L.K. Warren

Effect of dietary omega-3 fatty acid source on plasma and red blood cell membrane composition and immune function in yearling horses

To determine the effect of different sources of dietary n-3 fatty acids (FA) on plasma and red blood cell (RBC) FA composition and immune response, 18 Quarter Horse yearlings were randomly and equally assigned to 1 of 3 treatments: encapsulated fish oil (n = 6), milled flaxseed (n = 6), or no supplementation (control, n = 6). Fish oil contained 15 g of eicosapentaenoic acid (C20:5n-3) and 12.5 g of docosahexaenoic acid (C22:6n-3), and flaxseed contained 61 g of alpha-linolenic acid (C18:3n-3) per 100 g of FA. Horses had free access to bahiagrass pasture during the active growing season and were individually fed a grain mix concentrate at 1.5% BW/d. Fish oil and flaxseed were mixed into the concentrate in amounts to provide 6 g of total n-3/100 kg of BW. Horses were fed their respective treatments for 70 d. Blood samples were obtained to determine plasma and RBC FA composition and for isolation of peripheral blood mononuclear cells. Peripheral blood mononuclear cells were stimulated with concanavalin A and phytohemagglutinin (PHA) to determine lymphocyte proliferation and were challenged with lipopolysaccharide to determine PGE(2) production. In vivo inflammatory response was assessed on d 70 by measuring skin thickness and area of swelling in response to intradermal injection of PHA. Treatment did not affect BW gain, which averaged 0.6 +/- 0.03 kg/d. Horses fed fish oil had greater (P < 0.05) proportions of eicosapentaenoic acid, docosahexaenoic acid, and sum of n-3 in plasma and RBC compared with those in the flaxseed and control treatments. In addition, plasma arachidonic acid was greater (P < 0.05) and plasma linoleic and alpha-linolenic acids were less (P < 0.05) in the fish oil treatment compared with the flaxseed and control treatments. Dietary treatment did not affect lymphocyte proliferation or PGE(2) production. Across treatments, the peak increase in skin thickness was observed 4 to 8 h after PHA injection. At 4 h postinjection, horses fed fish oil and those fed flaxseed had a greater increase in skin thickness than those in the control treatment (P < 0.05) and horses fed fish oil had a larger area of swelling than those in the control treatment (P < 0.05). Skin thickness remained greater (P < 0.05) in horses fed flaxseed than in control horses 6 h after injection. Although fed to supply a similar amount of n-3 FA, fish oil had a greater impact on plasma and RBC n-3 FA content than did flaxseed. However, supplementing horses with both fish oil and flaxseed resulted in a more pronounced early inflammatory response to PHA injection as compared with nonsupplemented horses.

Digestibility and nutrient retention of perennial peanut and bermudagrass hays for mature horses1

