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Effects of Dietary Aflatoxin in Aflatoxin-Resistant and Control Lines of Chickens1
Effects of Dietary Aflatoxin in Aflatoxin-Resistant and Control Lines of Chickens 1 R. O. MANNING,2 R. D. WYATT,3 H. L. MARKS,3 and O. J. FLETCHER4 The University of Georgia, Athens, Georgia 30602 (Received for publication June 20, 1989)
1990 Poultry Science 69:922-928 INTRODUCTION
Numerous studies have shown that significant differences in resistance to aflatoxin exist between different breeds and strains of chickens (Brown and Abrams, 1965; Camaghan et al., 1967; Gumbmann et al, 1970; Smith and Hamilton, 1970; Washburn et al., 1978; Lanza et al., 1980a, 1982). Marks and Wyatt (1979) used survival of a single oral aflatoxin dose as the criterion of selection to develop lines of aflatoxin resistant Japanese quail. Differences in blood parameters, hepatic enzymes, and the in vivo distribution and disposition of aflatoxin Bi between the lines of Japanese quail were presented in a subsequent report (Pegram, 1986). Because the survivor selection technique used by Marks and Wyatt (1979) allowed rapid genetic progress and provided an excellent model to study aflatoxin toxicity and metabolism in the Japanese quail, a similar technique was used to establish aflatoxinresistant lines of a commercial broiler stock and the Athens-Canadian (AC) broiler popula-
1 Supported by state and Hatch funds allocated to the Georgia Agricultural Experiment Stations of The University of Georgia. Department of Pharmacology and Toxicology. department of Poultry Science. TJepartment of Avian Medicine.
tion (Wyatt et al., 1987). In that study little progress was made in selecting for aflatoxin resistance in the broiler stock, but progress was rapid in the AC population. The purpose of the present study was to further characterize the resistance to a single oral dose of aflatoxin and dietary aflatoxin exposure of the resistant and control, AC broiler lines. MATERIALS AND METHODS
Chicks used in the present study were sixth generation progeny from lines of AC chickens selected for five generations for resistance to a single oral dose of aflatoxin (AR line) or maintained as an unselected control line (C line) as described by Wyatt et al. (1987). Dayold chicks were placed in electrically heated batteries with raised wire floors under continuous illumination. Equal numbers of males and females were used for all determinations in the study, but chicks were separated into replicates of 10 chicks of the same line and sex per pen, and replicates were distributed randomly throughout the batteries. The University of Georgia (Athens, GA) standard, unmedicated, broiler-starter ration (aflatoxin-free) served as the basal ration. Aflatoxin contaminated rice powder (Marks and Wyatt, 1979) was incorporated into the basal diet to attain dietary aflatoxin concentrations of 0 and 2.5 mg per
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ABSTRACT The resistance to a single oral dose (12 mg of aflatoxin per kg of BW) and 4 wk of dietary aflatoxin (2.5 mg per kg of feed) were investigated in chicks selected for five generations for resistance to acute aflatoxicosis (AR) and unselected control (C) chicks. The AR chicks were more resistant to a single oral dose of aflatoxin and had significantly decreased sodium pentobarbital sleeping time compared to C chicks. Four-weeks exposure to dietary aflatoxin did not result in any significant change in BW or feed conversion ratios of chicks from either the C or AR line. However, more sensitive indicators of aflatoxicosis including plasma total protein, albumin, cholesterol concentrations, and gamma glutamyl transferase activity were significantly altered in C chicks but not in AR chicks fed aflatoxin. Percentages of liver lipid and liver hyperplasia score were also significantly altered as a result of dietary aflatoxin treatment in C but not AR chicks. These data indicate that selection-associated differences exist between the C and AR lines of chickens that convey resistance to not only a single oral dose of aflatoxin but also to a more chronic dietary exposure to aflatoxin. (Key words: aflatoxin, resistance, selection, blood parameters, liver lipids)
GENETIC SELECTION AND AFLATOXICOSIS
Collected liver tissues were embedded in paraffin and sections were cut and stained with hematoxylin and eosin as described by Luna (1960). Coded sections were evaluated by light microscopy and graded on 13 criteria. Criteria indicating inflammation were fibrin, heterophils, lymphocytes, giant cells, and fibrosis. Criteria related to cellular change included vacuoles and necrosis as indicators of cellular degeneration and death. Hyperplasia, metaplasia, atrophy, and neoplasia were scored as indicators of alterations in cellular growth. Sections were also scored for parasites and mineral deposits. Scores for each criterion were recorded as: 1 = absence of lesion; 2 = focal; 3 = multiple foci; 4 = diffuse. Individual criterion scores for each slide were summed to obtain a total lesion score per slide. The total lesion scores were averaged for all criteria. Scores for each criterion were also averaged within treatments and lines. Using this system the minimum and maximum scores possible for total lesions were 13 and 52, respectively,
Pathologists' Service Professional Associates, Inc., Atlanta, GA 30340: SMAC II, Technicon Instruments, Tarrytown, NJ 07018.
and for individual criteria were 1 and 4, respectively. Sodium pentobarbital sleeping-time was determined for 30 chicks from each line; the chicks were maintained on the basal diet but were not used in the feeding study. The values were obtained when the chicks were 27 days of age. A single injection of sodium pentobarbital (25 mg per kg of BW) was administered via the left wing vein of each bird and time was measured from the injection until the bird could right itself. As an indication of the resistance of the two different lines to an acute aflatoxin stress, 40 chicks from each line fed the basal diet were administered a single oral dose of aflatoxin (12 mg per kg of BW) on Day 30 and mortality was recorded for 7 days. Body weight of the survivors was also measured four days postdosing. Differences between sexes of the same line were not apparent for the parameters measured in the present study. Therefore, all data for males and females of the same line were combined. Mortality values were analyzed using a chi-square test with the appropriate contingency tables (Bhattacharyya and Johnson, 1977). Nonparametric histological data were compared using a Kruskal-Wallis test and the means were partitioned using a MannWhitney test as described by Zar (1974). All other data were compared by a two-way ANOVA and the means partitioned using a t test for differences among several means as described by Bruning and Kintz (1968). Statements of significance are based on P<.05. RESULTS
Body weight on the day of hatching was significantly greater for chicks of the C line than the AR line (Table 1). There was a significant line effect evident during the first 2 wk of the study, but by Day 21 it was no longer apparent. Dietary aflatoxin (2.5 mg per kg of feed) did not cause a significant decrease in BW for either the C or AR line during the first 3 wk of the experiment. However, by the end of the 28-day trial there was a significant aflatoxin effect. There were no significant differences in feed conversion ratios between lines fed the basal diet or as a result of dietary aflatoxin treatment during the 4-wk study. Feed conversion ratios ranged from 1.52 ± .02 (x ± SEM) during the first week of the study to
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kg of feed. The diets were available ad libitum for 4 wk to 4 replicates (2 male and 2 female) of 10 chicks from each line. A total of 160 birds were used in the feeding study. Individual BW and replicate feed consumption were measured weekly. On Day 28, a 3-mL blood sample was obtained via cardiac puncture and placed into heparinized tubes in an ice bath. All birds were killed by cervical dislocation and the liver was excised, blotted, and weighed. Total liver lipid was determined as described by Smith and Hamilton (1970). Liver samples were taken from 5 individual birds of each line, sex, and dietary treatment and fixed by immersion in 10% buffered formalin for 3 days. Individual plasma samples were obtained from heparinized blood by low speed centrifugation and combined into four replicate pools (10 birds per pool) by line, sex, and dietary treatment. Pooled plasma samples were analyzed for total protein, albumin, globulin, glucose, cholesterol, uric acid, Fe, Ca, P, Na, and K concentrations as well as glutamic-oxaloacetic transaminase and gamma glutamyl transferase activity by an independent laboratory5 The procedures which were used followed guidelines for human analyses.
