Superovulatory treatment with exogenous gonadotrophins adversely affects the uterus through the disruption of the delicate balance of ovarian steroid (estrogens, progestins, androgens) secretion rates. To examine the uterine effects of this treatment, 189 animals were given 4, 20 or 40 IU pregnant mare serum gonadotrophin (PMSG) at 28 days of age and sacrificed every 24 h until day 10 (D10) post injection. To study the long term uterine effects, 12 rats were treated with 4 or 40 IU PMSG and killed on D30. The morphological and histological changes of control (4 IU) uteri mimicked those of the adult on a comparable time course from D2 to D5. Administration of superovulatory doses (2 0, 4 0 IU) of PMSG produced stromal hypertrophy by D2 and focal papillary hyperplasia of the luminal epithelia by D3. It is suggested that previous exposure to high levels of estrogen and androgens, secondary to superovulation, are possible causes for this pathology. Levels of 17B-estradiol following 2 0 and 40 IU PMSG treatment were significantly (p<0.005, p<0.05) elevated above those of controls from DI to early D3 and at D2, respectively. Androgen levels of both groups (20 IU, 40 IU) significantly (p<0.05, p<0.005) increased from baseline at DI to maxima by D2 and D3, respectively. In the 20 IU PMSG group, the hyperplasia gradually regressed after D3 and was absent by D10. The hyperplasia in the 40 IU PMSG group, however, had diffused by D6. It is suspected that preceding elevated levels of estrogen may be responsible for this progressive change. At D4, levels of 17B-estradiol reached a maximum, which was significantly (p<0.001) greater than those of controls and 20 IU PMSG treated rats. Between D6 and D10, the hyperplasia partially regressed. Examination of uteri from D30 revealed no evidence of pathology. In addition to these structural effects, superovulation induced secretion of a mucinous substance in both 20 IU and 40 IU PMSG groups at D5-D6 and D6-D7, respectively. These results suggest that abnormal changes in the uterine histology and metabolism may result following administration of superovulatory doses of PMSG. Although these dose-dependent alterations appear reversible, they may interfere with preparations associated with implantation and thus require further investigation. / Medicine, Faculty of / Obstetrics and Gynaecology, Department of / Graduate

Introduced cool-season grasses are dominant in Virginia's grasslands, but their high digestible energy and non-structural carbohydrate (NSC) levels pose a risk for horses prone to obesity and laminitis. Native warm-season grasses (NWSG) have lower digestible energy and NSC levels that may be more suitable for horses susceptible to laminitis. The overall objectives of this research were to 1) assess voluntary intake, toxicological response, and apparent digestibility of NWSG hays fed to horses; 2) evaluate the characteristics of three NWSG species under equine grazing; and 3) evaluate establishment strategies for NWSG and wildflowers in Virginia. For the first objective, a hay feeding trial was conducted with 9 Thoroughbred geldings in a 3 x 3 Latin square design. Voluntary dry matter intake of indiangrass (Sorghastrum nutans) and big bluestem (Andropogon gerardii) hays by horses were 1.3% and 1.1% of BW/d, significantly lower than orchardgrass (Dactylis glomerata), an introduced cool-season grass, at 1.7% of BW/d. Biomarkers for toxicity remained within acceptable ranges for all treatments. Apparent DMD did not differ among hays, ranging from 39 to 43%. Non-structural carbohydrate levels were below the maximum recommended concentration for horses susceptible to laminitis. For the second objective, a grazing trial was conducted comparing indiangrass (IG), big bluestem (BB), and eastern gamagrass (Tripsacum dactyloides) (EG) yields, forage losses, changes in vegetative composition, and effects on equine bodyweight. Nine, 0.1-hectare plots were seeded with one of the three native grass treatments, and each plot was grazed by one Thoroughbred gelding in two grazing bouts, one in July and another in September 2019. Indiangrass had the highest available forage, at 4340 kg/ha, compared with 3590 kg/ha from BB (P < 0.0001). Eastern gamagrass plots established poorly, and had only 650 kg/ha available forage during the experiment. Grazing reduced standing cover of native grasses in IG and BB treatments by about 30%, and trampled forage constituted 36-68% of groundcover in those plots after each grazing bout. Horses lost weight on all treatments, but