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Effects of Feeding Deoxynivalenol (DON, Vomitoxin)-Contaminated Wheat to Female White Leghorn Chickens from Day Old Through Egg Production1
Effects of Feeding Deoxynivalenol (DON, Vomitoxin)-Contaminated Wheat to Female White Leghorn Chickens from Day Old Through Egg Production 1 L. F. KUBENA, R. B. HARVEY, D. E. CORRIER, and W. E. HUFF US Department of Agriculture, Agricultural Research Service, Veterinary Toxicology and Entomology Research Laboratory, College Station, Texas 77841 T. D. PHILLIPS Department of Veterinary Public Health, Texas A&M University, College Station, Texas 77843
ABSTRACT White Leghorn chickens one day of age were fed starter and grower diets containing either a control (noncontaminated) wheat diet or a naturally contaminated deoxynivalenol (DON) wheat diet (18 mg DON/kg) from 1 day of age to the onset of egg production. The hens were then placed on their respective layer diets of control wheat or DON-contaminated wheat (18 mg DON/kg) for six 28-day egg production periods. Feeding the DON-contaminated diet did not significantly influence body weights during the growing or the laying phases. Overall, hen-day egg production and egg weights were significantly higher for hens receiving the DON diet. Feeding DON contaminated wheat caused no significant changes in percent shell, albumen height, percent fertility, percent hatch of fertile eggs, percent hatch of eggs set, or weight of chicks at hatch. There were slight, although significant, changes in shell weight and shell thickness and in some serum chemistry values. There were no significant differences in the hematology parameters measured or in prothrombin times. None of the eggs collected from hens fed the control and the DON-contaminated wheat diet contained detectable quantities of DON. Microscopic examination of sections of the liver, kidney, and proventriculus of control and treated hens revealed no unusual histopathology. The results indicate that feeding DON at relatively high levels beginning at 1 day of age and continuing through six egg production periods had only slight effects on the parameters measured. (Key words: deoxynivalenol, wheat diet, growth, laying hens, egg production, fertility, hatchability) 1987 Poultry Science 66:1612-1618 INTRODUCTION
There is considerable evidence on the natural occurrence of deoxynivalenol [3a,7a, 15trihydroxy-12, 13-epoxytrichothec-9-en-8-one; (DON, vomitoxin)] in grains in many areas of the world (Vesonder et al., 1978; Mirocha et al., 1979; Pathre and Mirocha, 1979; Trenholm et al., 1983; Cote et al., 1984; Hagler et al, 1984). Deoxynivalenol is a mold metabolite which causes feed refusal and emesis in swine (Vesonder et al., 1973, 1976). Forsyth et al. (1977) demonstrated a dose-response relationship in swine between the level of DON administered either orally or intraperitoneally and feed refusal and emesis.
'Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.
Reports in the literature on the effects of DON in growing chicks and mature chickens are limited. Huff et al. (1981) observed no mortality in day-old broiler chicks until oral doses reached 70 mg/kg of body weight and found the approximate oral LD50 dose to be 140 mg/kg, suggesting a relatively low acute toxicity. Hulan and Proudfoot (1982) found no significant effects on body weight gains from feeding naturally contaminated wheat-based diets containing as much as 1.87 mg DON/kg to broiler cockerels for 28 days. Moran et al. (1982) fed broiler cockerels from 6 to 11 days of age diets containing 1.7 to 210.0 mg DON/kg from corn purposely inoculated with Fusarium graminearum. These workers found growth and feed consumption were not reduced until feeding exceeded 116 mg DON/kg of diet. Hamilton et al. (1983, 1985b) and Trenholm et al. (1984) reported no detrimental effects in Leghorn and broiler chicks fed naturally contaminated wheat diets containing as much as 4.9 mg DON/kg. Kubena et al.
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(Received for publication September 22, 1986)
LONG-TERM FEEDING OF DEOXYNIVALENOL
on the effects of DON on reproductive performance of laying hens include short-term studies at high DON levels (50 mg/kg), longer term studies at low DON levels (<5 mg/kg), and long-term exposure at relatively high levels (18 mg/kg) that might be found in naturally contaminated feeds. The purpose of the research reported here was to describe the major effects of a relatively high level of DON (18 mg/kg) fed from 1 day of age through several egg production periods. MATERIALS AND METHODS
Sixty female one-day-old white Leghorn chicks were individually weighed; sequentially numbered aluminum wing bands were randomly assigned and affixed to the wing web. Thirty chicks were placed into each of two groups (two replicates of 15 chicks per treatment) according to the wingband number sequence and placed in electrically heated battery brooders. One group received a control (noncontaminated) wheat starter-grower diet and the other group received a naturally contaminated wheat startergrower diet. Diets contained or exceeded the levels of critical nutrients recommended by the National Research Council (1977) and consisted of 68.5% wheat, 28.5% soybean meal-based concentrate (including vitamins and minerals), and 3.0% corn oil. The contaminated and noncontaminated wheat were analyzed for DON by the procedure of Scott et al. (1981). The wheat was found to contain 26 mg DON/kg and no detectable levels of zearalenone, T-2 toxin, diacetoxyscirpenol, aflatoxin, or ochratoxin. Diets were calculated to contain 0 and 18 mg DON/kg and analyses of diets for DON periodically during the study were in good agreement with calculated values. The chickens received their respective starter-grower diets and water ad libitum from one day to 20 wk of age. At 4 wk of age, chickens were moved into growing batteries until 14 wk of age, at which time they were transferred into individual cages in laying batteries. At 18 wk of age, as no treatment differences in body weights were present, 24 chickens from each dietary treatment were selected for the production phase based on similar body weights (the three lightest and three heaviest chickens from each treatment were removed). Lighting was gradually increased until the chickens (hens) were receiving 16 h of light and was maintained at this level for the remainder of the study. At 20 wk of age, hens were