Mature horses were used to determine apparent DM, OM, NDF, and CP digestibility values of 2 bermudagrass (BG; Cynodon dactylon) hays, Coastal (CB) and Tifton 85 (T85), and Florigraze perennial peanut (PP; Arachis glabrata) hay. In addition, N, Ca, and P balances were determined in horses fed those hays. Five mature Thoroughbred geldings and 1 Quarter Horse gelding (mean initial BW = 542 +/- 37 kg) were used (5 horses for the last period) in a 3 x 3 repeated Latin square design, with 2 horses per hay and 3 adjustment and collection periods. Horses were randomly assigned to pairs and the initial hay to be fed. Each period consisted of a 10-d adjustment phase, followed by a 4-d total fecal and urine collection phase. Horses were fed at 1.5 (period 1), 1.7 (period 2), or 2% (period 3) of their BW daily (DM basis). The 2 BG were grown under similar conditions, with CB and T85 being cut at 4 and 5 wk of regrowth, respectively, and PP being of a late first cutting. The compositions (DM basis) of PP, CB, and T85, respectively, were 93, 94, and 93% DM; 92, 94, and 94% OM; 46, 73, and 77% NDF; 34, 37, and 42% ADF; 11, 10, and 8% CP; 1.10, 0.28, 0.27% Ca; and 0.19, 0.15, and 0.19% P, respectively. Least squares means (pooled SE) for apparent digestibility of PP, CB, and T85, respectively, were 65, 53, and 52% (1) DM digestibility; 67, 53, and 52% (1) OM digestibility; 44, 50, and 46% (4) NDF digestibility; and 66, 60, and 57% (1) CP digestibility. Digestibility values of DM and OM were greater (P < 0.001) for PP than for the BG. Digestibility of CP was greater (P = 0.001) for PP than for CB or T85, with no difference (P = 0.37) between PP and BG hays in NDF digestibility. There were no differences between CB and T85 for DM digestibility (P = 0.67), OM digestibility (P = 0.59), CP digestibility (P = 0.11), and NDF digestibility (P = 0.48). Nitrogen (P = 0.01) and P balances (P = 0.04) were greater for PP than BG hays, whereas N balance of CB was greater (P = 0.01) than that of T85. There were no differences among all hays in Ca balance (P = 0.54) and between the BG in P balance (P = 0.34). Results indicated that PP, CB, and T85 are suitable forages for horses.

Oral L-arginine supplementation impacts several reproductive parameters during the postpartum period in mares.

L-arginine is an amino acid which can alter pituitary function and increase blood flow to the reproductive tract. The objective was to determine the effect of supplementing 100g of L-arginine on plasma arginine concentrations, follicular dynamics and ovarian and uterine artery blood flow during the estrus that occurs subsequent to foaling. In Experiment 1, mares were fed 100g L-arginine for 1 day during the last 3 weeks of pregnancy and plasma samples taken for every hour for the first 4h and every other hour until 12h.L-arginine supplementation elevated plasma arginine concentrations from 1 to 8h post feeding; arginine peaked at 6h (arginine: 515±33μmol/L; control: 80±33μmol/L). In Experiment 2, mares received either 100g L-arginine or control diets beginning 21 d before the expected foaling date and continued for 30 d postpartum. The reproductive tract was evaluated by transrectal Doppler ultrasonography from Day 1 postpartum through Day 30. There were no differences in ovarian follicular dynamics, ovarian or uterine resistance indices between groups. Vascular perfusion of the F1 follicular wall was greater in L-arginine supplemented mares (37.3±2.6%) than controls (25.4±2.7%; P<0.05). L-arginine supplemented mares had a smaller uterine body and horns and accumulated less uterine fluid than controls (P<0.05). The combination of reducing uterine fluid accumulation, while not altering follicular development, raises the possible use of L-arginine supplementation as a breeding management tool during the postpartum period to increase reproductive success.

Effects of aging on mitochondrial function in skeletal muscle of Quarter Horses

Skeletal muscle function, aerobic capacity, and mitochondrial (Mt) function have been found to decline with age in humans and rodents. However, not much is known about age-related changes in Mt function in equine skeletal muscle. Here, we compared fiber-type composition and Mt function in gluteus medius and triceps brachii muscle between young (age 1.8 ± 0.1 yr, n = 24) and aged (age 17-25 yr, n = 10) American Quarter Horses. The percentage of myosin heavy chain (MHC) IIX was lower in aged compared with young muscles (gluteus, P = 0.092; triceps, P = 0.012), while the percentages of MHC I (gluteus; P < 0.001) and MHC IIA (triceps; P = 0.023) were increased. Mass-specific Mt density, indicated by citrate synthase activity, was unaffected by age in gluteus, but decreased in aged triceps (P = 0.023). Cytochrome-c oxidase (COX) activity per milligram tissue and per Mt unit decreased with age in gluteus (P < 0.001 for both) and triceps (P < 0.001 and P = 0.003, respectively). Activity of 3-hydroxyacyl-CoA dehydrogenase per milligram tissue was unaffected by age, but increased per Mt unit in aged gluteus and triceps (P = 0.023 and P < 0.001, respectively). Mt respiration of permeabilized muscle fibers per milligram tissue was unaffected by age in both muscles. Main effects of age appeared when respiration was normalized to Mt content, with increases in LEAK, oxidative phosphorylation capacity, and electron transport system capacity (P = 0.038, P = 0.045, and P = 0.007, respectively), independent of muscle. In conclusion, equine skeletal muscle aging was accompanied by a shift in fiber-type composition, decrease in Mt density and COX activity, but preserved Mt respiratory function.

