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Variation with Age in Response of Broilers to Aflatoxin
Variation with Age in Response of Broilers to Aflatoxin G. M. LANZA, K. W. WASHBURN, and R. D. WYATT Department of Poultry Science, University of Georgia, Athens, Georgia 30602 (Received for publication November 20, 1978)
1980 Poultry Science 59:282-288
INTRODUCTION
Dietary aflatoxin has been shown to be highly toxic when fed to young broiler chicks from 1 to 21 days of age, depressing body weight (Smith and Hamilton, 1970), altering liver lipid transport (Tung et al, 1972), depressing packed cell volume (Tung et al, 1975), and decreasing serum cholesterol (Tung et al, 1972). The inhibition of secretory proteins of the liver, such as plasma proteins and lipoproteins, during aflatoxicosis has been demonstrated in several studies (Brown and Abrams, 1965; Datta and Gajan, 1965; Tung et al, 1972; Tung et al, 1975). Uniform decreases of plasma proteins have been reported in ducklings (Brown and Abrams, 1965; Datta and Gajan, 1965; Nemeth and Juhasz, 1968), turkeys (Magwood et al, 1966), and chickens (Brown, 1966) and have been proposed as a diagnostic indicator of aflatoxicosis. Tung et al. (1975) verified the incidence of hypoproteinemia, but suggested that production of some plasma proteins were inhibited more than others. The disruption of hepatic lipid metabolism and transport has been demonstrated by Kato et al. (1969) and Tung et al. (1972). Incorporation of
C-acetate cholesterol (Kato et al., 1969) and the subsequent extra cellular transport of endogenous cholesterol from parenchymal hepatocytes (Tung et al, 1972) have been reported to be inhibited during aflatoxicosis. In a program to develop lines of broilers which would be genetically resistant to aflatoxin, a preliminary study of the variability in response of commercial broiler sire families to dietary aflatoxin was conducted (Washburn et al, 1978). When levels of aflatoxin comparable to those previously shown to produce a response in young broilers were fed to broilers from 18 to 32 days of age, neither body weight depression nor anemia occurred. Since the body weight and packed cell volume response to aflatoxin feeding initiated after 18 days of age was atypical, a program was developed to ascertain the effect of age on physiological criteria useful in assessing genetic variability in broiler response. The present study investigated the differential response in body weight, blood components, total plasma protein, and plasma cholesterol of broilers fed dietary aflatoxin at different ages from 1 to 42 days of age. 282
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ABSTRACT This study investigated the effects on growth and composition of the blood of dietary aflatoxin fed at levels of 0, 2.5, or 5.0 ug/g of diet for a three week period beginning at 1, 7, 14, and 21 days of age in commercial broilers. Packed cell volume (PCV), erythrocyte counts, mean corpuscular volume, hemoglobin content and mean corpuscular hemoglobin concentration, body weight, and mortality were measured weekly. Plasma cholesterol and total plasma protein levels were determined weekly. Dietary aflatoxin at levels of 2.5 and 5.0 ug of aflatoxin per gram of diet when fed to young chicks for three weeks, beginning at either one or seven days, depressed body weight and PCV in a dose related fashion. Body weight and PCV continued to be depressed by the 5.0 ug/g diets in chicks treated from 2 to 5 weeks of age but not at the lower dosages. The feeding of aflatoxin at levels of up to 5.0 Mg aflatoxin per gram of diet from 3 to 6 weeks of age did not significantly depress body weight or PCV from control levels. Plasma cholesterol and total protein were found to be more sensitive to aflatoxin treatment at the later ages than body weight and other blood values. Plasma cholesterol was significantly depressed by the 5.0 Mg/g level of aflatoxin in all treatment periods, but the 2.5 Mg/g level of aflatoxin did not significantly reduce cholesterol during the 3 to 6 week treatment period. Plasma proteins were found to be the most sensitive criteria for detecting broiler susceptibility to aflatoxin, being depressed by all levels of aflatoxin for all age groups.
RESPONSE WITH AGE OF BROILERS TO AFLATOXIN MATERIALS AND METHODS
Six-hundred Cobb broilers were housed in floor pens under continuous lighting in trial 2. Three replicate groups of 50 chicks each received 5.0 /ig/g of dietary aflatoxin from either 0 to 3, 1 to 4, or 3 to 6 weeks of age. Controls consisted of 50 chicks which received the University of Georgia ration without aflatoxin. Each week during the aflatoxin treatment period a random sample of 4 birds was chosen from each replicate of a treatment combination for plasma protein and cholesterol determinations. Approximately 2.0 ml of blood were obtained from each bird via cardiac stab and placed in heparinized tubes which had been oven dried overnight. Total plasma proteins were determined by the biuret method (Wooton, 1964). Plasma cholesterol was determined by the method of Zlatkis et al. (1953).
1
Diamond Shamrock, Newark, NJ.
In trial 3, Cobb broiler chicks were housed in heated starting batteries under constant illumination. Each treatment combination, which consisted of 8 to 12 birds, was replicated three times. Nopstress 1 was placed in the drinking water for the first three days and early temperature was maintained at a slightly higher level than in trials 1 and 2. At the conclusion of each three week period, approximately 5.0 ml of blood were obtained by cardiac stab and placed into tubes containing a drop of heparin. Plasma protein and cholesterol determinations were performed by the methods previously described. Body weight and PCV were determined weekly. The aflatoxin used in these studies was produced by Aspergillus parasiticus NRRL 2999 on sterile polished rice by the method of Shotwell et al. (1966) as modified by West et al. (1973), using the flasks described by Smith and Hamilton (1969). The moldy rice was steamed to kill the fungus, dried, and ground to a fine powder. This powder was analyzed for aflatoxin content by the colorimetric method of Nabney and Nesbitt (1965) with the modifications of Wiseman et al. (1967). Appropriate amounts of dried powder were added to the University of Georgia broiler starter ration (non-medicated for coccidiosis control) to attain dietary levels. The data generated by these studies were analyzed using the General Linear Models procedure of Statistical Analysis System (SAS) (Barr et al, 1976) in conjunction with the Duncan separation of means option (Duncan, 1955).
RESULTS
The results of trials 1 and 3 presented in Table 1 show that the effect of aflatoxin on body weight at either a level of 2.5 or 5.0 Uglg diet diminishes with age. In agreement with results previously reported by Smith and Hamilton (1970), body weights were signficantly depressed in a dose-related fashion in broilers fed aflatoxin from 0 to 3 weeks of age in both trials. A similar dose-related depression was observed for both trials in birds which were fed aflatoxin from 1 to 4 weeks of age. Body weights of groups fed diets containing aflatoxin at 2.5 jug/g diet from 2 to 5 weeks of age were 98% of controls, while the weights of those receiving 5.0 /ig/g diet were 85% of controls. For the group fed aflatoxin from 3 to 6 weeks of age, the body weights of those receiving 2.5 Mg/g diet
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The effects on growth, clinical blood values, total plasma protein, and cholesterol of feeding aflatoxin for different age periods from 1 to 42 days of age were studied using Cobb broiler males. The chicks were fed dietary aflatoxin at levels of 0, 2.5, or 5.0 uglg diet for a 21 day period beginning at 1, 7, 14, or 21 days of age in each of three trials, except trial 2 in which the 14 to 35 day treatment groups and the 2.5 £(g/g levels were omitted. In all studies feed and water were provided ad libitum. In trial 1, which consisted of two replicates of ten birds for each treatment combination, chicks were reared in heated wire-floor batteries under constant illumination. The batteries were housed in a semi-environmentally controlled room with vertical lighting. One milliliter of blood was obtained by cardiac puncture for each bird each week of the three-week treatment period. For each blood sample the packed cell volume (PCV) was determined by the microhematocrit method (Johnson, 1955). Erythrocyte counts (RBC) and mean corpuscular volume (MCV) were measured with a Coulter Counter (Model F) electron particle counter using the Coulter Dual Diluter. Hemoglobin content (Hb) was quantitated using the acid-hematin method (Bankowski, 1942). Mean corpuscular hemoglobin concentration (MCHC) was calculated from Hb and PCV values. Body weights and mortality were determined weekly.