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MANNING ET AL.
TABLE 1. The effect of dietary aflatoxin on body weight of unselected control (C) and aflatoxin-resistant (AR) chicks with ANOVA summary
Line
c c
Body weight1
Dietary aflatoxin
0 days
(mg per kg) .0 2.5 .0 2.5
37.4 36.3 30.5 30.0
.8 a .6* .3 b .4 b
88 87 74 72
± ± ± ±
2a la 2b 2b
14 days (£) — 166 ± 5 a 157 ± 3* 146 ± 5 1 * 139 ± 5 b - Probability
<0001 .2502 .8825
<0001 .0421 .1974
21 days 250 234 234 214
.0010 .1033 .7877
± 15a ± -jab ± pab ±
.1079 .0993 .8108
28 days 336 305 323 287
± ± ± ±
.3303 .0488 .8727
a,b
Means ± SEM within the same column with no common superscripts are significantly different (P<.05). 'Values are means of four replicates of 10 chicks.
3.94 ± .07 during the last week. Only one Na, and K concentrations (data not shown). chick died during the study: the chick died There were no significant differences in during the last week of the study and was a C relative liver weight between the two lines female fed aflatoxin. with or without dietary aflatoxin treatment There were significant line effects in pro- (Table 4). There were significant aflatoxin tein, albumin, iron, Ca, and P concentrations as effects and interactions on liver lipid and liver well as gamma glutamyl transferase activity dry matter. Chicks of the AR line fed the basal (Tables 2 and 3). Significant aflatoxin effects diet had a significantly higher percentage of were evident in concentrations of total protein, liver lipid than did chicks of the NS line. albumin, and Ca only. Significant interactions Dietary aflatoxin resulted in a significant were observed for albumin and blood Ca increase in liver lipid of C chicks but not AR concentrations, and gamma glutamyl transfer- chicks. Liver dry matter did not differ between ase activity. There were no line, treatment, or the lines fed the basal diet, and dietary interaction effects for glutamic oxaloacetic aflatoxin resulted in a significant decrease in transaminase activity (Table 3), or uric acid, liver dry matter for C chicks only.
TABLE 2. The effect of dietary aflatoxin on blood parameters of unselected control (C) and aflatoxin-resistant (AR) chicks with ANOVA summary
Line
Dietary aflatoxin
(mg per kg) C .0 C 2.5 AR .0 AR 2.5 ANOVA summary Source of variation Line Aflatoxin level Interaction
Parameters1 Total protein
2.8 2.3 2.9 2.8
± ± ± ±
.0034 .0089 .0942
.la .lb .la .la
Albumin
1.3 1.0 1.4 1.3
± ± ± ±
Globulin
(g per 100 mL) .0* 1.5 ± . tab .lb 1.2 ± . .0 s 1.6 ± . .0" 1.5 ± .
.0011 .0011 .0152
.0501 .1284 .4301
Glucose
263 253 265 268
± ± ± ±
51:ab 6b 4ab 4a
.0934 .5322 .1735
Cholesterol (mg per 100 mL) 111 ± 4 a 97 ± lb 110 ± 4 a 112 ± 5a
.0965 .1493 .0612
^TOeans ± SEM within the same column with no common superscripts are significantly different (P<05). Values are means of individual assays of four different pooled (10 birds per pool) samples.
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AR AR ANOVA summary Source of variation Line Aflatoxin level Interaction
± ± ± ±
7 days
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GENETIC SELECTION AND AFLATOXICOSIS
TABLE 3. The effect of dietary aflatoxin on blood parameters of unselected control (C) and aflatoxin-resistant (AR) chicks with ANOVA summary Parameters
Line
Dietary aflatoxin
Iron
(mg per kg)
(Mg per 100 mL) 108 ± 3** 09
±
3 bc
121 ± 4 a 116 ± 4 a b
.0012 .0825 .5396
Phosphorus (mg per 100 mL)
10.4 .1* .l b 9.8 10.4 ± .l a 10.3 ± .1*
6.7 6.6 7.0 6.8
(U per L) —
± ab ± .l b ± .1* ± .2 a
— Probability .0394 .1400 .7219
.0435 .0020 .0438
Gamma glutamyl transferase
182 195 196 194
5507 .4657 .4497
± ± ± ±
3a 6a 10" 13a
29 32 28 27
D
±0 ±0° ± 1be ±0°
<0001 .0826 .0043
"Means ± SEM within the same column with no common superscripts are significantly different (P<05). ^Values are means of individual assays of four different pooled (10 birds per pool) samples.