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(1985) found no detrimental effects on body weights or feed efficiency in male Leghorn chicks fed naturally contaminated wheat diets containing 9 or 18 mg DON/kg from 1 to 35 days of age. These workers did observe a slight, yet significant, transitory anemia due to DON intoxication. A response, thought to be caused by irritation by DON or by the difference in the density of the diets, was found in the upper gastrointestinal tract, and various physiological measurements were slightly altered by DON. Huff et al. (1986) observed reduced growth rate, decreased feed efficiency, increased relative gizzard weights, anemia, decreased lactic dehydrogenase activity, and decreased serum triglycerides in male broilers fed naturally contaminated wheat diets containing approximately 16 mg DON/kg. Moran et al. (1983) fed artificially contaminated corn-based diets containing 50 mg DON/ kg to mature laying hens for 4 consecutive weeks and found no change in the hatch of total eggs. Trenholm et al. (1981) fed naturally contaminated DON wheat diets to adult female Leghorn chickens for 10 wk and observed no marked changes in feed consumption, weight gain, or egg production. Hamilton et al. (1981a,b, 1983, 1985a) reported that naturally contaminated wheat diets containing .35 or .70 mg DON/kg and fed for 10 wk caused a significant decrease in egg and shell weight, shell thickness, and percentage shell, whereas other parameters measured were not affected. Hamilton et al. (1983) in additional studies fed naturally contaminated wheat diets containing as much as 4.9 mg DON/ kg for 24 wk and observed no significant effect on these parameters; however, trends were noted toward lower values with higher DON feeding levels. Kubena et al. (1987) fed naturally contaminated wheat diets containing 18 mg DON/kg to mature laying hens for 16 wk and found no significant effects on body weights, hen day egg production, egg weights, or efficiency of feed utilization. Egg shell weight and percent egg shell were slightly, although significantly, decreased by feeding the DON-contaminated wheat diet. Lun etal.{\986) fed diets containing 82.8 mg DON/kg for 27 days and observed no detrimental effects on the performance of mature laying hens. The literature indicates that poultry may be relatively insensitive to DON, thus raising the possibility that grains contaminated with DON may be incorporated into poultry diets without affecting performance. Reports in the literature
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KUBENA ET AL. RESULTS AND DISCUSSION
There were no significant differences in body weights from 1 day to 18 wk of age, with the exception of significantly higher body weights for the DON-treated chickens at 10 wk of age (data not shown). These results are in agreement with reports of Hulan and Proudfoot (1982), Moran et al. (1982), Hamilton et al. (1983, 1985b), and Kubena et al. (1985) who observed no detrimental effects on performance of growing chicks fed diets containing DON. The report of Huff et al. (1986) and additional results obtained recently in our laboratory (Kubena et al., 1986) show that the performance of rapidly growing broilers was significantly reduced when fed DON-contaminated wheat diets containing 16 mg DON/kg. In the present study, significant differences in body weights due to dietary treatment, were not observed at any of the time intervals between 24 and 48 wk of age. These results agree with Hamilton et al. (1985a), Moran et al. (1983), and Kubena et al. (1987) who also found no significant differences in body weights at these ages. Effects of feeding DON-contaminated wheat diets on egg weights, egg production, and feed consumption and efficiency are shown in Table 1. There were no differences in the average age of chickens at onset of production (data not shown) and there were no significant differences in egg weights or egg production during the 1st 28-day production period. A significant increase in egg weights occurred during the 2nd 28-day period and, with the exception of the 3rd 28-day period, continued throughout the experiment. Beginning with the 3rd 28-day period and continuing for the remainder of the experiment, percent hen day egg production was significantly higher for hens in the DON treatment group. Kubena et al. (1987) also observed increased egg production (nonsignificant) for the hens fed DON. As the wheat diets used in the study reported herein were formulated with DON-contaminated wheat from the same shipment, the increase in egg weights and egg production may be due to wheat source. Hens fed the DON-contaminated wheat diet consumed 130.4 g of feed/ hen per day, whereas hens fed the control diet consumed 119.0 g/hen per day. The efficiency of feed utilization as measured by kilograms of feed required to produce a kilogram of eggs was almost identical (2.80 for control and 2.78 for DON-fed hens).
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placed on a laying diet that consisted of 67.0% wheat (noncontaminated for control and naturally contaminated for second treatment), 22.0% soybean meal-based concentrate (including vitamins and minerals), 7.8% calcium carbonate, 1.2% phosphate, and 2.0% corn oil. Hens were provided their respective laying diets and water ad libitum from 20 wk of age to the end of the study. Chickens were individually weighed at 1 day of age and at 4, 10, 14, 18, 24, 32, 40, and 48 wk of age. All eggs were recorded, collected, and individually weighed on a daily basis during six 28-day experimental periods. Mortality was recorded as it occurred and feed consumption was measured for each 28-day period throughout the experiment. Shell weight, shell thickness, and albumen height were measured on ail eggs collected on 3 consecutive days each week for 2 wk and 2 consecutive days for 1 wk during the last two 28-day experimental periods. All hens were inseminated with pooled semen from control males and the eggs produced during a 3 to 4-day period each week were incubated once a week for 11 wk. Fertility, hatchability, and chick weights at hatching were measured. After six 28-day experimental periods, 20 hens from each treatment were bled by cardiac puncture for hematology and serum chemistry analyses as previously described by Kubena et al. (1985). Prothrombin times were determined by the method as described by Doerr et al. (1974) with the exception that a ML A Electra 600, P 4 with animal clot time option (Medical Laboratory, Automation, Inc., Mount Vernon, NY) was used. Samples of the liver, kidney, and proventriculus were taken from five randomly selected chickens per treatment for microscopic examination. Six eggs from each treatment group were randomly selected during the last week of the study and analyzed for DON content according to the procedure of El-Banna et al. (1983). Data for all response variables except feed consumption were analyzed using the general linear model analysis of variance procedure in the SAS software (SAS Institute Inc., 1982). Variable means for treatments showing significant differences in the analysis of variance were compared and differences were tested using Duncan's new multiple range procedure (Duncan, 1955). All statements of significance are based on the probability level of .05.
