Impact of Oral Phytozen EQ Supplementation on Plasma Cortisol and Behavior Responses of Young Horses Exposed to Stressful Stimuli.

Calming supplements are common in the equine industry. This study tested the hypothesis that Phytozen EQ, a blend of citrus botanical oils, magnesium, and yeast would reduce startle response as well as reduce behavioral and physiological signs of stress in young (1.5-6 years of age) horses (n = 14) when tied in isolation and when trailered in isolation. During the 59-day trial, horses were assigned to either the control (CON; n = 7) or treatment (PZEN; n = 7) group that received 56 g of Phytozen EQ daily. Horses underwent a 10-minute isolation test on d 30 and a 15 minute individual trailering test on day 52 or 55. For both tests, blood samples were obtained pre, immediately after, and 1-hour post for analysis of plasma cortisol concentrations, which were analyzed by repeated measures ANOVA. On day 59, horses underwent a startle test, for which time to travel 3 m and total distance traveled were recorded. These data were analyzed using a T-test. During trailering, PZEN horses tended to have lower overall geometric mean (lower, upper 95% confidence interval) cortisol concentrations than CON (81 [67, 98] vs. 61 [48, 78] ng/mL; P = .071). For the startle test, PZEN horses tended to have longer geometric mean times to travel 3 m than CON horses (1.35 [0.39, 4.70] vs. 0.26 [0.07, 0.91 seconds, P = 0.064). Other data points were not different between treatments (P > .1). It is possible that this dietary supplement could have beneficial calming effects on horses undergoing trailering or in novel situations.

Short Communication: Supplementation with calcium butyrate causes an increase in the percentage of oxidative fibers in equine gluteus medius muscle.

Optimal athletic performance requires meeting the energetic demands of the muscle fibers, which are a function of myosin ATPase enzymatic activity. Skeletal muscle with a predominant oxidative metabolism underlies equine athletic success. Sodium butyrate, a short-chain fatty acid, can affect muscle fiber composition in pigs. To determine if a similar scenario exists in horses, 12 adult Thoroughbred geldings (7.4 ± 0.6 yr of age; mean ± SEM) were fed 16 g of calcium butyrate (CB) or an equivalent amount of carrier (CON) daily for 30 d in a crossover design. Middle gluteal muscle biopsies were collected before and after the feeding trial for immunohistochemical determination of fiber type, and RNA and protein isolation. After 30 d, CB increased (P < 0.05) the percentage of type IIA fibers and tended (P = 0.13) to reduce the numbers of type IIX fibers in comparison to control (CON). No changes (P > 0.05) in type I, IIA, or IIX fiber size were observed in response to CB. No differences (P > 0.05) were noted in the abundance of succinate dehydrogenase (SDH) protein or activity between horses receiving CB or CON. Myogenin mRNA abundance was unaffected (P > 0.05) by 30 d of CB supplementation. The increase in type IIA fibers in the absence of altered mitochondrial SDH enzymatic activity suggests that CB affects myosin ATPase expression independent of altered metabolism.

The largest tissue in the body, skeletal muscle, is a heterogeneous mix of fibers that are categorized based on their primary source of energy production and speed of contraction. Evidence suggests that Thoroughbred horses with a greater percentage of type IIA, fast-twitch, oxidative fibers are more successful than those with fewer. Pigs fed a diet supplemented with butyrate contained a greater percentage of oxidative muscle fibers. This study examined the ability of calcium butyrate (CB), a short-chain fatty acid, to alter muscle fiber composition in horses. Adult Thoroughbred geldings were supplemented with a placebo or CB for 30 d, and gluteus medius muscle biopsies were retrieved and analyzed for fiber type, myogenin expression, and succinate dehydrogenase (SDH) activity. Results demonstrate a small increase in the percentage of type IIA fibers without a change in SDH activity, a marker of oxidative metabolism. Myogenin expression remained unaffected by CB supplementation. These efforts underscore the need for further research to validate improved exercise performance in response to CB supplementation and identify a mechanism of action for the fatty acid in the equine skeletal muscle.

Impacts of DigestaWell NRG Supplementation on Post Exercise Muscle Soreness in Unconditioned Horses, a Pilot Study.

