Abstract
Twelve horses weighing 570 ± 35 kg were split into two groups (standard protein, STD, and balanced protein, BAL) to evaluate the effect of balanced vs. unbalanced protein on muscle mass. Groups were fed grass hay and a 12% CP feed (one formulated for balanced protein). Horses were out of work for 14 wk prior and participated in 1 to 2 h of light-to-moderate exercise 5 d/wk for 12 wk. A 4-d total urine and feces collection period was conducted at the start and end of the study. Urine and feces samples were analyzed for nitrogen (N) to calculate N balance. Muscle mass status was evaluated using histochemistry, muscle scoring, and body measurements at the start and end of the study. Muscle biopsies were analyzed for free amino acid concentrations. An ANOVA was done using the Proc MIXED function of SAS (2006). Nitrogen retention was greater and urine nitrogen as a percent of intake less for the STD group at the end of the study (P = 0.01). Muscle free concentrations of methionine, leucine, phenylalanine, and arginine were greater in the BAL group at the end of the study compared to the STD group (P < 0.04) while muscle free histidine, threonine, and valine were less for the STD group at the end of the study compared to the start of the study (P < 0.05). Muscle fiber diameters were greater at the start of the study for the STD group (P = 0.004) but greater for the BAL group at the end of the study (P = 0.001). There is some evidence of improved muscle mass for the BAL group with greater muscle free amino acids (arginine, leucine, methionine, phenylalanine, and valine) as well as greater type II muscle fiber areas and diameters. While changes did not appear evident between groups at the “whole body” level, changes at the tissue level appear more evident.
Keywords: balanced protein, exercise, horses, muscle
The use of balanced protein (relative to amino acid requirements) in horses may result in improvement of muscle mass development in exercising horses. Improvement in muscle free amino acids and type II muscle fiber diameters suggests an improvement in the utilization of the profile of dietary amino acids closely mimicking muscle amino acid profiles in these exercising horses.
INTRODUCTION
Dietary protein is the main supplier of amino acids for repair and development of tissues in the body. Protein synthesis in the body requires that all necessary amino acids for tissue synthesis be present at the same time. Therefore, high-quality dietary protein (that would provide all the essential amino acids and in adequate amounts) is key to maintaining proper tissue repair and synthesis (Wolfe, 2000). Horses’ bodies have a high proportion of muscle tissue compared to other animals (Gunn, 1987). Horses are primarily used for athletic performance. With muscle mass being key to athletic ability, development and maintenance of muscle tissue would be of paramount concern for exercising horses (Hiney and Potter, 1996).
Research in swine and chickens have employed the concept of ideal protein when determining high-quality dietary protein (NRC, 2001). The premise of this concept is that not only are amino acids required in particular amounts for protein synthesis but also in correct ratios to each other. This is particularly true for the essential amino acids (those that cannot be synthesized by the body in adequate amounts). The estimation of ideal protein in swine and chickens was determined through nitrogen balance studies as well as the evaluation of amino acid profiles in body tissues (Wang and Fuller, 1989). The premise of the ideal protein concept is that if the dietary amino acid profile matches the desired end product (body tissues), this would allow for the most efficient use of the dietary protein (and amino acids) and result in improved production and nitrogen balance. Studies in swine and chickens utilizing the concept of ideal protein have observed improved performance (such as growth and ADG), feed efficiency, and nitrogen balance (Soares et al., 2022; Wang et al., 2018).
Bryden (1991) was the first to speculate on the concept of ideal protein in the horse. The study evaluated muscle tissue and milk amino acid profiles to establish ideal protein for the horse. More recently, research was done analyzing the muscle amino acid profiles of horses in various functions (maintenance, growth, pregnancy, lactation, and exercise) to determine any relationships between dietary, plasma and muscle amino acid profiles (Graham-Thiers et al., 2012). The study found that muscle amino acid profiles do not change with function. Muscle amino acid profiles were similar between all groups (Graham-Thiers et al., 2012).
