- Email: [email protected]
Mineral balance in juvenile horses in race training
Scientific Papers Mineral Balance in Juvenile Horses in Race Training Tonya L. Stephens, MS,a Gary D. Potter, PhD,b Pete G. Gibbs, PhD,b and David M. Hood, DVM, PhDc REFEREED SUMMARY
Twenty-four long yearlings were fed rations containing differing amounts of calcium, phosphorus, and magnesium to further elucidate the requirements for these minerals during exercise-induced skeletal modeling and remodeling in juvenile racehorses. The animals were assigned randomly within gender subgroups to 1 of 4 diets. Total collections of feces and urine were performed on days 0, 64, and 128 of the trial for determination of mineral absorption and retention. Horses were maintained in a typical race-training protocol to mimic the nutritional stresses placed on long yearlings during strenuous exercise. Calcium absorption and retention were lower (P < .05) at day 64 than at day 0 and day 128. Also, the efficiency of retaining absorbed calcium was lower at day 64 than at day 0 or day 128. Thus, lower calcium retention at day 64 was due to both reduced absorption and reduced systemic demand. At day 64, calcium absorption and retention were not maximized at calcium intake of 160 mg/kg per day. At day 128, calcium absorption was maximal at a daily intake of 124 mg/kg per day, and retention was maximal at a daily intake of 123 mg/kg per day. These are in excess of current National Research Council (NRC)1 recommendations by 38% and 36%, respectively. There was no consistent, significant effect of days on trial on phosphorus absorption or retention, which may have been due to inadvertent limited phosphorus intake. The efficiency of phosphorus retention systemically was over 94% to 98%. Phosphorus absorption and retention were not maximized at the highest intake (66 mg/kg/d), which is 32% over current NRC1 rec-
From the Department of Animal Sciences, University of Florida,a and the Departments of Animal Scienceb and Veterinary Physiology and Pharmacology,c Texas A&M University. Reprint requests: Dr. Tonya L. Stephens, Department of Animal Sciences, University of Florida, P.O. Box 110910, Gainesville, FL 32611-0910. 0737-0806/$ - see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2004.09.006
438
ommendations. Similarly, there was no day effect on the efficiency of magnesium absorption or retention. Urinary excretion of magnesium was highest; thus, systemic efficiency of magnesium retention was lower on days 64 and 128 than on day 0. At day 64, magnesium retention was maximized at a daily intake of 35.6 mg/kg per day, which is 66% over NRC1 recommendations. However, at day 128, magnesium retention was not maximized even at its highest intake (44 mg/kg/d), which is over 2 times the current NRC1 recommendations. These data indicate that early race training affects the dietary requirements for calcium, phosphorus, and magnesium. INTRODUCTION
The need for a marketable product at an early age in both competition and in sales places significant pressure on horse owners to start training or conditioning early in a horse’s life. Training at an early age may exacerbate physiologic stress on young horses and increase nutritional requirements. The process of bone modeling/remodeling is necessary for the juvenile skeleton to adapt to the stresses of training. The horse must form new bone and remodel existing bone to bear the load being applied. During the demineralization process of bone remodeling, there is an increase in mineral excretion from the degradation of the bone matrix. Nielsen et al2 reported this demineralization of the third metacarpal in conjunction with the introduction of speed in juvenile horses on a race-training regimen. The lack of time routinely given to the stressed bone of a juvenile horse to model and/or remodel according to the stresses placed upon it can cause serious and career-ending injuries.3 While injury reduction may be achieved from a delay in the onset of training until the horse is mature, this is not feasible economically. Alterations in existing training methods and increasing physiologic stimulus to the skeleton are approaches being researched to minimize skeletal injuries to young horses. Mineral density increase and size of the third metacarpal would result in larger cortical bone mass, de-
Journal of Equine Veterinary Science
October 2004
Table 1
Formulated (F) versus analyzed (A) mineral concentrations in total diet (dry matter basis) % Calcium
Bermuda grass contribution at 40% of diet Formulated pellet concentration at 60% of diet Treatment #1 Treatment #2 Treatment #3 Treatment #4 Total diet concentration Treatment #1 Treatment #2 Treatment #3 Treatment #4
% Phosphorus
% Magnesium
F
A
F
A
F
A
0.12
0.13
0.08
0.06
0.04
0.04
0.29 0.33 0.49 0.51
0.34 0.38 0.41 0.42
0.15 0.18 0.26 0.31
0.11 0.15 0.17 0.19
0.07 0.09 0.13 0.14
0.07 0.08 0.11 0.11
0.41 0.45 0.61 0.63
0.46 0.50 0.53 0.55
0.23 0.26 0.34 0.39
0.17 0.21 0.23 0.25
0.11 0.13 0.17 0.18
0.11 0.12 0.15 0.15
Diets 1, 2, and 3 were experimental, with all diets identical except for concentrations of calcium, phosphorus, and magnesium. Diet 4 was a commercially available concentrate, Patriot 14, by Consolidated Nutrition, Omaha, NE.
creased strain on the bone, and decreased susceptibility for injury. Previous work in this area demonstrated that increased calcium and phosphorus intake above NRC1 recommendations for juvenile horses in training enhanced bone density, but no quantitative estimates of requirements were made.4 The NRC1 developed an approximation of the requirements for juvenile horses in training using mineral intakes based on extrapolations from research conducted with sedentary and mature horses. This study was conducted to verify previous findings and to further quantify the dietary requirement for calcium, phosphorus, and magnesium during the bone modeling/remodeling process in juvenile athletic horses. The specific objectives of this study were to determine calcium, phosphorus, and magnesium balances in young horses during race training. MATERIALS AND METHODS
Management of Animals Twenty-four long-yearling Quarter Horses were grouped according to age and sex, then randomly assigned to the diet treatments as shown in Table 1. Two horses from each group were randomly assigned to 1 of 4 diets with the condition that each treatment group would contain the same number of fillies and geldings. While at the Texas A&M University Horse Center, the horses were vaccinated against eastern and western equine encephalomyelitis, influenza, and tetanus and dewormed. All horses began the trial with a background period of at least 1 week, during which the horses were group-housed at the Texas A&M University Horse Center, fed hay ad lib, and offered concentrate at approximately 1% of body weight twice daily. Regular hoof care
Volume 24, Number 10
and deworming were provided throughout the course of the study. The horses were then moved from the Texas A&M University Horse Center in groups of 8 to Steephollow Farm in Bryan, Texas, a race-training facility, where they were separated and housed individually in 7 × 7-m stalls. The horses were moved to Steephollow Farm in 3 groups of 8 at an average age of 226 days for the group. Rations were formulated based on 60% concentrate and 40% Bermuda grass hay, with varying concentrations of calcium, phosphorus, and magnesium. The concentrate diets were provided by Consolidated Nutrition of Omaha, Nebraska (Table 1). All concentrates were mixed, pelleted, and bagged by Consolidated Nutrition and shipped to the Texas A&M University Horse Center. The horses were fed concentrate and hay at 12-hour intervals (7:00 AM and 7:00 PM) for the duration of the trial. They were given until the next feeding to consume all concentrate and hay, and any feed refused was weighed and recorded. Refusals were very infrequent. Feed intake was adjusted weekly as needed to allow for normal growth and to maintain a body condition score of 5 to 6,5 while maintaining a constant ratio of 60% concentrate and 40% hay. The protocol for management and treatment of the animals was approved by the Texas A&M University Agricultural Animal Care and Use Committee. Five horses were not able to complete the study because of lameness, injury, or sickness, and data from those horses were not included in the results of the study.