tended (P=0.09) greater weight loss on the indiangrass treatment at 1.5 kg/d compared to 0.5 kg/d in the BB and EG treatments. For the third objective, three experiments were conducted to evaluate different strategies for establishing NWSG and wildflowers. The first experiment compared large grazed plots with or without a 2 oz/acre rate of the herbicide imazapic. Imazapic led to higher biomass and percent cover in plots seeded only with NWSG. For plots seeded with a mix of NWSG and wildflowers, imazapic reduced wildflower establishment and resulted in higher biomass and percent cover of weeds over the course of the experiment. The second experiment examined four rates of imazapic application for NWSG and wildflower establishment in small plots seeded with either NWSG or a NWSG and wildflower mix, and found biomass and percent cover of weeds was lowest at a 6 oz/acre rate, while NWSG biomass and cover did not differ between treatments. Wildflower establishment was again reduced by imazapic. The third establishment experiment compared four site preparation strategies for wildflower establishment and found tillage resulted in the most cover and biomass of wildflowers. / Doctor of Philosophy / Introduced cool-season grasses are dominant in Virginia's grasslands, but their high digestible energy and non-structural carbohydrate (NSC) levels pose a risk for horses prone to obesity and laminitis. Native warm-season grasses (NWSG) have lower digestible energy and NSC levels that may be more suitable for horses susceptible to laminitis. The overall objectives of this research were to 1) assess voluntary intake, toxicological response, and apparent digestibility of NWSG hays fed to horses; 2) evaluate the characteristics of three NWSG species under equine grazing; and 3) evaluate establishment strategies for NWSG and wildflowers in Virginia. For the first objective, a hay feeding trial was conducted with 9 Thoroughbred geldings in a 3 x 3 Latin square design. Voluntary dry matter intake of indiangrass (Sorghastrum nutans) and big bluestem (Andropogon gerardii) hays by horses were 1.3% and 1.1% of BW/d, significantly lower than orchardgrass (Dactylis glomerata), an introduced cool-season grass, at 1.7% of BW/d. Biomarkers for toxicity remained within acceptable ranges for all treatments. Apparent DMD did not differ among hays, ranging from 39 to 43%. Non-structural carbohydrate levels were below the maximum recommended concentration for horses susceptible to laminitis. For the second objective, a grazing trial was conducted comparing indiangrass (IG), big bluestem (BB), and eastern gamagrass (Tripsacum dactyloides) (EG) yields, forage losses, changes in vegetative composition, and effects on equine bodyweight. Nine, 0.1-hectare plots were seeded with one of the three native grass treatments, and each plot was grazed by one Thoroughbred gelding in two grazing bouts, one in July and another in September 2019. Indiangrass had the highest available forage, at 4340 kg/ha, compared with 3590 kg/ha from BB (P < 0.0001). Eastern gamagrass plots established poorly, and had only 650 kg/ha available forage during the experiment. Grazing reduced standing cover of native grasses in IG and BB treatments by about 30%, and trampled forage constituted 36-68% of groundcover in those plots after each grazing bout. Horses lost weight on all treatments, but tended (P=0.09) greater weight loss on the indiangrass treatment at 1.5 kg/d compared to 0.5 kg/d in the BB and EG treatments. For the third objective, three experiments were conducted to evaluate different strategies for establishing NWSG and wildflowers. The first experiment compared large grazed plots with or without a 2 oz/acre rate of the herbicide imazapic. Imazapic led to higher biomass and percent cover in plots seeded only with NWSG. For plots seeded with a mix of NWSG and wildflowers, imazapic reduced wildflower establishment and resulted in higher biomass and percent cover of weeds over the course of the experiment. The second experiment examined four rates of imazapic application for NWSG and wildflower establishment in small plots seeded with either NWSG or a NWSG and wildflower mix, and found biomass and percent cover of weeds was lowest at a 6 oz/acre rate, while NWSG biomass and cover did not differ between treatments. Wildflower establishment was again reduced by imazapic. The third establishment experiment compared four site preparation strategies for wildflower establishment and found tillage resulted in the most cover and biomass of wildflowers.