Effects of aging on mitochondrial function in skeletal muscle of American Quarter Horses

Skeletal muscle function, aerobic capacity, and mitochondrial (Mt) function have been found to decline with age in humans and rodents. However, not much is known about age-related changes in Mt function in equine skeletal muscle. Here, we compared fiber-type composition and Mt function in gluteus medius and triceps brachii muscle between young (age 1.8 ± 0.1 yr, n = 24) and aged (age 17-25 yr, n = 10) American Quarter Horses. The percentage of myosin heavy chain (MHC) IIX was lower in aged compared with young muscles (gluteus, P = 0.092; triceps, P = 0.012), while the percentages of MHC I (gluteus; P < 0.001) and MHC IIA (triceps; P = 0.023) were increased. Mass-specific Mt density, indicated by citrate synthase activity, was unaffected by age in gluteus, but decreased in aged triceps (P = 0.023). Cytochrome-c oxidase (COX) activity per milligram tissue and per Mt unit decreased with age in gluteus (P < 0.001 for both) and triceps (P < 0.001 and P = 0.003, respectively). Activity of 3-hydroxyacyl-CoA dehydrogenase per milligram tissue was unaffected by age, but increased per Mt unit in aged gluteus and triceps (P = 0.023 and P < 0.001, respectively). Mt respiration of permeabilized muscle fibers per milligram tissue was unaffected by age in both muscles. Main effects of age appeared when respiration was normalized to Mt content, with increases in LEAK, oxidative phosphorylation capacity, and electron transport system capacity (P = 0.038, P = 0.045, and P = 0.007, respectively), independent of muscle. In conclusion, equine skeletal muscle aging was accompanied by a shift in fiber-type composition, decrease in Mt density and COX activity, but preserved Mt respiratory function.

Dietary selenium and prolonged exercise alter gene expression and activity of antioxidant enzymes in equine skeletal muscle

Untrained Thoroughbred horses (6 mares and 6 geldings; 11 yr [SE 1] and 565 kg [SE 11]) were used to evaluate antioxidant gene expression and enzyme activity in blood and skeletal muscle in response to prolonged exercise after receiving 2 levels of dietary selenium for 36 d: 0.1 (CON; = 6) or 0.3 mg/kg DM (SEL; = 6). Horses were individually fed 1.6% BW coastal bermudagrass hay, 0.4% BW whole oats, and a mineral/vitamin premix containing no Se. Sodium selenite was added to achieve either 0.1 or 0.3 mg Se/kg DM in the total diet. On d 35, horses underwent 2 h of submaximal exercise in a free-stall exerciser. Blood samples were obtained before (d 0) and after 34 d of Se supplementation and on d 35 to 36 immediately after exercise and at 6 and 24 h after exercise. Biopsies of the middle gluteal muscle were obtained on d 0, before exercise on d 34, and at 6 and 24 h after exercise. Supplementation with Se above the NRC requirement (SEL) increased serum Se ( = 0.011) and muscle thioredoxin reductase (TrxR) activity ( = 0.051) but had no effect on glutathione peroxidase (GPx) activity in plasma, red blood cell (RBC) lysate, or muscle in horses at rest. Serum creatine kinase activity increased ( < 0.0001) in response to prolonged exercise but was not affected by dietary treatment. Serum lipid hydroperoxides were affected by treatment ( = 0.052) and were higher ( = 0.012) in horses receiving CON than SEL immediately following exercise. Muscle expression of was unchanged at 6 h but increased ( = 0.005) 2.8-fold 24 h after exercise, whereas muscle TrxR activity remained unchanged. Glutathione peroxidase activity increased in plasma (P < 0.0001) and decreased in RBC lysate ( = 0.010) after prolonged exercise. A Se treatment × time interaction was observed for RBC GPx activity (P = 0.048). Muscle and expression and GPx activity did not change during the 24-h period after exercise. Level of dietary Se had no overall effect on expression of , , , , , , or in muscle following exercise. The impact of prolonged exercise on the activities of antioxidant enzymes varied. Furthermore, changes in enzyme activity did not necessarily align with enzyme gene expression following exercise. A higher level of Se intake elevated Se status of untrained horses, increased GPx activity, and lessened lipid peroxidation following exercise, suggesting that Se may be beneficial for mitigating oxidative muscle damage and aiding in postexercise recovery.

