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Changes of serum free amino acids in eventing horses at rest and during exercise in response to dietary protein
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Changes of serum free amino acids in eventing horses at rest and during exercise in response to dietary protein ⁎
C.A.A. Oliveiraa,b, , L.A.M. Kellerb,1, M.T. Ramosb, V.P. Silvaa, C.D. Baldanib, F.Q. Almeidab a b
Animal Science Institute, Universidade Federal Rural do Rio de Janeiro (UFRRJ), Br 465, km 7, 23890-000 Seropédica, Rio de Janeiro, Brazil Veterinary Institute, Universidade Federal Rural do Rio de Janeiro (UFRRJ), Seropédica, Rio de Janeiro, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords: Amino acid metabolism Amino acids concentration Diet Horses
Although there are published expertise about serum free amino acids, to the knowledge of the authors no data were reported on eventing horses. In this way, it is important to study the concentration of dietary protein and serum free amino acid during exercise in order to improve nutritional strategies. The aim of this study was to investigate the concentration of the serum free lysine (Lys), threonine (Thr), leucine (Leu), isoleucine (Ile) and valine (Val) of eventing horses fed diets with different levels of protein at before and during exercise. Twentyfour Brazilian Sport Horses trained for eventing were used in a randomized block design with 4 diets (7.5%, 9.0%, 11.0% and 13.0% of CP) and 6 repetitions (horses). Horses were blocked according to their experience in competitions. The test protocol consisted of a warm up of walking and trotting and then a gallop starting at 6.0 m/s with increases in speed of 1 m/s every minute up to 10 m/s. Venous blood samples were collected before the test and during exercise. Concentration of Lys, Thr, Leu, Ile and Val from diets and serum free amino acids were determined by HPLC analysis. Total intake values of Lys, Thr, Leu, Ile and Val were affected by dietary protein levels. Differences on the serum free concentrations of Lys, Leu, Ile and Val as a function of the sampling time were observed. The Lys requirement for athletic horses appear to be lower than what is currently proposed. Moreover, the effect of exercise on serum free Lys, Leu, Ile and Val concentration, may be interpreted as an indicator of these amino acids metabolic response.
1. Introduction In athletic horses, dietary protein:amino acid, especially lysine and threonine, ratios have an important role in the nitrogen balance, acidbase balance and muscle protein metabolism (Graham-Thiers and Kronfeld, 2005). Moreover, the serum free branched chain amino acids (BCAA) play a critical role during and after exercise on muscle recovery in horses (Matsui et al., 2006). The concentrations of free amino acids in horses’ serum or plasma can vary under different conditions, in particular with different nutritional programs, type and quality of food (Bergero et al., 2005; Graham-Thiers and Bowen, 2011; Urschel and Lawrence, 2013). Furthermore, AA were previously examined mostly in growing horses, rather than in athletic/exercising horses. Therefore, it is important to study the concentrations of dietary protein and serum free amino acids during exercise in order to improve nutritional strategies. In addition, lysine is essential to horses in the composition and growth of muscle, and leucine, isoleucine and valine
play a role in the muscle tissue metabolism. Dietary crude protein can change horses serum free amino acid concentrations during exercise. To our knowledge, these effects need to be more investigated considering intense exercising horses. The aim of this study was to investigate the concentration of serum free lysine, threonine, leucine, isoleucine and valine of eventing horses fed diets with different levels of crude protein, before and during exercise. 2. Material and methods The study was conducted at the Equine Performance Evaluation Laboratory at the Brazilian Army Cavalry School, Rio de Janeiro, Brazil. The chemical analyses were performed at the Equine Health Laboratory and at the Mycological and Mycotoxicologic Research Center both at the Universidade Federal Rural do Rio de Janeiro.
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Corresponding author at: Veterinary and Animal Science School, Universidade Federal da Bahia, Av. Ademar de Barros, 500, Ondina, 40170-110 Salvador, Bahia, Brazil. E-mail addresses: [email protected] (C.A.A. Oliveira), [email protected] (L.A.M. Keller), [email protected] (M.T. Ramos), [email protected] (V.P. Silva), [email protected] (C.D. Baldani), [email protected] (F.Q. Almeida). 1 Present address: Animal Science Institute, Universidade Federal Fluminense, Niterói, Rio de Janeiro, Brazil. http://dx.doi.org/10.1016/j.livsci.2017.03.008 Received 30 October 2016; Received in revised form 24 January 2017; Accepted 8 March 2017 1871-1413/ © 2017 Published by Elsevier B.V.
Please cite this article as: Oliveira, C.A.d.A., Livestock Science (2017), http://dx.doi.org/10.1016/j.livsci.2017.03.008
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3. Management of horses
concentrate 3 times daily in equal amounts.
Twenty-four Brazilian Sport Horses, 16 males and 8 females, aged between 8 and 15 yr, mean 488 kg BW (432–562 kg) and mean 5.3 BCS (5.0–5.5 BCS) (Henneke et al., 1983) were used. Horses were randomly distributed in individual 4×4 m box stalls, with a water dispenser, a feeder, and wood shaving bedding. Diet adaptation lasted 21d. All horses were trained according to the Brazilian Army Cavalry School´s protocol for eventing. The yearly program comprised 10 months of training divided into three cycles (initial, intermediate and final) of three months each. The study began at the middle of the intermediate cycle (day 130) and finished at the end of the final cycle (day 270). The weekly workload consisted of 60 min of daily training (6 d per week), including 30% walking, 30% trotting, 10% galloping, and 30% jumps on dirt and grass tracks. The training program targeted the eventing modality and was considered as intense physical activity (NRC, 2007). On the 24 h that preceded the exercise test, horses were not submitted to intense activities, but to flexing exercises (light exercise consisting of walking and trotting flexing exercises and slow canter).
6. Exercise test The exercise test was carried out on the 262nd day of the training period, at the end of final cycle, on a high-speed treadmill (Galloper 5500 - Sahinco, São Paulo, Brazil). The protocol consisted of a warm up of walking and trotting for 6 min and then a gallop starting at 6.0 m/s with increases in speed of 1 m/s every minute up to 10 m/s. The treadmill inclination was set to 4% during the test. The total test time was 11 min, covering 3.400 m, followed by 6 min of trot and walk to cool-down, without slope. 7. Blood samples Venous blood samples were collected from the left jugular vein of each horse on the following times: before the test (6 h after meal) and during exercise (last 15 s of the last gallop). Blood was collected into silicone tubes without anticoagulant and was processed immediately. The serum was transferred to a 2 mL Eppendorf and samples were kept at −18 °C until analyses of the free amino acids.
