Master of Science / Department of Animal Sciences and Industry / Teresa L. Douthit / Abruptly increasing concentrate in the ration of horses results in altered cecal dynamics which can culminate in digestive distress. Nine Quarter horses previously fitted with cecal cannulae were utilized for 3 consecutive 22-d experiments, each separated by 2 d of rest. During Exp. 1 and 2 horses were acclimated to the same ration for the initial 21 d of each period, followed by a concentrate challenge on d 22. The acclimation ration consisted of a morning meal of 0.5% BW concentrate (Omolene 200, Purina Animal Nutrition, LLC, Gray Summit, MO) fed with 1.5% BW prairie grass hay divided evenly between a morning and evening meal. On d 22 of Exp. 1, horses were fed a morning meal consisting solely of 1.0% BW concentrate while 1.25% BW concentrate was fed on d 22 of Exp. 2. Cecal samples were obtained through cecal cannulae from d 19 to 22 of each experiment every 4 h for h 24 following the morning meal each day. Cecal pH during Exp. 1 was recorded and decreased at h 12 following the concentrate meal on d 22 in comparison to cecal pH at h 12 on d 19 to 21 (P = 0.009). During Exp. 2 cecal pH increased at h 4 (P = 0.02) and decreased at h 12 and 20 (P < 0.0001) following this oncentrate challenge compared to cecal pH recorded at the same time points during the acclimation period. Experiment 3 differed from that of Exp. 2 only in the respect that during the acclimation period horses were fed, in addition to the acclimation ration, either a proprietary supplement (n = 5) or a placebo (n = 5). Cecal samples from d 19 to 22 were analyzed for pH, concentration of lactate, and concentration of VFA. Horses consuming the supplement had increased cecal pH at h 4 (P = 0.009), concurrently decreased cecal lactate (P = 0.02), increased ratio of (acetate+butyrate)/propionate at h 8 and 16 (P ≤ 0.006), and decreased VFA concentration at h 24 (P ≤ 0.05) compared to horses in the control group following the concentrate challenge.

Sarcopenia is a condition that is most common in aged animals, and is characterized by the loss of skeletal muscle mass and integrity, and can lead to physical disability and poor quality of life. Since skeletal muscle protein synthesis can be limited by the availability of amino acids, supplementation of limiting amino acids to ameliorate the progression of sarcopenia has become a topic of interest in companion animal research. Although there is some data to support the idea that amino acid supplementation improves maintenance of muscle mass in aged horses, the cellular mechanisms behind that improvement have yet to be elucidated. Therefore, the objective of this study was to examine the effect of amino acid supplementation in aged horses on markers of whole body and muscle protein metabolism. In a cross-over design, six old horses were studied while receiving each of three treatments in a replicated Latin square design. For all three treatments, horses received 1.8% BW/d of timothy hay cubes and 0.5% BW/d of experimental concentrate. The three treatments included a control (CON) treatment concentrate that was designed to meet all requirements of mature horses when fed in combination with the timothy hay cubes, and two supplemented concentrates, LYS/THR with additional lysine and threonine (40 mg/kg BW/d and 31 mg/kg BW/d, respectively), and LYS/THR/MET with additional lysine, threonine, and methionine (40 mg/kg BW/d, 31 mg/kg BW/d and 11mg/kg BW/d respectively). In each 15 d period, following a 9-day adaptation, horses were fitted with a collection harness, and total urine and feces were collected for 72 hours for assessment of nitrogen balance and creatinine output. Blood samples were taken directly before feeding and 30, 60, 90, 120, 150, 180, 210, and 240 minutes post-feeding for analysis of plasma urea nitrogen (PUN), glucose, insulin, and plasma amino acid concentrations. Muscle biopsy samples were taken for analysis of proteins in the mTOR pathway. Additionally, horses underwent stable isotope infusion procedures, and comparisons of phenylalanine kinetics were used to determine whole-body rates of protein synthesis and degradation. There was no significant effect of treatment on creatinine output (P=0.58), relative abundance of proteins in the mTOR pathway (P>0.05), nitrogen retention (P=0.70), or phenylalanine kinetics (P>0.05). PUN concentrations were significantly (P=0.0058) higher for LYS/THR and LYS/THR/MET than for CON. Atrogin-1 activation was significantly higher for the pre-feeding CON sample compared to the post-feeding CON sample. Lack of significant difference in creatinine output suggests that there were not significant differences in muscle mass between treatments. Lack of significant differences in mTOR protein activation suggests that amino acid supplementation did not result in improvements in protein synthesis. Lack of significant differences in nitrogen retention and phenylalanine kinetics suggests that whole-body protein metabolism was not improved. Additionally, higher PUN concentrations in the supplemented diets suggests that the supplemented amino acids being provided were catabolized. However, increased activation of Atrogin-1 in the pre-feeding CON samples, but not the pre-feeding samples of supplemented treatments, suggests amino acid supplementation may have reduced protein degradation in the post-absorptive state. Data from the present study suggests that amino acid availability may not have been limiting protein synthesis in the sedentary aged horses in the present study.