84.0 a 92.6 a
47.6 a 48.5 a
1
84.8 a 89.3 a
48.8D 50.3 a
2
85.9 b 92.4 a
51.5 a 52.8 a
3
84.3 b 89.6 a
52.9 b 54.9 a
4
81.8 b 86.4 a
54.0 b 56.0 a
5
1
DON = Deoxynivalenol.
a' b In the same vertical column within each time period and parameter, values with different superscripts are signific
DON
Feed efficiency, kg feed/kg eggs Control
DON
Feed consumed, g feed/hen/day Control
DON
Egg production, % Control
DON
Egg weights, g Control
Parameter and treatment
TABLE 1. Effects of feeding DON-contaminated wheat on production characteristics of fe in six 28-day periods1
from http://ps.oxfordjournals.org/ at University of North Dakota on May 26, 2015
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KUBENA ET AL.
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There was a significant increase in shell weight and shell thickness in the DON treatment group when compared with values for controls (Table 2). No significant differences were observed in albumen height or percent shell. Feeding DON-contaminated wheat did not significantly affect fertility, hatchability of fertile eggs, hatch of total eggs set, or chick weights at hatch. These data agree with the results of Hamilton et al. (1985a), Moran et al. (1983), and Kubena et al. (1987). Effects of feeding DON-contaminated wheat on serum chemistry values are shown in Table 3. There was a significant decrease in serum uric acid possibly associated with the efficiency of amino acid utilization, changes in other enzyme systems, altered renal filtration and reabsorption rates, or other unknown factors. Glucose, triglycerides, and cholesterol were significantly decreased in the DON treatment possibly due to liver involvement and a shift of concentrations from the blood to the liver. There were no significant changes in serum urea nitrogen, creatinine, total protein, or albumen. Gamma glutamyl transferase and alkaline phosphatase activities were significantly increased in the DON treatment, possibly indicating chronic liver involvement. There were no significant changes in the activities of cholinesterase, creatine kinase, aspartate aminotransferase, or lactic dehydrogenase. There were no significant differences observed in any of the hematological parameters measured (data not shown). Huff et al.(\986) found a slight anemia in broiler chicks and Kubena et al. (1985) observed a slight, yet significant, anemia in Leghorn chicks. The anemia in Leghorn chicks appeared transitory, being less pronounced at 5 wk than at 4 wk of age. The data from the present study support the fact that the anemia was transitory in nature. No changes in prothrombin times were observed. Eggs collected during the last week of the study from randomly selected hens fed the control and the DON-contaminated wheat diet were analyzed for DON content. None of these eggs were found to contain detectable quantities (detection limit: 10 |xg/kg) of DON. This finding agrees with the reports of El-Banna etal. (1983) and Lun et al. (1986) who found no detectable levels of DON in eggs from chickens fed DONcontaminated diets. No treatment-related differences in mortality were observed; two hens from each treatment died during the experiment. Microscopic exam-
LONG-TERM FEEDING OF DEOXYNIVALENOL
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ination of sections of the liver, kidney, and proventriculus from five randomly selected control and DON hens revealed no lesions in the proventriculus nor in the kidney. Mild to moderate lipidosis, a condition not uncommon in livers of high producing hens of this age, was observed in two of the five control treatment hens and two of the five DON treatment hens examined histopathologically. This is the first study reported wherein relatively high levels of DON (18 mg/kg) were fed beginning at 1 day of age and continuing through six egg production periods. The results show that DON caused only slight effects on the parameters measured and indicate that laying strain chicks and laying hens may be more tolerant of DON than rapidly growing broiler chickens and some other animals. These findings support the short-term studies of Moran et al. (1983) and Lun et al. (1986) using high levels of DON (50 and 83 mg DON/kg, respectively), the longer term studies of Hamilton et al. (1983, 1985a) using levels of DON less than 5 mg/kg, and the long-term study of Kubena et al. (1987) with relatively high levels of DON (18 mg/kg), in that DON had only slight effects on most of the parameters measured. The lack of agreement on some parameters may be due to quantity of DON in the diet, length of exposure, time in life-cycle of exposure, dietary factors (including source of wheat), breed or strain of chickens, environmental factors, or some other unidentified factors. No DON residues were detected in any eggs and no additional or unique lesions were observed in DON-treated hens when compared with controls. The biological significance of some of the small, although statistically significant changes observed, is not known. Poultry and livestock may be more susceptible to DON as well as other mycotoxins if nutritional, environmental, health, or other stress factors are involved and differences in susceptibility due to breed or strains might also occur, making it difficult to assess the safety of feeding DON-contaminated grains. REFERENCES
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Cote, L. M., J. D. Reynolds, R. F. Vesonder, W. B. Buck, S. P. Swanson, R. T. Coffey, and D. C. Brown, 1984. Survey of vomitoxin-contaminated feed grains in midwestern United States and associated health problems in swine. J. Am. Vet. Med. Assoc. 184:189-192. Doerr, J. A., W. E. Huff, H. T. Tung, R. D. Wyatt, and P. B. Hamilton, 1974. A survey of T-2 toxin, ochratoxin, and aflatoxin for their effects on the coagulation