4. Experimental design 8. Amino acid analysis Twenty-four horses were used in a randomized block design with 4 diets (protein levels) and 6 repetitions (horses). Horses were blocked according to their experience in competitions (eventing experience: level 1 and 1*).
Total concentration of lysine (Lys), threonine (Thr), leucine (Leu), isoleucine (Ile) and valine (Val) from diets (concentrate and coast-cross hay) and blood serum free amino acids were determined by highperformance liquid chromatography (HPLC). All analytical methods were compared with the reference amino acid external standard in triplicate evaluation. The standard curve for calibration method of AA evaluated was obtained with values of 100–800 µmol/L with correlation levels of ≥99% (Helrich, 1990). Diet and serum amino acid concentrations were determined using reverse-phase HPLC of phenylisothiocyanate derivatives. The chromatographic conditions mobile phase (methanol:acetonitrile:MilliQ water 10:20:70); the HPLC equipment was composed of a binary pump equipped with micro vacuum degasser, manual injection (30 µL injected), column compartment and fluorescence detector; for analysis use a Hypersil ODS(C18) column. The extraction of the solution with total amino acid of diet samples were performed according to Antonie et al. (1999) and the extraction of the solution with serum free amino acid, according to Hill et al. (1979). After extraction, the tubes were cooled and the samples was filtered using HPLC 13-mm syringe filters (0.45 µm, 30 mm)5; the filtrate was diluted with mobile phase (1:2 vol:vol) in amber glass vials. Before injection, AA was derivatized on-line using o-phthaldehyde (Antonie et al., 1999). The elution of samples were performed at a flow rate of 2.0 mL/min by gradient elution, and the total run time was 30 min. Fluorescence detection was carried out at 340 (excitation) and 450 nm (emission).
5. Experimental diets The horses were fed experimental diets from day 130–270. The intake was calculated for 1.8% of BW. Diets were formulated for intense exercising horses, according to NRC (2007) with 4 different levels of CP: 7.5%, 9.0%, 11.0%, and 13.0%, at a concentrate and hay (50:50) on a DM basis (Table 1). The concentrate was fed 3 times daily in equal amounts at 0400, 1300, and 2000 h, and the roughage was fed 2 times daily in equal amounts at 1100 and 1600 h. Soybean oil, previously weighed and calculated individually, was added directly to the Table 1 Ingredients and nutrients composition of total diets, concentrate and hay (50:50) on a DM basis. Item
Soybean meal (%) Rolled oats (%) Commercial concentrate (%) Soybean oil (%) Mineral supplement (%)a Calcium carbonate (%) L-lysine (%) Cynodon dactylon hay (%) Total Dry matter (%) Crude protein (%) Lysine (%) Digestible energy (Mcal/kgDM) Total amino acidsb Lysine (%) Threonine (%) Leucine (%) Isoleucine (%) Valine (%)
Protein diet level (%) 7.5
9.0
11.0
13.0
1.5 28.3 10.0 7.0 2.0 1.0 0.15 50 100 88.1 7.5 0.35 2.8
7.0 23.0 10.0 7.0 2.0 1.0 – 50 100 88.0 9.0 0.36 2.8
12.5 19.5 8.0 7.0 2.0 1.0 – 50 100 87.9 11.0 0.50 2.8
17.5 15.8 7.0 6.7 2.0 1.0 – 50 100 87.9 13.0 0.62 2.8
0.35 0.29 0.75 0.99 0.37
0.36 0.32 0.87 1.02 0.39
0.49 0.58 0.90 1.13 0.43
0.62 0.64 1.04 1.35 0.61
9. Statistical analyses A two way ANOVA (time and diet) was performed using the GLM procedure of SAS software (version 9.3). The Shapiro-Wilk test was used to verify the normality of the data set. Linear regression analyses as a function of dietary CP were performed to obtain protein and total amino acids intake data. Linear and quadratic regression analyses were also used to test the effect of dietary CP levels on serum free amino acids concentrations before and during exercise. Significance was declared when P < 0.05. 10. Results
a
Omolene-fós: Ca (Max) 150 g, P (Min) 70 g, S 10 g, Mg 10 g, Na 150 g, Fe 2.500 mg, Cu 820 mg, Zn 2500 mg, Mn 2124 mg,I 20 mg, Se 12,5 mg, Co 20 mg, Cr 6 mg. b The concentration of amino acid of diets was achieved after extracting the solution with total amino acid of ingredient sample by HPLC according to Antonie et al. (1999).
10.1. Dietary amino acid Total intake values of Lys, Thr, Leu, Ile and Val were affected by 2
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Table 2 Total intake of nutrients and amino acid by horses fed diets with different protein levels. Protein diet level (%)
P value
Item
7.5
9.0
11.0
13.0
SEM
L
DM (kg/d) CP (g/d) Lys (g/d) Thr (g/d) Leu (g/d) Ile (g/d) Val (g/d)
8.0+0.9 754.9+84.6 33.2+2.8 23.1+1.5 60.0+3.1 79.2+2.3 29.6+2.5
8.3+0.9 884.6+102.4 36.6+3.2 25.6+2.0 65.6+2.7 80.6+2.7 30.2+2.6
8.4+1.3 1.039.3+130.4 48.5+5.6 46.3+2.4 70.1+4.9 89.2+3.1 33.2+3.8
8.5+0.7 1.208.3+125.5 49.3+2.8 48.4+1.5 76.2+1.7 93.5+1.7 41.8+1.1
0.09 20.8 2.7 0.3 1.8 1.0 2.6
0.1745 < 0.0001 < 0.0001a < 0.0001b < 0.0001c < 0.001d < 0.0001e
Dry matter (DM), crude protein (CP), ether extract (EE), lysine (Lys), threonine (Thr), isoleucine (Ile), leucine (Leu) and valine (Val). SEM: Standard error of means. Linear regression (L) as a function of CP diet levels. a ˆ Lys =2.38+38.23x, R2=0.80; b ˆ Thr =−16.25+51.34x, R2=0.86; c ˆ Leu =39.58+28.36x, R2=0.97; d ˆ Ile =57.72+27.15x, R2=0.89; e ˆ Val =10.21+22.83x, R2=0.84.
