J. Anim. Sci. 2005. 83:2783-2788
© 2005 American Society of Animal Science
Amino acid supplementation improves muscle mass in aged and young horses1
P. M. Graham-Thiers*,2 and
D. S. Kronfeld
* Virginia Intermont College, Bristol 24201; and
and
Virginia Polytechnic Institute and State University, Blacksburg 24601-0606
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Abstract
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The objective of the study was to evaluate the effect of supplementary AA on the ability to support muscle mass in aging horses. Sixteen horses of light horse type were used in a 2 x 2 factorial arrangement of treatments with two age groups [
10 yr (average = 9.1 ± 0.29 yr) and 
20 yr (average = 22.4 ± 0.87 yr)] and two diet groups [no supplementation (N) or supplementary lysine and threonine (S; 20.0 and 15 g/d, respectively)]. Horses were fed the diets for 14 wk and received regular light exercise throughout the study. Body weight, BCS, and venous blood samples were taken every 2 wk. Plasma was analyzed for total protein, albumin, creatinine, urea N (PUN), and an AA profile, including 3-methyl histidine (3MH) and sulfur AA. Photographs of the horses taken at the start and at the end of the experiment were used to assign a subjective muscle mass score from 1 to 5 (1 = lowest to 5 = highest). There was no difference in BW caused by diet; however, the S-group horses tended (P = 0.064) to gain more weight (6.91 ± 2.3 kg), and in fact, the N-group horses lost weight (– 11.76 ± 5.2 kg) during the experiment. Repeated measures analysis revealed that BCS was lower for the aged vs. the young horses (P = 0.001) as well as for the S- vs. the N-group horses (P = 0.026). Subjective muscle mass scores were not different at the start of the experiment but were greater (P = 0.047) for the S-group horses (3.77 ± 0.13) at the end of the experiment compared with the N-group horses (3.28 ± 0.14). Plasma creatinine was greater (P = 0.032), and PUN was lower (P = 0.027), for S-group horses compared with N-group horses. Initial 3MH concentrations were not different; however, at the end of the experiment, 3MH was lower for the S-group horses (P = 0.016) compared with the N-group horses. Plasma lysine and threonine concentrations were greater for S-group horses at the end of the experiment than for N-group horses (P = 0.023 and 0.009, respectively). Both 3MH and PUN concentrations were negatively correlated to lysine (R2 = 0.57 and 0.65, respectively) and threonine intake (R2 = 0.56 and 0.60, respectively) at the end of the study. These data suggest that horses receiving supplementary AA were able to maintain muscle mass better than those without supplementation, regardless of age, as evidenced by the improvement in muscle mass scores, lower BCS with no difference in BW, greater creatinine, and lower 3MH and PUN concentrations in the S-group horses.
Key Words: Horse • Amino Acid • Muscle Mass • Age
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Introduction
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A common phenomenon in aging humans is the loss of muscle mass (Booth et al., 1994
). This phenomenon has also been documented in aging horses, which is due to a decline in activity, poor teeth, and a decline in the ability to digest protein (Rich, 1989; Hintz, 1995). The decline in protein digestibility could increase the dietary protein needs of the aged horse. To meet this need, the quantity of CP could be increased, which has metabolic disadvantages, or the protein quality could be improved, or limiting AA could be supplemented (Graham et al., 1994). Thus, proper AA supplementation may be necessary to improve the protein status of the aging horse to ensure an adequate AA pool for maintaining muscle mass. This could allow aged horses to maintain their vigor and stamina much longer.
The objective of the study was to determine whether supplementary AA minimize the loss of muscle in the aging, lightly exercised horse.
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Materials and Methods
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Horses
Sixteen healthy horses of light horse type (503 ± 12.5 kg, 12 geldings and 4 mares) were assigned to a 2 x 2 factorial arrangement of treatments by sex and age with two age groups [10 yr (9.1 ± 0.29 yr) and x20 yr (22.4 ± 0.87 yr)] and two AA supplement groups [no supplementation (N) or supplementary lysine and threonine (S; 20 and 15 g/d, respectively)] for 14 wk. Horses were housed individually in 3.7- x 3.7-m box stalls during the day and in a dry lot at night. Horses were maintained in a light exercise program throughout the study.
The protocol was approved by the Institutional Animal Care and Use Committee at Virginia Intermont College (protocol number 2003-1).