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TABLE 1. The effects of dietary aflatoxin on packed cell volume (PCV) and body weight at the conclusion of each three week treatment period Body weight (g)
PCV (%) Aflatoxin (Mg/g)
0-3
0 2.5 5.0 0 2.5 5.0 0 2.5 5.0 0 2.5 5.0
1-4
2-5
3-6
Trial 1
Trial 3
X
31.3* 28.0 b 26.9 b
28.2* 24.9 b 23.6 b
27.6* 24.1b 18.5 C 29.1* b 30.5* 27.0 b 26.8* 28.9 b 29.0 b
27.6* 24.2 b 24.1b 27.4* 26.4* 23.9 b 27.6* b 27.7* 25.9 b
29.8 26.4 25.2 27.6 24.2 21.3 28.2 28.4 25.4 27.2 28.3 27.4
%of Control
89 85 88 77 101 90 104 101
Trial 1
Trial 3
494* 419 h 357 c
468* 377 b 340 c
697* 629 b 553 c 904* 932* 826 b 1141* 1209 b 1135*
727* 633 b 544 c 1059* 988 b 805 c 1333* 1304* 1206 b
X
481 398 348 712 631 548 982 960 816 1237 1256 1170
%of Control
83 72 89 77 98 84 101 95
a ' ' c Means with different superscripts within the same treatment period and trial are significantly different (P<.05). (Duncan, 1955).
were 1 0 1 % of t h e controls, while those receiving 5.0 /ig/g diet were 95% of controls. Although t h e effect of aflatoxin clearly diminishes with age, an exact age at which aflatoxin can be fed with n o s u b s e q u e n t effects o n b o d y weight is n o t clear from t h e results of these trials. In trial 1, 2.5 £ig aflatoxin did n o t significantly depress weight when fed from 2 t o 5 or 3 to 6 weeks of age; in trial 3 t h e b o d y weights of groups fed 2.5 jug aflatoxin from 2
to 5 weeks were significantly depressed. For t h e g r o u p s fed t h e higher level of aflatoxin, b o d y weight was n o t significantly depressed if aflatoxin t r e a t m e n t was initiated at 3 weeks of age in trial 1, while in trial 3 b o d y weights of this age g r o u p were slightly b u t significantly depressed. T h e results presented in Table 2 show t h e times required for aflatoxin fed at different ages t o result in a significant response. F o r t h e
TABLE 2. Timing of effect of aflatoxin on body weight and packed cell volume response of broilers fed aflatoxin at different ages in Trial 1
Age (wk)
Aflatoxin (Mg/g)
0-3
0 2.5 5.0 0 2.5 5.0 0 2.5 5.0 0 2.5 5.0
1-4
2-5
3-6
a
Body weight (g)
Packed c<:11 volume (%)
Weeks of aflatoxin feeding
Weeks of aflatoxin feeding
0
1
2
141* 140* 140* 285* 278* 278* 494* 492* 492*
141* 142* 132* 285* 279* 271* 494* 496* 473* 697* 782 b 782 b
285* 264 b 214C 494* 475* 425b 697* 726* 645b
494* 419b 357 c 697* 629 b 553 c 904* 932* 826 b
904* 963b 890*
1141* 1209b 1135*
3
0
1
2
3
28.5* 28.0* 28.5* 29.2* 28.8* 29.0* 31.3* 31.9* 31.2*
28.6* 29.0* 29.8* 29.7* 29.5* 28.9* 31.3* 32.3* 32.4* 27.6* 28.8* 28.0*
29.7* 26.1b 24.4b 31.0* 31.6* 28.9 b 27.6* 25.8* 23.4b 29.1* b 30.2 b 27.7*
31.3* 28.0 b 26.9 b 27.6* 24.lb 18.5 C 29.1* b 30.5* 27.0 b 26.8* 28.9 b 29.0 b
' ' c Means with different superscripts within the same treatment period and trial are significantly different (P«.05). (Duncan, 1955).
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Age (wk)
RESPONSE WITH AGE OF BROILERS TO AFLATOXIN
of the influence of dietary aflatoxin on the hematopoietic system. In a previous study (Stino and Washburn, 1970) in which a single dose of the hemolytic agent phenylhydrazine was injected, a similar overcompensation of the erythropoiesis was observed to occur. The PCV was determined by the microhematocrit method (Johnson, 1955) for blood obtained by wing vein puncture and by cardiac stab. Both sets of results are reported in Table 3 and their differences are elaborated upon in a subsequent paper (Washburn and Lanza, unpublished). The basis for PCV depression was primarily a decrease in red blood cell numbers with a less severe, but statistically significant, decrease in mean corpuscular volume (MCV). As with body weight, PCV was most severely decreased in the birds fed aflatoxin from 1 to 4 weeks of age. For this time period the RBC counts and MCV were significantly depressed. The RBC counts in groups fed aflatoxin from 2 to 5 or 3 to 6 weeks of age were not significantly different from that of controls. The MCV was significantly depressed by the 5.0 jUg/g level in all age groups, while the 2.5 (ig/g level did not significantly reduce MCV in the 2 to 5 or 3 to 6 week groups. Hemoglobin concentration was depressed analogous to PCV, reflecting the change in cell number and size. The mean corpuscular hemoglobin concentration (MCHC) showed no significant changes in any group.
TABLE 3. The effects of dietary aflatoxin on clinical blood values at the conclusion of each three week treatment period in trial 1 Age (wk)
jug aflatoxin/ g of diet
Wing vein PCV
cardiac PCV 1
RBC (millions)
Hb (g/100 ml)
MCV (M3)
MCHC
0-3
0 2.5 5.0 0 2.5 5.0 0 2.5 5.0 0 2.5 5.0
31.3* 28.0 b 26.9 b 27.6* 24.1b 18.5 C 29.1* b 30.5* 27.0 b 26.8* 28.9 b 29.0 b
27.8* 22.8 b 22.6 b 28.0* 24.4 b 19.0 C 29.5* 28.3* 28.0* 27.0* 27.5* 27.6*
2.59* 2.32 b 2.32b 2.62* 2.42 b 2.06 c 2.78* 2.89* 2.82* 2.74* 2.83* 2.88*
7.86* 6.56 b 6.53 b 8.12* 7.15 b 5.75 c 9.28* 8.99* 8.80* 8.15*b 7.87 b 8.52*
109* 101b 98b 106* 103 b 101b 102* 101* 97b 100* 101* 98b
28.9* 28.4* 28.1* 30.3* 29.1* 28.5* 31.6* 31.8* 30.9* 30.5* 28.8 b 31.2*
1-4
2-5
3-6
(%)
' ' Means with different superscripts within the same treatment period and trial are significantly different; a,b,c indicate statistically significant differences (P<.05) between the three levels of aflatoxin for each treatment period. (Duncan, 1955). PCV = packed erythrocyte volume in percent, RBC = red blood cell counts in millions per ml. Hb = Hemoglobin content in g per 100 ml of blood, MCHC = mean corpuscular hemoglobin conentration in percent, MCV = mean corpuscular volume in cubic microns.