Training of Animals The 4 training periods were 28 days each. During the first week, horses were worked 6 days and rested 1 day. The riding began in the round pen and progressed to the track by the middle of the week, depending on the tem-
439
perament of the horse. During the initial days of training, the horses were walked 550 m, trotted 1100 m, and galloped 275 m. The gallop length was increased by 275 m each day after day 3; therefore, by day 6 the horses were galloped 1375 m. Horses were ridden 5 days a week during weeks 2 to 4. Some horses were started in the round pen through week 2 if needed before riding their assigned distances on the track. They continued to walk and trot the same distances as week 1 but were galloped a total of 8250 m per week. During the next 4 weeks in period 2, the horses were ridden 4 days per week. The total gallop distance was increased to 8440 m. During period 3, horses were ridden 4 days per week, with 1 of the 4 days being a sprint. On nonsprint days, the horses were trotted 550 m and galloped 1925 m. On sprint days, the horses were warmed up, sprinted 230 m, and galloped. During this period, the horses were galloped a total of 8210 m per week. During the last 4 weeks, period 4, the horses continued training as in period 3 except sprints were extended to 275 m and total gallop distance decreased to 8165 m per week. Throughout the entire trial, horses were walked at least 1 hour on a mechanical walker on rest days.
Sample Collection Total collections of feces and urine were made on days 4 through 0, 60 through 64, and 124 through 128. During total collections, horses were confined to tie stalls with rubber mats to prevent coprophagy and contamination of fecal samples. Feces were collected from the mats following each defecation throughout the 72-hour collection period. All feces collections were pooled, and a 10% representative sample was obtained and stored at –20°C for further analyses. A urine collection harness was secured to all horses and checked every 4 hours throughout the 72-hour total collection to prevent loss or contamination of urine. Urine collection containers were acidified and emptied every 4 hours; a 10% sample of the urine was taken and frozen at –20°C for subsequent analyses. Hay and concentrate feed were sampled at each feeding, then pooled to form a composite sample for each collection period. Horses were walked on the mechanical walker for at least 1 hour every day of the collection period. All fecal losses were gathered, weighed, and added to the total output amount but were not included in the composite sample.
Preparation and Analyses of Samples Feed and fecal samples were dried in an oven for 72 hours at 65°C, then ground in a Wiley mill with a 1-mm screen. The ground samples were mixed thoroughly, and
440
a representative of the total ground sample was obtained. Perchloric acid digestions were performed on all feed and fecal samples, and dilutions were made of these digestions and of urine and serum with double-distilled water. Cornstalk (Zea mays) reference material #8412 from the National Institute of Standards and Technology was used as a control standard in perchloric acid digestions and analyses of minerals. Concentrations of calcium and magnesium in samples were determined by atomic absorption spectrophotometry. Reference standards from Fisher Scientific of Pittsburgh, Pennsylvania, for atomic absorption spectroscopy were used to create the standards for calcium (SC191) and magnesium (SM51). Inorganic phosphorus was determined with an Ultraspec III spectrophotometer using methods of Fiske and Subbarow.6 A stock solution of potassium phosphate was used to create standards for phosphorus analyses. Lanthanum chloride was used as the suspension medium of acid digestions for calcium and magnesium analyses, and double-distilled water was used as the suspension medium of acid digestions for phosphorus analyses. Mineral absorption was determined by subtracting fecal output of mineral from mineral intake. Mineral retention was determined by subtracting mineral output in urine from mineral absorbed. STATISTICAL ANALYSES
Data were analyzed using 2-way analysis of variance appropriate for repeated measures for day, diet, and day × diet effects using general linear model procedures.7 In addition, regression analyses were performed on mineral balance data to establish the mineral intake at which mineral absorption and retention was maximized.7 RESULTS
Feed Intake, Weight, Height, and Body Condition Score Total feed intake remained constant throughout the study based on g/kg of body weight. Average concentrate intake was 4.38 ± 0.27 kg, and hay intake was 3.06 ± 0.11 kg. Horses had a significant increase (P < .05) in average weight over the entire experiment from 381 ± 8.3 kg at day 0 to 436 ± 7.7 kg by day 128. Wither height increased significantly from 148 ± 0.8 cm at days 0 to 152 ± 0.9 cm at day 128. Average body condition score was maintained at 5 throughout the entire project.
Calcium Balance As shown in Table 2, calcium intake ranged from 135.1 mg/kg per day at day 0 to 123.2 mg/kg per day at
Journal of Equine Veterinary Science
October 2004
Table 2
Apparent calcium (Ca) balance initially, at day 64 and at day 128 (mg/kg/d)
Day Ca Intake SEM Ca Fecal SEM Ca Urine SEM Ca Absorbed SEM Ca Absorbed % of intake SEM Ca Retained SEM Ca Retained % of intake SEM Ca Retained % of absorbed SEM a,bRow
0
64
128
135.1 6.1 62.5a 3.9 11.2 1.4 72.6a 4.1
124.2 6.2 78.7b 6.4 13.6 1.9 45.5b 5.0
123.2 4.2 59.6a 4.5 9.9 1.3 63.6a 4.1
53.8a 1.9 61.4a 3.7
37.3b 3.4 31.9b 5.4
51.8a 3.2 53.7 a 3.7
45.6a 2.0
24.7b 3.5
43.9 a 3.0
84.4a 1.7
70.1b 5.4
84.6 a 2.0
means not sharing superscript differ (P < .05).
