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J. Anim. Sci. 2005. 83:1267-1273
© 2005 American Society of Animal Science


ANIMAL NUTRITION

Effects of feeding a blend of grains naturally contaminated with Fusarium mycotoxins on feed intake, metabolism, and indices of athletic performance of exercised horses1

S. L. Raymond*, T. K. Smith{dagger},2 and H. V. L. N. Swamy{dagger},3

* Equine Guelph and and {dagger} Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, N1G 2W1, Canada


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
An experiment was conducted to determine the effect of feeding blends of grains naturally contaminated with Fusarium mycotoxins to mature, exercised horses, and to test the efficacy of a polymeric glucomannan mycotoxin adsorbent (GM polymer) in preventing Fusarium mycotoxicoses. Six mature, mixed-breed mares with an average BW of 530 kg were assigned to one of three dietary treatments for 21 d in a replicated 3 x 3 Latin square design. Feed consumed each day was a combination of up to 3.5 kg of concentrates and 5.0 kg of mixed timothy/alfalfa hay (as-fed basis). The concentrates fed included 1) manage; 2) blend of contaminated grains; and 3) contaminated grains + 0.2% GM polymer (MTB-100, Alltech Inc., Nicholasville, KY). Concentrates containing contaminated grains averaged 11.0 ppm deoxynivalenol, 0.7 ppm 15-acetyldeoxynivalenol, and 0.8 ppm zearalenone (as-fed basis). Feed intake and BW were monitored over a 21-d period. Horses were maintained on a fixed exercise schedule throughout the experiment. At the end of the experiment, each horse completed a time-to-fatigue treadmill step test. Variables measured during pretest, each step of the test, and 5 and 10 min posttest were as follows: 1) time-to-fatigue, 2) heart rate, 3) hematological variables, and 4) serum lactate concentration. Each step consisted of 2 min of fast trot with a 2% increase in incline after each 2 min. Feed intake by horses fed contaminated grains was decreased compared with controls throughout the experiment (P < 0.05). Supplementation of 0.2% GM polymer to the contaminated diet did not alter feed intake by horses compared with those fed the unsupplemented contaminated diet. All hay was consumed regardless of concentrate fed. Weight loss from 0 to 21 d was observed in horses fed contaminated grains compared with controls (P < 0.05). No effect of diet was seen on variables used to measure athletic ability, although the results showed an expected response to exercise for a fit horse. We conclude that exercised horses are susceptible to Fusarium mycotoxicoses as indicated by appetite suppression and weight loss.

Key Words: Deoxynivalenol • Fusaric Acid • Fusarium • Horses • Mycotoxins


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Mycotoxins are harmful secondary metabolites of fungi that can reduce performance of livestock (Smith, 1992Go). The occurrence of mold and mycotoxins in food and animal feed is a major problem globally. Fusarium fungi are commonly found in temperate climates, and Fusarium mycotoxins are likely the most prevalent on a global basis (Wood, 1992Go).

The feeding of blends of grains naturally contaminated with Fusarium mycotoxins has been shown to decrease livestock and poultry performance, leading to decreased feed intake and weight gain, and adverse metabolic, hematological, and neurochemical changes (Swamy et al., 2002Go; 2004aGo,bGo). Very little information is available, however, on the toxicity associated with the feeding of Fusarium mycotoxin-contaminated grains to horses. Johnson et al. (1997)Go found no effect on feed intake, serum chemistry, or hematology when horses were fed barley naturally contaminated with 36 to 44 ppm deoxynivalenol (DON). This contrasts with more recent findings, which described decreased feed intake in unexercised horses fed a combination of Fusarium mycotoxins (Raymond et al., 2003Go). Athletic ability is a major performance variable for the horse. Regular heavy exercise places metabolic stresses on the animal. A major concern, especially for elite athletes, is that the chronic feeding of low levels of mycotoxins may affect performance without the appearance of overt clinical symptoms.