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groups receiving aflatoxin from 0 to 3 weeks, a significant depression in body weight was noted after a 2 week period of aflatoxin feeding at either the 2.5 or 5.0 jug/g level. A similar period of time was required for a significant body weight depression in the groups fed aflatoxin for 1 to 4 weeks at the 5.0 /xg level, but significant differences were not observed in the groups fed the 2.5 Mg level until 3 weeks of feeding. No age groups showed significant decreases in body weight until 2 weeks of aflatoxin feeding. Some evidence of a growth stimulating effect by aflatoxin was observed for the lower levels and at the later ages. This was observed for the groups receiving the 2.5 /Ug level at 2 to 5 or 3 to 6 weeks of age after 1 or 2 weeks of aflatoxin feeding. The increased body weight of the aflatoxin groups was significantly increased only in the groups fed aflatoxin from 3 to 6 weeks of age. The effect of aflatoxin on PCV also diminished with age (Table 1). Aflatoxin fed after 2 weeks of age at the 2.5 Mg/g level or after 3 weeks of age at the 5.0 jug/g level did not significantly depress the PCV. Moreover, when aflatoxin was fed during the 3 to 6 week age period, the PCV was increased significantly above controls at 2.5 and 5.0 /ug/g level in trial 1. Increased PCV could be accounted for by a stimulation of erythropoietic activity as a result
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groups in trial 3. A similar response was observed in trial 2 but differences were not significant for the group fed aflatoxin from 3 to 6 weeks. Two factors contribute to the lack of significance for the 3 to 6 week protein differences. First, relatively small subclass numbers resulted in a lack of sensitivity. Although plasma proteins were depressed 14% from control levels, a 21% reduction was required to reach the P<.05 level of significance. Second, there was a decreasing effect of aflatoxin on plasma protein with age in both trials. This effect was more pronounced in trial 2, resulting in less differences between control and aflatoxin groups at the 3 to 6 week age. Plasma cholesterol levels were significantly reduced by the 5.0 /ig/g level of aflatoxin for all age groups in both trials. The effect of aflatoxin on plasma cholesterol diminished with age and the 2.5 jUg/g level of aflatoxin did not have a significant effect when fed from 3 to 6 weeks of age. Previous studies investigating the effects of dietary aflatoxin fed to young birds from 0 to 3 weeks of age have reported severe depressions of plasma proteins (Brown and Abrams, 1965; Datta and Gajan, 1965; Tung et al., 1975) and plasma cholesterol (Tung et al., 1972). Significant depressions in total plasma protein and total plasma cholesterol reaffirm these studies and, in addition, show that the
TABLE 4. The effect of dietary aflatoxin on plasma cholesterol and total plasma protein at the conclusion of each treatment period for trials 2 and 3
period (wk) 0-3
1-4
2-5
3-6
Aflatoxin level (Mg/g) 0 2.5 5.0 0 2.5 5.0 0 2.5 5.0 0 2.5 5.0
Total protein (mg%) T2
1
3815 a 2097 4106 a 2047 b
3996 a 3453a
T3 2877 a 1486 b 1150 c 3217a 1600 b 1331 c 3250 a 1980 b 183 5 b 3620 a 2810 b 2244 c
Cholesterol (mg%)
% Depression2 T2
T3
45
49 60
26
50 59
T2
T3
131.4a
133.8a 78.3 b 64.7 C 116.8 a 83.6 b 71.0 C 122.6 a 92.5b 77.1c 124.9a 118.8 a 87.0 b
47.5b 122.4 a 70.2 b
39 45 118.3a 14
22 38
72.0 b
% Depr ession T2
T3
64
42 52
43
28 39 25 37
39
5 30
' ' Means with different superscripts within the same treatment period and trial are significantly different (P<.05). (Duncan, 1955). J T2 = Trial 2, T3 = Trial 3. 2 Percent depression from control values. Data not obtained.
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Adverse effects of aflatoxin on avian blood constituents have been reported in turkeys (Wannop, 1961), ducklings, and New Hampshire chicks (Brown and Abrams, 1965). The first extensive study of aflatoxin related anemia in broilers was reported by Tung et al. (1975), who fed graded levels of aflatoxin to broiler chicks from 1 to 21 days of age. They observed dose related decreases of PCV, RBC, and Hb which agree with values reported here for the same age group. Based on decreased bone marrow lipid (Tung et al., 1975) and increased splenic weight (Smith and Hamilton, 1970), Tung et al. (1975) suggested that aflatoxin associated anemia was hemolytic. However, significant decreases in MCV reported in this study do not support the hemolytic anemia hypothesis. Since the MCHC of aflatoxin treated birds was not found to be significantly different from control values, significant dose related depressions of MCV are not supportive evidence for hemolytic anemia. Both chronic and acute hemolytic anemia, when induced by phenylhydrazine in broilers, resulted in increased MCV and decreased MCHC (Stino and Washburn, 1970). Total plasma proteins were depressed from control values by dietary aflatoxin at the conclusion of all 3 week treatment periods (Table 4). The depression was significant for all
287
RESPONSE WITH AGE OF BROILERS TO AFLATOXIN TABLE 5. The effect of dietary aflatoxin on plasma cholesterol weekly •within each treatment period for trial 2 Plasma cholesterol (mg% ± SEM) Weeks of aflatoxin feed ing
Aflatoxin level (Mg/g)
1
0-3
0 5.0 0 5.0 0 5.0
98.5 57.4 119.1 71.5 131.4 115.5
1-4 3-6
± 4.9 ± 5.0**1 ± 7.2 + 6.3** + 7.0 ± 11.0
2
3
119.1 ± 7.2 50.0+ 6.9** 131.4 ± 7.0 50.6+ 4.9** 1 1 7 . 0 + 11.9 8 9 . 0 ± 5.5*
131.4+ 47.5+ 122.4 ± 70.2+ 118.3 ± 72.0 ±
7.0 7.3** 13.3 3.1** 11.5 8.1**
1 Means for an aflatoxin level within a treatment period were significantly different (**P<.01; *P<.05). effects of aflatoxin on plasma protein and cholesterol diminish with age. Highly significant reductions in plasma cholesterol and plasma proteins were detected after one week of feeding in the 0 to 3 and 1 to 4 week treatment periods (Tables 5, 6). For the group fed aflatoxin from 3 to 6 weeks, aflatoxin did not affect plasma cholesterol significantly until after 2 weeks. It appears that with increasing age the magnitude of the effect is decreased as well as an increase in time required to affect these parameters. The sensitivity of these biochemical parameters is reflective of the mechanism by which aflatoxin exerts a biological effect. Although aflatoxin has been demonstrated to bind to DNA (Clifford and Rees, 1967; King and Nicholson, 1969; Edwards and Wogan, 1970) and to impair messenger RNA synthesis by the selective inhibition of the enzyme activity of RNA polymerase II (Akinrimis et al, 1974; Yu, 1977), the inhibition of protein synthesis for the production of export proteins