Exercising horses are commonly plagued by muscle fatigue and soreness, which can result in reduced performance ability. In the present study, ten unconditioned horses were fed 200g per day DigestaWell NRG, a commercial dietary supplement containing Yucca schidigera and Trigonella foenum-graecum, two herbs shown in other species to reduce post-exercise muscle pain and soreness. A control, unsupplemented group contained ten horses of similar age, breed, and gender. Horses completed a 50 minutes, ridden standardized exercise test of moderate intensity immediately prior to (Period1) and after 28 days of supplementation (Period2). Muscle soreness and tightness were evaluated 24 hours prior to and after each exercise test and used to determine the percent increase in post-exercise muscle soreness and tightness. Blood samples were collected before, and at 10 and 30 minutes, and 1, 4, and 24 hours post exercise. Plasma was analyzed for glucose, lactate, non-esterified fatty acid, tumor necrosis factor-α, and interleukin-1ß concentrations. Data were analyzed by repeated measures ANOVA using SAS Enterprise Guide v. 7.1. No changes in plasma parameters were indicated between periods for unsupplemented horses (P > 0.1) during Period2, excepting glucose, which was greater during Period2 (P = 0.018). Supplemented horses had lesser concentrations of tumor necrosis factor-α (P = 0.016) and lactate (P = 0.058) during Period2 than during Period1. During Period2, supplemented horses experienced a smaller percent increase in post exercise muscle soreness (P = 0.031). DigestaWell NRG supplementation may benefit unconditioned horses undergoing moderate intensity exercise through reducing lactate production and inflammation.

Influence of Metabolic Status and Diet on Early Pregnant Equine Histotroph Proteome: Preliminary Findings.

Insulin resistance (IR) is characterized by an increase in biomarkers of systemic inflammation and susceptibility to laminitis in horses. Impacts on reproduction include a lengthened interovulatory period in horses. Dietary omega-3 (docosahexaenoic acid [DHA]) promotes anti-inflammatory processes, has been implicated in health benefits, and can reduce cytokine secretion. This preliminary study investigated the impact of IR as well as the influence of dietary supplementation (DHA) on the uterine fluid proteome in early pregnant horses. Mares were artificially inseminated; uterine fluid and embryos were collected on d 12.5 after ovulation. Uterine fluid was pooled for metabolic and diet categories (n = 8; n = 2 per metabolic and dietary status) and concentrated, and the proteome was analyzed using tandem mass spectrometry (iTRAQ). Five proteins met differential abundance criteria (±1.5-fold change, P < .05) in all comparisons (Control C, IS vs. C, IR; C, IS vs. DHA, IS; C, IR vs. DHA, IR). Serum amyloid A, afamin, and serotransferrin were upregulated in C, IR mares but downregulated in DHA, IR mares when compared to C, IS and C, IR, respectively. Quantitative PCR supported mass spectrometry results. The presence of serum amyloid A and serotransferrin in histotroph of IR mares potentially indicates an inflammatory response not seen in IS counterparts. These preliminary findings provide novel evidence on the potential impact of insulin resistance and DHA supplementation on the secreted equine uterine proteome during early pregnancy.

Effects of dietary amino acid supplementation on measures of whole-body and muscle protein metabolism in aged horses.

The objective of this study was to examine markers of whole-body and muscle protein metabolism in aged horses fed a diet typical for North American aged horses, supplemented with amino acids. In a replicated Latin square design, six aged horses (20 ± 1.1 years) were studied while receiving each of three isocaloric, isonitrogenous diets, a control treatment concentrate (CON; 100 mg/kg-1 BW day-1 lysine, 84 mg kg-1  day-1 threonine, 51 mg kg-1  day-1 methionine), LYS/THR (134 mg kg-1 BW day-1 lysine, 110 mg kg-1 BW day-1 threonine, 52 mg kg-1 BW day-1 methionine) and LYS/THR/MET (132 mg kg-1 BW day-1 lysine, 112 mg kg-1 BW day-1 threonine, 62 mg kg-1 BW day-1 methionine). In each 15-days period, urine and faeces were collected for assessment of nitrogen balance. Blood samples were collected before and after feeding for analysis of plasma urea nitrogen (PUN), glucose, insulin and plasma amino acid concentrations. Skeletal muscle samples were collected for measurement of proteins associated with muscle protein synthesis and degradation, and horses underwent stable isotope infusion procedures for comparison of differences in whole-body rates of protein synthesis and degradation. There was no effect of treatment on relative abundance of proteins involved in protein synthesis, nitrogen retention or phenylalanine kinetics. PUN concentrations tended to be higher for LYS/THR (p = 0.054) and were higher for LYS/THR/MET (p = 0.0056) than for CON. Atrogin-1 abundance tended to be higher in the post-absorptive state for the CON treatment (p = 0.07), indicating that amino acid supplementation resulted in less muscle protein degradation when horses were in the post-absorptive state. However, lack of differences in nitrogen retention and phenylalanine kinetics indicated that whole-body protein metabolism was not improved, and higher PUN concentrations in the supplemented diets suggest that the supplemented amino acids may have been catabolized. Amino acid availability was not limiting protein synthesis in the sedentary aged horses in this study when fed the CON diet.