Our objectives were to determine the effects of feeding grains naturally contaminated with Fusarium mycotoxins on feed intake, weight maintenance, serum chemistry, hematology, and athletic performance of exercised horses, and the efficacy of an organic, polymeric glucomannan (GM) mycotoxin adsorbent in the prevention of Fusarium mycotoxicoses. The GM polymer has been shown to prevent many adverse effects of grain naturally contaminated with Fusarium mycotoxins in horses (Raymond et al., 2003Go), pigs (Swamy et al., 2002Go), broilers (Swamy et al., 2004bGo), and layers (Chowdhury and Smith, 2004Go).


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Experimental Animals and Diets

Mature, mixed-breed mares with an average weight of 530 kg (n = 6) were assigned randomly to one of three dietary treatments for 21 d following a replicated 3 x 3 Latin square design with a 14-d treatment recovery interval, during which time the control diet was fed. Feed consumed each day was a combination of up to 3.5 kg of concentrates and 5.0 kg of 50:50 timothy/alfalfa hay (as-fed basis) formulated to meet the nutritional requirements of a mature, medium-working horse (NRC, 1989Go; Table 1Go). The concentrates fed included 1) a control; 2) a blend of contaminated grains; and 3) a blend of contaminated grains + 0.2% GM polymer (MTB-100, Alltech Inc., Nicholasville, KY). Water was provided on an ad libitum basis. The blend of contaminated grains consisted of (as-fed basis) 53.6% contaminated corn and 35.8% contaminated wheat.


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Table 1. Composition of diets, as-fed basis
 
The horses were housed at the University of Guelph (Guelph, Ontario, Canada) in individual 3.6-m2 box-stalls, with 8 h of group turnout on paddocks with minimal pasture a day. The paddocks contained minimal grass growth due to both overgrazing and the time of year that the study was conducted. Horses were examined daily for any adverse clinical signs.

The horses were fed concentrates twice a day (0800 and 1600). Any uneaten portion from the 0800 feeding was added to the 1600 feeding. One hour was allotted to the 0800 feeding before the uneaten portion was removed. The concentrates fed at 1600 remained in the stall until 0800 and were then removed and weighed. The quantity of concentrate consumed by each horse was recorded once per day. The horses were weighed weekly at 1500 during the trial (7, 14, and 21 d).

Horses were initially conditioned for a 3-wk period before the first supplementation phase. Horses were maintained on a fixed exercise schedule during the supplementation phase. This consisted of three 45-min workouts, including 25 min of fast trot (18 km/h) each week using an exerciser (Odyssey Performance Trainer, System Fencing, Guelph, Ontario, Canada). Each horse was contained in an individual gated area. Horses were encouraged to remain at the required pace using the gate located at the rear of the animal. At the end of the supplementation phase, each horse completed a time-to-fatigue treadmill step test. Horses were maintained on a reduced exercise schedule using an exerciser during each treatment recovery period. The reduced exercise schedule consisted of one 30-min workout of walking during the first week, followed by one 30-min workout, including 10 min of fast trot during the second week.

Animal Care

The experiment was reviewed and approved by the University of Guelph Animal Care Committee. Animals were managed and cared for according to the guidelines of the Canadian Council on Animal Care.

Analysis of Dietary Mycotoxins

Diets and hay were analyzed for DON, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, nivalenol, T-2 toxin, iso T-2 toxin, acetyl-T-2 toxin, HT-2 toxin, T-2 triol, T-2 tetraol, fusarenon-X, diacetoxyscirpenol, scirpentriol, 15-acetoxyscirpentriol, neosolaniol, zearalenone, zearalenol, and fumonisin using a combination of gas chromatography and mass spectrometry (Shimadzu QP 5050, Tokyo, Japan; North Dakota State Univ., Fargo) as described by Raymond et al. (2003)Go. The limit of detection for fumonison B1 was 2.0 ppm and 0.2 ppm for all other mycotoxins. Fusaric acid (FA) content (0.77 ppm limit of detection) was determined by the method of Matsui and Watanabe (1988)Go, as modified by Smith and Sousadias (1993)Go and confirmed by Porter et al. (1995)Go.