The amino acid requirements of growing horses are currently unknown, and studies suggest that threonine is a limiting amino acid in common horse diets. Thus, the objective of this study was to determine the threonine requirement of growing horses fed two different forage to concentrate ratios using the indicator amino acid oxidation method. The study consisted of a high concentrate phase (HC; 60% concentrate and 40% forage) and a high forage phase (HF; 25% concentrate and 75% forage). Within each phase, 6 female yearling Thoroughbred horses were randomly assigned each of 6 dietary treatments in a 6 x 6 Latin square design. All 6 treatments were identical, apart from varying equimolar ratios of threonine to glutamate. After 6 days of adaptation, blood samples were collected before and after the morning meal for plasma urea nitrogen (PUN) and amino acid analysis. On day 7, horses underwent the IAAO protocol, during which regular breath and blood samples were collected. Phenylalanine flux, oxidation, non-oxidative disposal, and release from body protein, as well as total carbon dioxide production were calculated using plateau enrichment of samples. There was a significant linear effect of threonine intake on plasma threonine concentrations, and PUN had a significant linear response during the HC phase. There was no significant effect of treatment on phenylalanine oxidation during either phase (P ≥ 0.05). It is unlikely that threonine was limiting in the experimental diets.

Skeletal muscle protein synthesis is regulated by the mammalian target of rapamycin (mTOR) signaling pathway. The first objective was to optimize the methodological procedures for assessing mTOR signaling in horses. The response of mTOR signaling (P-Akt Ser473, P-S6K1 Thr389, P-rpS6 Ser235/26 & 240/244, and P-4EBP1 Thr37/46 by Western blotting techniques) to meal consumption was determined at three gluteal muscle biopsy depths (6, 8, and 10 cm), and the repeatability of the contralateral side at 8 cm during 5 days of repeated biopsies. There was no effect (P > 0.05) of sampling side or biopsy depth on mTOR signaling in mature horses. During repeated biopsies there was an increase (P < 0.05) in downstream (P-S6K1 Thr389, P-rpS6 Ser235/236 & 240/244 and P-4EBP1 Thr389) mTOR signaling in response to feeding. The second objective was to characterize alterations in mTOR signaling throughout the equid lifespan. Adolescent horses (yearlings and two year olds) studied in the postprandial had a lowered (P < 0.05) activation of downstream mTOR signaling with aging. There was a lower (P < 0.05) abundance of P-S6K1 Thr389 in aged horses (23.5 years old) than in mature horses (11 years old) during the post-absorptive state. The final objective was to assess mTOR signaling during acute and chronic inflammation. Acute inflammation occurred during 5 days of repeated biopsies, and chronic inflammation is characteristic of the aged. During acute inflammation, characterized by increased muscle mRNA expression of inflammatory cytokines, there was an increase (P < 0.05) in downstream mTOR signaling. Chronic inflammation resulted in a decrease (P < 0.05) in the abundance of P-S6K1 Thr389. Phenylbutazone was administered to reduce (P < 0.05) acute and chronic inflammation in muscle. Phenylbutazone administration during acute inflammation reduced (P < 0.05) the activation of downstream mTOR signaling and trended to increase (P = 0.09) P-S6K1 Thr389 abundance during chronic inflammation. Whole-body protein synthesis determined using isotope infusion techniques increased (P < 0.05) when chronic inflammation was reduced due to phenylbutazone administration. This research provides new standards for muscle biopsy collection when examining mTOR signaling, and insight into management and feeding practices for adolescent and aging horses.