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KUBENA ET AL. Kubena, L. F., S. P. Swanson, R. B. Harvey, O. J. Fletcher, L. D. Rowe, and T. D. Phillips, 1985. Effects of feeding deoxynivalenol (vomitoxin)-contaminated wheat to growing chicks. Poultry Sci. 64:1649-1655. Lun, A. K., L. G. Young, E. T. Moran, Jr., D. B. Hunter, and J. P. Rodriguez, 1986. Effects of feeding hens a high level of vomitoxin-contaminated corn on performance and tissue residues.Poultry Sci. 65:1095-1099. Mirocha, C. J., B. Schauerhamer, C. M. Christensen, and T. Kommendahl, 1979. Zearalenone, deoxynivalenol, and T-2 toxin associated with stalk rot in corn. Appl. Environ. Microbiol. 38:557-558. Moran, E. T., Jr., P. R. Ferket, D. B. Hunter, and L. R. Young, 1983. Effect of vomitoxin contaminated corn on poultry production. Pages 61 -65 in: Proc. Maryland Nutr. Conf., College Park, MD. Moran, E. T., Jr., B. Hunter, P. Ferket, L. G. Young, and L. G. McGirr, 1982. High tolerance of broilers to vomitoxin from corn infected with Fusarium graminearum. Poultry Sci. 61:1828-1831. National Research Council, 1977. Nutrient Requirements ofPoultry. 7thed. Natl. Acad. Sci., Washington, DC. Pathre, S. V., and C. J. Mirocha, 1979. Trichothecenes: Natural occurrence and potential hazard. J. Am. Oil Chem. Soc. 56:820-823. Scott, P. M., P. Y. Lau, and S. R. Kanhere, 1981. Gas chromatography with electron capture and mass spectrometric detection of deoxynivalenol in wheat and other grains. J. Assoc. Off. Anal. Chem. 64:13641371 Statistical Analysis System Institute Inc., 1982. SAS User's Guide: Statistics. 1982 ed. SAS Institute Inc., Cary, NC. Trenholm, H. L., W. P. Cochrane, H. Cohen, J. I. Elliot, E. R. Farnworth, D. W. Friend, R.M.G. Hamilton, G. A. Neish, and J. F. Standish, 1981. Survey of vomitoxin contamination of the 1980 white winter wheat crop in Ontario, Canada. J. Am. Oil Chem. Soc. 58:992A-994A. Trenholm, H. L., W. P. Cochrane, H. Cohen, J. I. Elliot, E. R. Farnworth, D. W. Friend, R.M.G. Hamilton, J. F. Standish, and B. K. Thompson, 1983. Survey of vomitoxin contamination of 1980 Ontario white winter wheat crop: Results of survey and feeding trials. J. Assoc. Off. Anal. Chem. 66:92-97. Trenholm, H. L., R.M.G. Hamilton, D. W. Friend, B. K. Thompson, and K. E. Hartin, 1984. Feeding trials with vomitoxin (deoxynivalenol)-contaminated wheat: Effects in swine, poultry, and dairy cattle. J. Am. Vet. Med. Assoc. 185:527-531. Vesonder, R. F., A. Ciegler, and A. H. Jensen, 1973. Isolation of the emetic principle from Fusarium-'mfected corn. Appl. Microbiol. 26:1008-1010. Vesonder, R. F., A. Ciegler, A. H. Jensen, W. K. Rohwedder, andD. Weisleder, 1976. Co-identity of the refusal and emetic principle from Fusarium-'mkclzA corn. Appl. Environ. Microbiol. 31:280-285. Vesonder, R. F., A. Ciegler, R. F. Rogers, K. A. Burbridge, R. J. Bothast, and A. H. Jensen, 1978. Survey of 1977 crop year preharvest corn for vomitoxin. Appl. Environ. Microbiol. 36:885-888.
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of blood in young broiler chickens. Poultry Sci. 53:1728-1734. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics 11:1^12. El-Banna, A. A., R.M.G. Hamilton, P. M. Scott, and H. L. Trenholm, 1983. Nontransmission of deoxynivalenol (vomitoxin) to eggs and meat in chickens fed deoxynivalenol-contaminated diets. J. Agric. FoodChem. 31:1381-1384. Forsyth, D. M., L. Yoshizawa, N. Morooka, and J. Tuite, 1977. Emetic and refusal activity of deoxynivalenol to swine. Appl. Environ. Microbiol. 34:547-552. Hagler, W. M., Jr., K. Tyczkowska, and P. B. Hamilton, 1984. Simultaneous occurrence of deoxynivalenol, zearalenone, and aflatoxin in 1982 scabby wheat from the Midwestern United States. Appl. Environ. Microbiol. 47:151-154. Hamilton, R.M.G., B. K. Thompson, and H. L. Trenholm, 1981a. The effect of vomitoxin contaminated wheat on the palatability of laying diets by white Leghorn hens. Poultry Sci. 60:1665-1666. (Abstr.) Hamilton, R.M.G., B. K. Thompson, and H. L. Trenholm, 1981b. Feed intake, egg production, and shell quality of hens given diets that contained vomitoxin contaminated wheat. Poultry Sci. 60:1666. (Abstr.) Hamilton, R.M.G., B. K. Thompson, H. L. Trenholm, P. S. Fiser, and R. Greenhalgh, 1985a. Effects of feeding white Leghorn hens diets that contain deoxynivalenol (vomitoxin)-contaminated wheat. Poultry Sci. 64:1840-1852. Hamilton, R.M.G., H. L. Trenholm, and B. K. Thompson, 1983. Effects of feeding chicks and laying hens diets that contain vomitoxin (deoxynivalenol) contaminated wheat. Proc. Annu. Nutr. Conf. Feed Manuf. 19:48— 55. Hamilton, R.M.G., H. L. Trenholm, B. K. Thompson, and R. Greenhalgh, 1985b. The tolerance of white Leghorn and broiler chicks, and turkey poults to diets that contain deoxynivalenol (vomitoxin)-contaminated wheat. Poultry Sci. 64:273-286. Huff, W. E., J. A. Doerr, P. B. Hamilton, and R. F. Vesonder, 1981. Acute toxicity of vomitoxin (deoxynivalenol) in broiler chickens. Poultry Sci. 60:1412-1414. Huff, W. E., L. F. Kubena, R. B. Harvey, W. M. Hagler, Jr., S. P. Swanson, T. D. Phillips, and C. R. Creger, 1986. Individual and combined effects of aflatoxin and deoxynivalenol (DON, vomitoxin) in broiler chickens. Poultry Sci. 65:1291-1298. Hulan, H. W., and F. G. Proudfoot, 1982. Effects of feeding vomitoxin contaminated wheat on the performance of broiler chickens. Poultry Sci. 61:1653-1659. Kubena, L. F., R. B. Harvey, T. D. Phillips, G. M. Holman, and C. R. Creger, 1987. Effects of feeding mature white Leghorn hens diets that contain deoxynivalenol (DON, vomitoxin). Poultry Sci. 66:55-58. Kubena, L. F., W. E. Huff, R. B. Harvey, D. E. Corrier, T. D. Phillips, and C. R. Creger, 1986. Influence of ochratoxin A (OA) and deoxynivalenol (DON) on growing broiler chicks. Poultry Sci. 65 (Suppl. 1):73. (Abstr.)
