An Investigation of the Effect of Hesperidin Complex and Lemon Bioflavonoid Complex on Growth and Development of Thoroughbred Horses G. R.

Wooden, C. S. Crane and C. G. Beisel J ANIM SCI 1984, 59:1529-1535.

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AN INVESTIGATION OF THE EFFECT OF HESPERIDIN COMPLEX A N D LEMON B I O F L A V O N O I D COMPLEX ON GROWTH A N D DEVELOPMENT OF T H O R O U G H B R E D HORSES 1
G. R. Wooden 2, C. S. Crane 3 and C. G. Beisel 4

Summary

The objective of this study was to determine the effect of Hesperidin Complex and Lemon Bioflavonoid Complex (HC/LBC) on the growth and development of thoroughbred horses. The trial involved twenty-four foals (12 colts, 12 fillies) allotted to treatment shortly after weaning. The study was conducted for a 342-d growing phase and a 153-d training phase, or a total of 495-d. The HC/LBC was included in the test diet at a level calculated to supply the compound at 55 mg'kg body weighCl'd -1. During the growing phase, average daily gain of colts that received HC/ LBC was higher (P<.05) than gain of control fillies but not different from that of treated and control colts. No differences (P>.05) in daily gain due to treatment were observed during the growing phase, training phase, or the data for the combined growing and training phases. Differences between treatments in changes in height at the withers and knees during the growing phase were not significant. In the training phase, average daily height change values for colts was higher (P<.05) than that of fillies; however, these differences were not related to diet treatment. Radiographs were taken of the epiphyseal plate of the knee initially and at approximately 120-d intervals. No differences (P>.05) were noted between diet treatments through d 365 of the trial. Radiographs taken upon termination of the trail (495 d) showed a difference (P<.05)

favoring fillies over colts, but no differences related to diet treatment. Blood samples were taken initially and at periodic intervals throughout the trial. The various characteristics included in the blood study showed a few differences (P<.05) between treatment groups, but no strong trends related to diet treatment. The HC/LBC had no major effect on the growth of horses. It is planned to monitor the progress of the horses involved in the trial to evaluate the possible relationship between diet treatment during early growth, and performance later on at the racetrack. (Key Words: Horses, Growth, Bioflavonoids.)

I n'croduotion

Previous studies (Palmer, 1954, Bruner and Palmer, 1961; Bauer, 1978) have shown the effect of Hesperidin and other citrus bioflavonoids on human athletes that undergo periods of intense physical stress. These compounds have also been used in the treatment of patients subjected to other stress situations such as nutritional, physical, environmental and disease conditions (Fostvedt, 1956). A factor common to these seemingly unrelated conditions is based on the assumption that capillary weakness may be a contributing factor to the problem; thus, maintenance of capillary integrity should aid in the treatment or control of various stress conditions. Because growth, especially rapid growth, is considered to be a form of stress, it is reasonable to suggest that the addition of citrus bioflavonoids to the diet may aid in the reduction of stress-related problems (such as respira1Research supported by Sunkist Growers, Inc., tory infections) and therefore improve growth Ontario, CA 91762. rate and characteristics. 2Consulting Animal Nutritionist, Arroyo Grande, Biofiavonoids have also been used in the CA 93420. s Veterinary Practioner, Porterville, CA 93257. horse industry with varying degrees of success 4Food Technology Consultant, Rancho Cuca- as part of the treatment procedure for bleeding monga, CA 91730. in racehorses (J. Wheat, personal communicaReceived January 24, 1984. tion) and for laminitis (Horvath, 1974). Accepted June 7, 1984. 1529 JOURNAL OF ANIMALSCIENCE, Vol. 59, No. 6, 1984
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WOODEN ET AL. which the horses were prepared for race training. In the growing phase, the horses were maintained in pastures as separate groups according to experimental treatment. Grain was group-fed to each pasture twice daily and consumption of grain fed to each separate group was recorded throughout the trial. Average daily grain consumption per head was then calculated. Alfalfa hay was fed ad libitum. All horses were fed the same basal grain mixes throughout the trial (table 1). The grain mix was formulated to contain 17% crude protein (dry basis) for the first 150-d of the study and 14% protein throughout the remainder of the trial. The HC/LBC was added to the grain mix for the test horses by blending it with the protein supplement before mixing in the final feed. This supplied HC/LBC at a level of 5.5 g/kg of feed during the growing phase and 11.5 g/kg of feed during the training phase. Dose titration studies had not been conducted with this compound; however, it had been used in race horses that were known bleeders at the rate of 25 g/d or about 55 mg/kg body weight. The HC/LBC was added to the feed at levels calculated to provide approximately the same