Due to the detrimental side effects of NSAID administration, such as gastrointestinal ulceration and renal papillary necrosis, there is a profound need for clinical pain relief in horses with long term orthopedic disease whereby gastrointestinal side effects are obviated. Acetaminophen is one of the most commonly used analgesic drugs in humans, and is readily available as an inexpensive generic over-the-counter preparation. Acetaminophen has a number of mechanisms of action that differ from NSAIDs, including actions on the serotonergic, opioid, endocannabinoid and lipoxygenase pathways. These alternate pathways may provide greater efficacy against chronic or neuropathic pain in equine patients. Acetaminophen was preferred by physicians over COX-2 and nonselective NSAIDs, even when those drugs were coupled with proton-pump inhibitors to reduce gastrointestinal side effects; due to cost considerations and the occurrence of adverse side effects from those drugs. In horses, acetaminophen has been reported to be efficacious as an adjunct treatment for laminitis in one pony, and was an effective analgesic agent when combined with NSAIDs in a model of inducible foot pain. However, no studies have been performed to validate a dose-response curve in horses. A study recently completed by our group demonstrated rapid absorption following oral administration of acetaminophen. Reported human therapeutic plasma concentrations were achieved within 30 minutes of administration, with no clinical or clinicopathologic evidence of adverse side effects after two weeks of repeated dosing. Dose simulation trials indicate that a change in dosage schedule may be required in order to provide adequate plasma concentrations. / Master of Science / The use of non-steroidal anti-inflammatory drugs (NSAIDs) such as phenylbutazone in horses is widespread, and can be associated with detrimental side effects such as gastrointestinal ulceration and kidney damage. The clinical need for pain relief in horses with long-term lameness that minimizes gastrointestinal side effects has led to the development of cyclooxygenase-2 (COX-2) selective NSAIDs, such as firocoxib, but the expense of this therapy is often a major consideration limiting its use and few alternatives are available. Acetaminophen is one of the most commonly used analgesic drugs in humans, and is readily available as an inexpensive generic over-the-counter preparation. Despite the lower efficacy of acetaminophen in trials of human patients with chronic osteoarthritis, acetaminophen remains the preferred analgesic in humans due to its increased tolerance and improved cost-benefit analysis when compared to nonselective and COX2 selective NSAIDs. Acetaminophen has a number of mechanisms of action that differ from the current mainstays of equine analgesic therapy, which may provide greater efficacy against chronic or neuropathic pain in equine patients. A recent study of acetaminophen in horses has shown rapid absorption and achievement of levels reported to be therapeutic in humans, with no adverse side effects after two weeks of repeated dosing. In horses, acetaminophen has demonstrated efficacy as an adjunct treatment for laminitis in one pony, and was an effective analgesic agent when combined with NSAIDs in a model of inducible foot pain.