12. Discussion
dietary protein levels. Linear increasing responses were observed for Lys, Thr, Leu, Ile and Val (P < 0.01) as a function of the diets (Table 2). Concerning to the dietary total amino acid, the NRC (2007) recommends for a 488 kg BW horse in intense exercise, a daily intake of 36.1 g/d of Lys. Horses from the 7.5% CP experimental diet, intake 33.2 g/d of Lys, 8.3% less Lys than the recommendations.
In the present study, the dietary CP linearly increased the AA intake of the horses (P < 0.01). Furthermore, the amounts of Thr, Ile, Leu and Val consumed by the horses were enough or higher to meet the requirements (NRC, 2007) for horses in intense physical activities. The exception was the 7.5% CP diet group, the average intakes were 100 and 3 g/d, 8.3% less than recommended by NRC (2007), for CP and Lys, respectively, to the corresponding intense exercise horse category. However, according to Coenen et al. (2011) and Urschel and Lawrence (2013), horses' requirements described in the NRC (2007) are expressed in CP and Lys concentrations and could be better evaluated if expressed in metabolizable protein (French and German systems). However, based on the total amino acid intake and clinical evaluations of the horses during the experiment, no deficiencies of amino acids capable of impairing their performance were noted. During the experimental study, the horses maintained their body weight (497 ± 23 kg BW) and body score (5.0). According to Assenza et al. (2004) and Bergero et al. (2005), the intensity of exercise influenced the serum concentrations of BCAA,
11. Serum free amino acid The dietary CP levels significantly influenced (P=0.001) the serum free AA concentration. The serum concentrations of free Lys, Leu, Ileu and Val were affected by sampling time, increasing with exercise (Table 3). Both linear (P=0.001) and quadratic (P=0.016) responses were observed on the serum free Lys, Leu, Ile and Val concentrations during exercise as a function of the increasing CP in the diets. The concentrations of Thr did not show significant changes as a function of the dietary CP and the serum free Thr concentration decreased with exercise. The exercise induced changes of the serum free amino acids concentrations in the horses.
Table 3 Serum free amino acid concentrations (µmol/L) before and during exercise in horses fed diets with different protein levels. Serum free AAs (µmol/L)
Protein diet level (%)
P value
Moment
7.5
9.0
11.0
13.0
SEM
L
Q
Lys
Before Exercise
135.6 ± 4.2 137.1 ± 10.8
140.5 ± 5.4 170.2 ± 15.8
154.5 ± 15.7 145.3 ± 7.8
137.5 ± 7.8 147.9 ± 10.8
4.4 4.0
0.018 0.030
0.024a 0.030b
Thr
Before Exercise
160.5 ± 31.6 109.5 ± 29.9
141.9 ± 40.8 122.1 ± 31.5
155.6 ± 26.5 127.9 ± 21.6
116.5 ± 27.1 140.1 ± 4.5
20.6 15.5
0.711 0.797
0.488 0.984
Leu
Before Exercise
145.4 ± 4.9 145.9 ± 11.2
149.5 ± 5.6 180.3 ± 16.6
164.0 ± 16.3 156.1 ± 7.6
148.3 ± 7.6 156.9 ± 11.2
5.1 4.1
0.032 0.019
0.045c 0.020d
Ile
Before Exercise
212.3 ± 83.7 138.5 ± 10.9
185.2 ± 84.5 226.4 ± 86.3
200.5 ± 88.6 248.7 ± 99.2
177.2 ± 91.1 167.8 ± 35.8
27.7 20.2
0.887 0.008
0.960 0.010e
Val
Before Exercise
210.1 ± 72.7 104.6 ± 73.4
263.6 ± 58.9 321.1 ± 73.2
243.3 ± 77.1 260.0 ± 76.0
270.5 ± 48.8 280.4 ± 48.3
26.6 70.7
0.515 0.004
0.656 0.011f
Lysine (Lys), threonine (Thr), isoleucine (Ile), leucine (Leu) and valine (Val). SEM: Standard error of means. Linear (L) and quadratic regression (Q) as a function of CP diet levels. a ˆ Lys 209.65+29.42x −5.49x2, R2 =0.24; b ˆ Lys =210.22+38.77x −7.60x2, R2 =0.21; c ˆ Leu =221.12+27.18x – 4.97x2, R2 =0.21; d ˆ Leu =215.91+42.62x −8.35x2, R2 =0.23; e ˆ Ile =56.88+221.92x −42.17x2, R2 =0.29; f ˆ Val =− 20.27+291.82x −49.03x2, R2 =0.44.
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excesses are eliminated as nitrogen (N) in urine, however demanding larger water uptake and special attention to possible dehydration. Besides, studies about N balance in horses consuming different CP levels (Oliveira et al., 2015) indicated that the absorption of N increases with the inclusion of dietary CP. However, the ratio Nretained:Nabsorbed is directly influenced by the quality of dietary CP, but not by the CP quantity. Thus, the use of good quality protein to formulate horses‘ diets can reduce the quantity of CP included leading to lower costs for formulation and diminishing the N excretion (Urschel and Lawrence, 2013) promoting an environmental benefit.