The concept of ideal protein sets the ratios of essential amino acids in reference to lysine. Lysine is considered the first limiting amino acid in most animals for growth. Research in horses also has determined that lysine is most commonly the first limiting amino acid when fed typical diets (NRC, 2007). Using the muscle amino acid profiles from Graham-Thiers et al. (2012), an ideal or balanced protein was calculated determining ratios to the muscle lysine concentration. The ideal protein ratios of Bryden (1991) and Graham-Thiers et al. (2012) are in good agreement with each other. Once a lysine requirement has been established, using the ideal protein ratios, all other essential amino acid requirements could be estimated. This could be useful in equine nutrition given the limited data on essential amino acid requirements for the horse. While the concept of ideal protein does not take into account differences in digestibility as well as the amino acid needs of other tissues besides muscle, the idea of a more balanced protein has its merits in attempting to achieve a dietary amino acid profile that matches the animal’s amino acid requirements.
The purpose of this study was to evaluate muscle development, nitrogen balance, and muscle amino acids of exercising horses fed either a standard protein diet (typical amino acid profile with soybean meal as the main protein source) or a balanced protein diet with the amino acid profile manipulated (via synthetic lysine supplementation) to mimic the amino acid profile of muscle tissue in the horse.
MATERIALS AND METHODS
This study was approved by the institution’s IACUC committee and followed the USDA Guide for Care and Use of Agricultural Animals in Research and Teaching. Twelve gelding horses of light horse type were used for the study (570 ± 35 kg BW; age 16 ± 0.3 yr). Horses were out of work for 14 wk prior to the start of the study. Horses were divided into two groups: standard protein (STD) and balanced protein (BAL). All horses were fed a mixed grass hay (timothy/orchardgrass). The STD group received a grain concentrate based on typical horse feed formulations (Country Acres, Purina Mills, St. Louis, MO). The grain concentrate was selected to be the same CP as the customized concentrate with no supplemental lysine in the formula. The BAL group received a grain concentrate customized to mimic muscle amino acid ratios in horses thus reflecting a balanced protein (in regards to amino acid ratios) by utilizing supplemental synthetic lysine. Ideal/balanced protein is expressed in terms of a ratio to lysine therefore manipulating lysine concentrations in the diet will change the ratios with other amino acids. Diets were formulated to be isocaloric and isonitrogenous and to meet the requirements of horses in light-to-moderate exercise (NRC, 2007). Daily rations were divided into two equal meals fed in the morning and early evening. Analysis of concentrates and hay fed to the horses is shown in Table 1. All horses participated in light exercise 5 d/wk, 1 to 2 h/d in the college’s riding program. Body weight (BW) and body condition score (BCS) were evaluated weekly as an indicator of overall health. The BW was measured using an electronic scale (Model AL660-LA, Cambridge Scaleworks, Honey Brook, PA) while BCS was scored using experienced evaluators on a 1 to 9 scale using a standardized system (Henneke et al., 1983).
Table 1.
Analysis of the standard protein (STD) concentrate, balanced protein (BAL) concentrate, and hay
| Amino acid, % | STD1 | BAL2 | Hay3 |
|---|---|---|---|
| Arginine | 0.86 | 0.81 | 0.34 |
| Histidine | 0.35 | 0.33 | 0.13 |
| Isoleucine | 0.44 | 0.45 | 0.28 |
| Leucine | 0.88 | 0.91 | 0.54 |
| Lysine | 0.59 | 1.27 | 0.37 |
| Methionine | 0.19 | 0.19 | 0.12 |
| Phenylalanine | 0.54 | 0.58 | 0.34 |
| Threonine | 0.42 | 0.42 | 0.30 |
| Valine | 0.64 | 0.62 | 0.38 |
| Dietary component | |||
| Dry matter, % | 90.20 | 90.10 | 93.65 |
| DE4, Mcal/kg | 3.10 | 3.00 | 2.00 |
| CP5, % | 14.20 | 14.80 | 7.70 |
1Country Acres, Purina Mills, St. Louis, MO.
2Custom Feed mix (manufactured by Purina Mills, St. Louis, MO).
3Timothy/orchardgrass mix.
4Digestible energy.
5Crude protein.
Prior to the start of the study all horses were being fed a typical grain product (Strategy, Purina Mills, St. Louis, MO) and grass hay. A prestudy collection period was conducted. All horses were fitted with total collection harnesses that allowed for the separate collection of feces and urine (Equisan, S. Melbourne, Australia). Horses wore the harnesses for 4 d. Harnesses were emptied three times daily. Total daily urine and feces output were measured for each 24-h period with a 10% aliquot frozen for later analysis. Following this prestudy collection period, horses were divided into their respective groups. Horses were fed their respective diets for 12 wk followed by another collection period. Nitrogen was analyzed in feces (AOAC, 1996) and urine (APHA, 1998) in order to calculate N balance.
