2014

J. L. HEATH

Steel, R. F. D., and J. H. Torrie. 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York.

United States Department of Agriculture Egg Grading Manual. 1968. Agriculture Handbook 75. U.S. Government Printing Office, Washington, D.C.

Effects of Dietary Aflatoxin on Certain Egg Yolk Parameters1,2-3 W . E . H U F F , R. D . WYATT AND P . B . HAMILTON

Department of Poultry Science and Department of Microbiology, North Carolina State Raleigh, North Carolina 27607

University,

ABSTRACT Aflatoxicosis was induced in laying hens by incorporating graded amounts of aflatoxin into a commercial layer ration (0, 1.25, 2.5, 5.0, and 10.0 ji-g./g. of feed). After four weeks liver size and liver lipid were increased by aflatoxin while egg production and egg size were decreased (P < 0.05). Total yolk weight and the yolk as percent of total egg weight were lowered (P < 0.05). The dry weight and lipid content of the yolk were not affected. Yolk and plasma carotenoid concentrations were elevated (P < 0.05). The data suggest that plasma and yolk lipids respond to the inhibition of lipid synthesis and transport from the liver by aflatoxin, but plasma and yolk carotenoids which are dietary in origin necessarily increase when egg production decreases during aflatoxicosis. POULTRY SCIENCE 54: 2014-2018, 1975

INTRODUCTION FLATOXIN causes in laying hens an enlarged, friable, fatty liver and a decrease in egg production which are characteristic of the "Fatty Liver Syndrome" (Hamilton and Garlich, 1971). The decrease in egg production does not occur immediately after aflatoxin is introduced into the diet but rather occurs after a 10 to 14 day lag period (Hamilton and Garlich, 1972; Garlich et al., 1973). This drop in egg production is preceded

A

1. Paper Number 4626 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina. 2. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Experiment Station, nor criticism of similar ones not mentioned. 3. A preliminary report of part of this paper was presented at the 63rd Annual Meeting of the Poultry Science Association, Morgantown, West Virginia, 1974.

several days by a drop in serum proteins and lipids which are the precursors of egg constituents (Garlich et al., 1973). The eggs which are produced during af latoxicosis are smaller than normal (Hamilton and Garlich, 1971). The objective of the present study was to determine whether the eggs produced during aflatoxicosis had abnormalities other than a smaller size.

MATERIALS AND METHODS Single Comb White Leghorn hens were obtained at 27 weeks of age and were selected at 30 weeks on the basis of body weight and uniform rate of egg production. The basal diet was commercial layer ration which was analyzed to assure freedom from extraneous aflatoxin and it was fed during the three week equilibration period. The experiment was four weeks in duration and graded doses of dietary aflatoxin (0, 1.25, 2.5, 5.0, and 10.0 |xg./g. of feed) were fed to 10 birds per treatment.

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(Received for publication March 17, 1975)

2015

AFLATOXIN AND EGG YOLKS

TABLE 1.—Response of egg production, liver weight and liver lipid content during aflatoxicosis in laying hens Dose (n-g-/g-) 0.0 1.25 2.5 5.0 10.0

Egg production'

(%) 81.7 74.6 85.2 72.2 26.8

± ± ± ± ±

4.4a 5.3a 3.2a 5.1a 8.4b

Liver size (g-) 32.4 ± 1.5a 31.4 ± 2.0a 40.4 ± 1.5a 52.3 ± 2.7b 64.6 ± 5.8b

Liver lipid

(%) 21.2 24.4 32.7 35.6 46.5

± ± ± ± ±

1 Egg production during the last week of the experiment. Values in table are means error of mean and values in a column with different small letters differ (P < 0.05).