day 128 but was not significantly different. Fecal calcium increased from day 0 to day 64 (P < .05) and returned to day 0 amounts at day 128 (P < .05). This increase in fecal calcium at day 64 has been correlated previously with bone resorption occurring during this time period, followed by a subsequent increase in calcium absorption for bone formation and remineralization.4 Urinary excretion of calcium was not reduced in parallel fashion with reduced calcium absorption. This is contrary to previous reports, which indicated a decrease in urinary calcium excretion in exercised versus non-exercised horses.8 Further, Nielsen et al4 reported a dramatic reduction in urinary calcium excretion over the course of their study with a 7-fold decrease from day 0 to day 112. The efficiency of calcium absorption (Table 3) was reduced from day 0 to day 64 (P < .05) and returned to day 0 values by day 128. Calcium retention, retention as a percent of intake, and retention as a percent of absorption all decreased from day 0 to day 64 (P < .05) and returned to day 0 values by day 128. Depressed calcium retention on day 64 in comparison with both day 0 and day 128 indicates an increased systemic supply of calcium, probably from bone resorption, thus causing downregulation of calcium absorption and retention by the calcium homeostatic system. Feed intake, and thus mineral intake, did not follow diet treatments consistently. Some horses fed diets with
Volume 24, Number 10
Table 3
Apparent phosphorus (P) balance initially, at day 64 and at day 128 (mg/kg/d)
Day P Intake SEM P Fecal SEM P Urine SEM P Absorbed SEM P Absorbed % of intake SEM P Retained SEM P Retained % of intake SEM P Retained % of absorbed SEM a,bRow
0
64
128
51.4a 2.3 13.9 3.0 1.9a 0.6 37.5 3.5
44.6b 2.8 15.5 2.0 1.6a,b 0.6 29.1 3.5
45.26 b 2.5 15.8 2.3 0.4 b 0.1 29.4 3.3
72.9 5.8 35.6 3.2
62.2 5.2 27.5 3.4
63.5 5.4 29.0 3.3
69.4 5.5
58.9 5.2
62.6 5.4
95.3a,b 1.1
94.4a 1.7
98.1b 0.5
means not sharing superscript differ (P < .05).
lower mineral concentration actually consumed more minerals than other horses on diets with higher mineral concentrates, and vice versa. Also, variation in mineral intake among horses was quite high. Regression analyses were conducted to determine responses to varying mineral intakes. At day 64, there was a trend (P < .1) for increased calcium absorption with increasing intake, but absorption was variable at the highest intakes (Fig 1). At day 64, a significant increase in calcium retention (P < .05) was seen with increasing intake (Fig 2). At day 128, calcium absorption was maximized at an intake of 124 mg/kg per day (Fig 3), but maximum retention was not realized at the highest intake of 147 mg/kg per day, which is 163% of current NRC1 recommendations. Also at day 128, maximum calcium retention was observed at 123 mg/kg per day (Fig 4), which is 136% of current NRC1 recommendations. Although the variation among the horses by day and the correlations for the regressions were fairly low, the data tended to follow earlier indications4 that NRC1 recommendations were insufficient to meet the demands for calcium during bone modeling and remodeling in exercised, juvenile horses.
Phosphorus Balance Lower than expected concentrations of phosphorus in the concentrates resulted in a total dietary calcium/ phosphorus ratio of 3:1 compared with the planned cal-
441
Figure 1. Calcium absorption versus intake d 64.
Figure 2. Calcium retention versus intake d 64.
cium/phosphorus ratio of 1.75:1.00. As a result, the average phosphorus intake was near NRC1 recommendations at all 3 days and did not allow any potential response to higher levels of phosphorus. Therefore, no changes in
442
phosphorus balance were evident at the intakes fed (Table 3). Phosphorus intake was significantly higher at day 0 than all other days owing to the assignment of treatments after the initial total collection period. There were no sig-
Journal of Equine Veterinary Science
October 2004
Figure 3. Calcium absorption versus intake d 128.
Figure 4. Calcium retention versus intake d 128.
Volume 24, Number 10
443
Figure 5. Phosphorus absorption versus intake d 64.
Figure 6. Phosphorus retention versus intake d 64.
444
Journal of Equine Veterinary Science
October 2004
Figure 7. Phosphorus absorption versus intake d 128.
Figure 8. Phosphorus retention versus intake d 128.
Volume 24, Number 10
445
Figure 9. Phosphorus retention versus absorption d 64.
Figure 10. Phosphorus retention versus absorption d 128.
446
Journal of Equine Veterinary Science
October 2004
Figure 11. Magnesium absorption versus intake d 64.
Figure 12. Magnesium absorption versus intake d 128.
Volume 24, Number 10
447
Figure 13. Magnesium retention versus intake d 64.
Figure 14. Magnesium retention versus intake d 128.
448
Journal of Equine Veterinary Science
October 2004
Table 4
Apparent magnesium (Mg) balance initially, at day 64 and at day 128 (mg/kg/d)
Day Mg Intake SEM Mg Fecal SEM Mg Urine SEM Mg Absorbed SEM Mg Absorbed % of intake SEM Mg Retained SEM Mg Retained % of intake SEM Mg Retained % of absorbed SEM a,bRow
0
64
128
34.6a 1.5 18.7a 1.2 4.5a 0.3 16.3 0.9
30.3b 1.9 15.9b 1.4 5.3a, b 0.4 14.4 1.1
29.5b 1.5 14.7b 1.1 5.8b 0.5 14.8 0.9
47.0 2.1 11.8a 0.8
48.4 2.7 9.1b 0.9
50.8 2.7 9.1b 0.9
33.7 1.7
29.8 2.3
30.5 2.3
71.5a 1.6
61.0b 2.8
60.3b 3.5
means not sharing superscript differ (P < .05).
nificant differences in fecal phosphorus, absorption of phosphorus, phosphorus absorption as a function of intake, phosphorus retention, or phosphorus retention as a function of intake (Table 2). High demand for phosphorus was evidenced by low urinary phosphorus and significantly decreased phosphorus from day 0 to day 128 (P < .05). This corresponds with Caple et al,9 who reported that urinary phosphorus remained relatively low until dietary concentrations exceed 2 g/kg. Urinary excretion also increased when dietary concentrations of calcium were low as mineral was removed from bone to maintain calcium homeostasis.10 In this study, systemic efficiency of phosphorus retention was very high, remained constant from day 0 to day 64, and was increased significantly (P < .05) at day 128, thus reiterating the previously noted high demand for phosphorus at day 128. Other trends seen in calcium absorption and retention were followed loosely by phosphorus absorption and retention, with decreasing values from day 0 to day 64. However, because of limited intake of phosphorus and retention of calcium, there was no increase in phosphorus absorption and retention from day 64 to day 128. Phosphorus balance was analyzed by regression to determine effects of intake. At neither day 64 (Figs 5 and 6) nor day 128 (Figs 7 and 8) was phosphorus absorption or retention maximized at the highest intake of 66 mg/kg per day. Efficiency of systemic retention was high at day
Volume 24, Number 10
64 and significantly higher at day 128 (P < .05) at almost 100% (Figs 9 and 10). This indicates the horses retained almost all of the absorbed phosphorus for bone formation, which one would expect in a growing horse.10 Systemic demands for phosphorus may not have been maximized at the highest intake of 66 mg/kg per day. Thus, it appears the phosphorus requirement for the juvenile horse in training is at least 66 mg/kg per day, which is 132% of current NRC1 recommendations.