The objective of this study was to determine the effect of Hesperidin Complex and Lemon Bioflavonoid Complex (HC/LBC) on the growth and development of Thoroughbred horses. No particular emphasis was placed on the compound with respect to its previous use in the treatment of bleeders or in cases of laminitis.
Experimental Procedure

Thirty-three weanling Thoroughbred foals born between January and May, 1980 were allotted to treatments in a 2 x 2 factorial experiment. The foals were allotted to the controI or HC/LBC treatments on the basis of sex, age, uniformity, and age of dam and sire. During the course of the trial, nine animals were withdrawn because of transfer to another breeding farm, illness or injury. Of the 33 animals initially allotted to the trial, 24 were maintained to termination of the study, equally divided among the respective cells of the experimental design. The 495-d study consisted of a growing phase (342-d) and a training phase (153-d) in

TABLE 1. COMPOSITIONOF EXPERIMENTAL DIETS Diet numbera Ingredient Oats, crimped (IFN 4-0%999), % Barley, steam rolled (IFN 4-07-939), % Cane molasses (IFN 4-04-696), % Protein supplement b, % Dried milk supplement c, % Calculated analysis (as-fed basis) Dry matter, % Digestible energy, Mcal/kg Crude protein, % Crude fat, % Crude fiber, % Calcium, % Phosphorus, % Potassium, % aDiet 1, d 1 through 151; diet 2, d 152 through 501. bprovided the following to the diet: soybean meal (IFN 5-04-600) 6.25%; cottonseed meal (IFN 5-07-872) 3.5%; dicalcium phosphate (IFN 6-01-O80) .88%; soybean oil (IFN 4-07-983) .25%; cane molasses (IFN 4-04-696) .25%; salt .75%; vitamin and trace mineral premix .63% that supplied (per kg of feed): vitamin A, 8,800 IU; vitamin D, 4,400 IU; vitamin E, .22 IU; Se, .10 rag; Mn, 22 rag; Zn, 110 rag; Fe, 5.5 rag; Cu, 2.2 rag; C1, 1.65 rag; Co, 1.10 rag. CStart to Finish, Milk Specialties Co., Dundee, IL.
1

45.00 22.50 10.00 12.50 10.00 100.00 88.20 3.06 15.05 3.96 7.22 .45 .60 .99

50.00 29.50 10.00 10.50 100.00 87.84 3.06 12.58 3.25

7.89

.27 .49 .83

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BIOFLAVONOIDS FOR GROWING HORSES amount to the growing horses on an equal body weight basis. The animals were individually weighed at approximately 28-d intervals throughout the study. Measurements were also taken to determine the height at the withers (the highest protruding thoracic vertebra) and the height at the knee (the accessory carpal bone was the reference point). The horses were photographed while standing behind a wire grid. The original intent for the photographs was to make additional body measurements; however, results of this procedure were not satisfactory because it was difficult to stand the young horses squarely enough each time to obtain accurate measurements from the photographs. In order to monitor bone development, radiographs of the epiphyseal plate at the knee were taken initially, at d 109, 229, 365 and upon termination of the trail. Blood samples were collected by jugular venipuncture from all animals at the start of the trial, at the end of the growing phase and at the end of the trial. Blood samples were also collected from one-half of the animals in each group at each weigh period. The animals selected for sampling were alternated at each collection period. Blood analyses performed included: white blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), serum glutamic oxalic transaminase (SGOT), alkaline phosphatase (Alk. Phos.), total bilirubin, Ca, P, K, Na, lactic dehydrogenase (LDH), C1, CO2,

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estimated sedimentation rate (ESR), differential, platelets, fibrinogen, red blood cell morphology and red blood cell osmotic fragility. On the last day of the trial, 12 animals (three from each group) were subjected to a punch biopsy of the skin and subcutaneous tissue was taken from the anterior cervical area under the mane (for cosmetic purposes). Histocytologic examination of the samples was performed to determine if any changes in capillary wall structures could be observed. Pertinent data were analyzed statistically by analysis of variance (Snedecor and Cochran, 1967; significant means were separated by Duncan's new multiple range test (Steel and Torrie, 1960).
Results and Discussion

The data in table 2 shows the average animal weights, grain consumption and actual HC/LBC intake values for the growing and training phases, respectfully. The intake data has been calculated to show HC/LBC intake on a mg/kg of body weight (BW) basis. As indicated previously, dose titration studies have not been conducted with this material, but the intended feeding level of 55 mg/kg BW was based on levels previously used with mature horses. Moderate diviations from this feeding level were noted at various points throughtout the trial; however, overall average HC/LBC intake was 53.4 and 58.9 mg/kg BW for the colts and fillies, respectively. These values compare favorably with the target level of 55 mg/kg BW. No problems related to palatability or handling of the product were encountered throughout the trial.