Determinations of cardiac output (CO) by M-mode echocardio-graphy were compared with simultaneous determinations by thermodilution in 2 conscious and 5 anesthetized horses. A range of cardiac outputs was induced by use of a pharmacological protocol (dopamine, 4 ug/kg/min, dobutamine, 4 ug/kg/min, and 10 ug/kg detomidine plus 20 ug/kg butorphanol, in sequence). Changes from baseline CO in response to each drug were evaluated, and data was analyzed to determine whether there were any interactions between drug treatment and measurement method. The mathematical relationship between CO as determined by M-mode echocardio-graphy (COecho) and as determined by thermodilution (COTD) was described and used to predict COTD from COecho. The 2 methods were compared with respect to bias and variability in order to determine the suitability of COecho as a substitute for COTD . Sources of the variability for each method were determined. Determination of CO by either method in standing horses was prohibitively difficult due to patient movement. The pharmacologi-cal protocol was satisfactory for inducing a range of cardiac outputs for the purpose of method comparison; however, use of dopamine did not offer any additional benefit over the use of dobutamine and was generally less reliable for increasing CO. Inclusion of detomidine provided an additional change in CO but did not increase the overall range of CO over that produced by halothane and dobutamine. COecho and COTD were significantly related by the predictive equation COTD = (0.63 +/- 0.157) x COecho + (16.6 +/- 3.22). The relatively large standard errors associated with COecho measurements resulted in a broad 95% prediction interval such that COecho would have to change by more than 100% in order to be 95% confident that the determined value represents true hemodynamic change. COecho underestimated COTD by a mean of 10 +/- 6.3 l/min/450 kg. The large standard deviation of the bias resulted in broad limits of agreement (-22.3 to +2.3 l/min/450 kg). Measurement-to-measurement variability accounted for 28% of the total variation in COTD values and 64% of the total variation in COecho values. Results might be improved if the mean of 3-5 consecutive beats was used for each measurement, but as determined in this experiment, COecho is too variable to have confidence in its use for precise determinations of CO. / Master of Science

Establishment and maintenance of a successful pregnancy requires signaling from the embryo to the mare, a process known as maternal recognition. Six days after fertilization, the trophectoderm (TE), a placenta precursor is formed. Signals emanating from the TE to the uterine environment are critical to maternal recognition of pregnancy. The identity of factors necessary for this process remain unknown. A novel equine induced trophoblast cell line (iTr) that closely mimics the genotype and phenotype of native equine TE was created. Transcriptome analysis of iTr revealed increased expression of growth factor (GF) receptors for Epidermal GF (EGF), Hepatocyte GF (HGF), Fibroblast GF-2 (FGF-2) and Insulin GF (IGF-1), suggesting these GF may be important targets during TE development in the early embryo. We hypothesized that treatment of iTr cells with these GF would induce changes in cell proliferation and expression of genes likely involved in maternal recognition. The objectives of this experiment were to evaluate the effect of these GFs on iTr mitotic response and regulation of genes involved in steroidogenesis. Equine iTr cells (n = 3) were cultured with 10 ng/mL EGF, HGF, FGF-2 or IGF-1 for 24 hr, with 5-ethynyl-2'-deoxyuridine (EdU) supplementation during the final 2 hr. Subsequently, cells were fixed and EdU positive and total nuclei were enumerated. A parallel plate of iTr cells was treated in a similar manner and lysed for total RNA isolation. Quantitative PCR using gene-specific primers for CYP11A1, PTGS2, PTGES2, and PTGES3 was performed. Data were analyzed by ANOVA with Tukey's post hoc adjustment using the GLM procedure of SAS. Treatment with EGF, FGF-2, HGF, and IGF-1 increased (P < 0.05) iTr proliferation from control levels of 25.33 ± 1.03% to 38.58 ± 1.61%, 45.50 ± 2.94%, and 38.23 ± 2.01% respectively. The 2-&#916;&#916;CT method was used to calculate the fold change (FC) using GAPDH as the reference gene for normalization. Expression of CYP11A2, PTGES2, and PTGES3 was not affected by GF, as measured by qPCR. By contrast, PTGS2 transcript abundance increased (P < 0.05) following FGF-2 (FC = 3.327 ± 0.8291) and HGF (FC = 11.88 ± 4.572) treatment. These results indicate that FGF-2 and HGF may simultaneously induce proliferation and prostaglandin production by TE cells. The combined results of these experiments will improve our understanding of TE morphogenesis and its response to uterine-derived growth factors. / Master of Science / Establishment and maintenance of a pregnancy requires that the mare uterus recognize the