There were no significant differences between lines fed the basal diet for any of the histological criteria measured in this study. Between the lines fed aflatoxin there were also no significant differences in fibrin, giant cells, fibrosis, necrosis, metaplasia, atrophy, neoplasia, parasites, or mineral deposits. Dietary aflatoxin treatment did not affect heterophils and lymphocytes (Table 5). Variolation was increased significantly after aflatoxin treatment. Hyperplasia was increased significantly as a result of dietary aflatoxin in C chicks but not AR chicks. The total lesion score was increased significantly after aflatoxin treatment
in both lines, but the increase was greater in C chicks than in AR chicks. Sodium pentobarbital sleeping time, measured in chicks fed the basal diet for 27 days, was significantly longer for C chicks than AR chicks (Table 6). Predose BW of chicks fed the basal diet for 30 days was greater for C chicks than for AR chicks. Four days after a single oral dose of aflatoxin (12 mg per kg of BW) there was a significant difference in weight gain between the lines. The C survivors had lost weight whereas AR survivors had continued to gain weight. Between the lines, the difference in mortality after a single oral dose
TABLE 4. 77ie effect of dietary aflatoxin on liver weight, liver lipid, and liver dry matter of unselected control (C) and aflatoxin resistant (AR) chicks with ANOVA summary'
Line
Dietary aflatoxin
Liver weight
C C AR AR
(mg per kg) .0 2.5 .0 2.5
2.41 2.37 2.35 2.37
(g per 100 g BW) ± ± ± ±
.071>a .08° .09" .08a
Liver lipid
Liver dry matter
(% of dry wt) 12.7 ± .7° 18.9 ± .7 a 15.0 ± .3 b 15.5 ± .3 b
(%) 35.1 29.0 32.0 32.0
± 1.8* ± .7 b ± 2th ± 2a>
ANOVA summary Line Aflatoxin level Interaction a,b
.7121 .9268 .6889
.3265 <0001 .0002
.8934 .0277 .0107
Means ± SEM within the same column with no common superscripts are significantly different (P<.05). Values are means of four replicates of 10 chicks each.
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C .0 C 2.5 AR .0 AR 2.5 ANOVA summary Source of variation Line Aflatoxin level Interaction
Calcium
Glutamic oxaloacetic transaminase
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MANNING ET AL.
TABLE 5. The effect of dietary qflatoxin on liver histological criteria of unselected control (C) and aflatoxinresistant (AR) chickr
Line
c c AR AR
Dietary anatoxin
Heterophils
Lymphocytes
(mg per kg) .0 2.5 .0 2.5
1.3 2.1 1.3 1.9
1.9 2.4 1.8 2.0
± ± ± ±
.2* .4a .2a .2*
± ± ± ±
.l a .2a .l a .l a
Vacuoles 1.2 3.6 1.5 2.9
± ± ± ±
.2 b .2 a .2 b .3 a
Hyperplasia
1.8 3.1 2.0 2.1
± .2b ± .3 a ± jab ± 2 ab
Total lesion score
15.3 20.2 15.7 17.9
± ± ± ±
.3C .7a
.5°b .4
a-c
Means ± SEM within the same column with no common superscripts are significantly different (P<05). 1 Values are means of lesion scores of 10 chicks.
TABLE 6. Mortality from acute aflatoxicosis1 and sodium pentobarbital? sleeping time of unselected control (C) and qflatoxin-resistant (AR) chicks
Lline
Sleeping time
C AR
(min) 48 ± 2 a 37 ± 2 b
Predose body weight
380 ± l l a 327 ± 14b
Postdose weight gain (4 days) (g) -12.0 ± 9.8b +19.3 ± 1.8*
Mortality (no. dead per no. dosed) 14/40* l/40 b
^"•Means ± SEM or mortality values within the same column with no common superscripts are significantly different (P<05). Single oral dose of 12 mg of aflatoxin per kg of BW on Day 30. Intravenous injection of 25 mg of sodium pentobarbital per kg of BW on Day 27. 3 Sleeping time determined on 30 chicks per line and BW determined on 4 replicates of 10 chicks per line.
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appears that there was a difference in the relative aflatoxin resistance between lines of the fifth and sixth generation progeny. The aflatoxin challenges in the previous study were DISCUSSION administered to chicks 14 days of age and in Mortality values of approximately 35% and the present study to chicks 30 days of age. 3% for C and AR chicks, respectively, indicate Therefore, the relative differences in aflatoxin that there was a large line difference in the resistance between the lines from the fifth and resistance of the sixth generation of progeny to sixth generation stock may be age-related or a single dose of aflatoxin. A direct comparison due to environmental variation between expericannot be made between mortality values ments or both. The primary site of aflatoxin metabolism resulting from the single oral dose of aflatoxin in the present study and the LD50 values and biotransformation is the microsomal reported for fifth generation progeny of the C mono-oxygenase system (MMO) of the liver and AR lines by Wyatt et al. (1987), because (Busby and Wogan, 1981). Sodium pentobarthose values were calculated from six different bital sleeping time is often used as an indicator doses of aflatoxin administered to each line. of the ability of the MMO system to metaboHowever, mortality values of 63% and 30% lize xenobiotics or drugs (Domer, 1971). for the C and AR lines, respectively, were Decreased sodium pentobarbital sleeping-time obtained when fifth generation progeny were response and increased cytochrome P-450 administered 12 mg of aflatoxin per kg of BW content were observed in chicks that were (Wyatt et al, 1987). From these data, it resistant to acute aflatoxicosis as a result of of aflatoxin was large, with the most mortality occurring in the C line.
GENETIC SELECTION AND AFLATOXICOSIS
Decreased feed consumption and BW are considered to be secondary effects of aflatoxin, whereas disruption of carbohydrate and lipid metabolism, and inhibition of protein synthesis are considered to be primary effects (Hsieh, 1979). Several of the more sensitive indicators of aflatoxicosis measured in the present study indicated a large difference in the response of C and AR chicks to dietary aflatoxin. Differences in circulating total protein and albumin of C and AR chicks, measured as indicators of protein synthesis (Tung et al., 1975), indicated that AR chicks were more resistant to inhibi-
tion of protein synthesis than were C chicks. The accumulation of lipid in liver, a result of altered transport, and the inhibition of cholesterol synthesis are considered to be two of the most sensitive indicators of aflatoxicosis in chickens (Donaldson et al., 1972; Tung et ah, 1972). In the present study, circulating cholesterol was decreased and percent liver lipid increased as a result of dietary aflatoxin only in C chicks, suggesting that AR chicks were resistant to alterations of lipid metabolism caused by aflatoxin. Certain histological criteria including vacuoles, hyperplasia, and total lesion score, as well as gamma glutamyl transferase activity, indicated that the extent of hepatic injury in AR chicks consuming dietary aflatoxin was less severe than in C chicks. Portman et al. (1970) studied the rate of aflatoxin transport into rat and mouse liver hepatocytes and concluded that the rate of cellular transport was a considerably more important aspect of the pathological effects of aflatoxin than was the subsequent rate of metabolism. Decreased cellular transport of aflatoxin into hepatocytes, independent of any alterations in hepatic metabolic capabilities, could play a role in the decreased hepatic injury observed in AR chicks, but no study of cellular uptake of aflatoxin in the NS and AR chicks has been conducted thus far. From these results, it appears that there is a significant selection-related difference in the resistance of the C and AR lines to dietary aflatoxin. Recent studies with temperature acclimation (Manning and Wyatt, 1990; Manning et al., 1990) have shown an increase in the resistance of cold-acclimated broiler chicks to a single oral dose of aflatoxin but not to dietary aflatoxin. In contrast to those reports, the present findings are particularly interesting: namely, that five generations of selection for resistance to a single oral dose of aflatoxin have resulted in a line of chicks resistant to not only an acute exposure but also to the more chronic dietary exposure to aflatoxin. The mechanism(s) responsible for the resistance of AR chicks to aflatoxin cannot be determined from the present work. REFERENCES Bhattacharyya, G. K., and R. A. Johnson, 1977. Statistical Concepts and Methods. John Wiley and Sons, Inc., New York, NY. Brown, J.M.M., and L. Abrams, 1965. Biochemical studies on aflatoxicosis. Onderstepoort J. Vet. Res. 32: 119-145.
