Eight veterinarian students from Lincoln Memorial University (LMU), in Harrogate, Tennessee, spent their summer collaborating with researchers in the University of Kentucky (UK) Department of Veterinary Science in Lexington. Each student focused on a specific project and then reported on their project during an LMU Student Research Presentations day at the end of July at the UK Veterinary Diagnostic Laboratory.
Ana Weiland completed her summer project, “Non-invasive Method for Sexing Equine Embryo During Embryo Transfer,” with Alejandro Esteller-Vico, PhD, assistant professor, in the reproductive health laboratory at the Gluck Equine Research Center.
Embryo transfer involves flushing an embryo from one, often valuable, mare and transferring it to a recipient mare’s uterus. This procedure is becoming common in most breeds except for Thoroughbreds (The Jockey Club still prohibits any type of assisted reproductive techniques in Thoroughbred breeding). It has been helping increase the number of foals from mares that are in competition or having reproduction problems. Weiland’s project examined whether embryos obtain any genetic material before it is transferred into the recipient mare.
She said current methods of finding gender determination, such as an ultrasound, are not ideal because they’re not useful until too far along in the pregnancy. Current preimplantation genetic diagnosis use a micromanipulator to biopsy the trophectoderm (outer layer of the embryo), but her team recently began using the micromanipulator to sample the blastocoel fluid (which contains the embryo’s DNA), which is not yet an established method but is expected to help the team determine how much DNA/RNA is present in free fluid within the embryo.
She said, however, this method is not practical and expensive, which is why the laboratory is looking into other non-invasive methods for obtaining this genetic material. Researchers conducting studies in humans and mice noticed genetic material (DNA/RNA) leaking into the culture media during in vitro embryo culture. The researchers then used the DNA and RNA for PGD (pre-implantation genetic diagnosis) and sexing. Weiland and colleagues would like to mimic this procedure in equine embryos, which are much larger than mice or human embryos.
Potential PGD uses include avoiding detrimental diseases such as HYPP (hyperkalemic periodic paralysis disease). Weiland said the study could help with selecting traits in the foal such as sex, size, color, and gait.
“My favorite part of the summer was discovering the many ways to collect the embryo depending on the size embryo, as it changes drastically with rapid growth, doubling in size almost every two days with each week,” she said.
Brittany Jones completed her summer project, “Combination Equine Deworming vs. Multi-drug Resistant Parasites,” with Martin Nielsen, DVM, PhD, Dipl. ACVM, associate professor, Schlaikjer professor of equine infectious disease, in the Gluck Center’s parasitology laboratory.
She used a herd of Miniature Horses from UK’s Maine Chance Farm that has been maintained since 1974 and harbors multidrug-resistant parasites. The Nielsen laboratory collected fecal samples from the herd every two weeks. They used those fecal samples to look at combination deworming treatments and parasite resistance. The study established baseline efficacies of the dewormers being evaluated, and Jones’ role was to establish the efficacy of the first combination treatment of these. Her preliminary results indicated no differences between the tested deworming regimens.
Jones said this study is important because there are not many dewormers on the market and equine parasites are becoming more resistant to the ones available. There are also no new products coming on the market, so researchers must find a way to use the current drugs without promoting further resistance.
In addition to doing her research project, Jones also helped with various other projects and assisted Ashley Steuer, graduate research assistant, with diagnostic samples from dogs, cats, bears, and others received at the UK Veterinary Diagnostic Laboratory.
Jones also attended the AAVP (American Association of Veterinary Parasitologists) conference, which took place July 22-25 in Indianapolis, Indiana. While there, she had the opportunity to visit the Indianapolis Zoo, tour its veterinarian hospital, and talk to the zoo veterinarians about parasite problems.
Kaitlin Walton completed her summer project, “Effects of Endocrine Disease on Cytokine Production in Geriatric Horses,” with Amanda Adams, PhD, assistant professor, in the Gluck Center’s immunology laboratory. Her work focused on PPID (pituitary pars intermedia dysfunction), also known as equine Cushing’s disease.
“PPID affects 20% to 30% of all horses as they age, and more than 85% of these horses are 15 years old or older,” she said.
This disease is caused by a decrease in dopaminergic inhibition, which increases the activity of the pars intermedia and leads to increased adrenocorticotropic hormone (ACTH) levels. Because of ACTH’s effects, PPID horses are commonly thought to have decreased immune function. Older horses are being used more for competition and breeding purposes, which makes it essential for researchers to better understand how older horses’ immune systems function.
“I have been able to see a lot of things, such as blood collection for basal ACTH and TRH (thyrotropin-releasing hormone) stimulation testing, and have also been able to help with other projects, including oral sugar tests at the farm, and PBMC (peripheral blood mononuclear cell) isolation and ELISAs in the lab,” Walton said.