Magnesium Balance As seen in Table 4, magnesium intake decreased from day 0 to day 64 owing to the assignment of treatments after the initial total collection on day 0. Fecal excretion of magnesium decreased significantly from day 0 to day 64 (P < .05) and remained low for the duration of the trial, reflecting decreased intake. This is consistent with data from Nielsen et al,4 in which fecal magnesium seemed to be influenced primarily by dietary magnesium intake. Fractional absorption of magnesium did not change during the study. Conversely, urinary magnesium increased significantly from day 0 to day 128 (P < .05), with a slight increase from day 0 to day 64 and again from day 64 to day 128. Perhaps the increase in urinary magnesium excretion in the presence of reduced magnesium intake is in response to an increased rate of bone turnover during training. Retention of magnesium at both day 64 and day 128 was significantly lower than at day 0 (P < .05), reflecting reduced intake. However, the efficiency of systemic magnesium retention was also decreased significantly from day 0 to both day 64 and day 128 (P < .05) owing to increased urinary excretion, which could have been due to an increased rate of bone turnover. From regression analyses, it was determined that at day 64, absorption of magnesium was maximized at an intake of 39 mg/kg per day (Fig 11). However, at day 128 magnesium absorption was not maximized at the highest intake of 44 mg/kg per day (Fig 12), indicating a high demand for magnesium during bone remineralization. Magnesium retention was maximized at day 64 at an intake of 36 mg/kg per day (Fig 13); however, maximal retention of magnesium was not reached at the highest intake of 44 mg/kg per day at day 128 (Fig 14). Current NRC1 magnesium requirements for the long yearling in training is 22 mg/kg per day. This data indicates that current NRC1 recommendations for magnesium are inadequate for the juvenile horse in training. These findings are consistent with those of Nielsen et al,4 where increased need for magnesium at day 84 likely occurred in response to a higher demand for bone remineralization.
449
CONCLUSIONS AND IMPLICATIONS
This study was conducted to determine the influence of training on calcium, phosphorus, and magnesium absorption and retention. This was done to better understand the calcium, phosphorus, and magnesium requirements for growth and bone modeling/remodeling in the juvenile horse in training. The authors recognize the tremendous variation among animals in this study, and some of the regression correlations lack fit. Even so, it is obvious that training affects the absorption and retention of calcium, phosphorus, and magnesium in the juvenile racehorse. The NRC1 recommendation for calcium is 90 mg/kg per day for a long yearling in training. From these and other data, it appears that current NRC1 recommendations for calcium, phosphorus, and magnesium are too low relative to the demand during bone modeling and remodeling. In this study, maximum calcium retention of 123 mg/kg per day during bone remineralization represents a 36% increase over current recommendations. This is within the range that Nielsen et al4 suggested (between 125% and 148% of current NRC1 recommendations) for increased requirement of calcium in juvenile horses in training. Likewise, phosphorus requirements for the juvenile horse in training as suggested by the NRC1 may not be adequate. At the highest phosphorus intake of 66 mg/kg per day in this study, neither absorption nor retention was maximized, indicating that phosphorus requirements are at least that high, if not higher. This is in contrast with the findings of Nielsen et al,4 who suggested that current NRC1 recommendations for phosphorus may be adequate. Consequently, estimated phosphorus requirements at any time in this study were deficient, since intakes were not high enough to identify maximum phosphorus retention. The recommended requirements for magnesium suggested by the NRC1 do not appear to be adequate to support young horses during the bone modeling/remodeling process. At day 128, maximum magnesium retention was
450
not achieved at intakes 2 times greater than current recommendations. At day 64, maximum retention of magnesium was realized at an intake of 36 mg/kg per day, indicating that magnesium demand changes with the different stages of bone modeling/remodeling. From this study and previous research conducted with juvenile horses in training, it appears that adjustments need to be made in expressed requirements for calcium, phosphorus, and magnesium during the bone modeling/remodeling process. It appears that the demand for calcium, phosphorus, and magnesium changes during bone modeling/remodeling. Clearly, additional work is required to more accurately define the mineral requirements at the different stages of bone formation and turnover in the juvenile horse in training. This study points to the real need for further investigation into the exact mineral requirements of juvenile horses in training.
REFERENCES 1.
NRC. Nutrient requirements of horses, 5th ed. National Research Council. Washington, DC: National Academy Press; 1989. 2. Nielsen BD, Potter GD, Morris EL, Odom TW, Senor DM, Reynolds JA, et al. Changes in the third metacarpal bone and frequency of bone injuries in young Quarter Horses during race training: observations and theoretical considerations. J Equine Vet Sci 1997;17:541. 3. Jones WE. Racetrack breakdown epidemiology. Equine Vet Data 1989;10:190. 4. Nielsen BD, Potter GD, Greene LW, Morris EL, Murray-Gerzik M, Smith WB, et al. Response of young horses in training to varying concentrations of dietary calcium and phosphorus. J Equine Vet Sci 1998;18:397. 5. Henneke DR, Potter GD, Kreider JL, Yeates BF. A condition scoring system comparing body condition in horses. Equine Vet J 1983;15:371. 6. Fiske CH, Subbarow Y. The colorimetric determination of phosphorus. J Biol Chem 1925;66:375. 7. SAS. Statistical analysis system manual. Cary (NC); 1994. 8. Schryver HF, Hintz HF, Lowe JE. Calcium metabolism, body composition and sweat losses of exercised horses. Am J Vet Res 1978;39:245. 9. Caple IW, Bourke JM, Ellis PG. An examination of the calcium and phosphorus nutrition of Thoroughbred racehorses. Austr Vet J 1982;58:132. 10. Schryver HF, Hintz HF, Lowe JE. Calcium and phosphorus in ponies fed varying levels of phosphorus. J Nutr 1974;101:955..