Exercise
Exercise consisted mainly of participating in the college’s riding program. Records were utilized to determine the number of hours worked and the type of work for each horse. The data were used to determine consistency in the amount and type of work for each horse. Four times (0, 5, 9, and 14 wk) during the study, horses completed a standardized exercise test (SET) to ensure similar fitness among all horses. The SET consisted of 5 min of walking at 1.5 m/s, trotting for 5 min at 3.5 m/s, cantering for 3 min at 7 m/s, trotting for 5 min at 3.5 m/s, cantering for 3 min at 7 m/s, and walking for 5 min at 1.5 m/s. The speeds were determined by observing average paces in typical riding classes at the college. The perimeter of the riding arena was measured, and an optimum time was determined to maintain the necessary pace. The same rider exercised all horses to reduce rider variation. The necessary perimeter to follow was outlined in the arena, and a timer was used to keep the speeds consistent.
Horses wore heart rate monitors (V-Max, Equine Performance Technology, Reliance, TN) during regular exercise as well as during the SET to monitor exercise intensity.
Diets
Horses were fed a commercially available grain mix (Table 1) along with a mixed timothy/orchardgrass hay in three separate meals. Diluted corn syrup (10 mL) was mixed with the supplemental AA along with the grain mix and was hand-fed to the S-group horses to ensure consumption. A similar amount of corn syrup was added to the grain mix for horses in the N group. Horses were fed individually in box stalls at a rate of approximately 1.8% BW for forage and 0.5% BW for grain mix. Grain and hay refusals were weighed and recorded for determination of daily intakes.
Sampling and Analysis
The BW, BCS, and venous blood samples were taken every 2 wk. The blood sample was obtained 3 to 4 h after the morning feed was consumed. The BW was measured using an electronic scale (Model AL660-LA, Cambridge Scaleworks, Honey Brook, PA) while BCS was evaluated on a 1- to 9-point scale using a standardized system (Henneke et al., 1983). Plasma was separated from whole blood and analyzed for total protein (TP; Proc. No. 541, Sigma Diagnostics, St. Louis, MO), albumin (Proc. No. 631; Sigma Diagnostics), creatinine (Proc. No. 557; Sigma Diagnostics), and plasma urea nitrogen (PUN; Proc. No. 67-U; Sigma Diagnostics). Initial and final plasma samples were analyzed for AA concentrations including 3-methyl histidine (3MH) and sulfur AA. Amino acid analysis was performed using an HPLC (Hitachi L-8800A AA analyzer, Hitachi High Technologies America, Inc., San Jose, CA) following the procedure described by Miller-Graber et al. (1990). Photographs were taken of the horses before the start of the experiment and at the conclusion of the experiment for individuals (blind to the treatments) to assign a subjective muscle mass score from 1 to 5 (1 = lowest to 5 = highest) based on definition and mass of major muscle groups such as the gluteal, pectoral, and complexus muscles. These data were used to evaluate visual changes in muscle mass.
Statistics
Effects of diet and age on TP, albumin, PUN, creatinine, heart rate, BW, and BCS were analyzed using the mixed model with repeated measures; sources of variation included age, diet, diet x age, horse(diet), and the residual error horse x age x diet. Horse(diet) was used as the error term to test effects of age, and means were compared using the Tukey test (SAS Inst., Inc., Cary, NC). Effects of diet and age on feed and AA intake, plasma AA concentrations, BCS change, BW change, and muscle mass scores were analyzed by ANOVA using GLM procedure of SAS (SAS Inst., Inc.). Horse within diet was the error term for the effect of diet, and horse within age was the error term for the effect of age with the residual error as horse x age x diet. Initial data (BCS) that were different at the start of the study were analyzed using initial values as a covariate. Correlations were also estimated to determine relationships between treatments and measured variables (SAS Inst., Inc.). Data were summarized as least squares means with standard errors. Statistical significance was set at a level P < 0.05.
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Results
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All horses maintained good health throughout the experiment. There was no difference in grain mix intake, hay intake, or overall feed intake between groups for age (P = 0.62, 0.46, and 0.51, respectively) or diet (P = 0.42, 0.77, and 0.59, respectively). There was also no difference in the intake of any of the essential AA for age and diet with the exception of lysine and threonine in the S-group horses (P = 0.001 and 0.005, respectively) because of the addition of lysine and threonine to these diets (Table 2). There were no age x diet interactions for grain intake (P = 0.51), hay intake (P = 0.69), or overall intake (P = 0.59) as well as no interactions of age and diet for any of the AA intakes. Horses maintained similar levels of fitness throughout the study as evidenced by a lack of difference in heart rates during the SET (data not shown).