Influence of L-arginine supplementation on reproductive blood flow and embryo recovery rates in mares

Supplementation with L-arginine can increase uterine arterial blood flow and vascular perfusion of the preovulatory follicle in mares. Increased vascular perfusion of the preovulatory follicle has been correlated with successful pregnancy in mares. The objective of this study was to determine if supplemental L-arginine would increase ovarian arterial blood flow, vascular perfusion of the preovulatory follicle, and embryo recovery rates in mares. Mares were blocked by age and breed and assigned at random within block to L-arginine supplementation or control groups. Mares were fed L-arginine beginning 17 days before and through the duration of the study. Transrectal Doppler ultrasonography was used to measure ovarian arterial blood flow and vascular perfusion of the preovulatory follicle daily when it reached 35 mm and subsequent CL on Days 2, 4, and 6. Mares, on achieving a follicle of 35 mm or more were bred via artificial insemination and an embryo collection was attempted 7 days after ovulation. Treatment did not affect interovulatory interval (arginine-treated, 18.1 ± 2.6 days; control, 20.7 ± 2.3 days) or embryo recovery rate (arginine-treated, 54%; control, 48%). Mares treated with l-arginine had a larger follicle for the 10 days preceding ovulation than control mares (30.4 ± 1.2 and 26.3 ± 1.3 mm, respectively; P < 0.05) and vascular perfusion of the dominant follicle tended (P = 0.10) to be greater for the 4 days before ovulation. No differences were observed between groups in diameter or vascular perfusion of the CL. Resistance indices, normalized to ovulation, were not significantly different between groups during the follicular or luteal phase. Oral l-arginine supplementation increased the size and tended to increase perfusion of the follicle 1, but had no effect on luteal perfusion or embryo recovery rates in mares.

Orally supplemented l-arginine impairs amino acid absorption depending on dose in horses1

The beneficial effect of L-arginine (L-Arg) supplementation, on the physiology of several species, has generated an interest in the use of L-Arg as a nutraceutical in horses, but dosage and absorption of orally supplemented L-Arg must be inferred from other species. The study objective was to determine the effect of 2 oral L-Arg doses on plasma arginine concentrations and the effect on absorption of other amino acids in mares. In Experiment 1, mares were blocked by age and breed and were fed L-Arg supplemented (supplemented with 0.025% BW L-Arg; n=6) or control (no supplement; n=6) concentrate on a single day with blood samples taken at 0, 0.5, 1, 2, 3, 4, and 5 h relative to feeding. In Experiment 2, mares (n=6) were used in a 3×3 Latin square design with L-Arg (0.0125% of BW), urea (0.0087% of BW), and control (no supplement) fed mixed into a grain concentrate as single meal with blood samples taken at 0, 1,2, 4, 6, 8,10, and 12 h relative to feeding. In Experiment 1, L-Arg supplementation increased (P<0.05) plasma L-Arg and ornthine concentrations and decreased (P<0.05) lysine and methionine concentrations compared with the control group. At 1 h post feeding, L-Arg mares had lower (P<0.05) plasma concentrations of histidine, glutamic acid, proline, isoleucine, threonine, phenylalanine, leucine, valine, alanine, and taurine. In Experiment 2, L-Arg supplementation increased (P<0.05) arginine and ornithine concentrations compared with urea and control; there was no difference among other amino acids. These experiments indicate that L-Argis absorbed and, dependent on the dose, alters the absorption of other amino acids in mares.