Eggs were collected the last week of the experiment and the production rate, egg weight, dry shell weight, and yolk wet and dry weight were determined. At the end of the experiment plasma was collected and its lipid content (Friedman, 1968) and carotenoid content (Kimble, 1938) were measured. The livers were excised, weighed, and their lipid content determined (Smith and Hamilton, 1970). The lipid content of the egg yolk was measured by the same method. The data were analyzed by an analysis of variance, and if the F-ratio were significant, the least significant difference among means was calculated (Bruning and Kintz, 1968). RESULTS The responses of egg production, liver size,

standard

and liver lipid content during the experiment are presented in Table 1. Egg production was decreased by about 70% from the control value at the dose level of 10.0 |xg./g. The liver size was increased significantly (P < 0.05) by doses of 5 and 10.0 p-g./g. and the liver lipid was increased dramatically by a smaller dose of 2.5 p-g./g. The response of egg size and plasma lipids are given in Table 2. There was a significant (P < 0.05) decrease in egg size by doses of 2.5 u,g./g. and above. Egg shell thickness and shell as percent of egg were determined but there were no significant effects, which is in agreement with an earlier report (Hamilton and Garlich, 1971). Plasma lipids were decreased significantly (P < 0.05) 26 and 33% at doses of 5.0 and 10.0 p.g. / g . , respectively. When the eggs were broken out, the egg yolks were separated from the whites and weighed. Total yolk weight as a function of dietary aflatoxin is shown in Fig. 1. There was a significant (P < 0.05) decrease of 34%

TABLE 2.—Response of egg size and plasma lipid during aflatoxicosis in laying hens Dose (M-g-/g-) 0.0 1.25 2.5 5.0 10.0

Egg weight' (g.) 56.7 ± 0.9a 57.2 ± 1.1a 55.2 ± 1.1a 55.6 ± 1.1a 51.3 ± 0.9b

Plasma lipid (g-/100) 2.6 ± 0.3a 2.6 ± 0.4a 2.1 ± 0.2a,b 1.9 ± 0.1b 1.7 ± 0.2b

'Weight of eggs produced the last ten days of experiment. Values in the table are means ± standard errors of means, and values in a column with different small letters differ (P < 0.05).

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The hens were housed in individual wire cages under continuous lighting at 22° C. with feed and water available ad libitum. The experimental design was completely randomized. Aflatoxin was produced by growing Aspergillus parasiticus NRRL 2999 on rice according to the method of Shotwell et al. (1966) with the modification of West et al. (1973). The moldy rice was steamed to kill the fungus, dried, and ground to a fine powder which was analyzed for aflatoxin content by the colorimetric method of Nabney and Nesbitt (1965) with the modification of Wiseman et al. (1967). Weighed amounts of the powder to furnish the desired concentrations of aflatoxin were incorporated into the basal ration, which was free of all medications.

1.1a 1.8a 1.8b 4.9b 4.7c

2016

W. E. HUFF, R. D. WYATT AND P. B. HAMILTON

20.0

18.0

5.0 AFLATOXIN (jjgVg.)

10.0

FIG. 1. Total yolk weight of eggs from hens fed graded dose of aflatoxin. Vertical bars on the data points are the standard errors of the means. from the control values at 10 (J-g./g. Because the total egg weight was decreased (Table 2), the yolk weight relative to egg weight was calculated. A similar result was obtained with a significant (P < 0.05) decrease (24%) at 10 fxg./g. The dry weight of the yolk and the lipid content of the yolk were determined (Table 3). Dietary aflatoxin had no significant effect on either character. The viscosity of the yolk appeared to be increased at the higher levels of aflatoxin, but this character was not quantitated. The yolks from hens fed the higher levels

10.0

FIG. 2. Yolk carotenoids in eggs from hens given dietary aflatoxin. Vertical bars on the data points are the standard errors of the means. of aflatoxin appeared yellower than those from the control hens. Since carotenoids are responsible for yolk color (Marusich, 1971; 27.0

TABLE 3.—Response of yolk dry weight and yolk lipid content to dietary aflatoxin' Dose (|J-g-/g-)

0.0 1.25 2.5 5.0 10.0

Yolk dry weight

(%) 49.2 48.8 49.2 50.0 49.6

± ± ± ± ±

Lipid content

(%) 0.6 0.3 0.6 0.7 0.7

29.9 30.6 30.4 30.4 31.3

± ± ± ± ±

0.7 0.3 0.6 0.7 0.3

'Values in the table are the means ± standard errors of the means. Values in a column did not differ significantly (P < 0.05).