Obesity is the most prevalent nutritional disorder encountered in small animal medicine. Problems related with obesity are the higher incidence of morbidity and mortality. Nutritional and physical activity interventions have been common strategies employed; however, they have shown low compliance rates. Because of it more attention has been given to the nutrient composition of diets. Using the canine model, three experiments were conducted to examine the effect of fish oil or barley on protein and lipid metabolism, as well as postprandial glycemia, and nutrient digestibility in mature and in young adult dogs. In Exp. 1, seven female dogs were randomly assigned to one of two isonitrogenous and isocaloric diets, control (CO) or fish oil (FO), in a crossover design. Animals fed the FO diet tended to be more sensitive to glucose, showing a lower glucose half life. Cholesterol and HDL decreased (p<0.05) on the FO treatment. Overall, the supplementation of fish oil may improve glucose clearance rate and is effective in decreasing cholesterol in mature overweight dogs. In Exp. 2, eight female Beagles were randomly assigned to one of two isonitrogenous and isocaloric diets, control (CO) or fish oil (FO), in a crossover design. Overall, feeding a FO containing diet showed a protective effect against the rise of plasma CHOL and it increased plasma ghrelin levels. However, it did not appear to improve protein metabolism or postprandial glycemia in adult lean dogs. In Exp. 3, sixteen female dogs were randomly assigned to four experimental diets; control (40% corn) or three levels of barley (10, 20, 40%). The data suggest that inclusion of barley up to 40% in diets for adult dogs is well tolerated and does not negatively impact nutrient digestibility of the diets. However, inclusion of barley did not improve aspects related to fecal odor, postprandial glycemia, or plasma cholesterol. Overall, the research presented herein suggests that different nutritional strategies - dietary lipid or carbohydrate manipulation - may be beneficial in ameliorating health issues (e.g., hyperlipidemia) or in improving the health status of dogs (e.g., gut health by increased SCFA production).

The objectives of these experiments were to evaluate the effects of selenium (Se) and vitamin E (Vit.E) supplementation in maternal and progeny diets on the performance of breeder hens and the performance and meat quality characteristics of progeny. Inclusion of Se, as Se yeast, in the diets of developing broiler breeder pullets resulted in greater Se accumulation of Se (P<0.01) in liver, pancreas, and breast tissues than when Se yeast was not provided. Improving the overall Se status of breeder pullets in the early stages may help maintain adequate tissue Se concentrations during egg production. Maternal supplementation of Se yeast and Vit.E increased the liver and breast Se concentration (P<0.05) of newly hatched chicks compared to the chicks originating from hens not receiving dietary Se. At 7d of age, Se yeast supplementation in either the chick or maternal diet increased breast and liver Se concentrations (P<0.01). At 14d of age, breast and liver Se concentrations remained the highest for chicks supplemented with Se yeast (P<0.01), however there was no effect of maternal Se supplementation. Vitamin E supplementation in either the chick or maternal diets did not affect the liver Vit.E concentrations of chicks at 7 or 14d of age. Supplementing broiler diets with Se yeast and Vit.E improved the meat quality characteristics of raw and marinated breast fillets. The Se content of breast meat from broilers fed Se yeast was higher (P<0.01) than those from broilers that were not fed Se yeast. Antioxidant supplementation improved the drip loss (P<0.05) and oxidative stability (P<0.10) of raw breast fillets after 7d of refrigerated storage. Marination appeared to increase the susceptibility for lipid oxidation of the marinated breast fillets. Dietary supplementation of Se yeast and Vit.E reduced lipid oxidation (P<0.01) of marinated breast fillets after prolonged refrigerated storage, thus improving oxidative stability. Overall, dietary supplementation of Se yeast can increase the accumulation of Se in the tissues of broiler breeder hens and their subsequent progeny. Improvements in the avian antioxidant system may have beneficial effects on the performance of broiler breeder hens, broilers, and the meat quality characteristics of broiler breast fillets.