Muscle measurements were taken at the start and conclusion of the study. Measurements of the neck (circumference at three equally spaced points determined by overall neck length), topline curvature (using the difference between curvature of the back and a straight line measure from the point of the withers to the point of the croup), groin circumference, forearm circumference, gaskin circumference, and the curvature of the musculature between the hip and stifle (gluteal) were all taken from marked (shaved) reference points at the start and end of the study.
Muscle biopsies were taken at the start and end of the study. The biopsy was taken from the semitendinosus muscle 13 cm below the point of the buttock. Horses were administered a mild sedative and lidocaine was administered at the biopsy site. A small incision through the skin was made at the sampling site. A skin punch measuring 7 mm was used to obtain the tissue sample approximately 3 to 4 cm deep. The muscle tissue was weighed, split into two aliquots, treated with optimal temperature cutting compound, immediately frozen in isopentane chilled with liquid nitrogen, and stored at −80 °C until analysis. One aliquot of muscle tissue was evaluated via enzyme histochemistry at three pHs (10, 4.2, and 4.5) to detect ATPase for muscle fiber typing (Barlow et al., 1984). Muscle fiber measurements such as muscle fiber area and diameters were also evaluated using Image-Pro Plus 7.0 (2011, Media Cybernetics, Rockville, MD). One aliquot of muscle tissue was analyzed for muscle free amino acids as well as 3-methyl-histidine (3MH). Muscle tissue was homogenized in 0.02 N HCL containing 3.75% sulfosalicylic acid with an internal standard. Muscle protein was removed by centrifugation (10 min at 16,100 × g at 22 °C) followed by 0.22-µm filtration, and the remaining protein was analyzed for free amino acids. Amino acid analysis was performed by HPLC (Hitachi L-880A amino acid analyzer, HTA Corporate, Schaumburg, IL) following the procedure described by Miller-Graber et al. (1990).
Statistical analysis was performed using the PROC MIXED procedure in SAS evaluating the effect of diet, time, and any interactions with horse as the random effect. Data are presented as LSMeans with SEs. Differences were considered significant at a level of P < 0.05.
RESULTS
All horses remained healthy and maintained BW throughout the study. There were no differences due to time, treatment, or interactions for BW or BCS. Horses in the STD group consumed 2.3 ± 0.1 kg grain mix and 8.0 ± 0.3 kg hay while the BAL group consumed 2.2 ± 0.1 kg grain mix and 8.2 ± 0.4 kg hay. Amino acid intakes are shown in Table 2. The ratios of amino acids in the diets (to evaluate ideal/balanced protein) based on intakes are shown in Table 3.
Table 2.
Daily amino acid intake for the standard protein (STD) and balanced protein (BAL) groups
| Amino acid | STD, g/d | BAL, g/d | SE |
|---|---|---|---|
| Arginine | 47 | 46 | 2.3 |
| Histidine | 16 | 18 | 0.9 |
| Isoleucine | 33 | 33 | 1.6 |
| Leucine | 63 | 64 | 3.0 |
| Lysine | 43 | 58 | 1.9 |
| Methionine | 14 | 14 | 0.6 |
| Phenylalanine | 40 | 41 | 1.9 |
| Threonine | 34 | 34 | 1.7 |
| Valine | 45 | 45 | 2.1 |
Table 3.
Amino acid ratios for the standard protein (STD) and balanced protein (BAL) diets
| Amino acid | STD | BAL | Bryden (1991) | Graham-Thiers et al. (2012) |
|---|---|---|---|---|
| Arginine | 109 | 75 | 74 | 63 |
| Histidine | 37 | 31 | 58 | 35 |
| Isoleucine | 77 | 57 | 54 | 54 |
| Leucine | 147 | 110 | 107 | 111 |
| Lysine | 100 | 100 | 100 | 100 |
| Methionine | 33 | 24 | 28 | 30 |
| Phenylalanine | 93 | 68 | 60 | 44 |
| Threonine | 79 | 59 | 61 | 64 |
| Valine | 105 | 70 | 62 | 73 |
All ratios set to lysine (LYS = 100).