Experimental Variables Studied

Hematology and Serum Biochemical Analyses. Blood samples were drawn by jugular venipuncture (1500, 7, 14 and 21 d). Samples were drawn into a 7-mL sterile Vacutainer containing EDTA with an 18-gauge needle. Red blood cell count (RBC; 0.0 to 7.0 109/L limit of detection), mean corpuscular volume (MCV; 76 to 100 fL expected value), and hematocrit (33 to 57% expected value) were determined, and mean corpuscular hemoglobin (MCH; 24 to 31 pg expected value) and mean corpuscular hemoglobin concentrations (MCHC; 28 to 34 g/L expected value) were calculated. Hemoglobin (0.0 to 225 g/L limit of detection) was measured as cyanomethemoglobin after lysing the red blood cells using an Advia 120 hematology system (Bayer Inc., Healthcare Division, Toronto, Ontario, Canada). Complete bloodcell counts (differential leukocyte count) with limits of detection were performed manually to test for changes in absolute numbers of leukocytes (WBC; 0.02 to 400 x 109/L), lymphocytes (16 to 44%), segmented neutrophils (40 to 77%), banded neutrophils (40 to 77%), monocytes (4 to 9%), eosinophils (1 to 7%), and basophils (0 to 1%).

Blood samples were drawn into a 7-mL sterile silicone-coated evacuated tube. Serum concentrations and limits of detection of total protein (0 to 150 g/L), albumin (0 to 80 g/L), globulin (0 to 80 g/L), glucose (0 to 25 mmol/L), ß-hydroxybutyrate (0 to 3,200 µmol/L), haptoglobin (0 to 100 g/L), urea (0 to 50 µmol/L), cholesterol (0 to 20 mmol/L), creatinine (0 to 1770 µmol/L), bilirubin (0 to 513 µmol/L), Ca (0 to 5 mmol/L), P (0 to 6.46 mmol/L), Mg (0 to 2 mmol/L), Na (0 to 300 mmol/L), K (0 to 200 mmol/L), Cl (0 to 300 mmol/L), and activities of alkaline phosphatase (AP; 0 to 500 U/L), glutamate dehydrogenase (GLDH; 0 to 80 U/L), aspartate aminotransferase (AST; 0 to 800 U/L), gamma glutamyltransferase (GGT; 0 to 1,200 U/L), and creatine kinase (CK; 0 to 2,300 U/L) (Roche Diagnostics, Hoffman-La Roche Ltd., Montreal, Quebec, Canada) were determined using a Hitachi 911 autoanalyzer (Tokyo, Japan).

Time-to-Fatigue Treadmill Step Test. At the end of the supplementation phase, each horse completed a time-to-fatigue step test on a high-speed treadmill. The step test consisted of a 5 min warm up at a walk, followed by 2 min of slow trot (approximately 7 km/h), 2 min of fast trot (approximately 15 km/h), and then consecutive steps. Each step consisted of 2 min of fast trot with a 2% increase in incline after each 2 min. Time to fatigue was determined by the handler as the time when the horse was unwilling to maintain position on the treadmill for the second time. The test was terminated at this time and concluded with 10 min of cool down at a walk. Variables measured during pretest, each step of the test, and 5 and 10 min posttest were time to fatigue, heart rate, hematology, and serum lactate concentrations. Heart rate was recorded with a digital monitor (Polar heart rate monitor, Equine Performance Group, Guelph, Ontario, Canada). A 14-gauge, 150-cm catheter was inserted into the left jugular vein before the time-to-fatigue treadmill step test. Once the catheter was inserted, it was secured with sutures and a 5-mL extension set was attached to facilitate blood sampling during exercise. The catheter was kept patent with heparinized saline solution (10 U/mL). The catheter remained in place for approximately 2 h.