Estradiol (Compudose®, COM) implants are extensively used in beef cattle production systems to alter body composition and feed efficiency. Little information exists about the physiological mechanisms affected by COM treatment in growing, mature, and senescent female cattle. Moreover, no reports describe the level of blood estradiol resulting from COM treatment. The effect of COM on levels of plasma estradiol and blood metabolites and proteins, and relative content of glutamine synthetase (GS) and other amino acid nitrogen-metabolizing enzymes in liver tissue, was studied using three experimental models relevant to cow-calf production regimens: senescent cows (Trial 1), young mature (young) versus senescent (old) cows (Trial 2), and growing heifers (Trial 3). In Trial 1, plasma estradiol concentrations were 222 % more after 14 and 28 d in COM-implanted than sham implanted (Control) cows. COM treatment did not affect measured blood metabolites and enzymes, but increased hepatic GS protein expression by 350% after 14 d and 200% after 28 d of implantation. In contrast, protein expression of alanine transaminase, aspartate transaminase, glutamate dehydrogenase, and two glutamate transporters was not affected by COM. In Trial 2, plasma estradiol concentrations of COM implanted young and old cows were 48% higher than Control groups, whereas blood metabolites were not affected. COM implantation did not affect GS protein expression in young cows, but tended to increase GS expression in the old cows by 283% after 14 d and 41% after 28 d. GS mRNA content was increased about 38% in both young and old COM-treated cows. Hepatic content of beta-catenin and G protein-coupled receptor 30 (GPR30) content was not affected by COM treatment, indicating that estradiol-mediated GS expression was not regulated by beta-catenin- or GPR30-controlled pathways. In Trial 3, plasma estradiol levels in COM-treated heifers were 70% higher in COM heifers, concomitant with increased levels of total bilirubin and creatine kinase, and decreased creatinine. Correlation analysis of plasma estradiol levels and blood constituents only identified a positive correlation between plasma estradiol and potassium. Collectively, these data describe positive estradiol-mediated effects on hepatic metabolism and blood parameters in female cattle.

Four studies were conducted to evaluate the effects of supplemental cooling and niacin on heat stress (HS) responses in vivo and in vitro in lactating dairy cows. For experiment one, lactating dairy cows were fed four levels of dietary niacin, NIASHURE® (0,4,8,12 g/d) for 21 d. Heat stress decreased niacin levels in whole blood, red blood cells and plasma as compared to thermoneutral. Water intake, plasma and milk niacin concentrations increased linearly with increasing dietary niacin in HS cattle. In thermoneutral, but not HS cows, niacin increased skin temperature compared to controls suggesting niacin increased skin blood flow and sensible heat loss. In experiment 2, lactating cows were used to evaluate the impact of feed-line soaking (FLS) combined with niacin supplementation. In evaporative cooled barns, FLS reduced body temperatures; however the addition of niacin did not improve heat status of these cows. For experiment 3, 200 lactating dairy cows were used to determine the effects of conductively cooled bedding (CC) compared to feed-line soaking with fans (FLSF). Conductively cooled bedding can reduce skin and vaginal temperatures in cows after nighttime rest. However, FLSF were more effective in decreasing body temperature, as cows had lower heat parameter indices, higher milk yield and longer rest time. For experiment 4, three cell types were used to evaluate niacin in vitro. Niacin induced heat shock proteins (HSP) that protected cells during HS in transformed bovine mammary epithelial cells but not in primary bovine mammary epithelial cells (BMEC) or bovine endometrial cells (BEND). Effect of niacin on HSP may depend on enzymes involved in DNA-binding activity of heat shock factor 1. These results suggest that niacin may be involved in whole body metabolism during heat stress and is cell dependent. We did not find dietary niacin to be commercially efficacious in treating HS in cows. Further research is warranted to improve efficacy of CC and FLSF under high temperature humidity index conditions.