Nitrogen balance was affected by time-by-diet interaction. Nitrogen digestibility was greater for the BAL group at the start of the study compared to the STD group (P = 0.04) while fecal nitrogen (FN) was less for the BAL group compared to the STD group at the start of the study. Nitrogen absorbed and urine nitrogen as a percentage of intake were greater at the start of the study compared to the end of the study for the STD group while nitrogen retention (g/d, as a percentage of intake and as a percentage of absorbed) was greater at the end of the study for the STD group compared to the start of the study (P = 0.01). The nitrogen balance data are shown in Table 4.
Table 4.
Nitrogen balance data for the standard protein (STD) and balanced protein (BAL) groups at the beginning of the study (pre) and at the end of the study (post; diet × time interactions)
| STD | BAL | SE | |||
|---|---|---|---|---|---|
| Pre | Post | Pre | Post | ||
| Nitrogen intake (NI), g/d | 172.1 | 186.2 | 180.5 | 186.5 | 7.0 |
| Fecal nitrogen (FN), g/d | 72.5 | 72.8 | 66.4 | 72.2 | 5.0 |
| Fecal nitrogen (FN), % of intake | 41.3c | 38.7 | 35.9d | 38.7 | 1.8 |
| Nitrogen absorbed, g/d | 71.6a | 50.3b | 68.8 | 58.3 | 5.4 |
| Digestibility, % | 58.7c | 61.3 | 64.1d | 61.3 | 1.8 |
| Urine nitrogen (UN), g/d | 73.2 | 57.5 | 80.2 | 65.6 | 6.0 |
| Urine nitrogen, % of intake | 41.9a | 30.7b | 43.9 | 35.1 | 3.3 |
| Nitrogen retention (NR), g/d | 29.7a | 56.4b | 37.9 | 48.8 | 6.0 |
| Nitrogen retention, % of intake | 16.8a | 30.5b | 20.2 | 26.2 | 3.6 |
| Nitrogen retention, % of absorbed | 28.5a | 49.7b | 31.2 | 41.7 | 5.4 |
a,bDifferences between pre and post (P < 0.05).
c,dDifferences between diets (P < 0.05).
Muscle free amino acids had several diet-by-time interactions. Muscle free concentrations of arginine, leucine, methionine, and phenylalanine were greater for the BAL group compared to the STD group at the end of the study (P < 0.02). Muscle free concentrations of histidine, threonine, and valine were less at the end of the study compared to the start of the study for the STD group (P < 0.05). Muscle free amino acid data are shown in Table 5.
Table 5.
Muscle free amino acid concentrations for the standard protein (STD) and balanced protein (BAL) groups at the beginning of the study and the end of the study (diet × time interactions)
| Amino acid, mmol/kg tissue | STD | BAL | SE | ||
|---|---|---|---|---|---|
| Pre | Post | Pre | Post | ||
| Arginine | 0.25 | 0.28c | 0.23 | 0.33d | 0.04 |
| Histidine | 0.18a | 0.14b | 0.18 | 0.17 | 0.02 |
| Isoleucine | 0.11 | 0.12 | 0.11 | 0.14 | 0.02 |
| Leucine | 0.26 | 0.19c | 0.27 | 0.32d | 0.03 |
| Lysine | 0.66 | 0.70 | 0.51 | 0.60 | 0.10 |
| Methionine | 0.08 | 0.07c | 0.08 | 0.11d | 0.01 |
| Phenylalanine | 0.14 | 0.13c | 0.15 | 0.19d | 0.02 |
| Threonine | 0.32a | 0.21b | 0.30 | 0.25 | 0.03 |
| Valine | 0.34a | 0.22b | 0.36 | 0.32 | 0.04 |
| 3-Methyl-histidine | 16.1 | 18.7 | 15.5 | 20.2 | 2.53 |
a,bDifferent pre and post (P < 0.05).
c,dDifferent between diet (post; <0.04).