and lipid transport is also a function of the dissociation of parenchymal hepatocyte ribosomes from the endoplasmic reticulum (Sarasin and Moule, 1973). Protein synthesis, prior to the inhibition of transcription impairment, has been found to be disrupted by the blockage of protein synthesis directly at the polysome level. The translation of export proteins, such as plasma proteins and the apoprotein moiety of very low density lipoproteins, has been shown to be dependent on a close association of ribosomes with the endoplasmic reticulum (Hicks et al, 1969; Redman, 1969). Fatty livers (Smith and Hamilton, 1970), altered lipid transport (Tung et al, 1972), and decreased plasma proteins (Brown and Abrams, 1965; Datta and Gajan, 1965; Nemeth and Juhasz, 1968; Tung et al, 1972) reported in young broilers during aflatoxicosis can all be explained by the disaggregation of ribosomes from the endoplasmic reticulum and are compounded by transcriptional interferences. The depression on body weight and PCV known to
TABLE 6. The effect of dietary aflatoxin on total plasma protein weekly within each treatment period for trial 2 T o t a l plasma p r o t e i n (g% i: SEM;I Weeks of aflatoxin feeding
Treatment period (wk)
Aflatoxin level (/xg/g)
1
2
3
0-3
0 5.0
3.82 ± .34 2.10 ± . 4 1 * * 1
3.75 ± .22 2.00 ± . 2 5 * *
3.50 ± .41 2.06 ± 2 9 * *
1-4
0 5.0 0 5.0
3.50 1.89 4.11 3.85
3.82 1.83 3.72 3.01
4.11 3.05 4.00 3.45
3-6
+ ± ± ±
.41 .23** .26 .19
± ± + ±
.34 .18** .33 .32
± ± ± ±
.26 .22** .34 .31
Means for aflatoxin levels within a treatment period were significantly different (* *P<-01; *P<.05).
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Treatment period (wk)
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King, A. M. Q., and B. H. Nicholson, 1969. The interaction of aflatoxin B, with polynucleotides and its effect on ribonucleic acid polymerase. Biochem. J. 114:679-687. Magwood, S. E., E. Annav, and A. H. Corner, 1966. Induced tolerance in turkeys to aflatoxin poisoning. Can. J. Comp. Med. Vet. Sci. 30:17-25. Nabney, J., and B. F. Nesbitt, 1965. A spectrophotometric method of determining the aflatoxins. Analyst 90:155-160. Nemeth, I., and S. Juhasz, 1968. Effects of aflatoxin on serum protein fractions of day-old ducklings. Acta Vet. (Scandinavia) 18:95-105. Redman, C. M., 1969. Biosynthesis of serum proteins and ferritin by free and attached ribosomes of rat liver. J. Biol. Chem. 244: 4308-4315. Sarasin, A., and Y. Moule, 1973. Inhibition of in vitro protein synthesis by aflatoxin S1 derivatives. FEBSLett. 32:347-350. Shotwell, O. L., C. W. Hesseltine, R. D. Stubblefield, and W. G. Sorenson, 1966. Production of aflatoxin on rice. Appl. Microbiol. 14:425—428. REFERENCES Smith, J. W., and P. B. Hamilton, 1969. Technique for the aseptic addition of liquid to flask cultures. Arkinrimis, E. O., B. J. Benecke, and K. H. Seifart, Appl. Microbiol. 17:317. 1974. Inhibition of rat liver RNA polymerase in vitro by aflatoxin B, in the presence of a micro- Smith, J. W., and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49:207—215. somal fraction. Europ. J. Biochem. 42:333—339. Bankowski, R. A., 1942. Studies on hemoglobin Stino, F. K. R., and K. W. Washburn, 1970. Response of chickens with different hemoglobin genotypes content of chicken blood and evaluation of to phenylhydrazine-induced anemia. 2. Hemamethods for its determination. Amer. J. Vet. Res. tology. Poultry Sci. 4 9 : 1 1 4 - 1 2 1 . 3:373-381. Barr, A. J., J. H. Goodnight, J. P. Sail, and J. J. Tung, H. T., F. W. Cook, R. D. Wyatt, and P. B. Helwig, 1976. A users guide to SAS 76. SAS Hamilton, 1975. The anemia caused by aflatoxin. Institute, Inc., Raleigh, NC. Poultry Sci. 54:1962-1969. Brown, J. M. M., 1966. Biochemical changes in the Tung, H. T., W. E. Donaldson, and P. B. Hamilton, livers of domestic birds poisoned with aflatoxin. 1972. Altered lipid transport during aflatoxicosis. South Africa Med. J. 39:778-788. Toxicol. Appl. Pharmacol. 15:97-104. Brown, J. M. M., and L. Abrams, 1965. Biochemical Wannop, C. C , 1961. The histopathology of turkey studies on aflatoxicosis. Onderstepoort J. Vet. "X" disease in Great Britain. Avian Dis. 5:371 — Res. 32:119-146. 381. Clifford, J. I., and K. R. Rees, 1967. The action of Washburn, K. W., G. M. Lanza, and R. D. Wyatt, 1978. aflatoxin B, on the rat liver. Biochem. J. 102: Selection procedures for developing genetic 65-75. resistance to dietary aflatoxin. World Poultry Sci. Datta, P. R., and R. J. Gajan, 1965. Plasma protein J. 10:1773-1778. index of aflatoxin fed ducklings. Life Sci. 4: West, S., R. D. Wyatt, and P. B. Hamilton, 1973. 1791-1795. Improved yield of aflatoxin by incremental Duncan, D. B., 1955. Multiple range and multiple F increases in temperature. Appl. Microbiol. tests. Biometrics 11:1—42. 25:1018-1019. Edwards, G. S., and G. N. Wogan, 1970. Aflatoxin Wiseman, H. G., W. C. Jacobson, and W. C. Harmeyer, inhibition of template activity of rat liver chro1967. Note on removal of pigments from chloromatin. Biochem. Biophys. Acta. 224:597-607. form extracts of aflatoxin cultures with copper Hicks, S. J., J. W. Drysdale, and H. N. Munro, 1969. carbonate. J. Ass. Of fie. Agr. Chem. 50:982— Preferential synthesis of ferritin and albumin by 983. different populations of liver polysomes. Science Wooton, I. D. P., 1964. Micro-analysis in medical 164:584-585. biochemistry. Grune and Stratton, New York. Johnson, P. M., 1955. Hematocrit values for the chick Yu, F. L., 1977. Mechanism of aflatoxin B[ inhibition embryo at various ag*es. Amer. Physiol. 180:361 — of rat hepatic nuclear RNA synthesis. J. Biol. 363. Chem. 252:3245-3251. Kato, R., K. Onoda, and Y. Omari, 1969. Effect of Zlatkis, A., B. Azk, and A. J. Boyle, 1953. A new aflatoxin B, on the incorporation of "C-acetate method for the direct determination of serum into cholesterol by rat liver. Experientia 25:1026. cholesterol. J. Lab. Clin. Med. 41:486-487. occur after 2 weeks of age in young chicks may be secondary effects to the primary effect of protein synthesis inhibition, since plasma protein and cholesterol values were significantly depressed soon after aflatoxin administration, while body weight and PCV depressions occurred later, possibly resulting from the accumulative effect of severe hepatic dysfunction. Although plasma proteins and cholesterol are both depressed at later ages, the severity of the decreases are not as dramatic as observed at early ages. Therefore, secondary effects such as body weight and PCV appear to return to normal, while primary biochemical disruption, though lessened, remains significantly affected.