Skeletal muscle regeneration is dependent upon the influences of intrinsic and extrinsic factors that stimulate satellite cells. Regenerating proteins are upregulated at the onset of trauma or inflammation in the pancreas, gastrointestinal tract, liver, neural cells and other tissues. Studies have shown that Reg proteins have a mitogenic, anti-apoptotic and anti-inflammatory function in damaged tissues and is necessary for normal progression of regeneration. As skeletal muscle is also able to regenerate itself at a rapid rate, it seems highly likely that Reg proteins function to promote myogenesis in skeletal muscle regeneration. Therefore, the goal of our research was to characterize the expression of the Reg proteins and receptor in regenerating skeletal muscle and satellite cells, investigate the effect of exogenous Reg protein on myogenesis, and to examine direct Reg protein effect on satellite cell activity. To determine whether Reg proteins participate in skeletal muscle regeneration, mice were injected with marcaine in their tibialis anterior muscles to induce skeletal muscle damage. The gene expression analysis of undamaged and marcaine-damaged tibialis anterior muscles and mice satellite cells showed that Reg I, II, IIIα, IIIγ, IV and EXTL3 genes are present during skeletal muscle regeneration and satellite cells significantly express Reg I, IIIα, IIIγ and EXTL3. As Reg I and IIIα are most prevalent in vivo and in vitro respectively, we advocate these isoforms as the predominant candidates in skeletal muscle regeneration. To determine the effect of exogenous Reg protein on myogenesis, we performed gene expression and muscle morphometry analysis of Reg IIIα or PBS injected tibialis anterior muscles. Interestingly, our results indicate that the addition of Reg IIIα to damaged muscles inhibited myogenesis. To determine the direct effect of Reg protein on myogenic stem cell activity, Reg proteins were added to mice satellite cells and C2C12 cells. Results from these studies were inconclusive due to the failure of known positive and negative controls. Overall, our studies suggest that Reg proteins contribute to skeletal muscle regeneration; however, as an overabundance of Reg IIIα in regenerating tissues may have inhibited myogenesis, it is imperative that other isoforms or lower concentrations be investigated.

There has been some concern recently about the ability of technicians to accurately measure and rank prospective replacement heifers and herd bulls for pelvic area. Two studies were conducted to determine the repeatabilities between and within technicians with varying levels of experience in measuring pelvic areas. In both studies, virgin yearling heifers were measured for pelvic height and width to determine pelvic area. In each study, technicians included a veterinarian with several years experience in reproductive physiology practice and Western Kentucky University students with varying levels of experience in rectal examinations. In the first study , the veterinarian used both the “Krautmann-Litton” pelvimeter and the “Rice” pelvimeter, but in the second study, the veterinarian used only the “Krautmann-Litton” Pelvimeter. In both studies, the students used a “Rice” pelvimeter. Coefficients of correlation and Spearman’s coefficients of rank correlation were calculated to determine repeatabilities between and within technicians. In the first experiment, the correlations between technicians for pelvic area ranged from .66 to .92 and Spearman’s rank correlations between technicians ranged from .65 to .92. In the second experiment, the correlations between and within technicians for pelvic area ranged from .78 to .97 and Sparman’s rank correlations between and within technicians ranged from .77 to .95. The results of these studies show clearly that cattle breeders can become proficient at measuring the pelvic area of heifers and bulls and in ranking them for selection purposes.