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cold acclimation (Manning and Wyatt, 1989), and the authors suggested that the resistance was due to a cold-induced stimulation of the MMO system via increased protein synthesis. Increased cytochrome P-450 content and altered aflatoxin metabolism have also been reported in Japanese quail selected for resistance to acute aflatoxicosis (Pegram, 1986). In the present study, sodium pentobarbital sleeping time of C chicks was significantly longer (30%) than that of AR chicks, which suggests that altered hepatic metabolism of aflatoxin may be associated with the resistance of the AR line to acute aflatoxicosis. However, because sodium pentobarbital sleeping-timeresponse was the only indicator of MMO activity measured in the present study, the authors cannot hypothesize whether increased aflatoxin resistance of the AR line is related to an alteration in the content or activity of MMO enzymes. Body weight data collected during the aflatoxin feeding trial indicated that five generations of selection for resistance to acute aflatoxicosis resulted in a significant decrease in BW of the chicks at hatching. This may have been due to egg size, or the age of breeders, or both. Body weight and feed conversion ratios also indicated that there was no significant effect of 2.5 mg of aflatoxin per kg of feed in chicks of either line. These findings are surprising, because this dietary concentration of aflatoxin has been well documented to result in significant growth depression and increased feed conversion ratios when fed to broiler chicks (Smith and Hamilton, 1970; Lanza et al, 1980b). The lack of an effect significant on BW of C and AR chicks fed 2.5 mg of aflatoxin per kg of feed in the present study indicates that these lines are more resistant to dietary aflatoxin than commercial-type broiler chicks.
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MANNING ET AL. Genet. 63:207-212. Luna, L., ed., 1960. Armed Forces Institute of Pathology Manual of Histologic and Special Staining Technics. McGraw-Hill Inc., New York, NY. Manning, R. O., and R. D. Wyatt, 1990. Effect of cold acclimation on the broiler chicks' resistance to acute aflatoxicosis. Poultry Sci. 69:388-396. Manning, R. O., R. D. Wyatt, and O. J. Fletcher, 1990. Effect of cold acclimation on the broiler chicks' resistance to dietary aflatoxin. Poultry Sci. 69: (in press). Marks, H. L., and R. D. Wyatt, 1979. Generic resistance to aflatoxin in Japanese quail. Science 206:1329-1330. Pegram, R. A., 1986. Characterization of genetic resistance to aflatoxin in Japanese quail. Ph.D. diss., The University of Georgia, Athens, GA. Portman, R. S., K. M. Plowman, and T. C. Campbell, 1970. On mechanisms affecting species susceptibility to aflatoxin. Biochem. Biophys. Acta 208:487-495. Smith, J. W„ and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49:207-215. Tung, H. T., W. E. Donaldson, and P. B. Hamilton, 1972. Altered lipid transport during aflatoxicosis. Toxicol. Appl. Pharmacol. 22:97-104. Tung, H. T., R. D. Wyatt, P. Thaxton, and P. B. Hamilton, 1975. Concentrations of serum proteins during aflatoxicosis. Toxicol. Appl. Pharmacol. 34:320-326. Washburn, K. W., G. M. Lanza, and R. D. Wyatt, 1978. Selection procedures for developing genetic resistance to dietary aflatoxin. World's Poult. Sci. J. 10: 1773-1778. Wyatt, R. D., R. O. Manning, and H. L. Marks, 1987. Selection for resistance to aflatoxin in chickens. Poultry Sci. 66:1901-1904. Zar, J. H„ 1974. Biostatistical Analysis. Prentice-Hall Inc., Englewood Cliffs, NJ.
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Bruiting, J. L., and B. L. Kintz, 1968. Computational Handbook of Statistics. Scott, Foresman and Company, Glenview, IL. Busby, W. F., Jr., and G. N. Wogan, 1981. Aflatoxins. Pages 3-27 in: Mycotoxins and N-Nitroso Compounds: Environmental Risks, Vol. 1. R. C. Shank, ed. CRC Press, Boca Raton, FL. Carnaghan, R B A , C. N. Herber, D.S.P. Patterson, and D. Sweasy, 1967. Comparative biological and biochemical studies in hybrid chicks. 2. Susceptibility to aflatoxin and effects on serum protein constituents. Br. Poult. Sci. 8:279-284. Domer, F. R., 1971. Animal Experiments in Pharmacological Analysis. Charles C. Thomas, Publisher, Springfield, JL. 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. 41:843-847. Gumbmann, M. R., S. N. Williams, A. N. Booth, P. Vohra, R. A. Ernst, and M. Bethard, 1970. Aflatoxin susceptibility in various breeds of poultry. Fed. Proc. 134: 683-688. Hsieh, DJ\H., 1979. Basic metabolic effects of mycotoxins. Pages 43-55 in: Interaction of Mycotoxins in Animal Production. Natl. Acad. Sci., Washington, DC. Lanza, G. M., K. W. Washburn, and R. D. Wyatt, 1980a. Strain variation in hematological response of broilers to dietary aflatoxin. Poultry Sci. 59:2686-2691. Lanza, G. M., K. W. Washburn, and R. D. Wyatt, 1980b. Variation with age in response of broilers to aflatoxin. Poultry Sci. 59:282-288. Lanza, G. M., K. W. Washburn, R. D. Wyatt, and H. L. Marks, 1982. Genetic variation of physiological response to aflatoxin in Gallus domesticus. Theor. Appl.