Using repeated measures analysis, there was no difference in BW caused by diet (P = 0.52) or age (P = 0.32) as well as no interaction between age and diet (P = 0.52). However, the S-group horses tended to gain more weight (6.91 ± 2.3 kg; P = 0.064), and in fact, the N-group horses lost weight (– 11.76 ± 5.2 kg) when evaluating the change in BW over the course of the experiment (Table 3). Body condition score was lower for the aged group of horses (P = 0.001) compared with the young horses over the course of the study. Body condition score was also lower for the S-group horses compared with the N-group horses (P = 0.026) over the duration of the experiment (Table 3). Body condition score was not affected by an age x diet interaction (P = 0.41). Subjective muscle mass scores were not different at the start of the experiment because of diet (P = 0.61), but they were greater for the S-group horses (3.77 ± 0.13; P = 0.047) at the conclusion of the experiment compared with the N-group horses (3.28 ± 0.14). Muscle mass scores were not affected by age or an age x diet interaction at the beginning of the experiment (P = 0.26 and 0.32, respectively); nor were muscle mass scores affected by age or by an age x diet interaction at the conclusion of the experiment (P = 0.61 and 0.13, respectively). The change in muscle mass score was greater for the S-group horses (+1.02) compared with the N-group horses (+0.52; P = 0.05) but was not affected by age or an age x diet interaction (P = 0.59 and 0.12, respectively).
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Table 3. Body weight, BCS, and muscle score values for horses with and without AA supplementation by age and dieta
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There were no differences in plasma albumin (P = 0.35) or TP (P = 0.27) caused by diet; nor was there an effect of age on plasma albumin (P = 0.62) or TP (P = 0.66) over the course of the experiment. Interactions between age and diet were also not observed for plasma albumin (P = 0.28) or TP (P = 0.25). Values observed for albumin and TP were all within the normal range for horses. Plasma creatinine (P = 0.67) and PUN (P = 0.27) were not affected by age or by an interaction between age and diet (P = 0.22 and 0.94, respectively). Creatinine was greater (173.1 ± 6.3 µmol/L) for S-group horses (P = 0.032) than for N-group horses (143.1 ± 6.3 µmol/L); PUN, however, was lower (5.59 ± 0.31 mmol/L) for S-group horses (P = 0.027) than for N-group horses (7.44 ± 0.31 mmol/L) over the duration of the experiment. Plasma AA including 3MH were not different at the start of the experiment and were not affected by age, diet, or an interaction of age and diet. Plasma lysine and threonine were greater for S-group horses (141.1 ± 3.3 and 307.8 ± 6.5 µmol/L, respectively) at the conclusion of the experiment (P = 0.023 and 0.009, respectively) compared with the N-group horses (86.9 ± 3.3 and 118.6 ± 6.5 µmol/L, respectively). At the conclusion of the experiment, 3MH was lower (14.3 ± 2.7 nmol/mL) for S-group horses (P = 0.016) than for N-group horses (22.5 ± 2.7 nmol/mL). Plasma lysine, threonine, and 3MH were not affected by age (P = 0.36, 0.39, and 0.80, respectively); nor were plasma lysine, threonine, or 3MH affected by an interaction of age and diet (P = 0.19, 0.95, and 0.74, respectively) at the conclusion of the experiment. All other plasma AA concentrations (arginine, histidine, leucine, isoleucine, phenylalanine, methionine, and valine) were within normal ranges.
There was a negative correlation between 3MH concentrations and lysine and threonine intakes (R2 = 0.57, P = 0.022 and R2 = 0.56, P = 0.025, respectively) at the conclusion of the experiment. There was a positive correlation between 3MH and PUN concentrations in the plasma (R2 = 0.55, P = 0.026) at the conclusion of the experiment. And finally, plasma PUN concentrations were negatively correlated with lysine and threonine intakes (R2 = 0.65, P = 0.007 and R2 = 0.60, P = 0.015, respectively) at the conclusion of the experiment.
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Discussion
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The results of this study suggest an improvement in maintenance and/or a reduction in muscle mass loss in horses that received supplemental lysine and threonine. The speculation is that the balance of AA in the diet was improved, presumably supporting the maintenance of the horses’ muscle mass as opposed to a loss of muscle mass. This conclusion is supported by lower BCS and greater muscle mass scores combined with no difference in overall weight between groups. Observations of lower PUN and 3MH as well as greater creatinine contribute to these conclusions as well.