Interactions between diet and time were observed with type II muscle fiber areas being greater for the BAL group and less for the STD group when comparing the muscle fiber areas between the start and end of the study (P = 0.009). Type II muscle fiber areas were also greater for the BAL group compared to the STD group at the end of the study (P = 0.001). Muscle fiber area data are shown in Table 6. Changes in muscle fiber areas from the start and end of the study were calculated. There was an increase in type I and type II muscle fiber areas (change from baseline) for the BAL group compared to the STD group (P = 0.066 and 0.009, respectively). Muscle fiber area changes data are shown in Table 7. There was an interaction of time and diet for muscle fiber diameters with the BAL group having greater muscle fiber diameter at the end of the study compared to the STD group (P = 0.001); however, the STD group had greater muscle fiber diameter at the start of the study compared to the BAL group at the start of the study (P = 0.004). Changes to muscle fiber diameter were also greater for the BAL group compared to the STD group (P < 0.0001). These changes represent a 7.1% decrease in fiber diameter for the STD group and a 2.6% increase in fiber diameter for the BAL group. Muscle fiber diameter data are shown in Table 8.
Table 6.
Muscle fiber areas at the start of the study and at the end of the study for the standard protein (STD) group and the balanced protein (BAL) group (areas in µm2)
| Fiber Type | STD Group | BAL Group | SE | ||
|---|---|---|---|---|---|
| Pre | Post | Pre | Post | ||
| Type I1 | 2,466.3 | 2,263.7 | 2,441.0 | 2,482.7 | 218 |
| Type IIA2 | 2,465.0 | 2,551.0 | 2,425.2 | 3,065.8 | 247 |
| Type IIB2 | 5,739.3 | 5,481.0 | 5,026.5 | 5,965.5 | 458 |
| Type II3 | 4,939.0a | 3,917.8b,c | 4,794.5a | 5,812.2b,d | 277 |
a,bDifferent pre, post (P = 0.017).
c,dDifferent STD vs. BAL post (P < 0.0001).
1Stained at pH 4.2 (3 fields of 10 fibers evaluated and averaged together).
2Stained at pH 4.5 (3 fields of 10 fibers evaluated and averaged together).
3Stained at pH 10.2 (3 fields of 10 fibers evaluated and averaged together).
Table 7.
Muscle fiber area changes (pre vs. post) for the standard protein (STD) and balanced protein (BAL; diet effect; units are µm2)
| Fiber type | STD | BAL | SE | P value |
|---|---|---|---|---|
| Type I1 | −25 | 219 | 224 | 0.460 |
| Type IIA2 | −39 | 514 | 330 | 0.260 |
| Type IIB2 | −454 | 226 | 639 | 0.470 |
| Type II3 | −145 | 1,894 | 449 | 0.009 |
P value in bold denotes a statistically significant difference.
1Original data from muscle stained at pH 4.2 (3 fields of 10 fibers evaluated and averaged together).
2Original data from muscle stained at pH 4.5 (3 fields of 10 fibers evaluated and averaged together).
3Original data from muscle stained at pH 10.2 (3 fields of 10 fibers evaluated and averaged together).
Table 8.
Muscle fiber diameters from the beginning of the study (pre) and the end of the study (post) as well as for the standard protein (STD) and balanced protein (BAL; N = 100)
| Diameter, µm | STD | BAL | SE | ||
|---|---|---|---|---|---|
| Pre | Post | Pre | Post | ||
| 84.8a | 78.9c | 81.6b | 83.2d | 0.80 | |
a,bSTD vs. BAL (pre; P = 0.004).
c,dSTD vs. BAL (post; P = 0.0001).
Measurements taken to evaluate muscle mass development at the “whole body” level Gluteal muscle measurement was less at the end of the study compared to the start of the study for both the STD group and BAL group (P < 0.002) while forearm circumference was greater at the end of the study compared to the start of the study for both the STD and BAL groups (P = 0.0001). Changes (calculated from pre and post measurements) in body measurements were affected by diet with the first two (out of three) neck measurements increasing in the BAL group compared to the STD group (first neck measurement change: −0.92 and +0.33 for the STD and BAL group respectively, P = 0.036; second neck measurement change: −1.33 and +1.00 for the STD and BAL group respectively, P = 0.0002). Muscle development measurements are shown in Table 9.
Table 9.