1990 Poultry Science 69:922-928 INTRODUCTION
Numerous studies have shown that significant differences in resistance to aflatoxin exist between different breeds and strains of chickens (Brown and Abrams, 1965; Camaghan et al., 1967; Gumbmann et al, 1970; Smith and Hamilton, 1970; Washburn et al., 1978; Lanza et al., 1980a, 1982). Marks and Wyatt (1979) used survival of a single oral aflatoxin dose as the criterion of selection to develop lines of aflatoxin resistant Japanese quail. Differences in blood parameters, hepatic enzymes, and the in vivo distribution and disposition of aflatoxin Bi between the lines of Japanese quail were presented in a subsequent report (Pegram, 1986). Because the survivor selection technique used by Marks and Wyatt (1979) allowed rapid genetic progress and provided an excellent model to study aflatoxin toxicity and metabolism in the Japanese quail, a similar technique was used to establish aflatoxinresistant lines of a commercial broiler stock and the Athens-Canadian (AC) broiler popula-
1 Supported by state and Hatch funds allocated to the Georgia Agricultural Experiment Stations of The University of Georgia. Department of Pharmacology and Toxicology. department of Poultry Science. TJepartment of Avian Medicine.
tion (Wyatt et al., 1987). In that study little progress was made in selecting for aflatoxin resistance in the broiler stock, but progress was rapid in the AC population. The purpose of the present study was to further characterize the resistance to a single oral dose of aflatoxin and dietary aflatoxin exposure of the resistant and control, AC broiler lines. MATERIALS AND METHODS
Chicks used in the present study were sixth generation progeny from lines of AC chickens selected for five generations for resistance to a single oral dose of aflatoxin (AR line) or maintained as an unselected control line (C line) as described by Wyatt et al. (1987). Dayold chicks were placed in electrically heated batteries with raised wire floors under continuous illumination. Equal numbers of males and females were used for all determinations in the study, but chicks were separated into replicates of 10 chicks of the same line and sex per pen, and replicates were distributed randomly throughout the batteries. The University of Georgia (Athens, GA) standard, unmedicated, broiler-starter ration (aflatoxin-free) served as the basal ration. Aflatoxin contaminated rice powder (Marks and Wyatt, 1979) was incorporated into the basal diet to attain dietary aflatoxin concentrations of 0 and 2.5 mg per
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ABSTRACT The resistance to a single oral dose (12 mg of aflatoxin per kg of BW) and 4 wk of dietary aflatoxin (2.5 mg per kg of feed) were investigated in chicks selected for five generations for resistance to acute aflatoxicosis (AR) and unselected control (C) chicks. The AR chicks were more resistant to a single oral dose of aflatoxin and had significantly decreased sodium pentobarbital sleeping time compared to C chicks. Four-weeks exposure to dietary aflatoxin did not result in any significant change in BW or feed conversion ratios of chicks from either the C or AR line. However, more sensitive indicators of aflatoxicosis including plasma total protein, albumin, cholesterol concentrations, and gamma glutamyl transferase activity were significantly altered in C chicks but not in AR chicks fed aflatoxin. Percentages of liver lipid and liver hyperplasia score were also significantly altered as a result of dietary aflatoxin treatment in C but not AR chicks. These data indicate that selection-associated differences exist between the C and AR lines of chickens that convey resistance to not only a single oral dose of aflatoxin but also to a more chronic dietary exposure to aflatoxin. (Key words: aflatoxin, resistance, selection, blood parameters, liver lipids)
GENETIC SELECTION AND AFLATOXICOSIS
Collected liver tissues were embedded in paraffin and sections were cut and stained with hematoxylin and eosin as described by Luna (1960). Coded sections were evaluated by light microscopy and graded on 13 criteria. Criteria indicating inflammation were fibrin, heterophils, lymphocytes, giant cells, and fibrosis. Criteria related to cellular change included vacuoles and necrosis as indicators of cellular degeneration and death. Hyperplasia, metaplasia, atrophy, and neoplasia were scored as indicators of alterations in cellular growth. Sections were also scored for parasites and mineral deposits. Scores for each criterion were recorded as: 1 = absence of lesion; 2 = focal; 3 = multiple foci; 4 = diffuse. Individual criterion scores for each slide were summed to obtain a total lesion score per slide. The total lesion scores were averaged for all criteria. Scores for each criterion were also averaged within treatments and lines. Using this system the minimum and maximum scores possible for total lesions were 13 and 52, respectively,
Pathologists' Service Professional Associates, Inc., Atlanta, GA 30340: SMAC II, Technicon Instruments, Tarrytown, NJ 07018.
and for individual criteria were 1 and 4, respectively. Sodium pentobarbital sleeping-time was determined for 30 chicks from each line; the chicks were maintained on the basal diet but were not used in the feeding study. The values were obtained when the chicks were 27 days of age. A single injection of sodium pentobarbital (25 mg per kg of BW) was administered via the left wing vein of each bird and time was measured from the injection until the bird could right itself. As an indication of the resistance of the two different lines to an acute aflatoxin stress, 40 chicks from each line fed the basal diet were administered a single oral dose of aflatoxin (12 mg per kg of BW) on Day 30 and mortality was recorded for 7 days. Body weight of the survivors was also measured four days postdosing. Differences between sexes of the same line were not apparent for the parameters measured in the present study. Therefore, all data for males and females of the same line were combined. Mortality values were analyzed using a chi-square test with the appropriate contingency tables (Bhattacharyya and Johnson, 1977). Nonparametric histological data were compared using a Kruskal-Wallis test and the means were partitioned using a MannWhitney test as described by Zar (1974). All other data were compared by a two-way ANOVA and the means partitioned using a t test for differences among several means as described by Bruning and Kintz (1968). Statements of significance are based on P<.05. RESULTS
Body weight on the day of hatching was significantly greater for chicks of the C line than the AR line (Table 1). There was a significant line effect evident during the first 2 wk of the study, but by Day 21 it was no longer apparent. Dietary aflatoxin (2.5 mg per kg of feed) did not cause a significant decrease in BW for either the C or AR line during the first 3 wk of the experiment. However, by the end of the 28-day trial there was a significant aflatoxin effect. There were no significant differences in feed conversion ratios between lines fed the basal diet or as a result of dietary aflatoxin treatment during the 4-wk study. Feed conversion ratios ranged from 1.52 ± .02 (x ± SEM) during the first week of the study to
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kg of feed. The diets were available ad libitum for 4 wk to 4 replicates (2 male and 2 female) of 10 chicks from each line. A total of 160 birds were used in the feeding study. Individual BW and replicate feed consumption were measured weekly. On Day 28, a 3-mL blood sample was obtained via cardiac puncture and placed into heparinized tubes in an ice bath. All birds were killed by cervical dislocation and the liver was excised, blotted, and weighed. Total liver lipid was determined as described by Smith and Hamilton (1970). Liver samples were taken from 5 individual birds of each line, sex, and dietary treatment and fixed by immersion in 10% buffered formalin for 3 days. Individual plasma samples were obtained from heparinized blood by low speed centrifugation and combined into four replicate pools (10 birds per pool) by line, sex, and dietary treatment. Pooled plasma samples were analyzed for total protein, albumin, globulin, glucose, cholesterol, uric acid, Fe, Ca, P, Na, and K concentrations as well as glutamic-oxaloacetic transaminase and gamma glutamyl transferase activity by an independent laboratory5 The procedures which were used followed guidelines for human analyses.