For muscle mass to be maintained, the correct supply of AA needs to be available in the AA pool. The AA pool is maintained by a supply of AA in the diet, synthesis of AA in the liver, and catabolism of body tissues such as muscle. There also needs to be a stimulus to repair and maintain muscle such as exercise. With a decline in protein digestibility with age caused by poor teeth, parasite damage, or both (Hintz, 1995), the ability to maintain a proper supply of the essential AA to the AA pool in the body may become more difficult. Improving the AA profile of the diet may provide the horse with the necessary AA supply to ensure adequate levels of essential AA in the AA pool. This can improve the body’s efficiency in maintaining muscle mass as well as prevent the need to catabolize muscle to provide AA for other functions in the body. Providing additional essential AA in a synthetic form may be advantageous as well as free AA are readily available and could increase absorption of these AA compared with protein-bound AA, as has been documented in swine with synthetic lysine (Batterham, 1978). The supplementation of diets with lysine and threonine in this study might have improved the diet’s AA profile as well as improved the availability of these AA to the horse, as they were provided in the synthetic form in this experiment.
Body weight is an initial indicator of health and vigor, and BCS is an evaluator of body fat deposits. Weight should be considered in combination with BCS to help determine the proportion of fat to muscle. The lack of difference in overall weight in this study caused by age, diet, or an interaction shows that all horses maintained their health. The change in weight for the S-group horses, revealing a gain compared with a loss of weight in the N-group horses, combined with a lower BCS for the S-group horses suggests the change in weight was due to a shift in muscle mass and fat proportions in the body composition of horses in the S group. A reduction in BCS suggests a lower fat proportion in the body for the S-group horses and, without a concurrent reduction in BW, suggests an increase in muscle mass. Greater muscle mass scores in the S-group horses also suggest that this shift was visually perceived by the individuals scoring the horses as an increase in muscle mass. Graham et al. (1994) concluded that an increase in BW for yearling horses was most likely an increase in muscle because body fat was not different between treatments. O’Connor et al. (2002) also concluded that a lack of difference in BW between treatments combined with a decrease in BCS for one of the treatment groups supported the conclusion that muscle mass was increased.
Creatinine is a by-product of the creatine phosphate reaction in muscle. Therefore, it is used as a marker of muscle mass (Finco, 1997). Also, 3MH is a by-product of the breakdown of actin and myosin proteins in muscle fibers. Therefore, 3MH is a marker of the level of muscle catabolism occurring in the body. Natural turnover in muscle occurs in response to diet and exercise. An increase in muscle turnover can result because of a lack of essential AA or an increase in strenuous exercise (Gallagher et al., 1999). Greater creatinine in the S-group horses supports the suggestion that muscle mass increased for these groups. Without a difference in overall BW, lower 3MH in the S-group horses suggests less muscle catabolism, which would tend to support the theory that the AA pool was better able to supply the necessary essential AA for muscle maintenance as both groups received similar exercise. Plasma urea nitrogen concentrations have been used to judge the quality of dietary protein (Eggum, 1970). Those horses fed the supplementary AA had lower PUN concentrations compared with nonsupplemented groups. This supports the conclusion that the AA profile of the diet was improved with the supplementation of lysine and threonine. Both dietary groups consumed similar amounts of CP (1,129 g/d for the N group and 1,213 g/d for the S group; P > 0.05), yet the S-group horses had lower PUN concentrations. Reduction of PUN concentrations have been used to justify an improvement in dietary protein quality for yearling horses fed diets supplemented with lysine and threonine (Graham et al., 1994). This conclusion is further supported by the negative correlations observed between PUN concentrations and lysine and threonine intakes in this experiment
The dietary requirement for various AA correlates closely with the concentration of the same AA in muscle tissue in many species such as chickens and swine (Buttery and Lindsay, 1980). Examining the concept of ideal protein in chickens (the optimum balance of essential AA that provides for maximum utilization of the protein as a whole) finds a close relationship between AA ratios for the end products of protein synthesis (egg and tissue) and AA ratios recommended for ideal protein in the chicken (Cole and Van Lunen, 1994).