Journal of Equine Veterinary Science
October 2004
Twenty-four long yearlings were fed rations containing differing amounts of calcium, phosphorus, and magnesium to further elucidate the requirements for these minerals during exercise-induced skeletal modeling and remodeling in juvenile racehorses. The animals were assigned randomly within gender subgroups to 1 of 4 diets. Total collections of feces and urine were performed on days 0, 64, and 128 of the trial for determination of mineral absorption and retention. Horses were maintained in a typical race-training protocol to mimic the nutritional stresses placed on long yearlings during strenuous exercise. Calcium absorption and retention were lower (P < .05) at day 64 than at day 0 and day 128. Also, the efficiency of retaining absorbed calcium was lower at day 64 than at day 0 or day 128. Thus, lower calcium retention at day 64 was due to both reduced absorption and reduced systemic demand. At day 64, calcium absorption and retention were not maximized at calcium intake of 160 mg/kg per day. At day 128, calcium absorption was maximal at a daily intake of 124 mg/kg per day, and retention was maximal at a daily intake of 123 mg/kg per day. These are in excess of current National Research Council (NRC)1 recommendations by 38% and 36%, respectively. There was no consistent, significant effect of days on trial on phosphorus absorption or retention, which may have been due to inadvertent limited phosphorus intake. The efficiency of phosphorus retention systemically was over 94% to 98%. Phosphorus absorption and retention were not maximized at the highest intake (66 mg/kg/d), which is 32% over current NRC1 rec-
From the Department of Animal Sciences, University of Florida,a and the Departments of Animal Scienceb and Veterinary Physiology and Pharmacology,c Texas A&M University. Reprint requests: Dr. Tonya L. Stephens, Department of Animal Sciences, University of Florida, P.O. Box 110910, Gainesville, FL 32611-0910. 0737-0806/$ - see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2004.09.006
438
ommendations. Similarly, there was no day effect on the efficiency of magnesium absorption or retention. Urinary excretion of magnesium was highest; thus, systemic efficiency of magnesium retention was lower on days 64 and 128 than on day 0. At day 64, magnesium retention was maximized at a daily intake of 35.6 mg/kg per day, which is 66% over NRC1 recommendations. However, at day 128, magnesium retention was not maximized even at its highest intake (44 mg/kg/d), which is over 2 times the current NRC1 recommendations. These data indicate that early race training affects the dietary requirements for calcium, phosphorus, and magnesium. INTRODUCTION
The need for a marketable product at an early age in both competition and in sales places significant pressure on horse owners to start training or conditioning early in a horse’s life. Training at an early age may exacerbate physiologic stress on young horses and increase nutritional requirements. The process of bone modeling/remodeling is necessary for the juvenile skeleton to adapt to the stresses of training. The horse must form new bone and remodel existing bone to bear the load being applied. During the demineralization process of bone remodeling, there is an increase in mineral excretion from the degradation of the bone matrix. Nielsen et al2 reported this demineralization of the third metacarpal in conjunction with the introduction of speed in juvenile horses on a race-training regimen. The lack of time routinely given to the stressed bone of a juvenile horse to model and/or remodel according to the stresses placed upon it can cause serious and career-ending injuries.3 While injury reduction may be achieved from a delay in the onset of training until the horse is mature, this is not feasible economically. Alterations in existing training methods and increasing physiologic stimulus to the skeleton are approaches being researched to minimize skeletal injuries to young horses. Mineral density increase and size of the third metacarpal would result in larger cortical bone mass, de-
Journal of Equine Veterinary Science
October 2004
Table 1
Formulated (F) versus analyzed (A) mineral concentrations in total diet (dry matter basis) % Calcium
Bermuda grass contribution at 40% of diet Formulated pellet concentration at 60% of diet Treatment #1 Treatment #2 Treatment #3 Treatment #4 Total diet concentration Treatment #1 Treatment #2 Treatment #3 Treatment #4
% Phosphorus
% Magnesium
F
A
F
A
F
A
0.12
0.13
0.08
0.06
0.04
0.04
0.29 0.33 0.49 0.51
0.34 0.38 0.41 0.42
0.15 0.18 0.26 0.31
0.11 0.15 0.17 0.19
0.07 0.09 0.13 0.14
0.07 0.08 0.11 0.11
0.41 0.45 0.61 0.63
0.46 0.50 0.53 0.55
0.23 0.26 0.34 0.39
0.17 0.21 0.23 0.25
0.11 0.13 0.17 0.18
0.11 0.12 0.15 0.15
Diets 1, 2, and 3 were experimental, with all diets identical except for concentrations of calcium, phosphorus, and magnesium. Diet 4 was a commercially available concentrate, Patriot 14, by Consolidated Nutrition, Omaha, NE.
creased strain on the bone, and decreased susceptibility for injury. Previous work in this area demonstrated that increased calcium and phosphorus intake above NRC1 recommendations for juvenile horses in training enhanced bone density, but no quantitative estimates of requirements were made.4 The NRC1 developed an approximation of the requirements for juvenile horses in training using mineral intakes based on extrapolations from research conducted with sedentary and mature horses. This study was conducted to verify previous findings and to further quantify the dietary requirement for calcium, phosphorus, and magnesium during the bone modeling/remodeling process in juvenile athletic horses. The specific objectives of this study were to determine calcium, phosphorus, and magnesium balances in young horses during race training. MATERIALS AND METHODS
Management of Animals Twenty-four long-yearling Quarter Horses were grouped according to age and sex, then randomly assigned to the diet treatments as shown in Table 1. Two horses from each group were randomly assigned to 1 of 4 diets with the condition that each treatment group would contain the same number of fillies and geldings. While at the Texas A&M University Horse Center, the horses were vaccinated against eastern and western equine encephalomyelitis, influenza, and tetanus and dewormed. All horses began the trial with a background period of at least 1 week, during which the horses were group-housed at the Texas A&M University Horse Center, fed hay ad lib, and offered concentrate at approximately 1% of body weight twice daily. Regular hoof care
Volume 24, Number 10
and deworming were provided throughout the course of the study. The horses were then moved from the Texas A&M University Horse Center in groups of 8 to Steephollow Farm in Bryan, Texas, a race-training facility, where they were separated and housed individually in 7 × 7-m stalls. The horses were moved to Steephollow Farm in 3 groups of 8 at an average age of 226 days for the group. Rations were formulated based on 60% concentrate and 40% Bermuda grass hay, with varying concentrations of calcium, phosphorus, and magnesium. The concentrate diets were provided by Consolidated Nutrition of Omaha, Nebraska (Table 1). All concentrates were mixed, pelleted, and bagged by Consolidated Nutrition and shipped to the Texas A&M University Horse Center. The horses were fed concentrate and hay at 12-hour intervals (7:00 AM and 7:00 PM) for the duration of the trial. They were given until the next feeding to consume all concentrate and hay, and any feed refused was weighed and recorded. Refusals were very infrequent. Feed intake was adjusted weekly as needed to allow for normal growth and to maintain a body condition score of 5 to 6,5 while maintaining a constant ratio of 60% concentrate and 40% hay. The protocol for management and treatment of the animals was approved by the Texas A&M University Agricultural Animal Care and Use Committee. Five horses were not able to complete the study because of lameness, injury, or sickness, and data from those horses were not included in the results of the study.