Muscle development measurements for the standard protein (STD) group and the balanced protein (BAL) group at the beginning of the study and the end of the study (diet × time interaction)
| Measurement | STD | BAL | SE | ||
|---|---|---|---|---|---|
| Pre | Post | Pre | Post | ||
| Neck circumference (1), cm | 80.00 | 77.67 | 78.94 | 79.81 | 1.30 |
| Neck circumference (2), cm | 103.30 | 99.49 | 98.63 | 101.17 | 1.85 |
| Neck circumference (3), cm | 116.64 | 116.41 | 116.64 | 117.04 | 2.54 |
| Groin circumference, cm | 205.74 | 206.17 | 204.90 | 206.58 | 5.08 |
| Back curvature difference, cm | 2.97 | 3.18 | 3.60 | 3.18 | 0.46 |
| Gluteal curvature, cm | 42.55a | 37.47b | 42.34a | 37.80b | 0.69 |
| Forearm circumference, cm | 47.63a | 56.08b | 46.15a | 53.77b | 1.09 |
| Gaskin circumference, cm | 48.90 | 51.64 | 46.46 | 49.10 | 1.07 |
Neck measurement 1 (closest to poll), neck measurement 2 (center of the neck), neck measurement 3 (closest to the withers).
a,bPre vs. post different (P < 0.002).
DISCUSSION
Both groups of horses consumed similar amounts of all of the essential amino acids with the exception of lysine. With an increased concentration of lysine in the concentrate for the BAL group, lysine intake was greater than that for the STD group. A greater lysine intake would be expected in this case, and changes the ratios of the essential amino acid intake in relation to lysine. The ratios for the BAL group were more in line/agreement with the proposed idea of ideal or balanced protein for horses (Bryden, 1991; Graham-Thiers et al., 2012). Using the muscle amino acid ratios and the known lysine requirement for exercising horses, it is estimated that all amino acid requirements would have been met in both groups. Supplementation of diets with lysine have observed improved performance for growth such as ADG and exercise in horses (Graham et al., 1994; Graham-Thiers and Kronfeld, 2005). Although it was not the objective of the previously mentioned studies, by increasing lysine in the diet, the ratios of the amino acids were altered and those groups amino acid intake would be more in line with ideal or balanced protein. These groups also had better performance in the studies represented by improved growth and ADG (Graham et al., 1994) as well as improved nitrogen balance and muscle mass scores (Graham-Thiers and Kronfeld, 2005).
Nitrogen retention was greater at the end of the study compared to the beginning of the study for the STD group. Since there was no difference in nitrogen retention for the BAL group between the start and end of the study, the STD group changes are most likely attributed to variation in CP intake prior to the start of the actual treatments. The BAL group had greater digestibility at the start of the study and this is attributed to the lower FN that was also observed at the start of the study. The difference in digestibility at the start of the study may also be attributed to the randomization of the horses into their respective groups for the study. This is problematic when comparing pre and post study data for this study. It is not possible to say why the BAL group horses had greater digestibility prior to the treatments other than reduced FN which resulted in greater digestibility calculations. It is also not possible to say that the greater digestibility prior to the treatments influenced the results observed in the BAL group compared to the STD group post treatment 14 wk later. The premise of comparing pre and post data is to evaluate the effect of the treatment. Therefore, considering only the post study data when horses would have been on their respective treatments for several weeks, there were no differences in nitrogen balance data.
A time-by-diet interaction was observed for muscle free amino acid concentrations of arginine, leucine, methionine, and phenylalanine with greater concentrations for the BAL group compared to the STD group suggesting a better supply of these amino acids to muscle tissue. Since there were no differences in 3MH which is a marker of muscle protein breakdown, we can speculate that the greater amino acid concentrations represent supply to the muscle tissue more than breakdown. We cannot, however, say whether this contributed to the differences observed in muscle fiber hypertrophy but does suggest that compared to the STD diet, the BAL diet was able to provide a better supply of these amino acids to the muscle tissues. Other studies have found increased muscle protein synthesis with greater amino acid concentrations (Urschel et al, 2010; Deboer et al., 2018). Essential amino acids and especially leucine have been found to stimulate the mTOR pathway and increase protein synthesis (Urschel et al., 2010). Interestingly, there was a decrease in muscle free concentrations of histidine, threonine, and valine in the STD group comparing the start of the study to the end of the study. When protein amino acid profiles are unbalanced they create a difference in the availability of amino acids with respect to limiting amino acids and result in the deamination of excesses (Eugenio et al., 2022). While the authors are unsure why these particular amino acids increased or decreased in the BAL and STD respectively, what is of interest is that there was an increase in free amino acid concentrations in the BAL group and a decrease in free amino acid concentrations in the STD group.