presence of the embryo, a process known as maternal recognition of pregnancy. The trophectoderm (TE) are cells on the outer layer of the embryo formed six days after fertilization, which later give origin to the placenta. The TE sends signals from the embryo to the uterus, that are very important for the mare’s recognition of the embryo’s presence. The specific nature of these signals are still unknown in the horse. A cell line (iTr) very similar in aspect and genes to the horse’s native TE has been created in our laboratory. A set of comparative assays have showed that, during the developmental stage of maternal recognition, both the horse TE and the iTr cells share significant identity, and have receptors for the same set of growth factors (GF), suggesting these GF are important for early embryo development and potentially involved in the signaling process of maternal recognition. We proposed that treatment with these GF would induce iTr cells to proliferate and express signals likely involved in maternal recognition in horses. The objectives of this experiments were to evaluate the effect of EGF, HGF, FGF-2 and IGF-1 on iTr cells by measuring proliferation and cellular mechanisms of maternal recognition already established in in other species. Equine iTr cells were cultured with different GF and right before analysis a fluorescent dye that stain dividing cells was added in order to measure the proliferation. Equivalent cell cultures were used to evaluate if the treatment affected the production of hormones involved in signaling maternal recognition. Treatment with all GF induced higher cell proliferation, but HGF also increased the production of one enzyme that participates in producing a very important hormone (prostaglandin E2). The combined results of these experiments add to our understanding of maternal recognition in horses.

There are conflicting results from previous research evaluating the effects of dietary omega-3 fatty acid supplementation on IgG concentration of colostrum, milk and foal blood. No research has been done on the effect of omega-3 fat supplementation on the placental efficiency of horses or the nitrite concentrations of the placenta as an indicator of vascularization. This study examined the effect of dietary omega-3 supplementation on composition of milk, mare and foal serum and if it will result in transfer of fatty acid in utero as well as providing the foal with adequate IgG concentrations in the milk and colostrum. Omega-3 supplementation’s effect on concentration of glucose and insulin in blood of the mares and foal will also be determined. This study will determine if omega-3 supplementation has an effect on the placental efficiency or nitrite concentrations in the placenta.

The suspensory apparatus of the distal phalanx (SADP) is an intricate adaptation of dermal and epidermal tissue that has a specialized role in the horse to absorb concussion while suspending the weight of the horse from within the hoof. The integrity of the hoof-bone connection is critical to the health of the horse and it can be affected by numerous disorders that cause it to fail. Accurate data on the ultimate tensile strength and other material properties of the SADP are important in modeling the behavior of the tissue under load and selecting appropriate prevention and treatment strategies for disorders of the SADP. The tensile load orientation and tissue sample size both have a profound effect on obtaining representative estimates for the material properties of a tissue. Consideration of the collagen fiber axis is important when selecting both. The purposes of this investigation were 1) to morphometrically determine the true collagen fiber axis in the SADP, 2) determine the most appropriate test sample size and tensile load orientation for materials testing, 3) to determine the ultimate tensile strength and other material properties of the SADP in healthy digits of adult horses, 4) to determine the site of tissue failure during testing, and 5) to determine whether freezing the SADP tissue samples prior to testing alters the material properties. Results of this investigation indicated that the true collagen fiber axis of the SADP in the toe region was predominantly vertical, in alignment with gravity. A vertical tensile load most closely matched the collagen fiber axis and was appropriate to model the load in the SADP for standing horses. A radial tensile load was appropriate to model the load in the SADP near the break over portion of the stride. Tissue blocks with a proximal-to-distal dimension of 1 cm tested less than 30% of the vertically oriented collagen fibers during radial tension testing and significantly underrepresented the ultimate tensile strength. The tissue failed in the deep dermis during radial tension testing and at the dermo epidermal junction during vertical tension testing. Freezing the tissue prior to testing significantly reduced the ultimate tensile strength.