especially in long duration and low intensity mostly aerobic exercises, in which BCAAs may be used as a source for energy production. In a previous study, Essén-Gustavsson et al. (2010) evaluated horses during short and intense exercises (mostly anaerobic) on a high-speed treadmill reporting that the diet did not affect the plasma AA concentration, but there was an interaction between exercise and dietary CP for Lys and Val plasma concentrations. In contrast, in our study, the dietary CP affected the horses' serum free AA concentration. The same response that were reported by Essén-Gustavsson et al. (2010) was observed in the present study not only for Lys and Val serum free concentrations but also for Leu and Ile, that were influenced by mostly anaerobic exercise and diet. In both papers, authors used good quality diets with a good AA profile. Essén-Gustavsson et al. (2010) forage-only diets were offered as silages, while in the present study horses were feed mixed diets with concentrateds and hay (50:50), and soybean meal as the main source of CP. It is possible that the different AA sources may have influenced the blood AA concentration responses differently. Diet formulations for athletic horses are focused on energy content and sometimes the AA concentration can be undervalued. Horses are very sensitive to the type of diet as well as to the quality of dietary protein provided (Harris et al., 2013) and, therefore, it is justifiable the need for knowledge on the sources of protein with high quality AA profile. According to Westermann et al. (2011), AAs should not be considered as limiting to athletic performance in horses, except for aspartic acid. However, the minimum requirements of the dietary CP for athletic horses is yet to be determined. Therefore, the mostly anaerobic exercise, performed on a highspeed treadmill, induced measurable changes in serum concentrations of free amino acids in eventing horses. However, it is not possible to claim that these changes are due to muscle catabolism because no discernible trend in the amino acid concentrations was observed either before or during exercise. More studies should be developed with observations before, during and after exercise for better evaluation of the dietary AA and their relation with the AA's turnover (Matsui et al., 2006). According to Graham-Thiers et al. (1994), Lys is the first-limiting amino acid for growing horses and Thr is presumed to be the second for lactating mares (Wilson and Graham-Thiers, 2009). Moreover, according to Graham-Thiers and Kronfeld (2005), exercising horses need the correct supply of Lys to repair and maintain muscle mass, as a natural turnover in muscle occurs in response to dietary AA and exercise. Likewise, in a previous study, evaluating light exercise in horses fed different amino acid concentration, Graham-Thiers and Bowen (2011) reported that Lys plays a fundamental role in the metabolism of athletic horses. However, it is a theoretical question whether the discrepancies between studies on blood AAs occur because of different types (aerobic and anaerobic) and intensities of exercises, training status and age of horses, or due the muscles utilization of BCAA as an alternative energy source (Hackl et al., 2009). There are few studies concerning amino acid requirements of horses and also few published data on CP and AA pre-cecal digestibility (Zeyner et al., 2010; Coenen et al., 2011). According to Zeyner et al. (2010), the knowledge of the protein fraction that can suffer enzyme digestion is essential, since equine amino acidic absorption takes place only in the small intestine. However, if an amount of protein is not digested in this segment, it will serve as an important substrate for microbial. Moreover, Zeyner et al. (2010) and Coenen et al. (2011) recommend the utilization of the Cornell Net Carbohydrate and Protein System for determination of the fraction of protein digestible in the small intestine, trying to focus on AA% intake x AA% digestibility. The Lys requirements for athletic horses appear to be lower than what is currently proposed and therefore it seems possible to formulate less expensive diets and yet with a better amino acid profile and smaller excretion of nitrogen. According to Harris et al. (2013), the CP or AA excessive intake would not represent a problem for horses, since the
13. Conclusions Protein diet levels affect the serum free amino acid concentration during exercise. The effect of exercise on serum free Lys, Leu, Ile and Val concentration, may be interpreted as an indicator of these amino acids metabolic response and demand further investigation. Authors' declaration of interests No competing interests have been declared. Ethical animal research The study was approved by the Ethics in Research Board of Universidade Federal Rural do Rio de Janeiro, number 029/2009. Authorship C. A. A. Oliveira and F. Q. Almeida contributed to study design, study execution, data analysis and interpretation. M. T. Ramos contributed to study execution, data analysis, structure and text translation. V. P. Silva and C. D. Baldani contributed to study execution and data analysis. L. A. M. Keller contributed to data analysis and interpretation. All authors contributed to the preparation of the manuscript and gave final approval of the manuscript. Acknowledgements The authors acknowledge the support of the Brazilian Army Cavalry School, the CNPQ (Conselho Nacional de Desenvolvimento Científico e Tecnológico), the FAPERJ (Fundação de Amparo a Ciência do Estado do Rio de Janeiro), and the laboratories of the Universidade Federal Rural do Rio de Janeiro, the Equine Health Laboratory and the Mycological and Mycotoxicologic Research Center. References Antonie, F.R., Wei, C.I., Littell, R.C., Marshall, M.R., 1999. HPLC method for analysis of free amino acids in fish using o-phthaldialdehyde precolumn derivatization. J. Agric. Food Chem. 47, 5100–5107. Assenza, A., Bergero, D., Tarantola, M., Piccione, G., Caola, G., 2004. Blood serum branched chain amino acids and tryptophan modifications in horses competing in long-distance rides of different length. J. Anim. Physiol. Anim. Nutr. 88, 172–177. Bergero, D., Assenza, A., Schiavone, A., Piccione, G., Peroan, G., Caola, G., 2005. Amino acid concentrations in blood serum of horses performing long lasting low-intensity exercise. J. Anim. Physiol. Anim. Nutr. 89, 146–150. Coenen, M., Kienzle, E., Vervuert, I., Zeyner, A., 2011. Recent German developments in the formulation of energy and nutrient requirements in horses and the resulting feeding recommendations. J. Equine Vet. Sci. 31, 219–229. Essén-Gustavsson, B., Connysson, M., Jansson, A., 2010. Effects of crude protein intake from forage-only diets on muscle amino acids and glycogen levels in horses in training. Equine Vet. J. 42, 341–346. Graham-Thiers, P.M., Kronfeld, D.S., 2005. Amino acid supplementation improves muscle mass in aged and young horses. J. Anim. Sci. 83, 2783–2788. Graham-Thiers, P.M., Ott, E.A., Brendemuhl, J.H., Tenbroeck, S.H., 1994. The effect of supplemental lysine and threonine on growth and development of yearling horses. J. Anim. Sci. 72, 380–386. Graham-Thiers, P.M., Bowen, L.K., 2011. Effect of protein source on nitrogen balance and plasma amino acids in exercising horses. J. Anim. Sci. 89, 729–735. Hackl, S., Hoven, Van Den, Zickl, R., Spona, M., Zentek, J, J., 2009. The effects of short
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National Research Council – NRC, 2007. Nutrient requirements of horses. National Academies Press, Washington, D.C, pp. 341. Oliveira, C.A.A., Azevedo, J.F., Martins, J.A., Barreto, M.P., Silva, V.P., Julliand, V., Almeida, F.Q., 2015. The impact of dietary protein levels on nutrient digestibility and water and nitrogen balances in eventing horses. J. Anim. Sci. 93, 229–237. Westermann, C.M., Dorland, L., Wijnberg, I.D., de Sain-van der Velden, M.G.M., Van Breda, E., Barneveld, A., de Graaf-Roelfsema, E., Keizer, H.A., Van der Kolk, J.H., 2011. Amino acid profile during exercise and training in Standardbreds. Res. vet. Sci. 91, 144–149. Wilson, J.A., Graham-Thiers, P.M., 2009. Muscle and plasma amino acids in pregnant/ lactating mares and their weanling foals. J. Equine Vet. Sci. 29, 370–371. Urschel, K.L., Lawrence, L.M., 2013. Amino acids and protein. In: Coenen, M., Harris, P., Geor, R.J. (Eds.), Equine Applied and Clinical Nutrition: Health, Welfare and Performance. Elsevier, London, pp. 113–135. Zeyner, A., Kirchhof, S., Susenbeth, A., Südekum, K. H., Kienzle, E., 2010. Protein evaluation of horse feed: a novel concept. The impact of nutrition on the health and welfare of horses. EAAP publication, 128, pp. 40–42.