l-Arginine supplementation 0.5% of diet during the last 90 days of gestation and 14 days postpartum reduced uterine fluid accumulation in the broodmare

L-Arginine is an essential amino acid in many species that has been shown to influence reproduction. However, in horses a dose of 1% L-arginine of total dietary intake impaired absorption of other amino acids, whereas a dose of 0.5% did not. The objectives of this experiment were to evaluate postpartum parameters on mares supplemented with 0.5% L-arginine through the last 90d of gestation and 14d postpartum. Sixteen light-horse mares were randomly divided in two groups: 8 mares supplemented with 0.5% L-arginine and 8 mares fed an isonitrogenous equivalent. Gestation length, days to uterine clearance and days to first ovulation were compared. Uterine body depth, diameter of uterine horns, and length of largest pocket of uterine fluid were recorded daily via transrectal ultrasound. Measurements of foal weight, height, and cannon bone circumference were recorded for 9 weeks. Arginine treatment had no effect on gestation length (P=0.58). Supplemented mares cleared fluid quicker postpartum (6.8±0.53d; P=0.026) compared to control (9.0±0.38d). Mares supplemented with L-arginine had smaller diameter of fluid present in the postpartum uterus (P≤0.05). Days to first postpartum ovulation were not affected by treatment nor any influence on uterine involution. Finally, treatment had no effect on any foals measured parameters. L-Arginine supplementation fed at 0.5% of daily intake during the last 90d of gestation and early postpartum in mares decreased uterine fluid accumulation, yet did not appear to have any effect on any other parameters measured.

Skeletal muscle from aged American Quarter Horses shows impairments in mitochondrial biogenesis and expression of autophagy markers

&NA; Aging is associated with decreased mitochondrial content and function in skeletal muscle, possibly due to compromised biogenesis and autophagic removal of dysfunctional mitochondria. The aim of this study was to compare markers of mitochondrial content and biogenesis and of autophagy between skeletal muscle from young and aged American Quarter Horses. Citrate synthase protein and mtDNA copy number were decreased in triceps brachii (TB) muscle (P < 0.05) from aged horses, suggesting an age‐related decline in mitochondrial content. Concomitantly, mRNA expression of PGC‐1&agr; and TFAM, regulators of mitochondrial biogenesis, was lower in aged compared to young TB (P < 0.05). Expression of autophagy markers suggested an age‐associated decline of autophagy. The autophagosomal cargo protein SQSTM/p62 accumulated with age in both muscles (P < 0.05). Expression of Autophagy‐related protein Atg5 (P < 0.05) and the autophagosome‐bound form of Microtubule‐associated protein light chain 3 (LC3‐II; P < 0.05) were lower in aged compared to young TB. While LC3 transcript level was elevated in aged compared to young GM (P < 0.001), protein expression of LC3‐II was unaffected. Gene expression of Lysosomal Membrane‐Associated Protein 2 (LAMP2) was not affected by age in either muscle. However, LAMP2 protein expression declined with age (P < 0.05), suggesting a decline in autophagosome‐lysosome fusion. Taken together, our data indicate that equine skeletal muscle mitochondrial content and biogenesis were impaired with age. Further, autophagosome formation and lysosomal degradation were negatively affected in aged TB and GM, respectively. Future research needs to explore whether interventions targeting these cellular processes can prolong health and performance of aging American Quarter Horses. HighlightsMitochondrial content is impaired in aged equine skeletal muscle.Markers of autophagy and mitochondrial biogenesis are affected by age.The cellular response to aging differs between muscle types.