ABSTRACT White Leghorn chickens one day of age were fed starter and grower diets containing either a control (noncontaminated) wheat diet or a naturally contaminated deoxynivalenol (DON) wheat diet (18 mg DON/kg) from 1 day of age to the onset of egg production. The hens were then placed on their respective layer diets of control wheat or DON-contaminated wheat (18 mg DON/kg) for six 28-day egg production periods. Feeding the DON-contaminated diet did not significantly influence body weights during the growing or the laying phases. Overall, hen-day egg production and egg weights were significantly higher for hens receiving the DON diet. Feeding DON contaminated wheat caused no significant changes in percent shell, albumen height, percent fertility, percent hatch of fertile eggs, percent hatch of eggs set, or weight of chicks at hatch. There were slight, although significant, changes in shell weight and shell thickness and in some serum chemistry values. There were no significant differences in the hematology parameters measured or in prothrombin times. None of the eggs collected from hens fed the control and the DON-contaminated wheat diet contained detectable quantities of DON. Microscopic examination of sections of the liver, kidney, and proventriculus of control and treated hens revealed no unusual histopathology. The results indicate that feeding DON at relatively high levels beginning at 1 day of age and continuing through six egg production periods had only slight effects on the parameters measured. (Key words: deoxynivalenol, wheat diet, growth, laying hens, egg production, fertility, hatchability) 1987 Poultry Science 66:1612-1618 INTRODUCTION
There is considerable evidence on the natural occurrence of deoxynivalenol [3a,7a, 15trihydroxy-12, 13-epoxytrichothec-9-en-8-one; (DON, vomitoxin)] in grains in many areas of the world (Vesonder et al., 1978; Mirocha et al., 1979; Pathre and Mirocha, 1979; Trenholm et al., 1983; Cote et al., 1984; Hagler et al, 1984). Deoxynivalenol is a mold metabolite which causes feed refusal and emesis in swine (Vesonder et al., 1973, 1976). Forsyth et al. (1977) demonstrated a dose-response relationship in swine between the level of DON administered either orally or intraperitoneally and feed refusal and emesis.
'Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.
Reports in the literature on the effects of DON in growing chicks and mature chickens are limited. Huff et al. (1981) observed no mortality in day-old broiler chicks until oral doses reached 70 mg/kg of body weight and found the approximate oral LD50 dose to be 140 mg/kg, suggesting a relatively low acute toxicity. Hulan and Proudfoot (1982) found no significant effects on body weight gains from feeding naturally contaminated wheat-based diets containing as much as 1.87 mg DON/kg to broiler cockerels for 28 days. Moran et al. (1982) fed broiler cockerels from 6 to 11 days of age diets containing 1.7 to 210.0 mg DON/kg from corn purposely inoculated with Fusarium graminearum. These workers found growth and feed consumption were not reduced until feeding exceeded 116 mg DON/kg of diet. Hamilton et al. (1983, 1985b) and Trenholm et al. (1984) reported no detrimental effects in Leghorn and broiler chicks fed naturally contaminated wheat diets containing as much as 4.9 mg DON/kg. Kubena et al.
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Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on May 26, 2015
(Received for publication September 22, 1986)
LONG-TERM FEEDING OF DEOXYNIVALENOL
on the effects of DON on reproductive performance of laying hens include short-term studies at high DON levels (50 mg/kg), longer term studies at low DON levels (<5 mg/kg), and long-term exposure at relatively high levels (18 mg/kg) that might be found in naturally contaminated feeds. The purpose of the research reported here was to describe the major effects of a relatively high level of DON (18 mg/kg) fed from 1 day of age through several egg production periods. MATERIALS AND METHODS
Sixty female one-day-old white Leghorn chicks were individually weighed; sequentially numbered aluminum wing bands were randomly assigned and affixed to the wing web. Thirty chicks were placed into each of two groups (two replicates of 15 chicks per treatment) according to the wingband number sequence and placed in electrically heated battery brooders. One group received a control (noncontaminated) wheat starter-grower diet and the other group received a naturally contaminated wheat startergrower diet. Diets contained or exceeded the levels of critical nutrients recommended by the National Research Council (1977) and consisted of 68.5% wheat, 28.5% soybean meal-based concentrate (including vitamins and minerals), and 3.0% corn oil. The contaminated and noncontaminated wheat were analyzed for DON by the procedure of Scott et al. (1981). The wheat was found to contain 26 mg DON/kg and no detectable levels of zearalenone, T-2 toxin, diacetoxyscirpenol, aflatoxin, or ochratoxin. Diets were calculated to contain 0 and 18 mg DON/kg and analyses of diets for DON periodically during the study were in good agreement with calculated values. The chickens received their respective starter-grower diets and water ad libitum from one day to 20 wk of age. At 4 wk of age, chickens were moved into growing batteries until 14 wk of age, at which time they were transferred into individual cages in laying batteries. At 18 wk of age, as no treatment differences in body weights were present, 24 chickens from each dietary treatment were selected for the production phase based on similar body weights (the three lightest and three heaviest chickens from each treatment were removed). Lighting was gradually increased until the chickens (hens) were receiving 16 h of light and was maintained at this level for the remainder of the study. At 20 wk of age, hens were