intensive exercise on plasma free amino acids in Standardbred Trotters. J. Anim. Physiol. Anim. Nutr. 93, 165–173. Harris, P., Coenen, M., Geor, R.J., 2013. Controversial areas in equine nutrition and feeding management: The Editors' views. In: Coenen, M., Harris, P., Geor, R.J. (Eds.), Equine Applied and Clinical Nutrition: Health, Welfare and Performance. Elsevier, London, pp. 455–468. Helrich, K.C., 1990. Official Methods of Analysis of the AOAC 2. Association of Official Analytical Chemists Incpp. 15. Henneke, D.R., Potter, G.D., Kreider, J.L., Yeates, B.F., 1983. Relationship between condition score, physical measurements and body fat percentage in mares. Equine Vet. J. 15, 371–372. Hill, D.W., Walters, F.H., Wilson, T.D., Stuart, J.D., 1979. HPLC determination of amino acids in the picomole range. Anal. Chem. 51, 1338–1341. Matsui, A., Ohmura, H., Assait, Y., Takahashi, T., Hiraga, A., Okamura, K., Tokimura, H., Sugino, T., Obitsu, T., 2006. Effect of amino acid and glucose administration following exercise on turnover of muscle protein in the hindlimb femoral region of Thoroughbreds. Equine Vet. J. 36, 611–616.
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Changes of serum free amino acids in eventing horses at rest and during exercise in response to dietary protein ⁎
C.A.A. Oliveiraa,b, , L.A.M. Kellerb,1, M.T. Ramosb, V.P. Silvaa, C.D. Baldanib, F.Q. Almeidab a b
Animal Science Institute, Universidade Federal Rural do Rio de Janeiro (UFRRJ), Br 465, km 7, 23890-000 Seropédica, Rio de Janeiro, Brazil Veterinary Institute, Universidade Federal Rural do Rio de Janeiro (UFRRJ), Seropédica, Rio de Janeiro, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords: Amino acid metabolism Amino acids concentration Diet Horses
Although there are published expertise about serum free amino acids, to the knowledge of the authors no data were reported on eventing horses. In this way, it is important to study the concentration of dietary protein and serum free amino acid during exercise in order to improve nutritional strategies. The aim of this study was to investigate the concentration of the serum free lysine (Lys), threonine (Thr), leucine (Leu), isoleucine (Ile) and valine (Val) of eventing horses fed diets with different levels of protein at before and during exercise. Twentyfour Brazilian Sport Horses trained for eventing were used in a randomized block design with 4 diets (7.5%, 9.0%, 11.0% and 13.0% of CP) and 6 repetitions (horses). Horses were blocked according to their experience in competitions. The test protocol consisted of a warm up of walking and trotting and then a gallop starting at 6.0 m/s with increases in speed of 1 m/s every minute up to 10 m/s. Venous blood samples were collected before the test and during exercise. Concentration of Lys, Thr, Leu, Ile and Val from diets and serum free amino acids were determined by HPLC analysis. Total intake values of Lys, Thr, Leu, Ile and Val were affected by dietary protein levels. Differences on the serum free concentrations of Lys, Leu, Ile and Val as a function of the sampling time were observed. The Lys requirement for athletic horses appear to be lower than what is currently proposed. Moreover, the effect of exercise on serum free Lys, Leu, Ile and Val concentration, may be interpreted as an indicator of these amino acids metabolic response.
1. Introduction In athletic horses, dietary protein:amino acid, especially lysine and threonine, ratios have an important role in the nitrogen balance, acidbase balance and muscle protein metabolism (Graham-Thiers and Kronfeld, 2005). Moreover, the serum free branched chain amino acids (BCAA) play a critical role during and after exercise on muscle recovery in horses (Matsui et al., 2006). The concentrations of free amino acids in horses’ serum or plasma can vary under different conditions, in particular with different nutritional programs, type and quality of food (Bergero et al., 2005; Graham-Thiers and Bowen, 2011; Urschel and Lawrence, 2013). Furthermore, AA were previously examined mostly in growing horses, rather than in athletic/exercising horses. Therefore, it is important to study the concentrations of dietary protein and serum free amino acids during exercise in order to improve nutritional strategies. In addition, lysine is essential to horses in the composition and growth of muscle, and leucine, isoleucine and valine
play a role in the muscle tissue metabolism. Dietary crude protein can change horses serum free amino acid concentrations during exercise. To our knowledge, these effects need to be more investigated considering intense exercising horses. The aim of this study was to investigate the concentration of serum free lysine, threonine, leucine, isoleucine and valine of eventing horses fed diets with different levels of crude protein, before and during exercise. 2. Material and methods The study was conducted at the Equine Performance Evaluation Laboratory at the Brazilian Army Cavalry School, Rio de Janeiro, Brazil. The chemical analyses were performed at the Equine Health Laboratory and at the Mycological and Mycotoxicologic Research Center both at the Universidade Federal Rural do Rio de Janeiro.
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Corresponding author at: Veterinary and Animal Science School, Universidade Federal da Bahia, Av. Ademar de Barros, 500, Ondina, 40170-110 Salvador, Bahia, Brazil. E-mail addresses: [email protected] (C.A.A. Oliveira), [email protected] (L.A.M. Keller), [email protected] (M.T. Ramos), [email protected] (V.P. Silva), [email protected] (C.D. Baldani), [email protected] (F.Q. Almeida). 1 Present address: Animal Science Institute, Universidade Federal Fluminense, Niterói, Rio de Janeiro, Brazil. http://dx.doi.org/10.1016/j.livsci.2017.03.008 Received 30 October 2016; Received in revised form 24 January 2017; Accepted 8 March 2017 1871-1413/ © 2017 Published by Elsevier B.V.
Please cite this article as: Oliveira, C.A.d.A., Livestock Science (2017), http://dx.doi.org/10.1016/j.livsci.2017.03.008
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3. Management of horses
concentrate 3 times daily in equal amounts.