Three 14 day trials were used to compare granulated paper(GP) to pine pellets(PP) and wood shavings(WS) as bedding for horses. During days 1 through 5 stalls were cleaned daily of feces only, and stalls were cleaned of both feces and saturated areas from days 6 to 14. For stall cleanliness, PP was cleaner than both GP and WS. Initial pH was greater for both GP and WS compared to PP. The water holding capacity for WS was greater than PP and GP. On day 5 there was ammonia detected for WS during trial 1. GP and WS emitted more ammonia than PP when air flow was restricted. Bacteria found in the nasal cavity were not different among bedding types. GP had more fungal growth. Minerals were found within the maximum tolerance of horses. Results indicate the use of GP as a bedding material for horses has potential.

Traditional methods of blood glucose monitoring involve obtaining samples for measurement via laboratory methodology or point of care devices and require invasive collection techniques such as capillary stick, venipuncture, or the placement of intravenous catheters. Limitations of traditional methods include the limited information provided by intermittent testing and the stress associated with restraint and discomfort experienced by patients. The snapshot nature of the provided information restricts a clinician's ability to truly monitor trends in glucose concentrations over an extended period of time, influencing clinical decision making. The stress of invasive sampling can cause stress hyperglycemia in many veterinary species, complicating interpretation. Continuous interstitial glucose monitoring technology is widely used in the human medical field due to the expansive information provided in a minimally invasive manner. In recent years, the device technology has advanced and cost has improved, prompting application of these devices into the veterinary sector. Studies have shown good agreement between newer glucose monitoring systems and traditional methods in small animal patients with diabetes mellitus, allowing veterinarians to obtain comprehensive glucose data with minimal stress and discomfort to the patient. However, information regarding the use of this new technology in equine medicine is limited. The following study describes the evaluation of two widely available glucose monitoring systems, the Dexcom G6 and the FreeStyle Libre, in healthy adult horses. / Master of Science / Monitoring of glucose concentrations is essential for the diagnosis and monitoring of a variety of disorders within equine medicine. Traditional methods of obtaining samples for testing include capillary stick, venipuncture, or the placement of intravenous catheters, which can cause stress and discomfort to equine patients. The information obtained by this testing methodology only allows for intermittent assessment of glucose concentrations, limiting the amount of information available for clinicians to make clinical decisions. The use of continuous glucose monitoring systems in the human medical field have allowed clinicians to obtain continuous or near-continuous glucose concentrations, improving interpretation. These devices have nearly eliminated the need for blood sampling for glucose concentrations, instead relying on interstitial glucose concentrations which have been shown to compare favorably to blood concentrations. Studies in small animal veterinary species, such as dogs and cats, have shown good agreement between newer glucose monitoring systems and traditional methods in small animal patients with diabetes mellitus, allowing veterinarians to obtain comprehensive glucose data with minimal stress and discomfort to the patient. However, information regarding the use of this new technology in horses is limited. The study described in the manuscript following evaluates the use of two widely available glucose monitoring systems, the Dexcom G6 and the FreeStyle Libre, in healthy adult horse.

Current industry practices promote therapeutic use of pulsatile stimulation plates to increase muscle mass, strengthen bone density, reduce stress, and improve overall athletic performance of horses. The first objective of this study was to investigate the effect of vibration stimulation on muscle thickness of the extensor carpi radialis, extensor digitorum longus, gluteus medius, longissimus lumborum, semitendinosus, supraspinatus, and longissimus thoracis, as well as circumference and cross-sectional area of the extensor carpi radialis and extensor digitorum longus. The second objective was to evaluate changes in nutrient foramen thickness, circumference, and area, as well as dorsal cortical thickness of the left third metacarpal in response to vibration stimulation exposure over 56 d. Increases in thickness of the topline muscles and improvement of nutrient foramen parameters of treatment horses give evidence to conclude that vibration stimulation may be a viable therapeutic treatment for stock-type horses on stall rest.