Training of Animals The 4 training periods were 28 days each. During the first week, horses were worked 6 days and rested 1 day. The riding began in the round pen and progressed to the track by the middle of the week, depending on the tem-
439
perament of the horse. During the initial days of training, the horses were walked 550 m, trotted 1100 m, and galloped 275 m. The gallop length was increased by 275 m each day after day 3; therefore, by day 6 the horses were galloped 1375 m. Horses were ridden 5 days a week during weeks 2 to 4. Some horses were started in the round pen through week 2 if needed before riding their assigned distances on the track. They continued to walk and trot the same distances as week 1 but were galloped a total of 8250 m per week. During the next 4 weeks in period 2, the horses were ridden 4 days per week. The total gallop distance was increased to 8440 m. During period 3, horses were ridden 4 days per week, with 1 of the 4 days being a sprint. On nonsprint days, the horses were trotted 550 m and galloped 1925 m. On sprint days, the horses were warmed up, sprinted 230 m, and galloped. During this period, the horses were galloped a total of 8210 m per week. During the last 4 weeks, period 4, the horses continued training as in period 3 except sprints were extended to 275 m and total gallop distance decreased to 8165 m per week. Throughout the entire trial, horses were walked at least 1 hour on a mechanical walker on rest days.
Sample Collection Total collections of feces and urine were made on days 4 through 0, 60 through 64, and 124 through 128. During total collections, horses were confined to tie stalls with rubber mats to prevent coprophagy and contamination of fecal samples. Feces were collected from the mats following each defecation throughout the 72-hour collection period. All feces collections were pooled, and a 10% representative sample was obtained and stored at –20°C for further analyses. A urine collection harness was secured to all horses and checked every 4 hours throughout the 72-hour total collection to prevent loss or contamination of urine. Urine collection containers were acidified and emptied every 4 hours; a 10% sample of the urine was taken and frozen at –20°C for subsequent analyses. Hay and concentrate feed were sampled at each feeding, then pooled to form a composite sample for each collection period. Horses were walked on the mechanical walker for at least 1 hour every day of the collection period. All fecal losses were gathered, weighed, and added to the total output amount but were not included in the composite sample.
Preparation and Analyses of Samples Feed and fecal samples were dried in an oven for 72 hours at 65°C, then ground in a Wiley mill with a 1-mm screen. The ground samples were mixed thoroughly, and
440
a representative of the total ground sample was obtained. Perchloric acid digestions were performed on all feed and fecal samples, and dilutions were made of these digestions and of urine and serum with double-distilled water. Cornstalk (Zea mays) reference material #8412 from the National Institute of Standards and Technology was used as a control standard in perchloric acid digestions and analyses of minerals. Concentrations of calcium and magnesium in samples were determined by atomic absorption spectrophotometry. Reference standards from Fisher Scientific of Pittsburgh, Pennsylvania, for atomic absorption spectroscopy were used to create the standards for calcium (SC191) and magnesium (SM51). Inorganic phosphorus was determined with an Ultraspec III spectrophotometer using methods of Fiske and Subbarow.6 A stock solution of potassium phosphate was used to create standards for phosphorus analyses. Lanthanum chloride was used as the suspension medium of acid digestions for calcium and magnesium analyses, and double-distilled water was used as the suspension medium of acid digestions for phosphorus analyses. Mineral absorption was determined by subtracting fecal output of mineral from mineral intake. Mineral retention was determined by subtracting mineral output in urine from mineral absorbed. STATISTICAL ANALYSES
Data were analyzed using 2-way analysis of variance appropriate for repeated measures for day, diet, and day × diet effects using general linear model procedures.7 In addition, regression analyses were performed on mineral balance data to establish the mineral intake at which mineral absorption and retention was maximized.7 RESULTS
Feed Intake, Weight, Height, and Body Condition Score Total feed intake remained constant throughout the study based on g/kg of body weight. Average concentrate intake was 4.38 ± 0.27 kg, and hay intake was 3.06 ± 0.11 kg. Horses had a significant increase (P < .05) in average weight over the entire experiment from 381 ± 8.3 kg at day 0 to 436 ± 7.7 kg by day 128. Wither height increased significantly from 148 ± 0.8 cm at days 0 to 152 ± 0.9 cm at day 128. Average body condition score was maintained at 5 throughout the entire project.
Calcium Balance As shown in Table 2, calcium intake ranged from 135.1 mg/kg per day at day 0 to 123.2 mg/kg per day at
Journal of Equine Veterinary Science
October 2004
Table 2
Apparent calcium (Ca) balance initially, at day 64 and at day 128 (mg/kg/d)
Day Ca Intake SEM Ca Fecal SEM Ca Urine SEM Ca Absorbed SEM Ca Absorbed % of intake SEM Ca Retained SEM Ca Retained % of intake SEM Ca Retained % of absorbed SEM a,bRow
0
64
128
135.1 6.1 62.5a 3.9 11.2 1.4 72.6a 4.1
124.2 6.2 78.7b 6.4 13.6 1.9 45.5b 5.0
123.2 4.2 59.6a 4.5 9.9 1.3 63.6a 4.1
53.8a 1.9 61.4a 3.7
37.3b 3.4 31.9b 5.4
51.8a 3.2 53.7 a 3.7
45.6a 2.0
24.7b 3.5
43.9 a 3.0
84.4a 1.7
70.1b 5.4
84.6 a 2.0
means not sharing superscript differ (P < .05).