TABLE 2. SUMMARY OF AVERAGE GRAIN AND HC/LBC INTAKE FOR THE GROWING AND TRAINING PHASES Control Item Growing phase Avg weight, kg Avg grain intake/day, kg Avg HC/LBC intake, mg/kg Training phase Avg weight, kg Avg grain intake/day, kg Avg HC/LBC intake, mg/kg Colts Fillies Colts HC/LBC Fillies

349.9 3.5 0 476.6 2.7 0

348.7 3.7 0 472.9 2.7 0

351.4 3.6 51.8 485.1 2.7 44.4

352.4 3.8 58.1 477.1 2.7 46.0

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WOODEN ET AL.

TABLE 3. SUMMARYOF AVERAGE GROWTH DATA FOR THE GROWINGAND TRAINING PHASES Control Item No. of animals Weight, kg Initial End of growing phase Final Average daily gain, kg Growing phase (342 d) Training phase (153 d) Cumulative (495 d) Height at withers, cm Initial End of growing phase Final Average daily height change, cm Growing phase (342 d) Training phase (153 d) Cumulative (495 d) Height at knee, cm Initial End of growing phase Final Average daily change, cm Growing phase (342 d) Training phase (153 d) Cumulative (495 d) Colts 6 242.9 456.9 496.3 .63 ab .26 .52 133.88 154.00 158.85 .06 .03 a .05 a 47.82 51.92 52.76 .013 .005 .010 Fillies 6 246.2 451.2 494.6
.60 b .28 .50

HC/LBC Colts 6 237.0 465.8 504.4

.70 a .28 .57

Fillies 6 240.0 464.7 489.4

.66ab .16 .50

134.42 154.64 157.61 .06 .02b .04b 46.99 51.08 52.12 .013 .008 .010

133.78 154.74 159.87 .06 .03a .05 a 48.16 52.30 52.86 .010 .008 .010

133.88 151.77 156.01 .05 .02b .04b 46.56 49.86 50.85 .010 .008 .008

a'bMeans within rows with different superscripts differ (P<.05).

A summary of the average growth data is shown in table 3. During the entire 342-d growing period, the overall average daily gain of the horses was .64 kg/d. During the growing period, average daily gain (ADG) of colts that received HC/LBC was higher (P<.05) than that of the control fillies (.70 vs .60 kg/d). The ADG of colts that received HC/LBC appeared to be greater than that of the control colts; however, the differences were not significant. The gain data also tended to favor the treated fillies over control fillies; however, these differences also were not significant. As would be expected, ADG throughout the 153-d training phase was lower, with the overall value being .24 kg/d. The gain data tended to favor HC/LBC colts over control colts, and the opposite of this for fillies; however, no interaction was noted, nor were differences between the respective treatment means significant. The cumulative 495-d gain data also tended to favor the HC/LBC treatment, but these apparent differences were not significant.

Data for change in average daily height at the withers showed no differences (P>.05) between either diet treatment or sex during the growing phase. Differences in change of height during the training phase were higher (P<.05) for colts as compared with fillies. These data also tended to favor the HC/LBC treatment over the controls; however, the differences were not significant. The overall average daily height change data (495-d) showed an effect (P<.05) in favor of colts over fillies. The data also tended to favor the HC/LBC treatment; however, the apparent differences were not significant. No significant differences were found in values for average daily change in height at the knee either in the periodic or cumulative 495-d data. It was interesting to note that during the growing phase the data tended to favor the control animals, while in the training phase the data appeared to favor the HC/LBC treatment. As indicated above, the overall means for average daily change in height at the knee were