Nikolette Birky completed her summer project, “The Affect of Donor Age on the Osteogenic Potential of Equine Bone Marrow Stem Cells,” with James MacLeod, VMD, PhD, John S. and Elizabeth A. Knight chair, in the Gluck Center’s musculoskeletal science laboratory. She also worked with Jasmin Bagge, graduate research assistant, in MacLeod’s laboratory.
Birky compared bone marrow stem cells in foals, yearlings, seniors, and geriatric horses, focusing on optimizing an assay related to the synthesis of bone matrix. Bone marrow stem cells are multipotent, meaning that they can create several different cell types. The cells for her project were harvested from donor horses’ sternums.
The clinical significance of this study is that stem cells used to treat joint disease and other equine health conditions can potentially be collected from the patient itself. However, the importance of age as a variable is not well understood. Collecting stem cells from the patient might work well in a young horse, but could have lower success in an adult horse, and might not be a viable option in an older animal. There is much to learn and Birky’s research focused on stem cell performance as a function of donor age.
“I have learned several new skills that I can take back with me to veterinary school, such as cell culture, sterile technique, and how to discuss a subject without getting stuck on the heavy medical terminology,” Birky said.
Sofia Santacaterina and Natasha Marzolf completed their summer project, “Effect of Stabling on Surfactant and Plasma Lipidomic Profiles in Yearling Horses,” with David Horohov, PhD, chair of the Department of Veterinary Science, director of the Gluck Equine Research Center, and professor; and Undine Christmann, DVM, MSc, PhD, Dipl. ACVIM, associate professor of veterinary medicine at LMU, in the immunology laboratory.
Inflammatory airway disease, or mild equine asthma, is thought to be under diagnosed in horses because clinical signs present themselves after the disease has already begun its course. Clinical signs include coughing and overall decrease in performance. Dust and allergens associated with barn environment often promote the disease process. Santacaterina’s and Marzolf’s study focused on comparing the effect of barn environment or pasture environment on surfactant and plasma lipidomic profiles in young horses.
Previous studies have found an overall decrease in phospholipid content of surfactant in horses with asthma and an increased level of cyclic phosphatidic acid in horses with asthma with clinical signs.
“I didn’t have a lot of lab experience coming into this so it was great to work in a lab and see those kind of procedures,” Santacaterina said.
Marzolf echoed that statement.
“I didn’t have a whole lot of horse experience, other than what we were taught in veterinary school, so I really enjoyed being able to work with the horses,” she said.
Staci Palmer completed her summer project, “The Effect of Regumate (altrenogest) on the Immune Function of the Open Mare,” with Barry Ball, DVM, PhD, Dipl. ACT, Albert G. Clay endowed chair in equine reproduction, professor, in the reproductive health laboratory, and Adams in the immunology laboratory. She was mentored by Carleigh Fedorka, PhD, a postdoctoral scholar.
The effect of progestins on the immune response has been studied in humans and in mice. Palmer’s study primarily focused on cytokines (inflammatory mediators). Studies on humans and mice have revealed that when females receive progestins, pro-inflammatory cytokines decreased.
Palmer said Regumate is the most common progestin given to horses. It is used in pregnant mares to increase cervical tone and decrease myometrial contractility and in the nonpregnant mare to synchronize or suppress estrous cycles.
Tatiana Fraguela completed her summer project, “Magnetic Resonance Imaging of the Suspensory Apparatus in Thoroughbred Racehorses,” with Jennifer Janes, DVM, PhD, Dipl. ACVP, assistant professor and anatomic veterinary pathologist, and Laura Kennedy, DVM, DACVP, assistant professor and anatomic veterinary pathologist, at the UK Veterinary Diagnostic Laboratory.
Fraguela said catastrophic musculoskeletal injuries are the leading cause of equine deaths at racetracks. Suspensory apparatus failure is the most common catastrophic injury occurring in North America. The paired proximal sesamoid bones, suspensory ligament, intersesmoidean ligament, and the distal sesamoid ligaments are components of the suspensory apparatus. This apparatus functions as an energy-storing device to prevent excessive fetlock hyperextension.
Previous work has shown pre-existing bone and cartilage lesions at the sites of catastrophic injury, which supports the theory that these injuries are the result of cumulative stress and fatigue rather than a single “bad step.” Like stress fractures, catastrophic injuries can occur unilaterally, but the underlying disease is typically bilateral. Therefore, it is informative to examine both forelimbs when performing the postmortem examination. Fraguela spent her time comparing postmortem MRI studies between racehorses that were euthanized for suspensory apparatus failure and racehorses euthanized for reasons not related to suspensory apparatus failure. She evaluated a variety of bone and soft tissue parameters. Analysis of these parameters and identification of possible regions of interest is underway.
“The prospect of studying these type injuries and hopefully helping to prevent these kinds of injuries are exactly why I got into veterinary medicine,” Fraguela said.
Katie Lampert is a marketing and communications intern at the University of Kentucky Gluck Equine Research Center.