Twenty-four Brazilian Sport Horses, 16 males and 8 females, aged between 8 and 15 yr, mean 488 kg BW (432–562 kg) and mean 5.3 BCS (5.0–5.5 BCS) (Henneke et al., 1983) were used. Horses were randomly distributed in individual 4×4 m box stalls, with a water dispenser, a feeder, and wood shaving bedding. Diet adaptation lasted 21d. All horses were trained according to the Brazilian Army Cavalry School´s protocol for eventing. The yearly program comprised 10 months of training divided into three cycles (initial, intermediate and final) of three months each. The study began at the middle of the intermediate cycle (day 130) and finished at the end of the final cycle (day 270). The weekly workload consisted of 60 min of daily training (6 d per week), including 30% walking, 30% trotting, 10% galloping, and 30% jumps on dirt and grass tracks. The training program targeted the eventing modality and was considered as intense physical activity (NRC, 2007). On the 24 h that preceded the exercise test, horses were not submitted to intense activities, but to flexing exercises (light exercise consisting of walking and trotting flexing exercises and slow canter).
6. Exercise test The exercise test was carried out on the 262nd day of the training period, at the end of final cycle, on a high-speed treadmill (Galloper 5500 - Sahinco, São Paulo, Brazil). The protocol consisted of a warm up of walking and trotting for 6 min and then a gallop starting at 6.0 m/s with increases in speed of 1 m/s every minute up to 10 m/s. The treadmill inclination was set to 4% during the test. The total test time was 11 min, covering 3.400 m, followed by 6 min of trot and walk to cool-down, without slope. 7. Blood samples Venous blood samples were collected from the left jugular vein of each horse on the following times: before the test (6 h after meal) and during exercise (last 15 s of the last gallop). Blood was collected into silicone tubes without anticoagulant and was processed immediately. The serum was transferred to a 2 mL Eppendorf and samples were kept at −18 °C until analyses of the free amino acids.
4. Experimental design 8. Amino acid analysis Twenty-four horses were used in a randomized block design with 4 diets (protein levels) and 6 repetitions (horses). Horses were blocked according to their experience in competitions (eventing experience: level 1 and 1*).
Total concentration of lysine (Lys), threonine (Thr), leucine (Leu), isoleucine (Ile) and valine (Val) from diets (concentrate and coast-cross hay) and blood serum free amino acids were determined by highperformance liquid chromatography (HPLC). All analytical methods were compared with the reference amino acid external standard in triplicate evaluation. The standard curve for calibration method of AA evaluated was obtained with values of 100–800 µmol/L with correlation levels of ≥99% (Helrich, 1990). Diet and serum amino acid concentrations were determined using reverse-phase HPLC of phenylisothiocyanate derivatives. The chromatographic conditions mobile phase (methanol:acetonitrile:MilliQ water 10:20:70); the HPLC equipment was composed of a binary pump equipped with micro vacuum degasser, manual injection (30 µL injected), column compartment and fluorescence detector; for analysis use a Hypersil ODS(C18) column. The extraction of the solution with total amino acid of diet samples were performed according to Antonie et al. (1999) and the extraction of the solution with serum free amino acid, according to Hill et al. (1979). After extraction, the tubes were cooled and the samples was filtered using HPLC 13-mm syringe filters (0.45 µm, 30 mm)5; the filtrate was diluted with mobile phase (1:2 vol:vol) in amber glass vials. Before injection, AA was derivatized on-line using o-phthaldehyde (Antonie et al., 1999). The elution of samples were performed at a flow rate of 2.0 mL/min by gradient elution, and the total run time was 30 min. Fluorescence detection was carried out at 340 (excitation) and 450 nm (emission).
5. Experimental diets The horses were fed experimental diets from day 130–270. The intake was calculated for 1.8% of BW. Diets were formulated for intense exercising horses, according to NRC (2007) with 4 different levels of CP: 7.5%, 9.0%, 11.0%, and 13.0%, at a concentrate and hay (50:50) on a DM basis (Table 1). The concentrate was fed 3 times daily in equal amounts at 0400, 1300, and 2000 h, and the roughage was fed 2 times daily in equal amounts at 1100 and 1600 h. Soybean oil, previously weighed and calculated individually, was added directly to the Table 1 Ingredients and nutrients composition of total diets, concentrate and hay (50:50) on a DM basis. Item
Soybean meal (%) Rolled oats (%) Commercial concentrate (%) Soybean oil (%) Mineral supplement (%)a Calcium carbonate (%) L-lysine (%) Cynodon dactylon hay (%) Total Dry matter (%) Crude protein (%) Lysine (%) Digestible energy (Mcal/kgDM) Total amino acidsb Lysine (%) Threonine (%) Leucine (%) Isoleucine (%) Valine (%)
Protein diet level (%) 7.5
9.0
11.0
13.0
1.5 28.3 10.0 7.0 2.0 1.0 0.15 50 100 88.1 7.5 0.35 2.8
7.0 23.0 10.0 7.0 2.0 1.0 – 50 100 88.0 9.0 0.36 2.8
12.5 19.5 8.0 7.0 2.0 1.0 – 50 100 87.9 11.0 0.50 2.8
17.5 15.8 7.0 6.7 2.0 1.0 – 50 100 87.9 13.0 0.62 2.8
0.35 0.29 0.75 0.99 0.37
0.36 0.32 0.87 1.02 0.39
0.49 0.58 0.90 1.13 0.43
0.62 0.64 1.04 1.35 0.61
9. Statistical analyses A two way ANOVA (time and diet) was performed using the GLM procedure of SAS software (version 9.3). The Shapiro-Wilk test was used to verify the normality of the data set. Linear regression analyses as a function of dietary CP were performed to obtain protein and total amino acids intake data. Linear and quadratic regression analyses were also used to test the effect of dietary CP levels on serum free amino acids concentrations before and during exercise. Significance was declared when P < 0.05. 10. Results
a
Omolene-fós: Ca (Max) 150 g, P (Min) 70 g, S 10 g, Mg 10 g, Na 150 g, Fe 2.500 mg, Cu 820 mg, Zn 2500 mg, Mn 2124 mg,I 20 mg, Se 12,5 mg, Co 20 mg, Cr 6 mg. b The concentration of amino acid of diets was achieved after extracting the solution with total amino acid of ingredient sample by HPLC according to Antonie et al. (1999).