1980 Poultry Science 59:282-288
INTRODUCTION
Dietary aflatoxin has been shown to be highly toxic when fed to young broiler chicks from 1 to 21 days of age, depressing body weight (Smith and Hamilton, 1970), altering liver lipid transport (Tung et al, 1972), depressing packed cell volume (Tung et al, 1975), and decreasing serum cholesterol (Tung et al, 1972). The inhibition of secretory proteins of the liver, such as plasma proteins and lipoproteins, during aflatoxicosis has been demonstrated in several studies (Brown and Abrams, 1965; Datta and Gajan, 1965; Tung et al, 1972; Tung et al, 1975). Uniform decreases of plasma proteins have been reported in ducklings (Brown and Abrams, 1965; Datta and Gajan, 1965; Nemeth and Juhasz, 1968), turkeys (Magwood et al, 1966), and chickens (Brown, 1966) and have been proposed as a diagnostic indicator of aflatoxicosis. Tung et al. (1975) verified the incidence of hypoproteinemia, but suggested that production of some plasma proteins were inhibited more than others. The disruption of hepatic lipid metabolism and transport has been demonstrated by Kato et al. (1969) and Tung et al. (1972). Incorporation of
C-acetate cholesterol (Kato et al., 1969) and the subsequent extra cellular transport of endogenous cholesterol from parenchymal hepatocytes (Tung et al, 1972) have been reported to be inhibited during aflatoxicosis. In a program to develop lines of broilers which would be genetically resistant to aflatoxin, a preliminary study of the variability in response of commercial broiler sire families to dietary aflatoxin was conducted (Washburn et al, 1978). When levels of aflatoxin comparable to those previously shown to produce a response in young broilers were fed to broilers from 18 to 32 days of age, neither body weight depression nor anemia occurred. Since the body weight and packed cell volume response to aflatoxin feeding initiated after 18 days of age was atypical, a program was developed to ascertain the effect of age on physiological criteria useful in assessing genetic variability in broiler response. The present study investigated the differential response in body weight, blood components, total plasma protein, and plasma cholesterol of broilers fed dietary aflatoxin at different ages from 1 to 42 days of age. 282
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ABSTRACT This study investigated the effects on growth and composition of the blood of dietary aflatoxin fed at levels of 0, 2.5, or 5.0 ug/g of diet for a three week period beginning at 1, 7, 14, and 21 days of age in commercial broilers. Packed cell volume (PCV), erythrocyte counts, mean corpuscular volume, hemoglobin content and mean corpuscular hemoglobin concentration, body weight, and mortality were measured weekly. Plasma cholesterol and total plasma protein levels were determined weekly. Dietary aflatoxin at levels of 2.5 and 5.0 ug of aflatoxin per gram of diet when fed to young chicks for three weeks, beginning at either one or seven days, depressed body weight and PCV in a dose related fashion. Body weight and PCV continued to be depressed by the 5.0 ug/g diets in chicks treated from 2 to 5 weeks of age but not at the lower dosages. The feeding of aflatoxin at levels of up to 5.0 Mg aflatoxin per gram of diet from 3 to 6 weeks of age did not significantly depress body weight or PCV from control levels. Plasma cholesterol and total protein were found to be more sensitive to aflatoxin treatment at the later ages than body weight and other blood values. Plasma cholesterol was significantly depressed by the 5.0 Mg/g level of aflatoxin in all treatment periods, but the 2.5 Mg/g level of aflatoxin did not significantly reduce cholesterol during the 3 to 6 week treatment period. Plasma proteins were found to be the most sensitive criteria for detecting broiler susceptibility to aflatoxin, being depressed by all levels of aflatoxin for all age groups.
RESPONSE WITH AGE OF BROILERS TO AFLATOXIN MATERIALS AND METHODS
Six-hundred Cobb broilers were housed in floor pens under continuous lighting in trial 2. Three replicate groups of 50 chicks each received 5.0 /ig/g of dietary aflatoxin from either 0 to 3, 1 to 4, or 3 to 6 weeks of age. Controls consisted of 50 chicks which received the University of Georgia ration without aflatoxin. Each week during the aflatoxin treatment period a random sample of 4 birds was chosen from each replicate of a treatment combination for plasma protein and cholesterol determinations. Approximately 2.0 ml of blood were obtained from each bird via cardiac stab and placed in heparinized tubes which had been oven dried overnight. Total plasma proteins were determined by the biuret method (Wooton, 1964). Plasma cholesterol was determined by the method of Zlatkis et al. (1953).
1
Diamond Shamrock, Newark, NJ.
In trial 3, Cobb broiler chicks were housed in heated starting batteries under constant illumination. Each treatment combination, which consisted of 8 to 12 birds, was replicated three times. Nopstress 1 was placed in the drinking water for the first three days and early temperature was maintained at a slightly higher level than in trials 1 and 2. At the conclusion of each three week period, approximately 5.0 ml of blood were obtained by cardiac stab and placed into tubes containing a drop of heparin. Plasma protein and cholesterol determinations were performed by the methods previously described. Body weight and PCV were determined weekly. The aflatoxin used in these studies was produced by Aspergillus parasiticus NRRL 2999 on sterile polished rice by the method of Shotwell et al. (1966) as modified by West et al. (1973), using the flasks described by Smith and Hamilton (1969). The moldy rice was steamed to kill the fungus, dried, and ground to a fine powder. This powder was analyzed for aflatoxin content by the colorimetric method of Nabney and Nesbitt (1965) with the modifications of Wiseman et al. (1967). Appropriate amounts of dried powder were added to the University of Georgia broiler starter ration (non-medicated for coccidiosis control) to attain dietary levels. The data generated by these studies were analyzed using the General Linear Models procedure of Statistical Analysis System (SAS) (Barr et al, 1976) in conjunction with the Duncan separation of means option (Duncan, 1955).