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MANNING ET AL.
TABLE 1. The effect of dietary aflatoxin on body weight of unselected control (C) and aflatoxin-resistant (AR) chicks with ANOVA summary
Line
c c
Body weight1
Dietary aflatoxin
0 days
(mg per kg) .0 2.5 .0 2.5
37.4 36.3 30.5 30.0
.8 a .6* .3 b .4 b
88 87 74 72
± ± ± ±
2a la 2b 2b
14 days (£) — 166 ± 5 a 157 ± 3* 146 ± 5 1 * 139 ± 5 b - Probability
<0001 .2502 .8825
<0001 .0421 .1974
21 days 250 234 234 214
.0010 .1033 .7877
± 15a ± -jab ± pab ±
.1079 .0993 .8108
28 days 336 305 323 287
± ± ± ±
.3303 .0488 .8727
a,b
Means ± SEM within the same column with no common superscripts are significantly different (P<.05). 'Values are means of four replicates of 10 chicks.
3.94 ± .07 during the last week. Only one Na, and K concentrations (data not shown). chick died during the study: the chick died There were no significant differences in during the last week of the study and was a C relative liver weight between the two lines female fed aflatoxin. with or without dietary aflatoxin treatment There were significant line effects in pro- (Table 4). There were significant aflatoxin tein, albumin, iron, Ca, and P concentrations as effects and interactions on liver lipid and liver well as gamma glutamyl transferase activity dry matter. Chicks of the AR line fed the basal (Tables 2 and 3). Significant aflatoxin effects diet had a significantly higher percentage of were evident in concentrations of total protein, liver lipid than did chicks of the NS line. albumin, and Ca only. Significant interactions Dietary aflatoxin resulted in a significant were observed for albumin and blood Ca increase in liver lipid of C chicks but not AR concentrations, and gamma glutamyl transfer- chicks. Liver dry matter did not differ between ase activity. There were no line, treatment, or the lines fed the basal diet, and dietary interaction effects for glutamic oxaloacetic aflatoxin resulted in a significant decrease in transaminase activity (Table 3), or uric acid, liver dry matter for C chicks only.
TABLE 2. The effect of dietary aflatoxin on blood parameters of unselected control (C) and aflatoxin-resistant (AR) chicks with ANOVA summary
Line
Dietary aflatoxin
(mg per kg) C .0 C 2.5 AR .0 AR 2.5 ANOVA summary Source of variation Line Aflatoxin level Interaction
Parameters1 Total protein
2.8 2.3 2.9 2.8
± ± ± ±
.0034 .0089 .0942
.la .lb .la .la
Albumin
1.3 1.0 1.4 1.3
± ± ± ±
Globulin
(g per 100 mL) .0* 1.5 ± . tab .lb 1.2 ± . .0 s 1.6 ± . .0" 1.5 ± .
.0011 .0011 .0152
.0501 .1284 .4301
Glucose
263 253 265 268
± ± ± ±
51:ab 6b 4ab 4a
.0934 .5322 .1735
Cholesterol (mg per 100 mL) 111 ± 4 a 97 ± lb 110 ± 4 a 112 ± 5a
.0965 .1493 .0612
^TOeans ± SEM within the same column with no common superscripts are significantly different (P<05). Values are means of individual assays of four different pooled (10 birds per pool) samples.
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AR AR ANOVA summary Source of variation Line Aflatoxin level Interaction
± ± ± ±
7 days
925
GENETIC SELECTION AND AFLATOXICOSIS
TABLE 3. The effect of dietary aflatoxin on blood parameters of unselected control (C) and aflatoxin-resistant (AR) chicks with ANOVA summary Parameters
Line
Dietary aflatoxin
Iron
(mg per kg)
(Mg per 100 mL) 108 ± 3** 09
±
3 bc
121 ± 4 a 116 ± 4 a b
.0012 .0825 .5396
Phosphorus (mg per 100 mL)
10.4 .1* .l b 9.8 10.4 ± .l a 10.3 ± .1*
6.7 6.6 7.0 6.8
(U per L) —
± ab ± .l b ± .1* ± .2 a
— Probability .0394 .1400 .7219
.0435 .0020 .0438
Gamma glutamyl transferase
182 195 196 194
5507 .4657 .4497
± ± ± ±
3a 6a 10" 13a
29 32 28 27
D
±0 ±0° ± 1be ±0°
<0001 .0826 .0043
"Means ± SEM within the same column with no common superscripts are significantly different (P<05). ^Values are means of individual assays of four different pooled (10 birds per pool) samples.