Inhibition of tumorigenesis by peroxisome proliferator-activated receptor (PPAR)-dependent cell cycle blocks in human skin carcinoma cells.

To examine the functional role of peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) and PPARγ in skin cancer, stable cell lines were created in the A431 human squamous cell carcinoma cell line. Expression of PPAR target genes was greatly enhanced in response to ligand activation of PPARß/δ or PPARγ in A431 cells expressing these receptors. PPARß/δ expression blocked the cell cycle at the G2/M phase, and this effect was increased by ligand activation. Ligand activation of PPARß/δ markedly inhibited clonogenicity as compared to vehicle-treated controls. Similarly, ligand activation of PPARγ in A431 cells expressing PPARγ resulted in reduced clonogenicity. Expression of either PPARß/δ or PPARγ markedly reduced tumor volume in ectopic xenografts, while ligand activation of these receptors had little further influence on tumor volume. Collectively, these studies demonstrate that stable expression and activation of PPARß/δ or PPARγ in A431 cells led to reduced tumorigenicity. Importantly, PPAR expression or ligand activation had major impacts on clonogenicity and/or tumor volume. Thus, PPARß/δ or PPARγ could be therapeutically targeted for the treatment of squamous cell carcinomas.

Editor's Highlight: PPARß/δ and PPARγ Inhibit Melanoma Tumorigenicity by Modulating Inflammation and Apoptosis.

Skin tumorigenesis results from DNA damage, increased inflammation, and evasion of apoptosis. The peroxisome proliferator-activated receptors (PPARs) can modulate these mechanisms in non-melanoma skin cancer. However, limited data exists regarding the role of PPARs in melanoma. This study examined the effect of proliferator-activated receptor-ß/δ (PPARß/δ) and PPARγ on cell proliferation, anchorage-dependent clonogenicity, and ectopic xenografts in the UACC903 human melanoma cell line. Stable overexpression of either PPARß/δ or PPARγ enhanced ligand-induced expression of a PPARß/δ/PPARγ target gene in UACC903 cell lines as compared with controls. The induction of target gene expression by ligand activation of PPARγ was not altered by overexpression of PPARß/δ, or vice versa. Stable overexpression of either PPARß/δ or PPARγ reduced the percentage of cells in the G1 and S phase of the cell cycle, and increased the percentage of cells in the G2/M phase of the cell cycle in UACC903 cell lines as compared with controls. Ligand activation of PPARß/δ did not further alter the distribution of cells within each phase of the cell cycle. By contrast, ligand activation of PPARγ enhanced these changes in stable UACC903 cells overexpressing PPARγ compared with controls. Stable overexpression of either PPARß/δ or PPARγ and/or ligand activation of either PPARß/δ or PPARγ inhibited cell proliferation, and anchorage-dependent clonogenicity of UACC903 cell lines as compared with controls. Further, overexpression of either PPARß/δ or PPARγ and/or ligand activation of either PPARß/δ or PPARγ inhibited ectopic xenograft tumorigenicity derived from UACC903 melanoma cells as compared with controls, and this was likely due in part to induction of apoptosis. Results from these studies demonstrate the antitumorigenic effects of both PPARß/δ and PPARγ and suggest that targeting these receptors may be useful for primary or secondary melanoma chemoprevention.

Effects of advanced age on whole-body protein synthesis and skeletal muscle mechanistic target of rapamycin signaling in horses.