8.0 AFLATOXIN (p»/o.)

FIG. 3. Plasma carotenoids in hens fed graded doses of dietary aflatoxin. Vertical bars on the data points are the standard errors of the means.

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5.0 AFLATOXIN (pg./g.)

2017

AFLATOXIN AND EGG YOLKS

Hinton et al, 1974), the carotenoid content of the yolks was measured. There was a significant (P < 0.05) increase in yolk carotenoids at 5.0 and 10.0 jj-g./g. (Fig. 2). Because the immediate source of yolk carotenoids is the blood, the plasma carotenoids were measured (Fig. 3). Their concentration was found to be increased significantly (P < 0.05) at 10 (o-g./g- The liver carotenoids were measured and found to be unaffected, although the extreme variation (about a four fold difference in concentration) from hen to hen could have masked a change.

The results of this experiment combined with those of previous investigations appear to establish the following sequence of events during acute aflatoxicosis in the laying hen. Aflatoxin rapidly causes a liver lesion which results in impaired lipid transport (Tung et al., 1972), depressed fatty acid synthesis (Donaldson et al., 1972) and decreased protein synthesis (Tung et al., 1972). This liver malfunction results in an increase in liver lipid (Table 1) and a decrease in plasma lipids (Table 2) which are produced in the liver and which are precursors of the yolk lipids and proteins (Mclndoe, 1971). Egg production is not decreased as rapidly as plasma proteins and lipids (Garlich et al., 1973) and the hen apparently compensates by producing smaller eggs (Table 2) with a smaller yolk (Fig. 1). Another compensation would appear to be a preferential channeling of the reduced amounts of egg precursors to ova already committed to maturation, which explains the delayed effect of aflatoxin on egg production and which suggests that aflatoxin inhibits the commitment of ova to maturation (Garlich etal., 1973). It is noteworthy that the yolk composition as indicated by yolk dry weight and lipid content were not altered. This suggests that the accumulation of yolk components by the

ACKNOWLEDGMENTS We thank Sharon West, G. Whitaker, and Nancy Goodwin for technical assistance. REFERENCES Bruning, J. L., and B. L. Kintz, 1968. Computational Handbook of Statistics. Scott, Foresman and

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DISCUSSION

ovary is not altered by aflatoxin. The decrease in yolk size (Fig. 1) and the decrease in egg size (Table 2) were an identical 5.4 g. for the aflatoxin level of 10 |xg. / g . This indicates that the decrease in yolk weight accounts for the decrease in egg size and implies a differential effect of aflatoxin on lipid and protein metabolism with lipid metabolism being more sensitive. This implication is supported by the finding that lipid metabolism appears to be more sensitive to aflatoxin than protein metabolism in young broilers (Tung et al., 1972; Tung and Hamilton, unpublished results). Carotenoids are classed as lipids and it seemed paradoxical that they would be increased in the yolk (Fig. 2) and plasma (Fig. 3) while the other lipids were decreased. However, carotenoids are dietary in origin (Marusich, 1971; Hinton et al., 1974) while the other lipids are primarily synthesized de novo in the liver (O'Hea and Leveille, 1969). In a laying hen, the normal fate of carotenoids is to be deposited in the yolk and transported or excreted from the body in eggs. If egg production is decreased (69% in this experiment), the plasma and yolk carotenoids would necessarily increase because the hen continues to eat and absorb carotenoids despite a restriction in the normal route of excretion. Under field conditions it is common for hens out of production to show increased yellowing about the eye (M. Jones, personal communication). It is intriguing that carotenoids, which are an unessential component of eggs, yet so desirable to consumers, can be indicators of pathological conditions.