Statistical Analyses. Experimental animals were assigned to different treatments in a Latin square design. Data were analyzed by ANOVA using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC). The statistical model included diet, horse, and period. The effect of feeding Fusarium mycotoxin-contaminated diets was determined by contrasting the horses fed the control diet with those fed the mycotoxin-contaminated diet. The ability of the GM polymer to prevent Fusarium mycotoxin-induced effects was tested by a simple contrast between the horses fed the mycotoxin-contaminated diet with and without 0.2% GM polymer. Differences were considered significant at P < 0.05. Feed intake over a period of 1 wk for each treatment was added, and statistical analyses were made on this total amount per week. For cumulative feed intake, feed intake over the 21-d period was considered.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Dietary Mycotoxin Concentrations

The analyzed concentrations of mycotoxin in the concentrates and hay are given in Table 2Go. Deoxynivalenol was found in all concentrates, whereas zearalenone and 15-acetyl DON also were detected in the concentrate containing contaminated grains. Only FA was detected in the hay.


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Table 2. Mycotoxin content of concentrates and hay, as-fed basis
 
Feed Intake and Changes in Body Weight

Feed intake decreased in horses fed contaminated grain with or without 0.2% GM polymer compared with those fed the control diet throughout the study (P < 0.05; Table 3Go). All hay offered was consumed regardless of diet fed (data not shown). One horse was removed from the trial after one treatment period during the 14-d recovery period due to colic symptoms determined to be unrelated to the trial.


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Table 3. Feed intake (kg/horse) by horses fed blended grain contaminated with Fusarium mycotoxins on feed intake, as-fed basisa
 
Weight loss over 0 to 21 d was observed in horses fed contaminated grains compared with controls (P < 0.05; Table 4Go). All animals lost weight over 0 to 7 d and 0 to 14 d, whereas they gained weight over 7 to 14 d.


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Table 4. Changes in body weight of horses fed blended grain contaminated with Fusarium mycotoxinsa
 
Hematology and Serum Chemistry

Hematology and serum chemistry variables (7, 14, and 21 d) were not significantly affected by the dietary treatments (data not shown).

Time-to-Fatigue Treadmill Step Test

No variables were affected by diet. The mean resting heart rates for control, mycotoxin, and 0.2% GM horses were 40, 39, and 35 beats/min, respectively (SEM = 1.39). The starting heart rates for control, mycotoxin, and 0.2% GM horses were 125, 135, and 140 beats/min, respectively (SEM = 4.33). The time of fatigue heart rates for control, mycotoxin, and 0.2% GM horses were 173, 175, and 155 beats/min, respectively (SEM = 4.80). The 5-min recovery heart rates for control, mycotoxin, and 0.2% GM horses were 87, 82, and 79 beats/min, respectively (SEM = 2.68). The 10-min recovery heart rates for control, mycotoxin, and 0.2% GM horses were 71, 75, and 72 beats/min, respectively (SEM = 1.55). The times to fatigue for control, mycotoxin, and 0.2% GM horses were 12.32, 11.14, and 9.94 min, respectively (SEM = 0.64).