There were no significant differences between lines fed the basal diet for any of the histological criteria measured in this study. Between the lines fed aflatoxin there were also no significant differences in fibrin, giant cells, fibrosis, necrosis, metaplasia, atrophy, neoplasia, parasites, or mineral deposits. Dietary aflatoxin treatment did not affect heterophils and lymphocytes (Table 5). Variolation was increased significantly after aflatoxin treatment. Hyperplasia was increased significantly as a result of dietary aflatoxin in C chicks but not AR chicks. The total lesion score was increased significantly after aflatoxin treatment
in both lines, but the increase was greater in C chicks than in AR chicks. Sodium pentobarbital sleeping time, measured in chicks fed the basal diet for 27 days, was significantly longer for C chicks than AR chicks (Table 6). Predose BW of chicks fed the basal diet for 30 days was greater for C chicks than for AR chicks. Four days after a single oral dose of aflatoxin (12 mg per kg of BW) there was a significant difference in weight gain between the lines. The C survivors had lost weight whereas AR survivors had continued to gain weight. Between the lines, the difference in mortality after a single oral dose
TABLE 4. 77ie effect of dietary aflatoxin on liver weight, liver lipid, and liver dry matter of unselected control (C) and aflatoxin resistant (AR) chicks with ANOVA summary'
Line
Dietary aflatoxin
Liver weight
C C AR AR
(mg per kg) .0 2.5 .0 2.5
2.41 2.37 2.35 2.37
(g per 100 g BW) ± ± ± ±
.071>a .08° .09" .08a
Liver lipid
Liver dry matter
(% of dry wt) 12.7 ± .7° 18.9 ± .7 a 15.0 ± .3 b 15.5 ± .3 b
(%) 35.1 29.0 32.0 32.0
± 1.8* ± .7 b ± 2th ± 2a>
ANOVA summary Line Aflatoxin level Interaction a,b
.7121 .9268 .6889
.3265 <0001 .0002
.8934 .0277 .0107
Means ± SEM within the same column with no common superscripts are significantly different (P<.05). Values are means of four replicates of 10 chicks each.
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C .0 C 2.5 AR .0 AR 2.5 ANOVA summary Source of variation Line Aflatoxin level Interaction
Calcium
Glutamic oxaloacetic transaminase
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MANNING ET AL.
TABLE 5. The effect of dietary qflatoxin on liver histological criteria of unselected control (C) and aflatoxinresistant (AR) chickr
Line
c c AR AR
Dietary anatoxin
Heterophils
Lymphocytes
(mg per kg) .0 2.5 .0 2.5
1.3 2.1 1.3 1.9
1.9 2.4 1.8 2.0
± ± ± ±
.2* .4a .2a .2*
± ± ± ±
.l a .2a .l a .l a
Vacuoles 1.2 3.6 1.5 2.9
± ± ± ±
.2 b .2 a .2 b .3 a
Hyperplasia
1.8 3.1 2.0 2.1
± .2b ± .3 a ± jab ± 2 ab
Total lesion score
15.3 20.2 15.7 17.9
± ± ± ±
.3C .7a
.5°b .4
a-c
Means ± SEM within the same column with no common superscripts are significantly different (P<05). 1 Values are means of lesion scores of 10 chicks.
TABLE 6. Mortality from acute aflatoxicosis1 and sodium pentobarbital? sleeping time of unselected control (C) and qflatoxin-resistant (AR) chicks
Lline
Sleeping time
C AR
(min) 48 ± 2 a 37 ± 2 b
Predose body weight
380 ± l l a 327 ± 14b
Postdose weight gain (4 days) (g) -12.0 ± 9.8b +19.3 ± 1.8*
Mortality (no. dead per no. dosed) 14/40* l/40 b
^"•Means ± SEM or mortality values within the same column with no common superscripts are significantly different (P<05). Single oral dose of 12 mg of aflatoxin per kg of BW on Day 30. Intravenous injection of 25 mg of sodium pentobarbital per kg of BW on Day 27. 3 Sleeping time determined on 30 chicks per line and BW determined on 4 replicates of 10 chicks per line.
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appears that there was a difference in the relative aflatoxin resistance between lines of the fifth and sixth generation progeny. The aflatoxin challenges in the previous study were DISCUSSION administered to chicks 14 days of age and in Mortality values of approximately 35% and the present study to chicks 30 days of age. 3% for C and AR chicks, respectively, indicate Therefore, the relative differences in aflatoxin that there was a large line difference in the resistance between the lines from the fifth and resistance of the sixth generation of progeny to sixth generation stock may be age-related or a single dose of aflatoxin. A direct comparison due to environmental variation between expericannot be made between mortality values ments or both. The primary site of aflatoxin metabolism resulting from the single oral dose of aflatoxin in the present study and the LD50 values and biotransformation is the microsomal reported for fifth generation progeny of the C mono-oxygenase system (MMO) of the liver and AR lines by Wyatt et al. (1987), because (Busby and Wogan, 1981). Sodium pentobarthose values were calculated from six different bital sleeping time is often used as an indicator doses of aflatoxin administered to each line. of the ability of the MMO system to metaboHowever, mortality values of 63% and 30% lize xenobiotics or drugs (Domer, 1971). for the C and AR lines, respectively, were Decreased sodium pentobarbital sleeping-time obtained when fifth generation progeny were response and increased cytochrome P-450 administered 12 mg of aflatoxin per kg of BW content were observed in chicks that were (Wyatt et al, 1987). From these data, it resistant to acute aflatoxicosis as a result of of aflatoxin was large, with the most mortality occurring in the C line.
GENETIC SELECTION AND AFLATOXICOSIS
Decreased feed consumption and BW are considered to be secondary effects of aflatoxin, whereas disruption of carbohydrate and lipid metabolism, and inhibition of protein synthesis are considered to be primary effects (Hsieh, 1979). Several of the more sensitive indicators of aflatoxicosis measured in the present study indicated a large difference in the response of C and AR chicks to dietary aflatoxin. Differences in circulating total protein and albumin of C and AR chicks, measured as indicators of protein synthesis (Tung et al., 1975), indicated that AR chicks were more resistant to inhibi-
tion of protein synthesis than were C chicks. The accumulation of lipid in liver, a result of altered transport, and the inhibition of cholesterol synthesis are considered to be two of the most sensitive indicators of aflatoxicosis in chickens (Donaldson et al., 1972; Tung et ah, 1972). In the present study, circulating cholesterol was decreased and percent liver lipid increased as a result of dietary aflatoxin only in C chicks, suggesting that AR chicks were resistant to alterations of lipid metabolism caused by aflatoxin. Certain histological criteria including vacuoles, hyperplasia, and total lesion score, as well as gamma glutamyl transferase activity, indicated that the extent of hepatic injury in AR chicks consuming dietary aflatoxin was less severe than in C chicks. Portman et al. (1970) studied the rate of aflatoxin transport into rat and mouse liver hepatocytes and concluded that the rate of cellular transport was a considerably more important aspect of the pathological effects of aflatoxin than was the subsequent rate of metabolism. Decreased cellular transport of aflatoxin into hepatocytes, independent of any alterations in hepatic metabolic capabilities, could play a role in the decreased hepatic injury observed in AR chicks, but no study of cellular uptake of aflatoxin in the NS and AR chicks has been conducted thus far. From these results, it appears that there is a significant selection-related difference in the resistance of the C and AR lines to dietary aflatoxin. Recent studies with temperature acclimation (Manning and Wyatt, 1990; Manning et al., 1990) have shown an increase in the resistance of cold-acclimated broiler chicks to a single oral dose of aflatoxin but not to dietary aflatoxin. In contrast to those reports, the present findings are particularly interesting: namely, that five generations of selection for resistance to a single oral dose of aflatoxin have resulted in a line of chicks resistant to not only an acute exposure but also to the more chronic dietary exposure to aflatoxin. The mechanism(s) responsible for the resistance of AR chicks to aflatoxin cannot be determined from the present work. REFERENCES Bhattacharyya, G. K., and R. A. Johnson, 1977. Statistical Concepts and Methods. John Wiley and Sons, Inc., New York, NY. Brown, J.M.M., and L. Abrams, 1965. Biochemical studies on aflatoxicosis. Onderstepoort J. Vet. Res. 32: 119-145.