day 128 but was not significantly different. Fecal calcium increased from day 0 to day 64 (P < .05) and returned to day 0 amounts at day 128 (P < .05). This increase in fecal calcium at day 64 has been correlated previously with bone resorption occurring during this time period, followed by a subsequent increase in calcium absorption for bone formation and remineralization.4 Urinary excretion of calcium was not reduced in parallel fashion with reduced calcium absorption. This is contrary to previous reports, which indicated a decrease in urinary calcium excretion in exercised versus non-exercised horses.8 Further, Nielsen et al4 reported a dramatic reduction in urinary calcium excretion over the course of their study with a 7-fold decrease from day 0 to day 112. The efficiency of calcium absorption (Table 3) was reduced from day 0 to day 64 (P < .05) and returned to day 0 values by day 128. Calcium retention, retention as a percent of intake, and retention as a percent of absorption all decreased from day 0 to day 64 (P < .05) and returned to day 0 values by day 128. Depressed calcium retention on day 64 in comparison with both day 0 and day 128 indicates an increased systemic supply of calcium, probably from bone resorption, thus causing downregulation of calcium absorption and retention by the calcium homeostatic system. Feed intake, and thus mineral intake, did not follow diet treatments consistently. Some horses fed diets with
Volume 24, Number 10
Table 3
Apparent phosphorus (P) balance initially, at day 64 and at day 128 (mg/kg/d)
Day P Intake SEM P Fecal SEM P Urine SEM P Absorbed SEM P Absorbed % of intake SEM P Retained SEM P Retained % of intake SEM P Retained % of absorbed SEM a,bRow
0
64
128
51.4a 2.3 13.9 3.0 1.9a 0.6 37.5 3.5
44.6b 2.8 15.5 2.0 1.6a,b 0.6 29.1 3.5
45.26 b 2.5 15.8 2.3 0.4 b 0.1 29.4 3.3
72.9 5.8 35.6 3.2
62.2 5.2 27.5 3.4
63.5 5.4 29.0 3.3
69.4 5.5
58.9 5.2
62.6 5.4
95.3a,b 1.1
94.4a 1.7
98.1b 0.5
means not sharing superscript differ (P < .05).
lower mineral concentration actually consumed more minerals than other horses on diets with higher mineral concentrates, and vice versa. Also, variation in mineral intake among horses was quite high. Regression analyses were conducted to determine responses to varying mineral intakes. At day 64, there was a trend (P < .1) for increased calcium absorption with increasing intake, but absorption was variable at the highest intakes (Fig 1). At day 64, a significant increase in calcium retention (P < .05) was seen with increasing intake (Fig 2). At day 128, calcium absorption was maximized at an intake of 124 mg/kg per day (Fig 3), but maximum retention was not realized at the highest intake of 147 mg/kg per day, which is 163% of current NRC1 recommendations. Also at day 128, maximum calcium retention was observed at 123 mg/kg per day (Fig 4), which is 136% of current NRC1 recommendations. Although the variation among the horses by day and the correlations for the regressions were fairly low, the data tended to follow earlier indications4 that NRC1 recommendations were insufficient to meet the demands for calcium during bone modeling and remodeling in exercised, juvenile horses.
Phosphorus Balance Lower than expected concentrations of phosphorus in the concentrates resulted in a total dietary calcium/ phosphorus ratio of 3:1 compared with the planned cal-
441
Figure 1. Calcium absorption versus intake d 64.
Figure 2. Calcium retention versus intake d 64.
cium/phosphorus ratio of 1.75:1.00. As a result, the average phosphorus intake was near NRC1 recommendations at all 3 days and did not allow any potential response to higher levels of phosphorus. Therefore, no changes in
442
phosphorus balance were evident at the intakes fed (Table 3). Phosphorus intake was significantly higher at day 0 than all other days owing to the assignment of treatments after the initial total collection period. There were no sig-
Journal of Equine Veterinary Science
October 2004
Figure 3. Calcium absorption versus intake d 128.
Figure 4. Calcium retention versus intake d 128.
Volume 24, Number 10
443
Figure 5. Phosphorus absorption versus intake d 64.
Figure 6. Phosphorus retention versus intake d 64.
444
Journal of Equine Veterinary Science
October 2004
Figure 7. Phosphorus absorption versus intake d 128.
Figure 8. Phosphorus retention versus intake d 128.
Volume 24, Number 10
445
Figure 9. Phosphorus retention versus absorption d 64.
Figure 10. Phosphorus retention versus absorption d 128.
446
Journal of Equine Veterinary Science
October 2004
Figure 11. Magnesium absorption versus intake d 64.
Figure 12. Magnesium absorption versus intake d 128.
Volume 24, Number 10
447
Figure 13. Magnesium retention versus intake d 64.
Figure 14. Magnesium retention versus intake d 128.
448
Journal of Equine Veterinary Science
October 2004
Table 4
Apparent magnesium (Mg) balance initially, at day 64 and at day 128 (mg/kg/d)
Day Mg Intake SEM Mg Fecal SEM Mg Urine SEM Mg Absorbed SEM Mg Absorbed % of intake SEM Mg Retained SEM Mg Retained % of intake SEM Mg Retained % of absorbed SEM a,bRow
0
64
128
34.6a 1.5 18.7a 1.2 4.5a 0.3 16.3 0.9
30.3b 1.9 15.9b 1.4 5.3a, b 0.4 14.4 1.1
29.5b 1.5 14.7b 1.1 5.8b 0.5 14.8 0.9
47.0 2.1 11.8a 0.8
48.4 2.7 9.1b 0.9
50.8 2.7 9.1b 0.9
33.7 1.7
29.8 2.3
30.5 2.3
71.5a 1.6
61.0b 2.8
60.3b 3.5
means not sharing superscript differ (P < .05).
nificant differences in fecal phosphorus, absorption of phosphorus, phosphorus absorption as a function of intake, phosphorus retention, or phosphorus retention as a function of intake (Table 2). High demand for phosphorus was evidenced by low urinary phosphorus and significantly decreased phosphorus from day 0 to day 128 (P < .05). This corresponds with Caple et al,9 who reported that urinary phosphorus remained relatively low until dietary concentrations exceed 2 g/kg. Urinary excretion also increased when dietary concentrations of calcium were low as mineral was removed from bone to maintain calcium homeostasis.10 In this study, systemic efficiency of phosphorus retention was very high, remained constant from day 0 to day 64, and was increased significantly (P < .05) at day 128, thus reiterating the previously noted high demand for phosphorus at day 128. Other trends seen in calcium absorption and retention were followed loosely by phosphorus absorption and retention, with decreasing values from day 0 to day 64. However, because of limited intake of phosphorus and retention of calcium, there was no increase in phosphorus absorption and retention from day 64 to day 128. Phosphorus balance was analyzed by regression to determine effects of intake. At neither day 64 (Figs 5 and 6) nor day 128 (Figs 7 and 8) was phosphorus absorption or retention maximized at the highest intake of 66 mg/kg per day. Efficiency of systemic retention was high at day
Volume 24, Number 10
64 and significantly higher at day 128 (P < .05) at almost 100% (Figs 9 and 10). This indicates the horses retained almost all of the absorbed phosphorus for bone formation, which one would expect in a growing horse.10 Systemic demands for phosphorus may not have been maximized at the highest intake of 66 mg/kg per day. Thus, it appears the phosphorus requirement for the juvenile horse in training is at least 66 mg/kg per day, which is 132% of current NRC1 recommendations.