The following blood variables were measured during the time-to-fatigue treadmill step test. The WBC measured at rest for control, mycotoxin, and 0.2% GM horses were 8.32, 8.06, and 8.83 x 109/L, respectively (SEM = 0.34). The WBC measured at time of fatigue for control, mycotoxin, and 0.2% GM horses were 11.56, 11.56, and 11.88 x 109/L, respectively (SEM = 0.38). The WBC measured at 10-min recovery for control, mycotoxin, and 0.2% GM horses were 10.40, 10.56, and 11.18 x 109/L, respectively (SEM = 0.18). The LC measured at rest for control, mycotoxin, and 0.2% GM horses were 3.66, 2.88, and 2.70 x 109/L, respectively (SEM = 0.20). The LC measured at time of fatigue for control, mycotoxin, and 0.2% GM horses were 4.77, 4.42, and 4.31 x 109/L, respectively (SEM = 0.33). The LC measured at 10-min recovery for control, mycotoxin, and 0.2% GM horses were 4.76, 4.02, and 4.13 x 109/L, respectively (SEM = 0.17). The RBC measured at rest for control, mycotoxin, and 0.2% GM horses were 8.40, 7.84, and 8.08 x 109/L, respectively (SEM = 0.11). The RBC measured at time of fatigue for control, mycotoxin, and 0.2% GM horses were 11.41, 11.02, and 10.77 x 109/L, respectively (SEM = 0.08). The RBC measured at 10-min recovery for control, mycotoxin, and 0.2% GM horses were 9.66, 9.08, and 9.13 x 109/L, respectively (SEM = 0.09). The HB measured at rest for control, mycotoxin, and 0.2% GM horses were 142.2, 133.6, and 138.2 g/L, respectively (SEM = 1.90). The HB measured at time of fatigue for control, mycotoxin, and 0.2% GM horses were 195.6, 188.4, and 185.5 g/L, respectively (SEM = 1.64). The HB measured at 10-min recovery for control, mycotoxin, and 0.2% GM horses were 164.4, 156.2, and 158.0 g/L, respectively (SEM = 1.49). The HCT measured at rest for control, mycotoxin, and 0.2% GM horses were 37, 35, and 37%, respectively (SEM = 1%). The HCT measured at time of fatigue for control, mycotoxin, and 0.2% GM horses were 52, 49, and 48%, respectively (SEM = 1%). The HCT measured at 10-min recovery for control, mycotoxin, and 0.2% GM horses were 43, 40, and 40%, respectively (SEM = 0%).

The mean resting lactate concentrations for control, mycotoxin, and 0.2% GM horses were 1.16, 1.18, and 1.15 mmol/L, respectively (SEM = 0.05). The time of fatigue lactate concentrations for control, mycotoxin, and 0.2% GM horses were 3.78, 3.74, and 3.15 mmol/L, respectively (SEM = 0.08). The 5-min recovery lactate concentrations for control, mycotoxin, and 0.2% GM horses were 2.32, 2.10, and 1.97 mmol/L, respectively (SEM = 0.08). The 10-min recovery lactate concentrations for control, mycotoxin, and 0.2% GM horses were 2.04, 1.64, and 1.63 mmol/L, respectively (SEM = 0.05). The peak lactate concentrations for control, mycotoxin, and 0.2% GM horses were 3.78, 3.74, and 3.25 mmol/L, respectively (SEM = 0.08). The times to peak lactate for control, mycotoxin, and 0.2% GM horses were 12.35, 11.70, and 9.72 min, respectively (SEM = 0.66).


    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Dietary Mycotoxin Concentrations

Failure to achieve similar concentrations of DON (11.2 and 14.5 ppm) in the two contaminated diets might have been due to uneven distribution of mycotoxins in the grain and the limitations of mixing (Davis et al., 1980Go). Infestation of grains by Fusarium molds depends on appropriate microbial conditions for growth and can occur sporadically.

The presence of FA in the hay needs to be addressed in the context of possible synergistic interaction between FA and DON. Smith et al. (1997)Go reported a synergistic interaction between DON and FA on weight gains by pigs. Fusaric acid has a very low acute toxicity compared with trichothecene mycotoxins (Hidaka et al., 1969Go). Fusaric acid (Smith and MacDonald, 1991Go) and DON (Prelusky, 1993Go) have been shown to elevate pig brain concentrations of serotonin, albeit through different mechanisms, which can lead to lethargy, loss of appetite, and muscle incoordination. The absence of FA from the control and contaminated diets and its presence in the hay is an illustration of the variations in amounts of metabolites produced by different Fusarium strains (Bacon et al., 1996Go).

The dietary zearalenone content in the current experiment should not have caused metabolic effects in the horses tested. The levels found in the current study are similar to those found previously (Raymond et al., 2003Go). The minimum concentration of zearalenone in contaminated diets required to produce hyperestrogenism in swine according to literature reports is 1 ppm (James and Smith, 1982Go). A field outbreak of zearalenone toxicosis in horses was associated with the consumption of corn screenings containing approximately 2.6 ppm of zearalonene (Gimeno et al., 1983Go). The deoxynivalenol and FA contents were not reported. Fusarium graminearum fungi can produce deoxynivalenol and zearalenone simultaneously in infected corn and wheat (Cote et al., 1985Go). Toxicological synergism between DON and zearalenone has not been observed in swine (Cote et al., 1985Go) or mice (Forsell et al., 1986Go).