2018

W. E. HUFF, R. D. WVATT AND P. B. HAMILTON

cacy. Proc. North Carolina Anim. Nutr. Conf. 25: 50-68. Mclndoe, W. M., 1971 Yolk synthesis, p. 1209-1223. In: Physiology and Biochemistry of the Domestic Fowl, D. J. Bell and B. M. Freeman (ed.), Vol. 3. Academic Press Inc., New York. Nabney, J., and B. F. Nesbitt, 1965. A spectrophotometric method of determining the aflatoxins. Analyst, 90: 155-160. O'Hea, E. K., and G. A. Leveille, 1969. Lipid biosynthesis and transport in the domestic chick (Gallus domesticus). Comp. Biochem. Physiol. 30: 149-159. Shotwell, O. L., C. W. Hesseltine, R. D. Stubblefield and W. G. Sorenson, 1966. Production of aflatoxin on rice. Appl. Microbiol. 14: 425-428. Smith, J. W., and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49: 207-214. Tung, H. T., W. E. Donaldson and P. B. Hamilton, 1972. Altered lipid transport during aflatoxicosis. Toxicol. Appl. Pharmacol. 22: 97-104. West, S., R. D. Wyatt and P. B. Hamilton, 1973. Improved yield of aflatoxin by incremental increases of temperature. Appl. Microbiol. 25: 1018-1019. Wiseman, H. G., W. C. Jacobson and W. C. Harmeyer, 1967. Note on removal of pigments from chloroform extracts of aflatoxin cultures with copper carbonate. J. Assoc. Off. Agr. Chem. 50: 982-983.

NEWS AND NOTES (Continued from page 2006) honored for his generous contributions and support, as was Mr. Glen Norton. Two students at Colorado State have received $500 Pacific Egg and Poultry Association Scholarships— Beverley George and Don Jackson. Craig Godfrey, who will enter C.S.U. this fall, received a $250 scholarship. BACK ISSUES OF POULTRY SCIENCE For Sale Marlow W. Olsen, Box 82, Beltsville, Maryland 20705. Proceedings of the Meetings for the Years 1908, 1909, 1910, International Association of Instructors and Investigators in Poultry Husbandry, Volume 1; Journal of the American Association of Instructors in Poultry Husbandry, Volumes 1 to 7, inclusive (1914-1921) bound; Poultry Science, Volumes 1 to 46, inclusive, bound; and all single issues of Poultry

Science, Volumes 47 to 54, inclusive. Dr. Paul D. Sturkie, Department of Environmental Physiology, Cook College, Thompson Hall, Rutgers University, New Brunswick, New Jersey 08903— Bound Copies—Volume 16 (1937) to Volume 53 (1974), inclusive. ERRATA There are three errors in the "Poultry Science Association, Inc. Records 1970-1974," pages 948 to 951, in the May issue: The CPC International Award in 1972 was to A. W. Nordskog, not A. N. Fellows elected in 1974 should have included J. McGinnis, Washington State University, Pullman, Washington. Under Directors of the Poultry Science Association,

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Company, Glenview, Illinois. Donaldson, W. E„ H. T. Tung and P. B. Hamilton, 1972. Depression of fatty acid synthesis in chick liver (Gallus domesticus) by aflatoxin. Comp. Biochem. Physiol. 41B: 843-847. Friedman, H. S., 1968. Quantitative determination of total lipid in serum. Clin. Chem. Acta, 19: 291-295. Garlich, J. D., H. T. Tung and P. B. Hamilton, 1973. The effects of short term feeding of aflatoxin on egg production and some plasma components of the laying hen. Poultry Sci. 52: 2206-2211. Hamilton, P. B., and J. D. Garlich, 1971. Aflatoxin as possible cause of fatty liver syndrome in laying hens. Poultry Sci. 50: 800-804. Hamilton, P. B., and J. D. Garlich, 1972. Failure of vitamin supplementation to alter the fatty liver syndrome caused by aflatoxin. Poultry Sci. 51: 688-692. Hinton, C. T., J. L. Fry and R. H. Harms, 1974. Influence of a xanthophyll free pullet grower diet on subsequent egg yolk pigmentation. Poultry Sci. 53: 223-226. Kimble, M. S., 1938. The photocolorimetric determination of vitamin A and carotene in human plasma. J. Lab. Clin. Med. 24: 1055-1066. Marusich, W. L., 1971. Evaluation of broiler pigmentation sources and factors affecting their effi-

Effects of dietary aflatoxin on certain egg yolk parameters.

Aflatoxicosis was induced in laying hens by incorporating graded amounts of aflatoxin into a commercial layer ration (0, 1.25, 2.5, 5.0, and 10.0 mug...
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