RESULTS
The results of trials 1 and 3 presented in Table 1 show that the effect of aflatoxin on body weight at either a level of 2.5 or 5.0 Uglg diet diminishes with age. In agreement with results previously reported by Smith and Hamilton (1970), body weights were signficantly depressed in a dose-related fashion in broilers fed aflatoxin from 0 to 3 weeks of age in both trials. A similar dose-related depression was observed for both trials in birds which were fed aflatoxin from 1 to 4 weeks of age. Body weights of groups fed diets containing aflatoxin at 2.5 jug/g diet from 2 to 5 weeks of age were 98% of controls, while the weights of those receiving 5.0 /ig/g diet were 85% of controls. For the group fed aflatoxin from 3 to 6 weeks of age, the body weights of those receiving 2.5 Mg/g diet
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The effects on growth, clinical blood values, total plasma protein, and cholesterol of feeding aflatoxin for different age periods from 1 to 42 days of age were studied using Cobb broiler males. The chicks were fed dietary aflatoxin at levels of 0, 2.5, or 5.0 uglg diet for a 21 day period beginning at 1, 7, 14, or 21 days of age in each of three trials, except trial 2 in which the 14 to 35 day treatment groups and the 2.5 £(g/g levels were omitted. In all studies feed and water were provided ad libitum. In trial 1, which consisted of two replicates of ten birds for each treatment combination, chicks were reared in heated wire-floor batteries under constant illumination. The batteries were housed in a semi-environmentally controlled room with vertical lighting. One milliliter of blood was obtained by cardiac puncture for each bird each week of the three-week treatment period. For each blood sample the packed cell volume (PCV) was determined by the microhematocrit method (Johnson, 1955). Erythrocyte counts (RBC) and mean corpuscular volume (MCV) were measured with a Coulter Counter (Model F) electron particle counter using the Coulter Dual Diluter. Hemoglobin content (Hb) was quantitated using the acid-hematin method (Bankowski, 1942). Mean corpuscular hemoglobin concentration (MCHC) was calculated from Hb and PCV values. Body weights and mortality were determined weekly.
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TABLE 1. The effects of dietary aflatoxin on packed cell volume (PCV) and body weight at the conclusion of each three week treatment period Body weight (g)
PCV (%) Aflatoxin (Mg/g)
0-3
0 2.5 5.0 0 2.5 5.0 0 2.5 5.0 0 2.5 5.0
1-4
2-5
3-6
Trial 1
Trial 3
X
31.3* 28.0 b 26.9 b
28.2* 24.9 b 23.6 b
27.6* 24.1b 18.5 C 29.1* b 30.5* 27.0 b 26.8* 28.9 b 29.0 b
27.6* 24.2 b 24.1b 27.4* 26.4* 23.9 b 27.6* b 27.7* 25.9 b
29.8 26.4 25.2 27.6 24.2 21.3 28.2 28.4 25.4 27.2 28.3 27.4
%of Control
89 85 88 77 101 90 104 101
Trial 1
Trial 3
494* 419 h 357 c
468* 377 b 340 c
697* 629 b 553 c 904* 932* 826 b 1141* 1209 b 1135*
727* 633 b 544 c 1059* 988 b 805 c 1333* 1304* 1206 b
X
481 398 348 712 631 548 982 960 816 1237 1256 1170
%of Control
83 72 89 77 98 84 101 95
a ' ' c Means with different superscripts within the same treatment period and trial are significantly different (P<.05). (Duncan, 1955).
were 1 0 1 % of t h e controls, while those receiving 5.0 /ig/g diet were 95% of controls. Although t h e effect of aflatoxin clearly diminishes with age, an exact age at which aflatoxin can be fed with n o s u b s e q u e n t effects o n b o d y weight is n o t clear from t h e results of these trials. In trial 1, 2.5 £ig aflatoxin did n o t significantly depress weight when fed from 2 t o 5 or 3 to 6 weeks of age; in trial 3 t h e b o d y weights of groups fed 2.5 jug aflatoxin from 2
to 5 weeks were significantly depressed. For t h e g r o u p s fed t h e higher level of aflatoxin, b o d y weight was n o t significantly depressed if aflatoxin t r e a t m e n t was initiated at 3 weeks of age in trial 1, while in trial 3 b o d y weights of this age g r o u p were slightly b u t significantly depressed. T h e results presented in Table 2 show t h e times required for aflatoxin fed at different ages t o result in a significant response. F o r t h e
TABLE 2. Timing of effect of aflatoxin on body weight and packed cell volume response of broilers fed aflatoxin at different ages in Trial 1
Age (wk)
Aflatoxin (Mg/g)
0-3
0 2.5 5.0 0 2.5 5.0 0 2.5 5.0 0 2.5 5.0
1-4
2-5
3-6
a
Body weight (g)
Packed c<:11 volume (%)
Weeks of aflatoxin feeding
Weeks of aflatoxin feeding
0
1
2
141* 140* 140* 285* 278* 278* 494* 492* 492*
141* 142* 132* 285* 279* 271* 494* 496* 473* 697* 782 b 782 b
285* 264 b 214C 494* 475* 425b 697* 726* 645b
494* 419b 357 c 697* 629 b 553 c 904* 932* 826 b
904* 963b 890*
1141* 1209b 1135*
3
0
1
2
3
28.5* 28.0* 28.5* 29.2* 28.8* 29.0* 31.3* 31.9* 31.2*
28.6* 29.0* 29.8* 29.7* 29.5* 28.9* 31.3* 32.3* 32.4* 27.6* 28.8* 28.0*
29.7* 26.1b 24.4b 31.0* 31.6* 28.9 b 27.6* 25.8* 23.4b 29.1* b 30.2 b 27.7*
31.3* 28.0 b 26.9 b 27.6* 24.lb 18.5 C 29.1* b 30.5* 27.0 b 26.8* 28.9 b 29.0 b
' ' c Means with different superscripts within the same treatment period and trial are significantly different (P«.05). (Duncan, 1955).
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Age (wk)
RESPONSE WITH AGE OF BROILERS TO AFLATOXIN
of the influence of dietary aflatoxin on the hematopoietic system. In a previous study (Stino and Washburn, 1970) in which a single dose of the hemolytic agent phenylhydrazine was injected, a similar overcompensation of the erythropoiesis was observed to occur. The PCV was determined by the microhematocrit method (Johnson, 1955) for blood obtained by wing vein puncture and by cardiac stab. Both sets of results are reported in Table 3 and their differences are elaborated upon in a subsequent paper (Washburn and Lanza, unpublished). The basis for PCV depression was primarily a decrease in red blood cell numbers with a less severe, but statistically significant, decrease in mean corpuscular volume (MCV). As with body weight, PCV was most severely decreased in the birds fed aflatoxin from 1 to 4 weeks of age. For this time period the RBC counts and MCV were significantly depressed. The RBC counts in groups fed aflatoxin from 2 to 5 or 3 to 6 weeks of age were not significantly different from that of controls. The MCV was significantly depressed by the 5.0 jUg/g level in all age groups, while the 2.5 (ig/g level did not significantly reduce MCV in the 2 to 5 or 3 to 6 week groups. Hemoglobin concentration was depressed analogous to PCV, reflecting the change in cell number and size. The mean corpuscular hemoglobin concentration (MCHC) showed no significant changes in any group.