Magnesium Balance As seen in Table 4, magnesium intake decreased from day 0 to day 64 owing to the assignment of treatments after the initial total collection on day 0. Fecal excretion of magnesium decreased significantly from day 0 to day 64 (P < .05) and remained low for the duration of the trial, reflecting decreased intake. This is consistent with data from Nielsen et al,4 in which fecal magnesium seemed to be influenced primarily by dietary magnesium intake. Fractional absorption of magnesium did not change during the study. Conversely, urinary magnesium increased significantly from day 0 to day 128 (P < .05), with a slight increase from day 0 to day 64 and again from day 64 to day 128. Perhaps the increase in urinary magnesium excretion in the presence of reduced magnesium intake is in response to an increased rate of bone turnover during training. Retention of magnesium at both day 64 and day 128 was significantly lower than at day 0 (P < .05), reflecting reduced intake. However, the efficiency of systemic magnesium retention was also decreased significantly from day 0 to both day 64 and day 128 (P < .05) owing to increased urinary excretion, which could have been due to an increased rate of bone turnover. From regression analyses, it was determined that at day 64, absorption of magnesium was maximized at an intake of 39 mg/kg per day (Fig 11). However, at day 128 magnesium absorption was not maximized at the highest intake of 44 mg/kg per day (Fig 12), indicating a high demand for magnesium during bone remineralization. Magnesium retention was maximized at day 64 at an intake of 36 mg/kg per day (Fig 13); however, maximal retention of magnesium was not reached at the highest intake of 44 mg/kg per day at day 128 (Fig 14). Current NRC1 magnesium requirements for the long yearling in training is 22 mg/kg per day. This data indicates that current NRC1 recommendations for magnesium are inadequate for the juvenile horse in training. These findings are consistent with those of Nielsen et al,4 where increased need for magnesium at day 84 likely occurred in response to a higher demand for bone remineralization.
449
CONCLUSIONS AND IMPLICATIONS
This study was conducted to determine the influence of training on calcium, phosphorus, and magnesium absorption and retention. This was done to better understand the calcium, phosphorus, and magnesium requirements for growth and bone modeling/remodeling in the juvenile horse in training. The authors recognize the tremendous variation among animals in this study, and some of the regression correlations lack fit. Even so, it is obvious that training affects the absorption and retention of calcium, phosphorus, and magnesium in the juvenile racehorse. The NRC1 recommendation for calcium is 90 mg/kg per day for a long yearling in training. From these and other data, it appears that current NRC1 recommendations for calcium, phosphorus, and magnesium are too low relative to the demand during bone modeling and remodeling. In this study, maximum calcium retention of 123 mg/kg per day during bone remineralization represents a 36% increase over current recommendations. This is within the range that Nielsen et al4 suggested (between 125% and 148% of current NRC1 recommendations) for increased requirement of calcium in juvenile horses in training. Likewise, phosphorus requirements for the juvenile horse in training as suggested by the NRC1 may not be adequate. At the highest phosphorus intake of 66 mg/kg per day in this study, neither absorption nor retention was maximized, indicating that phosphorus requirements are at least that high, if not higher. This is in contrast with the findings of Nielsen et al,4 who suggested that current NRC1 recommendations for phosphorus may be adequate. Consequently, estimated phosphorus requirements at any time in this study were deficient, since intakes were not high enough to identify maximum phosphorus retention. The recommended requirements for magnesium suggested by the NRC1 do not appear to be adequate to support young horses during the bone modeling/remodeling process. At day 128, maximum magnesium retention was
450
not achieved at intakes 2 times greater than current recommendations. At day 64, maximum retention of magnesium was realized at an intake of 36 mg/kg per day, indicating that magnesium demand changes with the different stages of bone modeling/remodeling. From this study and previous research conducted with juvenile horses in training, it appears that adjustments need to be made in expressed requirements for calcium, phosphorus, and magnesium during the bone modeling/remodeling process. It appears that the demand for calcium, phosphorus, and magnesium changes during bone modeling/remodeling. Clearly, additional work is required to more accurately define the mineral requirements at the different stages of bone formation and turnover in the juvenile horse in training. This study points to the real need for further investigation into the exact mineral requirements of juvenile horses in training.
REFERENCES 1.
NRC. Nutrient requirements of horses, 5th ed. National Research Council. Washington, DC: National Academy Press; 1989. 2. Nielsen BD, Potter GD, Morris EL, Odom TW, Senor DM, Reynolds JA, et al. Changes in the third metacarpal bone and frequency of bone injuries in young Quarter Horses during race training: observations and theoretical considerations. J Equine Vet Sci 1997;17:541. 3. Jones WE. Racetrack breakdown epidemiology. Equine Vet Data 1989;10:190. 4. Nielsen BD, Potter GD, Greene LW, Morris EL, Murray-Gerzik M, Smith WB, et al. Response of young horses in training to varying concentrations of dietary calcium and phosphorus. J Equine Vet Sci 1998;18:397. 5. Henneke DR, Potter GD, Kreider JL, Yeates BF. A condition scoring system comparing body condition in horses. Equine Vet J 1983;15:371. 6. Fiske CH, Subbarow Y. The colorimetric determination of phosphorus. J Biol Chem 1925;66:375. 7. SAS. Statistical analysis system manual. Cary (NC); 1994. 8. Schryver HF, Hintz HF, Lowe JE. Calcium metabolism, body composition and sweat losses of exercised horses. Am J Vet Res 1978;39:245. 9. Caple IW, Bourke JM, Ellis PG. An examination of the calcium and phosphorus nutrition of Thoroughbred racehorses. Austr Vet J 1982;58:132. 10. Schryver HF, Hintz HF, Lowe JE. Calcium and phosphorus in ponies fed varying levels of phosphorus. J Nutr 1974;101:955..
Journal of Equine Veterinary Science
October 2004