10.1. Dietary amino acid Total intake values of Lys, Thr, Leu, Ile and Val were affected by 2
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Table 2 Total intake of nutrients and amino acid by horses fed diets with different protein levels. Protein diet level (%)
P value
Item
7.5
9.0
11.0
13.0
SEM
L
DM (kg/d) CP (g/d) Lys (g/d) Thr (g/d) Leu (g/d) Ile (g/d) Val (g/d)
8.0+0.9 754.9+84.6 33.2+2.8 23.1+1.5 60.0+3.1 79.2+2.3 29.6+2.5
8.3+0.9 884.6+102.4 36.6+3.2 25.6+2.0 65.6+2.7 80.6+2.7 30.2+2.6
8.4+1.3 1.039.3+130.4 48.5+5.6 46.3+2.4 70.1+4.9 89.2+3.1 33.2+3.8
8.5+0.7 1.208.3+125.5 49.3+2.8 48.4+1.5 76.2+1.7 93.5+1.7 41.8+1.1
0.09 20.8 2.7 0.3 1.8 1.0 2.6
0.1745 < 0.0001 < 0.0001a < 0.0001b < 0.0001c < 0.001d < 0.0001e
Dry matter (DM), crude protein (CP), ether extract (EE), lysine (Lys), threonine (Thr), isoleucine (Ile), leucine (Leu) and valine (Val). SEM: Standard error of means. Linear regression (L) as a function of CP diet levels. a ˆ Lys =2.38+38.23x, R2=0.80; b ˆ Thr =−16.25+51.34x, R2=0.86; c ˆ Leu =39.58+28.36x, R2=0.97; d ˆ Ile =57.72+27.15x, R2=0.89; e ˆ Val =10.21+22.83x, R2=0.84.
12. Discussion
dietary protein levels. Linear increasing responses were observed for Lys, Thr, Leu, Ile and Val (P < 0.01) as a function of the diets (Table 2). Concerning to the dietary total amino acid, the NRC (2007) recommends for a 488 kg BW horse in intense exercise, a daily intake of 36.1 g/d of Lys. Horses from the 7.5% CP experimental diet, intake 33.2 g/d of Lys, 8.3% less Lys than the recommendations.
In the present study, the dietary CP linearly increased the AA intake of the horses (P < 0.01). Furthermore, the amounts of Thr, Ile, Leu and Val consumed by the horses were enough or higher to meet the requirements (NRC, 2007) for horses in intense physical activities. The exception was the 7.5% CP diet group, the average intakes were 100 and 3 g/d, 8.3% less than recommended by NRC (2007), for CP and Lys, respectively, to the corresponding intense exercise horse category. However, according to Coenen et al. (2011) and Urschel and Lawrence (2013), horses' requirements described in the NRC (2007) are expressed in CP and Lys concentrations and could be better evaluated if expressed in metabolizable protein (French and German systems). However, based on the total amino acid intake and clinical evaluations of the horses during the experiment, no deficiencies of amino acids capable of impairing their performance were noted. During the experimental study, the horses maintained their body weight (497 ± 23 kg BW) and body score (5.0). According to Assenza et al. (2004) and Bergero et al. (2005), the intensity of exercise influenced the serum concentrations of BCAA,
11. Serum free amino acid The dietary CP levels significantly influenced (P=0.001) the serum free AA concentration. The serum concentrations of free Lys, Leu, Ileu and Val were affected by sampling time, increasing with exercise (Table 3). Both linear (P=0.001) and quadratic (P=0.016) responses were observed on the serum free Lys, Leu, Ile and Val concentrations during exercise as a function of the increasing CP in the diets. The concentrations of Thr did not show significant changes as a function of the dietary CP and the serum free Thr concentration decreased with exercise. The exercise induced changes of the serum free amino acids concentrations in the horses.
Table 3 Serum free amino acid concentrations (µmol/L) before and during exercise in horses fed diets with different protein levels. Serum free AAs (µmol/L)
Protein diet level (%)
P value
Moment
7.5
9.0
11.0
13.0
SEM
L
Q
Lys
Before Exercise
135.6 ± 4.2 137.1 ± 10.8
140.5 ± 5.4 170.2 ± 15.8
154.5 ± 15.7 145.3 ± 7.8
137.5 ± 7.8 147.9 ± 10.8
4.4 4.0
0.018 0.030
0.024a 0.030b
Thr
Before Exercise
160.5 ± 31.6 109.5 ± 29.9
141.9 ± 40.8 122.1 ± 31.5
155.6 ± 26.5 127.9 ± 21.6
116.5 ± 27.1 140.1 ± 4.5
20.6 15.5
0.711 0.797
0.488 0.984
Leu
Before Exercise
145.4 ± 4.9 145.9 ± 11.2
149.5 ± 5.6 180.3 ± 16.6
164.0 ± 16.3 156.1 ± 7.6
148.3 ± 7.6 156.9 ± 11.2
5.1 4.1
0.032 0.019
0.045c 0.020d
Ile
Before Exercise
212.3 ± 83.7 138.5 ± 10.9
185.2 ± 84.5 226.4 ± 86.3
200.5 ± 88.6 248.7 ± 99.2
177.2 ± 91.1 167.8 ± 35.8
27.7 20.2
0.887 0.008
0.960 0.010e
Val
Before Exercise
210.1 ± 72.7 104.6 ± 73.4
263.6 ± 58.9 321.1 ± 73.2
243.3 ± 77.1 260.0 ± 76.0
270.5 ± 48.8 280.4 ± 48.3
26.6 70.7
0.515 0.004
0.656 0.011f
Lysine (Lys), threonine (Thr), isoleucine (Ile), leucine (Leu) and valine (Val). SEM: Standard error of means. Linear (L) and quadratic regression (Q) as a function of CP diet levels. a ˆ Lys 209.65+29.42x −5.49x2, R2 =0.24; b ˆ Lys =210.22+38.77x −7.60x2, R2 =0.21; c ˆ Leu =221.12+27.18x – 4.97x2, R2 =0.21; d ˆ Leu =215.91+42.62x −8.35x2, R2 =0.23; e ˆ Ile =56.88+221.92x −42.17x2, R2 =0.29; f ˆ Val =− 20.27+291.82x −49.03x2, R2 =0.44.