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(1985) found no detrimental effects on body weights or feed efficiency in male Leghorn chicks fed naturally contaminated wheat diets containing 9 or 18 mg DON/kg from 1 to 35 days of age. These workers did observe a slight, yet significant, transitory anemia due to DON intoxication. A response, thought to be caused by irritation by DON or by the difference in the density of the diets, was found in the upper gastrointestinal tract, and various physiological measurements were slightly altered by DON. Huff et al. (1986) observed reduced growth rate, decreased feed efficiency, increased relative gizzard weights, anemia, decreased lactic dehydrogenase activity, and decreased serum triglycerides in male broilers fed naturally contaminated wheat diets containing approximately 16 mg DON/kg. Moran et al. (1983) fed artificially contaminated corn-based diets containing 50 mg DON/ kg to mature laying hens for 4 consecutive weeks and found no change in the hatch of total eggs. Trenholm et al. (1981) fed naturally contaminated DON wheat diets to adult female Leghorn chickens for 10 wk and observed no marked changes in feed consumption, weight gain, or egg production. Hamilton et al. (1981a,b, 1983, 1985a) reported that naturally contaminated wheat diets containing .35 or .70 mg DON/kg and fed for 10 wk caused a significant decrease in egg and shell weight, shell thickness, and percentage shell, whereas other parameters measured were not affected. Hamilton et al. (1983) in additional studies fed naturally contaminated wheat diets containing as much as 4.9 mg DON/ kg for 24 wk and observed no significant effect on these parameters; however, trends were noted toward lower values with higher DON feeding levels. Kubena et al. (1987) fed naturally contaminated wheat diets containing 18 mg DON/kg to mature laying hens for 16 wk and found no significant effects on body weights, hen day egg production, egg weights, or efficiency of feed utilization. Egg shell weight and percent egg shell were slightly, although significantly, decreased by feeding the DON-contaminated wheat diet. Lun etal.{\986) fed diets containing 82.8 mg DON/kg for 27 days and observed no detrimental effects on the performance of mature laying hens. The literature indicates that poultry may be relatively insensitive to DON, thus raising the possibility that grains contaminated with DON may be incorporated into poultry diets without affecting performance. Reports in the literature
1613
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KUBENA ET AL. RESULTS AND DISCUSSION
There were no significant differences in body weights from 1 day to 18 wk of age, with the exception of significantly higher body weights for the DON-treated chickens at 10 wk of age (data not shown). These results are in agreement with reports of Hulan and Proudfoot (1982), Moran et al. (1982), Hamilton et al. (1983, 1985b), and Kubena et al. (1985) who observed no detrimental effects on performance of growing chicks fed diets containing DON. The report of Huff et al. (1986) and additional results obtained recently in our laboratory (Kubena et al., 1986) show that the performance of rapidly growing broilers was significantly reduced when fed DON-contaminated wheat diets containing 16 mg DON/kg. In the present study, significant differences in body weights due to dietary treatment, were not observed at any of the time intervals between 24 and 48 wk of age. These results agree with Hamilton et al. (1985a), Moran et al. (1983), and Kubena et al. (1987) who also found no significant differences in body weights at these ages. Effects of feeding DON-contaminated wheat diets on egg weights, egg production, and feed consumption and efficiency are shown in Table 1. There were no differences in the average age of chickens at onset of production (data not shown) and there were no significant differences in egg weights or egg production during the 1st 28-day production period. A significant increase in egg weights occurred during the 2nd 28-day period and, with the exception of the 3rd 28-day period, continued throughout the experiment. Beginning with the 3rd 28-day period and continuing for the remainder of the experiment, percent hen day egg production was significantly higher for hens in the DON treatment group. Kubena et al. (1987) also observed increased egg production (nonsignificant) for the hens fed DON. As the wheat diets used in the study reported herein were formulated with DON-contaminated wheat from the same shipment, the increase in egg weights and egg production may be due to wheat source. Hens fed the DON-contaminated wheat diet consumed 130.4 g of feed/ hen per day, whereas hens fed the control diet consumed 119.0 g/hen per day. The efficiency of feed utilization as measured by kilograms of feed required to produce a kilogram of eggs was almost identical (2.80 for control and 2.78 for DON-fed hens).
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placed on a laying diet that consisted of 67.0% wheat (noncontaminated for control and naturally contaminated for second treatment), 22.0% soybean meal-based concentrate (including vitamins and minerals), 7.8% calcium carbonate, 1.2% phosphate, and 2.0% corn oil. Hens were provided their respective laying diets and water ad libitum from 20 wk of age to the end of the study. Chickens were individually weighed at 1 day of age and at 4, 10, 14, 18, 24, 32, 40, and 48 wk of age. All eggs were recorded, collected, and individually weighed on a daily basis during six 28-day experimental periods. Mortality was recorded as it occurred and feed consumption was measured for each 28-day period throughout the experiment. Shell weight, shell thickness, and albumen height were measured on ail eggs collected on 3 consecutive days each week for 2 wk and 2 consecutive days for 1 wk during the last two 28-day experimental periods. All hens were inseminated with pooled semen from control males and the eggs produced during a 3 to 4-day period each week were incubated once a week for 11 wk. Fertility, hatchability, and chick weights at hatching were measured. After six 28-day experimental periods, 20 hens from each treatment were bled by cardiac puncture for hematology and serum chemistry analyses as previously described by Kubena et al. (1985). Prothrombin times were determined by the method as described by Doerr et al. (1974) with the exception that a ML A Electra 600, P 4 with animal clot time option (Medical Laboratory, Automation, Inc., Mount Vernon, NY) was used. Samples of the liver, kidney, and proventriculus were taken from five randomly selected chickens per treatment for microscopic examination. Six eggs from each treatment group were randomly selected during the last week of the study and analyzed for DON content according to the procedure of El-Banna et al. (1983). Data for all response variables except feed consumption were analyzed using the general linear model analysis of variance procedure in the SAS software (SAS Institute Inc., 1982). Variable means for treatments showing significant differences in the analysis of variance were compared and differences were tested using Duncan's new multiple range procedure (Duncan, 1955). All statements of significance are based on the probability level of .05.