TABLE 3. The effects of dietary aflatoxin on clinical blood values at the conclusion of each three week treatment period in trial 1 Age (wk)
jug aflatoxin/ g of diet
Wing vein PCV
cardiac PCV 1
RBC (millions)
Hb (g/100 ml)
MCV (M3)
MCHC
0-3
0 2.5 5.0 0 2.5 5.0 0 2.5 5.0 0 2.5 5.0
31.3* 28.0 b 26.9 b 27.6* 24.1b 18.5 C 29.1* b 30.5* 27.0 b 26.8* 28.9 b 29.0 b
27.8* 22.8 b 22.6 b 28.0* 24.4 b 19.0 C 29.5* 28.3* 28.0* 27.0* 27.5* 27.6*
2.59* 2.32 b 2.32b 2.62* 2.42 b 2.06 c 2.78* 2.89* 2.82* 2.74* 2.83* 2.88*
7.86* 6.56 b 6.53 b 8.12* 7.15 b 5.75 c 9.28* 8.99* 8.80* 8.15*b 7.87 b 8.52*
109* 101b 98b 106* 103 b 101b 102* 101* 97b 100* 101* 98b
28.9* 28.4* 28.1* 30.3* 29.1* 28.5* 31.6* 31.8* 30.9* 30.5* 28.8 b 31.2*
1-4
2-5
3-6
(%)
' ' Means with different superscripts within the same treatment period and trial are significantly different; a,b,c indicate statistically significant differences (P<.05) between the three levels of aflatoxin for each treatment period. (Duncan, 1955). PCV = packed erythrocyte volume in percent, RBC = red blood cell counts in millions per ml. Hb = Hemoglobin content in g per 100 ml of blood, MCHC = mean corpuscular hemoglobin conentration in percent, MCV = mean corpuscular volume in cubic microns.
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groups receiving aflatoxin from 0 to 3 weeks, a significant depression in body weight was noted after a 2 week period of aflatoxin feeding at either the 2.5 or 5.0 jug/g level. A similar period of time was required for a significant body weight depression in the groups fed aflatoxin for 1 to 4 weeks at the 5.0 /xg level, but significant differences were not observed in the groups fed the 2.5 Mg level until 3 weeks of feeding. No age groups showed significant decreases in body weight until 2 weeks of aflatoxin feeding. Some evidence of a growth stimulating effect by aflatoxin was observed for the lower levels and at the later ages. This was observed for the groups receiving the 2.5 /Ug level at 2 to 5 or 3 to 6 weeks of age after 1 or 2 weeks of aflatoxin feeding. The increased body weight of the aflatoxin groups was significantly increased only in the groups fed aflatoxin from 3 to 6 weeks of age. The effect of aflatoxin on PCV also diminished with age (Table 1). Aflatoxin fed after 2 weeks of age at the 2.5 Mg/g level or after 3 weeks of age at the 5.0 jug/g level did not significantly depress the PCV. Moreover, when aflatoxin was fed during the 3 to 6 week age period, the PCV was increased significantly above controls at 2.5 and 5.0 /ug/g level in trial 1. Increased PCV could be accounted for by a stimulation of erythropoietic activity as a result
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groups in trial 3. A similar response was observed in trial 2 but differences were not significant for the group fed aflatoxin from 3 to 6 weeks. Two factors contribute to the lack of significance for the 3 to 6 week protein differences. First, relatively small subclass numbers resulted in a lack of sensitivity. Although plasma proteins were depressed 14% from control levels, a 21% reduction was required to reach the P<.05 level of significance. Second, there was a decreasing effect of aflatoxin on plasma protein with age in both trials. This effect was more pronounced in trial 2, resulting in less differences between control and aflatoxin groups at the 3 to 6 week age. Plasma cholesterol levels were significantly reduced by the 5.0 /ig/g level of aflatoxin for all age groups in both trials. The effect of aflatoxin on plasma cholesterol diminished with age and the 2.5 jUg/g level of aflatoxin did not have a significant effect when fed from 3 to 6 weeks of age. Previous studies investigating the effects of dietary aflatoxin fed to young birds from 0 to 3 weeks of age have reported severe depressions of plasma proteins (Brown and Abrams, 1965; Datta and Gajan, 1965; Tung et al., 1975) and plasma cholesterol (Tung et al., 1972). Significant depressions in total plasma protein and total plasma cholesterol reaffirm these studies and, in addition, show that the
TABLE 4. The effect of dietary aflatoxin on plasma cholesterol and total plasma protein at the conclusion of each treatment period for trials 2 and 3
period (wk) 0-3
1-4
2-5
3-6
Aflatoxin level (Mg/g) 0 2.5 5.0 0 2.5 5.0 0 2.5 5.0 0 2.5 5.0
Total protein (mg%) T2
1
3815 a 2097 4106 a 2047 b
3996 a 3453a
T3 2877 a 1486 b 1150 c 3217a 1600 b 1331 c 3250 a 1980 b 183 5 b 3620 a 2810 b 2244 c
Cholesterol (mg%)
% Depression2 T2
T3
45
49 60
26
50 59
T2
T3
131.4a
133.8a 78.3 b 64.7 C 116.8 a 83.6 b 71.0 C 122.6 a 92.5b 77.1c 124.9a 118.8 a 87.0 b
47.5b 122.4 a 70.2 b
39 45 118.3a 14
22 38
72.0 b
% Depr ession T2
T3
64
42 52
43
28 39 25 37
39
5 30
' ' Means with different superscripts within the same treatment period and trial are significantly different (P<.05). (Duncan, 1955). J T2 = Trial 2, T3 = Trial 3. 2 Percent depression from control values. Data not obtained.
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Adverse effects of aflatoxin on avian blood constituents have been reported in turkeys (Wannop, 1961), ducklings, and New Hampshire chicks (Brown and Abrams, 1965). The first extensive study of aflatoxin related anemia in broilers was reported by Tung et al. (1975), who fed graded levels of aflatoxin to broiler chicks from 1 to 21 days of age. They observed dose related decreases of PCV, RBC, and Hb which agree with values reported here for the same age group. Based on decreased bone marrow lipid (Tung et al., 1975) and increased splenic weight (Smith and Hamilton, 1970), Tung et al. (1975) suggested that aflatoxin associated anemia was hemolytic. However, significant decreases in MCV reported in this study do not support the hemolytic anemia hypothesis. Since the MCHC of aflatoxin treated birds was not found to be significantly different from control values, significant dose related depressions of MCV are not supportive evidence for hemolytic anemia. Both chronic and acute hemolytic anemia, when induced by phenylhydrazine in broilers, resulted in increased MCV and decreased MCHC (Stino and Washburn, 1970). Total plasma proteins were depressed from control values by dietary aflatoxin at the conclusion of all 3 week treatment periods (Table 4). The depression was significant for all
287
RESPONSE WITH AGE OF BROILERS TO AFLATOXIN TABLE 5. The effect of dietary aflatoxin on plasma cholesterol weekly •within each treatment period for trial 2 Plasma cholesterol (mg% ± SEM) Weeks of aflatoxin feed ing
Aflatoxin level (Mg/g)
1
0-3
0 5.0 0 5.0 0 5.0
98.5 57.4 119.1 71.5 131.4 115.5
1-4 3-6
± 4.9 ± 5.0**1 ± 7.2 + 6.3** + 7.0 ± 11.0
2
3
119.1 ± 7.2 50.0+ 6.9** 131.4 ± 7.0 50.6+ 4.9** 1 1 7 . 0 + 11.9 8 9 . 0 ± 5.5*