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excesses are eliminated as nitrogen (N) in urine, however demanding larger water uptake and special attention to possible dehydration. Besides, studies about N balance in horses consuming different CP levels (Oliveira et al., 2015) indicated that the absorption of N increases with the inclusion of dietary CP. However, the ratio Nretained:Nabsorbed is directly influenced by the quality of dietary CP, but not by the CP quantity. Thus, the use of good quality protein to formulate horses‘ diets can reduce the quantity of CP included leading to lower costs for formulation and diminishing the N excretion (Urschel and Lawrence, 2013) promoting an environmental benefit.
especially in long duration and low intensity mostly aerobic exercises, in which BCAAs may be used as a source for energy production. In a previous study, Essén-Gustavsson et al. (2010) evaluated horses during short and intense exercises (mostly anaerobic) on a high-speed treadmill reporting that the diet did not affect the plasma AA concentration, but there was an interaction between exercise and dietary CP for Lys and Val plasma concentrations. In contrast, in our study, the dietary CP affected the horses' serum free AA concentration. The same response that were reported by Essén-Gustavsson et al. (2010) was observed in the present study not only for Lys and Val serum free concentrations but also for Leu and Ile, that were influenced by mostly anaerobic exercise and diet. In both papers, authors used good quality diets with a good AA profile. Essén-Gustavsson et al. (2010) forage-only diets were offered as silages, while in the present study horses were feed mixed diets with concentrateds and hay (50:50), and soybean meal as the main source of CP. It is possible that the different AA sources may have influenced the blood AA concentration responses differently. Diet formulations for athletic horses are focused on energy content and sometimes the AA concentration can be undervalued. Horses are very sensitive to the type of diet as well as to the quality of dietary protein provided (Harris et al., 2013) and, therefore, it is justifiable the need for knowledge on the sources of protein with high quality AA profile. According to Westermann et al. (2011), AAs should not be considered as limiting to athletic performance in horses, except for aspartic acid. However, the minimum requirements of the dietary CP for athletic horses is yet to be determined. Therefore, the mostly anaerobic exercise, performed on a highspeed treadmill, induced measurable changes in serum concentrations of free amino acids in eventing horses. However, it is not possible to claim that these changes are due to muscle catabolism because no discernible trend in the amino acid concentrations was observed either before or during exercise. More studies should be developed with observations before, during and after exercise for better evaluation of the dietary AA and their relation with the AA's turnover (Matsui et al., 2006). According to Graham-Thiers et al. (1994), Lys is the first-limiting amino acid for growing horses and Thr is presumed to be the second for lactating mares (Wilson and Graham-Thiers, 2009). Moreover, according to Graham-Thiers and Kronfeld (2005), exercising horses need the correct supply of Lys to repair and maintain muscle mass, as a natural turnover in muscle occurs in response to dietary AA and exercise. Likewise, in a previous study, evaluating light exercise in horses fed different amino acid concentration, Graham-Thiers and Bowen (2011) reported that Lys plays a fundamental role in the metabolism of athletic horses. However, it is a theoretical question whether the discrepancies between studies on blood AAs occur because of different types (aerobic and anaerobic) and intensities of exercises, training status and age of horses, or due the muscles utilization of BCAA as an alternative energy source (Hackl et al., 2009). There are few studies concerning amino acid requirements of horses and also few published data on CP and AA pre-cecal digestibility (Zeyner et al., 2010; Coenen et al., 2011). According to Zeyner et al. (2010), the knowledge of the protein fraction that can suffer enzyme digestion is essential, since equine amino acidic absorption takes place only in the small intestine. However, if an amount of protein is not digested in this segment, it will serve as an important substrate for microbial. Moreover, Zeyner et al. (2010) and Coenen et al. (2011) recommend the utilization of the Cornell Net Carbohydrate and Protein System for determination of the fraction of protein digestible in the small intestine, trying to focus on AA% intake x AA% digestibility. The Lys requirements for athletic horses appear to be lower than what is currently proposed and therefore it seems possible to formulate less expensive diets and yet with a better amino acid profile and smaller excretion of nitrogen. According to Harris et al. (2013), the CP or AA excessive intake would not represent a problem for horses, since the
13. Conclusions Protein diet levels affect the serum free amino acid concentration during exercise. The effect of exercise on serum free Lys, Leu, Ile and Val concentration, may be interpreted as an indicator of these amino acids metabolic response and demand further investigation. Authors' declaration of interests No competing interests have been declared. Ethical animal research The study was approved by the Ethics in Research Board of Universidade Federal Rural do Rio de Janeiro, number 029/2009. Authorship C. A. A. Oliveira and F. Q. Almeida contributed to study design, study execution, data analysis and interpretation. M. T. Ramos contributed to study execution, data analysis, structure and text translation. V. P. Silva and C. D. Baldani contributed to study execution and data analysis. L. A. M. Keller contributed to data analysis and interpretation. All authors contributed to the preparation of the manuscript and gave final approval of the manuscript. Acknowledgements The authors acknowledge the support of the Brazilian Army Cavalry School, the CNPQ (Conselho Nacional de Desenvolvimento Científico e Tecnológico), the FAPERJ (Fundação de Amparo a Ciência do Estado do Rio de Janeiro), and the laboratories of the Universidade Federal Rural do Rio de Janeiro, the Equine Health Laboratory and the Mycological and Mycotoxicologic Research Center. References Antonie, F.R., Wei, C.I., Littell, R.C., Marshall, M.R., 1999. HPLC method for analysis of free amino acids in fish using o-phthaldialdehyde precolumn derivatization. J. Agric. Food Chem. 47, 5100–5107. Assenza, A., Bergero, D., Tarantola, M., Piccione, G., Caola, G., 2004. Blood serum branched chain amino acids and tryptophan modifications in horses competing in long-distance rides of different length. J. Anim. Physiol. Anim. Nutr. 88, 172–177. Bergero, D., Assenza, A., Schiavone, A., Piccione, G., Peroan, G., Caola, G., 2005. Amino acid concentrations in blood serum of horses performing long lasting low-intensity exercise. J. Anim. Physiol. Anim. Nutr. 89, 146–150. Coenen, M., Kienzle, E., Vervuert, I., Zeyner, A., 2011. Recent German developments in the formulation of energy and nutrient requirements in horses and the resulting feeding recommendations. J. Equine Vet. Sci. 31, 219–229. Essén-Gustavsson, B., Connysson, M., Jansson, A., 2010. Effects of crude protein intake from forage-only diets on muscle amino acids and glycogen levels in horses in training. Equine Vet. J. 42, 341–346. Graham-Thiers, P.M., Kronfeld, D.S., 2005. Amino acid supplementation improves muscle mass in aged and young horses. J. Anim. Sci. 83, 2783–2788. Graham-Thiers, P.M., Ott, E.A., Brendemuhl, J.H., Tenbroeck, S.H., 1994. The effect of supplemental lysine and threonine on growth and development of yearling horses. J. Anim. Sci. 72, 380–386. Graham-Thiers, P.M., Bowen, L.K., 2011. Effect of protein source on nitrogen balance and plasma amino acids in exercising horses. J. Anim. Sci. 89, 729–735. Hackl, S., Hoven, Van Den, Zickl, R., Spona, M., Zentek, J, J., 2009. The effects of short
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