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BIOFLAVONOIDS FOR GROWING HORSES very similar and differences between treatments were not significant. One of the objectives of the study was to evaluate the effect of HC/LBC when fed to young horses that are subjected to the stresses of rapid growth shortly after weaning. It was not possible to accomplish this in the study due to the fact that sickness (especially respiratory infections) was essentially nil in the entire group of horses. A few animals were treated for the normal types of injuries that usually occur in groups of growing horses; however, no health problems that could be related to diet treatment were noted throughout the trial. A summary of the average radiograph data is shown in table 4. Radiographs taken of the left knee initially and at approximately 120-d intervals were scored as per the system normally used at California racetracks to determine the physiological maturity of horses with respect to bone development. During the first 365 d, no significant differences were noted between treatments. During this time, all groups had "C" or open, immature knees. The last set of radiographs taken 501 d after the start of the trial showed higher (P<.05), or more mature scores for fillies as compared with colts; however, no significant differences due to diet treatment were observed. The average blood data values are summarized in table 5. A complete panel of tests were performed at each major sampling period. Because differences between treatments were minimal, it was decided that items to be statistically analyzed would be limited to RBC, Hb, Hct, SGOT, Alk. Phos., LDH and ESR. At the start of the trial, no significant differences in RBC counts were noted between

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treatments. At the end of the growing phase, RBC values for fillies were higher (P<.05) than for colts; however, differences due to diet treatment were not significant. Blood samples taken at the end of the training phase showed fillies to have higher (P<.01) RBC values than colts, and the HC/LBC group to be higher (P<.05) than that of the control animals. Duncan's new multiple range test was used to further test differences among means. This test showed RBC values for treated fillies to be higher (P<.05) than values for control colts; however, differences between treated and control fillies or treated colts were not significant, nor were differences between control or treated colts. Hemoglobin values for the respective groups were not different (P>.05) at the start of the trial or at the end of the growing phase. At the end of the training phase, Hb values for the HC/LBC fillies was higher (P<.05) than that for control colts; no other significant differences were noted between treatments. Differences in Hct values for the various treatment groups were not significant at either the start of the trial or at the end of the growing phase. At the end of the trial, Her values for HC/LBC fillies were higher (P<.05) than that for control or treated colts, but not higher than for control fillies. Serum glutamic oxaloacetic transaminase values for the respective groups were not significantly different at the initial or final sampling times. At the end of the growing phase, SGOT values for colts were higher (P<.01) than that for fillies. The SGOT values for HC/LBC colts were also higher (P<.05) than that for control colts and both colt groups were

TABLE 4. SUMMARYOF AVERAGE RADIOGRAPII DATA OF EPIPHYSEAL PLATE FOR THE GROWINGAND TRAINING PHASESa Control Day 0 109 229
365

HC/LBC Fillies 1.0 1.0 1.0 1.0 2.2c Colts 1.0 1.0 1.0 1.0 1.3b Fillies 1.0 1.0 1.0
1.0

Colts 1.0 1.0 1.0 1.0 1.5b

501

2.3 c

aEpiphyseal plate (left knee) scores: 3 = A (closed, mature), 2 = B (partially closed, 1 = C (open, immature). b'CMeanswithin rows with different superscripts differ (P<.05).

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WOODEN ET AL. TABLE 5. SUMMARY OF AVERAGE BLOOD DATA Control HC/LBC Fillies 6 10.90 11.10 b 9.92 ab 13.4 14.9 14.2 ab
38.0

Item No. of animals Red blood cells (xlO 6 ) Initial End growing phase End training phase Hemoglobin, g/liter Initial End growing phase End training phase Hernatocrit, % Initial End growing phase End training phase

Colts 6 10.53 9.73 a 8.42 a 13.3 13.9 13.3 a

38.2

Colts 6 11.03 10.38 a 9.43 ab 13.4 14.4 14.6 ab

38.3

Fillies 6 11.16 11.09 b 11.03 b 13.7 15.9 16.1 b 40.0 43.4 45.3 b 188 344 d 290 504 383 207 340 338 392 b n/t 9.88 9.87 n/t 9.06 b 9.04

38.7 36.5 a

41.7 39.8 ab 226

315 cd

41.2 38.4 a 221 423 b 343 444 351 192 316 337 332 a n/t 9.27
9.69

Serum glutamic oxaloacetic transaminase, mU/ml Initial 175 End growing phase 380 a End training phase 314 Alkaline phosphatase, mU/ml Initial End growing phase End training phase 501 326 198

341 454 346 212 326 297 378 ab n/t 9.81

9.86

Lactic acid dehydrogenase, IU/liter Initial 338 End growing phase 318 End training phase 339 ab Erythrocyte sed. rate (10 rain), mm/h Initial n/t e End growing phase 9.02 End training phase 9.55 Erthrocyte sed. rate (20 min), mm/h Initial n/t e End growing phase 6.98 a End training phase 8.43

n/t 8.8 b 8.57

n/t 6.97 a 8.75

a'b'C'dMeans within rows with different superscripts differ (P~.05). eNot tested at start of trial.