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cold acclimation (Manning and Wyatt, 1989), and the authors suggested that the resistance was due to a cold-induced stimulation of the MMO system via increased protein synthesis. Increased cytochrome P-450 content and altered aflatoxin metabolism have also been reported in Japanese quail selected for resistance to acute aflatoxicosis (Pegram, 1986). In the present study, sodium pentobarbital sleeping time of C chicks was significantly longer (30%) than that of AR chicks, which suggests that altered hepatic metabolism of aflatoxin may be associated with the resistance of the AR line to acute aflatoxicosis. However, because sodium pentobarbital sleeping-timeresponse was the only indicator of MMO activity measured in the present study, the authors cannot hypothesize whether increased aflatoxin resistance of the AR line is related to an alteration in the content or activity of MMO enzymes. Body weight data collected during the aflatoxin feeding trial indicated that five generations of selection for resistance to acute aflatoxicosis resulted in a significant decrease in BW of the chicks at hatching. This may have been due to egg size, or the age of breeders, or both. Body weight and feed conversion ratios also indicated that there was no significant effect of 2.5 mg of aflatoxin per kg of feed in chicks of either line. These findings are surprising, because this dietary concentration of aflatoxin has been well documented to result in significant growth depression and increased feed conversion ratios when fed to broiler chicks (Smith and Hamilton, 1970; Lanza et al, 1980b). The lack of an effect significant on BW of C and AR chicks fed 2.5 mg of aflatoxin per kg of feed in the present study indicates that these lines are more resistant to dietary aflatoxin than commercial-type broiler chicks.
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MANNING ET AL. Genet. 63:207-212. Luna, L., ed., 1960. Armed Forces Institute of Pathology Manual of Histologic and Special Staining Technics. McGraw-Hill Inc., New York, NY. Manning, R. O., and R. D. Wyatt, 1990. Effect of cold acclimation on the broiler chicks' resistance to acute aflatoxicosis. Poultry Sci. 69:388-396. Manning, R. O., R. D. Wyatt, and O. J. Fletcher, 1990. Effect of cold acclimation on the broiler chicks' resistance to dietary aflatoxin. Poultry Sci. 69: (in press). Marks, H. L., and R. D. Wyatt, 1979. Generic resistance to aflatoxin in Japanese quail. Science 206:1329-1330. Pegram, R. A., 1986. Characterization of genetic resistance to aflatoxin in Japanese quail. Ph.D. diss., The University of Georgia, Athens, GA. Portman, R. S., K. M. Plowman, and T. C. Campbell, 1970. On mechanisms affecting species susceptibility to aflatoxin. Biochem. Biophys. Acta 208:487-495. Smith, J. W„ and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49:207-215. Tung, H. T., W. E. Donaldson, and P. B. Hamilton, 1972. Altered lipid transport during aflatoxicosis. Toxicol. Appl. Pharmacol. 22:97-104. Tung, H. T., R. D. Wyatt, P. Thaxton, and P. B. Hamilton, 1975. Concentrations of serum proteins during aflatoxicosis. Toxicol. Appl. Pharmacol. 34:320-326. Washburn, K. W., G. M. Lanza, and R. D. Wyatt, 1978. Selection procedures for developing genetic resistance to dietary aflatoxin. World's Poult. Sci. J. 10: 1773-1778. Wyatt, R. D., R. O. Manning, and H. L. Marks, 1987. Selection for resistance to aflatoxin in chickens. Poultry Sci. 66:1901-1904. Zar, J. H„ 1974. Biostatistical Analysis. Prentice-Hall Inc., Englewood Cliffs, NJ.
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Bruiting, J. L., and B. L. Kintz, 1968. Computational Handbook of Statistics. Scott, Foresman and Company, Glenview, IL. Busby, W. F., Jr., and G. N. Wogan, 1981. Aflatoxins. Pages 3-27 in: Mycotoxins and N-Nitroso Compounds: Environmental Risks, Vol. 1. R. C. Shank, ed. CRC Press, Boca Raton, FL. Carnaghan, R B A , C. N. Herber, D.S.P. Patterson, and D. Sweasy, 1967. Comparative biological and biochemical studies in hybrid chicks. 2. Susceptibility to aflatoxin and effects on serum protein constituents. Br. Poult. Sci. 8:279-284. Domer, F. R., 1971. Animal Experiments in Pharmacological Analysis. Charles C. Thomas, Publisher, Springfield, JL. 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. 41:843-847. Gumbmann, M. R., S. N. Williams, A. N. Booth, P. Vohra, R. A. Ernst, and M. Bethard, 1970. Aflatoxin susceptibility in various breeds of poultry. Fed. Proc. 134: 683-688. Hsieh, DJ\H., 1979. Basic metabolic effects of mycotoxins. Pages 43-55 in: Interaction of Mycotoxins in Animal Production. Natl. Acad. Sci., Washington, DC. Lanza, G. M., K. W. Washburn, and R. D. Wyatt, 1980a. Strain variation in hematological response of broilers to dietary aflatoxin. Poultry Sci. 59:2686-2691. Lanza, G. M., K. W. Washburn, and R. D. Wyatt, 1980b. Variation with age in response of broilers to aflatoxin. Poultry Sci. 59:282-288. Lanza, G. M., K. W. Washburn, R. D. Wyatt, and H. L. Marks, 1982. Genetic variation of physiological response to aflatoxin in Gallus domesticus. Theor. Appl.