with private individuals who wanted to support advancements in this area and have cells from their horses used in the work. While reproductive cloning offered an opportunity for Hinrichs to further study the biology of the horse oocyte and early embryo, it also lead to her interests in endangered exotic equids.

Hinrichs hopes that her cloning research can eventually assist in saving endangered equids, such as Grevy’s zebra, by producing fertilized cloned embryos in the laboratory and then allowing the cloned animal to develop in a recipient mare. The cloning process would aid populations with extremely low numbers and low genetic diversity by cloning deceased or old individuals that had not reproduced in that population.

“A lot of people wonder why you would clone a horse,” Hinrichs said. “I’ve always been enthusiastic about cloning as a way to save endangered species or even endangered rare breeds. In fact, I have been in contact with people who work with rare breeds of horses who want to work with me on cloning and ICSI because there are only a few specimens left of certain breeds. For me, a major application of cloning is for saving endangered equids.”

A New Project: Saving the Northern and Southern White Rhino

In addition to hoping to save endangered equids, Hinrichs recently became involved with a project initiated by the San Diego Zoo that aims to replenish the populations of African northern and southern white rhinoceros. Only three northern white rhinos, which live in Kenya, remain in the world, while about 20,000 southern white rhinos remain in Africa. In the United States, there are approximately 100 southern white rhinos kept in captivity.

“I got involved with this project through my membership in the International Embryo Technology Society,” Hinrichs said. “At our annual meeting, there was a day on which the society had a course on exotic animal reproduction. One of the people that was at the course, Dr. Barbara Durrant, Director of Reproductive Physiology and Henshaw Chair at the San Diego Zoo, asked me if I wanted to meet with her to talk about assisted reproduction in the rhinoceros.  When we spoke, she invited me to a meeting they were planning at the Zoo’s Institute for Conservation Research in which scientists and conservationists from around the world were going to brainstorm on approaches to saving the rhino.”

It was no coincidence that Hinrichs was approached about assisted reproduction in rhinos. Although most people would not consider a domestic horse similar to a rhino, horses and rhinos are actually in the same family. Despite differences in physical appearance, the horse is accepted as being the best animal model for assisted reproduction in rhinos. Due to the threat of extinction in both the northern and southern white rhino, the San Diego Zoo has started an initiative to use assisted reproduction to save the two species, and Hinrichs’ work in the horse may pave the way for possible methods to accomplish this goal.

Since the 1970s, the San Diego Zoo has been collecting and freezing cells from the zoo’s deceased animals. This “frozen zoo” includes northern white rhino sperm and skin cells, and many other cells from animals that have died in zoos across the United States. By collaborating with Hinrichs, the San Diego Zoo hopes to produce rhino embryos through ICSI and cloning. However, there are many challenges in the way of this goal.

“Scientists in this project are trying to develop a way to get the eggs from rhinos that are still alive,” Hinrichs explained. “The rhino is so big that traditional methods of collecting oocytes, as used in other species including horses, can’t be performed. In addition, shipment of exotic animals and their sperm and cells is getting more difficult because of government regulations, so we cannot receive eggs from rhinos that die, say, in South Africa. This results in scientists trying to manage the population of southern white rhinos in the United States to maximize their numbers and genetic diversity, as a fallback if every single rhino in Africa is poached.”

Hinrichs and her research team are exploring the idea of how ICSI and cloning could play a key role in saving the northern and southern white rhino. Because of the genetic similarities between the rhinos, Hinrichs said it is highly likely that a northern white rhino’s DNA could develop normally inside a southern white rhino’s oocyte. “If we develop a way to get oocytes from the live southern white rhinos in the United States, then we could use the eggs for two purposes: first, we could increase the genetic diversity in the United States southern white rhino population by using southern white rhino sperm in the frozen zoo to produce southern embryos through ICSI. Second, we could use the southern white rhino eggs as “host” eggs, to clone northern white rhinos.” For embryos produced by both ICSI and cloning, the southern white rhino would serve as a recipient; but there is an additional challenge of finding a way to transfer the embryo without surgery to the female.  The size of the rhino makes both obtaining oocytes from female rhinos and placing them into a recipient a problem, one to which the rhino conservation group is now trying to find solutions.

“Obtaining oocytes is a big problem, and so is putting the embryo back into the rhino,” Hinrichs explained. “Nobody knows how to do either procedure, because the methods that we use in domestic species won’t work. Not only are rhinos too big for these methods, their cervix, the pathway between the vagina and the uterus that you have to get through to put the embryo into the uterus, is spiral. You also can’t perform surgery on rhinos easily, because their skin doesn’t heal well, so we would rather not place embryos in surgically.”


ICSI-in vitro produced blastocyst stained for three different cell types (Image courtesy of Dr. Katrin Hinrichs)

The San Diego Zoo’s initiative to save the northern and southern white rhino is a challenge, but Hinrichs and her research team are motivated and believe the methods of ICSI and cloning they have developed in the horse have the potential to save both species from extinction. “Every rhino is precious and irreplaceable,” Hinrichs said.

Hinrichs Honored for Her Research

In recognition for her efforts in assisted reproduction, Hinrichs will be awarded the third Simmet Prize in Assisted Reproduction at the International Congress in Animal Reproduction in 2016 in Tours, France. The award recognizes researchers for outstanding basic and applied research in the field of assisted reproductive technologies for animal production. The Simmet Prize is the most generous award of its type in the world and is presented every four years.

“I never thought I would actually be awarded the Simmet Prize, especially for work in horses, because there are some amazing researchers out there,” Hinrichs said. “However, my research is more applied and perhaps that was my strength. I have been able to do the research and then translate it to a successful clinical program in assisted reproduction in the horse. Maybe that is what interested the award committee.”

In addition to the Simmet Prize, Hinrichs has been recognized as a Texas A&M Regents Professor for her teaching, research, and service. As the highest honor in the Texas A&M University System for faculty members, the award recognizes faculty that have had a positive impact on their institution, their community, the state of Texas, and the nation.

As a newly recognized Regents Professor and Simmet Prize winner, Hinrichs looks forward to making even stronger connections with her students in the classroom, where she teaches reproductive physiology to first-year veterinary students. In addition, she hopes to continue advancing the study of equine assisted reproduction and cloning while contributing to her institution, community, and world through her research efforts.

What is in vitro fertilization?

Commonly referred to as IVF, in vitro fertilization refers to having the process of fertilization—that is, the combining of a sperm and a mature oocyte, or unfertilized egg—occur outside of the body in the laboratory. In standard IVF, an egg is placed with sperm together in a dish, and one sperm must penetrate the egg. Under the right conditions, an early embryo can develop. If the process is successful, the embryo is then transferred into the uterus for further development. Although this process has been successful in many species, it has not had repeatable success in equine species because the sperm do not penetrate into the egg.

What is ICSI?
Intracytoplasmic sperm injection (ICSI) involves manually inserting a single sperm into a mature oocyte via a pipette. This produces a fertilized egg, and if the laboratory provides the right conditions, an early embryo can develop. In theory, only one sperm is needed for each egg, so ICSI provides a method by which numerous offspring can be produced from a small store of frozen sperm. This process has proved successful for assisted reproduction in horses and Texas A&M is home to one of the world’s few laboratories that can successfully perform this procedure.

What is cloning?
In reproductive cloning, researchers remove all the DNA from a mature oocyte. Scientists collect a single somatic cell, any cell except sperm and eggs, from the donor animal. The DNA from the somatic cell is then transferred through a needle into the egg that has had its own DNA removed. Given the right laboratory conditions, an early-stage embryo can develop and will be placed into a mare’s uterus for further development.


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