h i g h e r ( P < . 0 5 ) t h a n c o n t r o l o r t r e a t e d fillies. D i f f e r e n c e s b e t w e e n c o n t r o l a n d test fillies were n o t significant. No s i g n i f i c a n t d i f f e r e n c e s were n o t e d in average s e r u m Alk. Phos. values. T h e overall m e a n value at t h e s t a r t o f t h e trial was 4 7 6 mU/ml. At the end of the growing phase the m e a n was 351 m U / m l a n d at t h e e n d o f t h e s t u d y , t h e overall m e a n h a d d r o p p e d t o 203 m U / m l . T h e s e values are w i t h i n n o r m a l ranges, a n d t h e d e c r e a s i n g values are in line w i t h t h e e x p e c t e d d e c r e a s e in Alk. Phos. a c t i v i t y w i t h increasing age.

Lactic acid d e h y d r o g e n a s e values for t h e respective t r e a t m e n t g r o u p s were n o t d i f f e r e n t ( P > . 0 5 ) at e i t h e r t h e start o f t h e trial or at t h e e n d o f t h e g r o w i n g phase. A t t h e e n d o f t h e t r a i n i n g p h a s e L D H values f o r t h e test fillies was h i g h e r ( P < . 0 5 ) t h a n for t h e test colts. D i f f e r e n c e s b e t w e e n H C / L B C fillies and t h e r e m a i n i n g g r o u p s w e r e n o t significant, n o r were d i f f e r e n c e s b e t w e e n c o n t r o l colts a n d fillies, or H C / L B C colts. Measurement of erthrocyte sedimentation r a t e was n o t m a d e at t h e s t a r t o f t h e trial. A f t e r t h e trial h a d b e e n s t a r t e d it was decided t h a t

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BIOFLAVONOIDS FOR GROWING HoRSEs

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this test be added to the items in the blood study, therefore the data for ESR readings are shown in table 5 for the end of the growing phase and the end of the training phase. The ESR data for the 10 rain reading for both the end of the growing and the end of the training phases tended to be somewhat higher for fillies as compared with colts; however, these apparent differences were not significant. At the end of the growing phase, data for the ESR reading at 20 min was higher (P<.05) for fillies as compared with colts. No significant differences were noted between diet treatments. The average data for the end of the training phase also tended to be higher for fillies as compared with colts, but these differences were not significant. Samples of skin and subcutaneous tissue were collected for histological examination, with particular emphasis on capillary wall structures. The number of observations were limited, therefore, no statistical analysis was conducted; however, results of the exzmination indicated no apparent histopathologic changes in any of the tissues examined. During the growing phase, the data presented above tended to favor average daily gain of animals that received HC/LBC; however, differences between treatments during the training phase and in the cumulative data were not significant. The remaining growth variables were in certain cases affected by sex (colts vs fillies) but not by diet treatment (HC/LBC). The variables included in the blood data also showed a few significant differences between treatment groups, but no strong trends related

to diet treatment were observed. The histological examination of skin and subcutaneous tissue also showed no apparent effect related to diet treatment. These observations indicate that under the conditions of this trial, HC/LBC had no major effect on the growth rate of horses. It should be pointed out that problems related to animal health in general were quite minimal, therefore, the stress factor related to health was minimized. This would make it more difficult to determine any positive effect that HC/LBC may have in the face of high stress conditions. It is planned to monitor, as closely as is practically possible, the racing performance of animals involved in this study for a period of 2 yr. It will be of definite interest to note any possible relationship between diet treatment and later performance at the racetrack.

Literature Cited

Bauer, W. F. 1978. Monograph on bioflavonoids. Informatics, Inc., Rockville, MD. Brunet, R. C. and G. H. Palmer. 1961. Citrus bioflavonoids in health and disease. Review. Sunkist Growers, Ontario, CA. Fostvedt, G. A. 1956. The clinical use of citrus bioflavonoids (A documented survey). Nutr. Res. 12:1. Horvath, A. A. 1974. Laminitis Therapy (letter). J. Amer. Vet. Med. Assoc. 165:776. Palmer, G. H. 1954. Bibliography of citrus bioflavoholds. Sunkist Growers, Ontario, CA. Snedecor, G. W. and W. G. Cochran. 1967. Statistical Methods (6th Ed.). Iowa State Univ. Press, Ames. Steel, R.G.D. and J. H. Torrie. 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York.

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