Ideal protein in horses has not been determined. Muscle AA profiles could be used to estimate ideal dietary AA ratios for the horse based on the evidence that, in other species, the tissue AA profile is correlated with the dietary requirement for the same AA. When expressing AA ratios, lysine is set to 100, as lysine is presumed to be the first-limiting AA for the horse (Ott et al., 1979), and all other AA would be relative to lysine. When previously reported data (total muscle AA) regarding muscle AA profiles in horses are examined, a relative estimate of muscle AA ratios for the horse would be arginine, 0.74; histidine, 0.58; isoleucine, 0.55; leucine, 1.07; lysine, 1.00; methionine, 0.27; phenylalanine, 0.60; threonine, 0.61; valine, 0.62; and tryptophan, data not available (Bryden, 1991).
Comparing the dietary AA ratios between the S-group diet and N-group diet in the current study yields different ratios. The ratios for the S-group diet are arginine, 0.82; histidine, 0.31; isoleucine, 0.57; leucine, 1.16; lysine, 1.00; methionine, 0.24; phenylalanine, 0.71; threonine, 0.88; valine, 0.76; and tryptophan, not analyzed. The ratios for the N-group diet are arginine, 1.24; histidine, 0.46; isoleucine, 0.86; leucine, 1.60; lysine, 1.00; methionine, 0.37; phenylalanine, 1.09; threonine, 0.96; valine, 1.15; and tryptophan, not analyzed. Comparing these ratios to muscle AA ratios estimated from Bryden (1991) reveals that the AA profile of the S-group diet more closely resembled the muscle AA ratios (with the exception of histidine) reported by Bryden (1991) and presumably resulted in a better balance and supply of AA to the horse in relation to the horses’ needs for muscle maintenance. This suggestion is speculation at this point and cannot be proven without muscle AA data from the present study because muscle AA profiles in horses have not been extensively studied.
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Implications
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Approximately 20% of the horse population is estimated to be "aged," which stimulates a need to understand the specific needs of this group of horses. Providing better quality protein may support muscle mass, which will allow horses to maintain their usefulness longer. These data suggest that exercising horses (regardless of age) may need additional lysine and threonine. Because of the observed responses in this study, it is apparent that the amino acid needs of the mature exercising horse are not well defined and require further research. The relationship between muscle amino acid profiles in horses and dietary amino acid profiles also warrants further attention.
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Footnotes
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1 The authors thank the students who assisted on the project: C. Hatsell, K. McCreight, K. Stevens, R. Skillicorn, D. Furler, and S. Holder. 
2 Correspondence: Campus Box S-604 (phone: 540-466-7168; fax: 540-669-5763; e-mail: thiers{at}vic.edu).
Received for publication September 29, 2004.
Accepted for publication September 8, 2005.
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Literature Cited
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Buttery, P. J., and D. B. Lindsay. 1980. Protein Deposition in Animals. Butterworth Press, Boston, MA.
Cole, D. J., and T. A. Van Lunen. 1994. Ideal Amino Acid Patterns. Pages 99–112 in Amino Acids in Farm Animal Nutrition. J. P. F. D’Mello, ed. CAB International, Wallingford, UK.
Eggum, B. 1970. Blood urea measurement as a technique for assessing protein quality. Br. J. Nutr. 24:983–988.[Medline]
Finco, D. R. 1997. Kidney function. Page 441 in Clinical Biochemistry of Domestic Animals. 5th ed. J. J. Kaneko, J. W. Harvey, and M. L. Bruss, eds. Academic Press, San Diego, CA.
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Graham, P. M., E. A. Ott, J. H. Brendemuhl, and S. H. TenBroeck. 1994. The effect of supplemental lysine and threonine on growth and development of yearling horses. J. Anim. Sci. 72:380–386.[Abstract]
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Hintz, H. F. 1995. Nutrition of the geriatric horse. Pages 8–10 in Proc. Cornell Nutr. Conf., Cornell Univ., Ithaca, NY.
Miller-Graber, P. A., L. M. Lawrence, E. Kurcz, R. Kane, K. Bump, M. Fisher, and J. Smith. 1990. The free amino acid profile in the middle gluteal before and after fatiguing exercise in the horse. Equine Vet. J. 22(3):209–210.[Medline]
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O’Connor, C. I., B. D. Nielsen, H. C. Schott, and H. M. Clayton. 2002. Effects of weight carrying, exercise and a myo-anabolic supplement on growth and muscle. Equine Vet. J. Suppl. 34:178–181.
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Abstract
Introduction