OBJECTIVE: To determine the effects of advanced age on whole-body protein synthesis and activation of the mechanistic target of rapamycin (mTOR) signaling pathway in skeletal muscle of horses. ANIMALS: Six 22- to 26-year-old (aged) and six 7- to 14-year-old (mature) horses. PROCEDURES: Whole-body protein synthesis was measured with a 2-hour primed constant infusion of (13)C sodium bicarbonate, followed by a 4-hour primed constant infusion of 1-(13)C phenylalanine. After the infusions, a biopsy specimen was obtained from a gluteus medius muscle and activation of protein kinase B (Akt), p70 riboprotein S6 kinase (S6K1), riboprotein S6 (rpS6), and eukaryotic initiation factor 4E binding protein 1 (4EBP1) was determined with western immunoblot analysis. For all horses, inflammatory cytokine expression in muscle and blood samples was measured with quantitative real-time PCR analysis. RESULTS: Advanced age had no effect on whole-body protein synthesis or the phosphorylation of Akt, rpS6, and 4EBP1; however, muscle specimens of aged horses had 42% lower phosphorylation of S6K1 than did those of mature horses. Aged and mature horses had similar inflammatory cytokine expression in muscle and blood samples. CONCLUSIONS AND CLINICAL RELEVANCE: The lower S6K1 activation for aged horses, compared with that for mature horses, could be indicative of low rates of muscle protein synthesis in aged horses. However, advanced age had no effect on any other indicators of whole-body or muscle protein synthesis or on measures of systemic or muscle inflammation, which suggested that protein metabolism and subsequently requirements may not differ between healthy mature and aged horses.

Effect of gluteus medius muscle sample collection depth on postprandial mammalian target of rapamycin signaling in mature Thoroughbred mares.

OBJECTIVE: To determine the effect of biopsy collection depth on the postprandial activation of mammalian target of rapamycin (mTOR) signaling factors, particularly protein kinase B, ribosomal protein S6 kinase, ribosomal protein S6, and eukaryotic initiation factor 4E binding protein 1 in middle-aged horses. ANIMALS: 6 healthy Thoroughbred mares (mean ± SD age, 13.4 ± 3.4 years). PROCEDURES: Horses were fed a high-protein feed at 3 g/kg. Sixty minutes after horses were fed, the percutaneous needle biopsy technique was used to collect biopsy specimens from the gluteus medius muscle at 6, 8, and 10 cm below the surface of the skin. Muscle specimens were analyzed for the activation of upstream and downstream mTOR signaling factors, myosin heavy chain (MHC) isoform composition, and amino acid concentrations. RESULTS: A 21% increase in MHC IIA isoform expression and a 21% decrease in MHC IIX isoform expression were identified as biopsy depth increased from 8 to 10 cm below the surface of the skin; however, no significant change was evident in the degree of MHC I expression with muscle depth. Biopsy depth had no significant effect on the phosphorylation of any of the mTOR signaling factors evaluated. CONCLUSIONS AND CLINICAL RELEVANCE: Postprandial mTOR signaling could be compared between middle-aged horses when biopsy specimens were collected between 6 and 10 cm below the surface of the skin. Optimization of muscle biopsy techniques for evaluating mTOR signaling in horses will facilitate the design of future investigations into the factors that regulate muscle mass in horses.

Developmental regulation of the activation of translation initiation factors of skeletal muscle in response to feeding in horses.

OBJECTIVE: To determine whether feeding-induced activation of translation initiation factors, specifically protein kinase B, ribosomal protein S6 kinase (S6K1), ribosomal protein S6 (rpS6), and eukaryotic initiation factor 4E binding protein 1, in horses is affected by age. ANIMALS: 6 yearlings, six 2-year-old horses, and 6 mature horses. PROCEDURES: After an 18-hour period of feed withholding, horses consumed a high-protein meal (2 g/kg) at time 0 and 30 minutes (postprandial state) or continued to have feed withheld (postabsorptive state). Blood samples were collected for the duration of the experimental procedures and used to determine plasma concentrations of glucose, insulin, and amino acids. At 90 minutes, biopsy specimens were collected from a gluteal muscle and used to measure phosphorylation of translation initiation factors. RESULTS: Plasma glucose, insulin, and amino acid concentrations were elevated for the postprandial state, compared with results for the postabsorptive state, regardless of age. Phosphorylation of protein kinase B, S6K1, rpS6, and eukaryotic initiation factor 4E binding protein 1 was increased for the postprandial state. There was an effect of age with increased phosphorylation of S6K1 at Thr(389) and rpS6 at Ser(235/236) in the yearlings and mature horses, compared with results for the 2-year-old horses. CONCLUSIONS AND CLINICAL RELEVANCE: Food consumption resulted in an increase in the activation of translation initiation factors, with the highest degree of responsiveness in the yearlings. This indicated that increased muscle accretion seen during growth could be a result of increased rates of muscle protein synthesis in response to a meal stimulus.