Feed Intake and Changes in Body Weight

Results of the current trial suggest a relatively high degree of decreased feed intake when horses are fed concentrates containing a blend of grains naturally contaminated with high levels of Fusarium mycotoxins. These results are in agreement with previous work (Raymond et al., 2003Go) but are in contrast to those reported by Johnson (1997)Go, who found no effect on feed intake when feeding barley naturally contaminated with 36 to 44 ppm DON. This may be attributable to the synergistic effect of feeding a combination of mycotoxins found in blends of contaminated grains, as has been observed for FA and DON in starter pigs (Smith et al., 1997Go). Johnson et al. (1997)Go fed only one source of contaminated grain compared with the current study in which a blend of naturally contaminated grains was used. Horses exhibited a lower degree of decreased feed intake (35%) of contaminated grains during the current trial compared with previous work (65%) (Raymond et al., 2003Go). This may be attributable to the increase in energy requirements due to exercise. The inability to maintain BW in horses fed contaminated grains is in contrast to previous work (Raymond et al., 2003Go), despite the consumption of greater amounts of contaminated grains. Weight loss exhibited in exercising horses when fed contaminated diets may affect athletic ability, but this was not evaluated at the beginning of this study and this conclusion cannot be made. Horses in the previous study were not maintained on an exercise schedule.

Time-to-Fatigue Treadmill Step Test

No effect of diet was seen on athletic ability. If the trial were longer or the exercise test more extreme, an effect might have been seen. As expected with exercise, there was an increase in serum lactate and heart rate followed by an appropriate decrease after recovery. This agrees with the findings of Sobotta et al. (2001)Go and Lindner et al. (2001)Go. In the current study, heart rate was reaching 180 beats/min at time of fatigue, indicating the approach of the threshold from aerobic to anerobic metabolism. Serum lactate concentrations increased during exercise and decreased during recovery, indicating the rapid removal of lactate by the recovering muscles and restoration of plasma volume. The results showed an expected response to exercise for a fit horse.

Hematology and Serum Chemistry

Serum activities of the hepatic membrane-associated enzyme, gamma-glutamyl transferase, were not different in horses fed the contaminated diet compared with the control diet. These findings are in contrast to earlier findings, where higher levels of these enzymes were found in horses fed contaminated grains when compared to controls (Raymond et al., 2003Go). Such differences may be due to an increase in liver function found in exercising horses compared with nonexercising animals, as exhibited by increased clearance rates of xenobiotics, specifically antipyrine as a means to measure changes in hepatic drug metabolism (Dyke et al., 1998Go). The feeding of similarly contaminated grains to pigs resulted in a reduction in liver and kidney weights (Swamy et al., 2002Go).

Effect of GM Polymer Supplementation

The use of adsorbents, such as activated charcoal, silicates, bentonites, clays, and zeolites, in preventing mycotoxicosis has been extensively studied in livestock exposed to dietary mycotoxins (Ramos et al., 1996Go). The feeding of GM polymer in previous studies resulted in increased consumption of Fusarium-contaminated equine concentrates (Raymond et al., 2003Go). This effect was not observed in the current study. The increase in dietary energy requirements due to the inclusion of the exercise regimen resulted in a lesser degree of feed refusal than was noted previously, which decreased the potential for the GM polymer to significantly increase concentrate intake.


    Footnotes
 
1 This study was supported in part by the Ontario Horse Racing Industry Association, Ontario Ministry of Agriculture and Food, and Alltech Inc., Nicholasville, KY. Back

3 Current address: Alltech Biotechnology Private Ltd., Bangalore, Karnataka 560 038, India. Back

2 Correspondence—phone: 519-824-4120, ext. 53746; fax: 519-822-7897; e-mail: tsmith{at}uoguelph.ca.

Received for publication August 24, 2004. Accepted for publication February 18, 2005.


    Literature Cited
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 


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