Muscle fiber areas for type II fibers in a time-by-diet interaction, were greater for the BAL group compared to the STD group which actually had a decrease in muscle fiber area for Type II fibers comparing the start and end of the study. This certainly suggests muscle hypertrophy for the BAL group. Changes in muscle fiber areas were calculated and there was a significant difference in muscle fiber area change for type II fibers for the BAL group compared to the STD group with the change being positive for the BAL group and changes being negative for the STD group. The changes observed for type II muscle fibers found a 20.7% decrease in area for the STD group and a 21.2% increase in type II muscle fiber area for the BAL group. This certainly reinforces the idea that the BAL diet supported muscle hypertrophy along with training. The STD group did not have the same response. The muscle fiber diameter for the BAL group was greater at the end of the study compared to the start of the study suggesting greater muscle fiber hypertrophy for the BAL group. Also, the muscle fiber diameters for the STD group were greater at the beginning of the study compared to the BAL group. Therefore, changes in muscle fiber diameters were calculated and were significantly greater in the BAL group compared to the STD group. These results also support greater muscle hypertrophy for the BAL group.
Muscle mass measurements present conflicting results. In a time-by-diet interaction, forearm circumference measurements were greater at the end of the study compared to the start of the study while gaskin circumference measurements were lesser at the end of the study compared to the start of the study for both the BAL and STD group (P = 0.002). These differences suggest an increase in muscle mass in one area but not another. These results along with no other differences in muscle mass measurements reflect the lack of a “whole body” response for the BAL group compared to the STD group. However, there were significant changes in two out of three of the neck measurements for the BAL group suggesting an improvement in muscle mass for the BAL group.
Some of the evaluations in this study are attempting to make whole body observations while others are trying to evaluate muscle changes at the cellular level. There were some muscle mass measurements suggesting changes in muscle mass between the treatment groups but those muscle groups were not specifically biopsied in this study in order to evaluate muscle fiber areas. Also, visible changes in muscle mass can be affected by the area of the body and the size of the muscle group. Changes to muscle groups on the forearm or gaskin may be visible sooner than areas such as the gluteals in the horse hindquarters. However, at the cellular level, it does appear that there were differences (muscle fiber areas and diameters) with these results supporting the conclusion that muscle hypertrophy was more evident at the cellular level in the BAL group. Again, these data are specifically for the semitendinosus muscle as other muscles were not biopsied in this study. Improved muscle free amino acids in the BAL group combined with decreases in muscle free amino acids for the STD group may help explain the difference in muscle fiber hypertrophy in the BAL group compared to the STD group. Marín-García et al. (2022) in their review of low protein diets for swine concluded that if the amino acid profiles of protein being synthesized (such as muscle protein) match the amino acids available at the end of digestion that protein synthesis will occur more efficiently. Feeding balanced vs. unbalanced amino acid profiles to pigs found that balanced diets had improved postprandial plasma amino acid profiles and protein synthesis. Pigs fed unbalanced diets had poor utilization of the dietary protein and increased nitrogen excretion (Eugenio et al., 2022). The results of this study also suggest better utilization of the BAL diet compared to the STD diet as a result of better balance of amino acids.
With the lack of difference at the “whole body” level, there is the possibility that the time frame of this study was not long enough to have the cellular changes translate into whole body changes. The changes in this study at the cellular level are supportive of the hypothesis that the BAL diet better supported muscle mass development. The idea of a balanced protein in the horse has merit and should continue to be evaluated.
ACKNOWLEDGMENTS
The authors wish to thank the staff of the Emory & Henry College Equestrian Center as well as the students enrolled in EQST 424 and EQST 360 for their assistance in caring for the horses and executing this project.
Contributor Information
Patty M Graham-Thiers, Department of Equine Studies, Emory & Henry College, Emory, VA 24327, USA.
L Kristen Bowen, Department of Equine Studies, Emory & Henry College, Emory, VA 24327, USA.
CONFLICT OF INTEREST STATEMENT
None declared.
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