84.0 a 92.6 a
47.6 a 48.5 a
1
84.8 a 89.3 a
48.8D 50.3 a
2
85.9 b 92.4 a
51.5 a 52.8 a
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52.9 b 54.9 a
4
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54.0 b 56.0 a
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DON = Deoxynivalenol.
a' b In the same vertical column within each time period and parameter, values with different superscripts are signific
DON
Feed efficiency, kg feed/kg eggs Control
DON
Feed consumed, g feed/hen/day Control
DON
Egg production, % Control
DON
Egg weights, g Control
Parameter and treatment
TABLE 1. Effects of feeding DON-contaminated wheat on production characteristics of fe in six 28-day periods1
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1616
KUBENA ET AL.
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There was a significant increase in shell weight and shell thickness in the DON treatment group when compared with values for controls (Table 2). No significant differences were observed in albumen height or percent shell. Feeding DON-contaminated wheat did not significantly affect fertility, hatchability of fertile eggs, hatch of total eggs set, or chick weights at hatch. These data agree with the results of Hamilton et al. (1985a), Moran et al. (1983), and Kubena et al. (1987). Effects of feeding DON-contaminated wheat on serum chemistry values are shown in Table 3. There was a significant decrease in serum uric acid possibly associated with the efficiency of amino acid utilization, changes in other enzyme systems, altered renal filtration and reabsorption rates, or other unknown factors. Glucose, triglycerides, and cholesterol were significantly decreased in the DON treatment possibly due to liver involvement and a shift of concentrations from the blood to the liver. There were no significant changes in serum urea nitrogen, creatinine, total protein, or albumen. Gamma glutamyl transferase and alkaline phosphatase activities were significantly increased in the DON treatment, possibly indicating chronic liver involvement. There were no significant changes in the activities of cholinesterase, creatine kinase, aspartate aminotransferase, or lactic dehydrogenase. There were no significant differences observed in any of the hematological parameters measured (data not shown). Huff et al.(\986) found a slight anemia in broiler chicks and Kubena et al. (1985) observed a slight, yet significant, anemia in Leghorn chicks. The anemia in Leghorn chicks appeared transitory, being less pronounced at 5 wk than at 4 wk of age. The data from the present study support the fact that the anemia was transitory in nature. No changes in prothrombin times were observed. Eggs collected during the last week of the study from randomly selected hens fed the control and the DON-contaminated wheat diet were analyzed for DON content. None of these eggs were found to contain detectable quantities (detection limit: 10 |xg/kg) of DON. This finding agrees with the reports of El-Banna etal. (1983) and Lun et al. (1986) who found no detectable levels of DON in eggs from chickens fed DONcontaminated diets. No treatment-related differences in mortality were observed; two hens from each treatment died during the experiment. Microscopic exam-
LONG-TERM FEEDING OF DEOXYNIVALENOL
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ination of sections of the liver, kidney, and proventriculus from five randomly selected control and DON hens revealed no lesions in the proventriculus nor in the kidney. Mild to moderate lipidosis, a condition not uncommon in livers of high producing hens of this age, was observed in two of the five control treatment hens and two of the five DON treatment hens examined histopathologically. This is the first study reported wherein relatively high levels of DON (18 mg/kg) were fed beginning at 1 day of age and continuing through six egg production periods. The results show that DON caused only slight effects on the parameters measured and indicate that laying strain chicks and laying hens may be more tolerant of DON than rapidly growing broiler chickens and some other animals. These findings support the short-term studies of Moran et al. (1983) and Lun et al. (1986) using high levels of DON (50 and 83 mg DON/kg, respectively), the longer term studies of Hamilton et al. (1983, 1985a) using levels of DON less than 5 mg/kg, and the long-term study of Kubena et al. (1987) with relatively high levels of DON (18 mg/kg), in that DON had only slight effects on most of the parameters measured. The lack of agreement on some parameters may be due to quantity of DON in the diet, length of exposure, time in life-cycle of exposure, dietary factors (including source of wheat), breed or strain of chickens, environmental factors, or some other unidentified factors. No DON residues were detected in any eggs and no additional or unique lesions were observed in DON-treated hens when compared with controls. The biological significance of some of the small, although statistically significant changes observed, is not known. Poultry and livestock may be more susceptible to DON as well as other mycotoxins if nutritional, environmental, health, or other stress factors are involved and differences in susceptibility due to breed or strains might also occur, making it difficult to assess the safety of feeding DON-contaminated grains. REFERENCES
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Cote, L. M., J. D. Reynolds, R. F. Vesonder, W. B. Buck, S. P. Swanson, R. T. Coffey, and D. C. Brown, 1984. Survey of vomitoxin-contaminated feed grains in midwestern United States and associated health problems in swine. J. Am. Vet. Med. Assoc. 184:189-192. Doerr, J. A., W. E. Huff, H. T. Tung, R. D. Wyatt, and P. B. Hamilton, 1974. A survey of T-2 toxin, ochratoxin, and aflatoxin for their effects on the coagulation
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KUBENA ET AL. Kubena, L. F., S. P. Swanson, R. B. Harvey, O. J. Fletcher, L. D. Rowe, and T. D. Phillips, 1985. Effects of feeding deoxynivalenol (vomitoxin)-contaminated wheat to growing chicks. Poultry Sci. 64:1649-1655. Lun, A. K., L. G. Young, E. T. Moran, Jr., D. B. Hunter, and J. P. Rodriguez, 1986. Effects of feeding hens a high level of vomitoxin-contaminated corn on performance and tissue residues.Poultry Sci. 65:1095-1099. Mirocha, C. J., B. Schauerhamer, C. M. Christensen, and T. Kommendahl, 1979. Zearalenone, deoxynivalenol, and T-2 toxin associated with stalk rot in corn. Appl. Environ. Microbiol. 38:557-558. Moran, E. T., Jr., P. R. Ferket, D. B. Hunter, and L. R. Young, 1983. Effect of vomitoxin contaminated corn on poultry production. Pages 61 -65 in: Proc. Maryland Nutr. Conf., College Park, MD. Moran, E. T., Jr., B. Hunter, P. Ferket, L. G. Young, and L. G. McGirr, 1982. High tolerance of broilers to vomitoxin from corn infected with Fusarium graminearum. Poultry Sci. 61:1828-1831. National Research Council, 1977. Nutrient Requirements ofPoultry. 7thed. Natl. Acad. Sci., Washington, DC. Pathre, S. V., and C. J. Mirocha, 1979. Trichothecenes: Natural occurrence and potential hazard. J. Am. Oil Chem. Soc. 56:820-823. Scott, P. M., P. Y. Lau, and S. R. Kanhere, 1981. Gas chromatography with electron capture and mass spectrometric detection of deoxynivalenol in wheat and other grains. J. Assoc. Off. Anal. Chem. 64:13641371 Statistical Analysis System Institute Inc., 1982. SAS User's Guide: Statistics. 1982 ed. SAS Institute Inc., Cary, NC. Trenholm, H. L., W. P. Cochrane, H. Cohen, J. I. Elliot, E. R. Farnworth, D. W. Friend, R.M.G. Hamilton, G. A. Neish, and J. F. Standish, 1981. Survey of vomitoxin contamination of the 1980 white winter wheat crop in Ontario, Canada. J. Am. Oil Chem. Soc. 58:992A-994A. Trenholm, H. L., W. P. Cochrane, H. Cohen, J. I. Elliot, E. R. Farnworth, D. W. Friend, R.M.G. Hamilton, J. F. Standish, and B. K. Thompson, 1983. Survey of vomitoxin contamination of 1980 Ontario white winter wheat crop: Results of survey and feeding trials. J. Assoc. Off. Anal. Chem. 66:92-97. Trenholm, H. L., R.M.G. Hamilton, D. W. Friend, B. K. Thompson, and K. E. Hartin, 1984. Feeding trials with vomitoxin (deoxynivalenol)-contaminated wheat: Effects in swine, poultry, and dairy cattle. J. Am. Vet. Med. Assoc. 185:527-531. Vesonder, R. F., A. Ciegler, and A. H. Jensen, 1973. Isolation of the emetic principle from Fusarium-'mfected corn. Appl. Microbiol. 26:1008-1010. Vesonder, R. F., A. Ciegler, A. H. Jensen, W. K. Rohwedder, andD. Weisleder, 1976. Co-identity of the refusal and emetic principle from Fusarium-'mkclzA corn. Appl. Environ. Microbiol. 31:280-285. Vesonder, R. F., A. Ciegler, R. F. Rogers, K. A. Burbridge, R. J. Bothast, and A. H. Jensen, 1978. Survey of 1977 crop year preharvest corn for vomitoxin. Appl. Environ. Microbiol. 36:885-888.
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of blood in young broiler chickens. Poultry Sci. 53:1728-1734. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics 11:1^12. El-Banna, A. A., R.M.G. Hamilton, P. M. Scott, and H. L. Trenholm, 1983. Nontransmission of deoxynivalenol (vomitoxin) to eggs and meat in chickens fed deoxynivalenol-contaminated diets. J. Agric. FoodChem. 31:1381-1384. Forsyth, D. M., L. Yoshizawa, N. Morooka, and J. Tuite, 1977. Emetic and refusal activity of deoxynivalenol to swine. Appl. Environ. Microbiol. 34:547-552. Hagler, W. M., Jr., K. Tyczkowska, and P. B. Hamilton, 1984. Simultaneous occurrence of deoxynivalenol, zearalenone, and aflatoxin in 1982 scabby wheat from the Midwestern United States. Appl. Environ. Microbiol. 47:151-154. Hamilton, R.M.G., B. K. Thompson, and H. L. Trenholm, 1981a. The effect of vomitoxin contaminated wheat on the palatability of laying diets by white Leghorn hens. Poultry Sci. 60:1665-1666. (Abstr.) Hamilton, R.M.G., B. K. Thompson, and H. L. Trenholm, 1981b. Feed intake, egg production, and shell quality of hens given diets that contained vomitoxin contaminated wheat. Poultry Sci. 60:1666. (Abstr.) Hamilton, R.M.G., B. K. Thompson, H. L. Trenholm, P. S. Fiser, and R. Greenhalgh, 1985a. Effects of feeding white Leghorn hens diets that contain deoxynivalenol (vomitoxin)-contaminated wheat. Poultry Sci. 64:1840-1852. Hamilton, R.M.G., H. L. Trenholm, and B. K. Thompson, 1983. Effects of feeding chicks and laying hens diets that contain vomitoxin (deoxynivalenol) contaminated wheat. Proc. Annu. Nutr. Conf. Feed Manuf. 19:48— 55. Hamilton, R.M.G., H. L. Trenholm, B. K. Thompson, and R. Greenhalgh, 1985b. The tolerance of white Leghorn and broiler chicks, and turkey poults to diets that contain deoxynivalenol (vomitoxin)-contaminated wheat. Poultry Sci. 64:273-286. Huff, W. E., J. A. Doerr, P. B. Hamilton, and R. F. Vesonder, 1981. Acute toxicity of vomitoxin (deoxynivalenol) in broiler chickens. Poultry Sci. 60:1412-1414. Huff, W. E., L. F. Kubena, R. B. Harvey, W. M. Hagler, Jr., S. P. Swanson, T. D. Phillips, and C. R. Creger, 1986. Individual and combined effects of aflatoxin and deoxynivalenol (DON, vomitoxin) in broiler chickens. Poultry Sci. 65:1291-1298. Hulan, H. W., and F. G. Proudfoot, 1982. Effects of feeding vomitoxin contaminated wheat on the performance of broiler chickens. Poultry Sci. 61:1653-1659. Kubena, L. F., R. B. Harvey, T. D. Phillips, G. M. Holman, and C. R. Creger, 1987. Effects of feeding mature white Leghorn hens diets that contain deoxynivalenol (DON, vomitoxin). Poultry Sci. 66:55-58. Kubena, L. F., W. E. Huff, R. B. Harvey, D. E. Corrier, T. D. Phillips, and C. R. Creger, 1986. Influence of ochratoxin A (OA) and deoxynivalenol (DON) on growing broiler chicks. Poultry Sci. 65 (Suppl. 1):73. (Abstr.)