131.4+ 47.5+ 122.4 ± 70.2+ 118.3 ± 72.0 ±
7.0 7.3** 13.3 3.1** 11.5 8.1**
1 Means for an aflatoxin level within a treatment period were significantly different (**P<.01; *P<.05). effects of aflatoxin on plasma protein and cholesterol diminish with age. Highly significant reductions in plasma cholesterol and plasma proteins were detected after one week of feeding in the 0 to 3 and 1 to 4 week treatment periods (Tables 5, 6). For the group fed aflatoxin from 3 to 6 weeks, aflatoxin did not affect plasma cholesterol significantly until after 2 weeks. It appears that with increasing age the magnitude of the effect is decreased as well as an increase in time required to affect these parameters. The sensitivity of these biochemical parameters is reflective of the mechanism by which aflatoxin exerts a biological effect. Although aflatoxin has been demonstrated to bind to DNA (Clifford and Rees, 1967; King and Nicholson, 1969; Edwards and Wogan, 1970) and to impair messenger RNA synthesis by the selective inhibition of the enzyme activity of RNA polymerase II (Akinrimis et al, 1974; Yu, 1977), the inhibition of protein synthesis for the production of export proteins
and lipid transport is also a function of the dissociation of parenchymal hepatocyte ribosomes from the endoplasmic reticulum (Sarasin and Moule, 1973). Protein synthesis, prior to the inhibition of transcription impairment, has been found to be disrupted by the blockage of protein synthesis directly at the polysome level. The translation of export proteins, such as plasma proteins and the apoprotein moiety of very low density lipoproteins, has been shown to be dependent on a close association of ribosomes with the endoplasmic reticulum (Hicks et al, 1969; Redman, 1969). Fatty livers (Smith and Hamilton, 1970), altered lipid transport (Tung et al, 1972), and decreased plasma proteins (Brown and Abrams, 1965; Datta and Gajan, 1965; Nemeth and Juhasz, 1968; Tung et al, 1972) reported in young broilers during aflatoxicosis can all be explained by the disaggregation of ribosomes from the endoplasmic reticulum and are compounded by transcriptional interferences. The depression on body weight and PCV known to
TABLE 6. The effect of dietary aflatoxin on total plasma protein weekly within each treatment period for trial 2 T o t a l plasma p r o t e i n (g% i: SEM;I Weeks of aflatoxin feeding
Treatment period (wk)
Aflatoxin level (/xg/g)
1
2
3
0-3
0 5.0
3.82 ± .34 2.10 ± . 4 1 * * 1
3.75 ± .22 2.00 ± . 2 5 * *
3.50 ± .41 2.06 ± 2 9 * *
1-4
0 5.0 0 5.0
3.50 1.89 4.11 3.85
3.82 1.83 3.72 3.01
4.11 3.05 4.00 3.45
3-6
+ ± ± ±
.41 .23** .26 .19
± ± + ±
.34 .18** .33 .32
± ± ± ±
.26 .22** .34 .31
Means for aflatoxin levels within a treatment period were significantly different (* *P<-01; *P<.05).
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Treatment period (wk)
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King, A. M. Q., and B. H. Nicholson, 1969. The interaction of aflatoxin B, with polynucleotides and its effect on ribonucleic acid polymerase. Biochem. J. 114:679-687. Magwood, S. E., E. Annav, and A. H. Corner, 1966. Induced tolerance in turkeys to aflatoxin poisoning. Can. J. Comp. Med. Vet. Sci. 30:17-25. Nabney, J., and B. F. Nesbitt, 1965. A spectrophotometric method of determining the aflatoxins. Analyst 90:155-160. Nemeth, I., and S. Juhasz, 1968. Effects of aflatoxin on serum protein fractions of day-old ducklings. Acta Vet. (Scandinavia) 18:95-105. Redman, C. M., 1969. Biosynthesis of serum proteins and ferritin by free and attached ribosomes of rat liver. J. Biol. Chem. 244: 4308-4315. Sarasin, A., and Y. Moule, 1973. Inhibition of in vitro protein synthesis by aflatoxin S1 derivatives. FEBSLett. 32:347-350. Shotwell, O. L., C. W. Hesseltine, R. D. Stubblefield, and W. G. Sorenson, 1966. Production of aflatoxin on rice. Appl. Microbiol. 14:425—428. REFERENCES Smith, J. W., and P. B. Hamilton, 1969. Technique for the aseptic addition of liquid to flask cultures. Arkinrimis, E. O., B. J. Benecke, and K. H. Seifart, Appl. Microbiol. 17:317. 1974. Inhibition of rat liver RNA polymerase in vitro by aflatoxin B, in the presence of a micro- Smith, J. W., and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49:207—215. somal fraction. Europ. J. Biochem. 42:333—339. Bankowski, R. A., 1942. Studies on hemoglobin Stino, F. K. R., and K. W. Washburn, 1970. Response of chickens with different hemoglobin genotypes content of chicken blood and evaluation of to phenylhydrazine-induced anemia. 2. Hemamethods for its determination. Amer. J. Vet. Res. tology. Poultry Sci. 4 9 : 1 1 4 - 1 2 1 . 3:373-381. Barr, A. J., J. H. Goodnight, J. P. Sail, and J. J. Tung, H. T., F. W. Cook, R. D. Wyatt, and P. B. Helwig, 1976. A users guide to SAS 76. SAS Hamilton, 1975. The anemia caused by aflatoxin. Institute, Inc., Raleigh, NC. Poultry Sci. 54:1962-1969. Brown, J. M. M., 1966. Biochemical changes in the Tung, H. T., W. E. Donaldson, and P. B. Hamilton, livers of domestic birds poisoned with aflatoxin. 1972. Altered lipid transport during aflatoxicosis. South Africa Med. J. 39:778-788. Toxicol. Appl. Pharmacol. 15:97-104. Brown, J. M. M., and L. Abrams, 1965. Biochemical Wannop, C. C , 1961. The histopathology of turkey studies on aflatoxicosis. Onderstepoort J. Vet. "X" disease in Great Britain. Avian Dis. 5:371 — Res. 32:119-146. 381. Clifford, J. I., and K. R. Rees, 1967. The action of Washburn, K. W., G. M. Lanza, and R. D. Wyatt, 1978. aflatoxin B, on the rat liver. Biochem. J. 102: Selection procedures for developing genetic 65-75. resistance to dietary aflatoxin. World Poultry Sci. Datta, P. R., and R. J. Gajan, 1965. Plasma protein J. 10:1773-1778. index of aflatoxin fed ducklings. Life Sci. 4: West, S., R. D. Wyatt, and P. B. Hamilton, 1973. 1791-1795. Improved yield of aflatoxin by incremental Duncan, D. B., 1955. Multiple range and multiple F increases in temperature. Appl. Microbiol. tests. Biometrics 11:1—42. 25:1018-1019. Edwards, G. S., and G. N. Wogan, 1970. Aflatoxin Wiseman, H. G., W. C. Jacobson, and W. C. Harmeyer, inhibition of template activity of rat liver chro1967. Note on removal of pigments from chloromatin. Biochem. Biophys. Acta. 224:597-607. form extracts of aflatoxin cultures with copper Hicks, S. J., J. W. Drysdale, and H. N. Munro, 1969. carbonate. J. Ass. Of fie. Agr. Chem. 50:982— Preferential synthesis of ferritin and albumin by 983. different populations of liver polysomes. Science Wooton, I. D. P., 1964. Micro-analysis in medical 164:584-585. biochemistry. Grune and Stratton, New York. Johnson, P. M., 1955. Hematocrit values for the chick Yu, F. L., 1977. Mechanism of aflatoxin B[ inhibition embryo at various ag*es. Amer. Physiol. 180:361 — of rat hepatic nuclear RNA synthesis. J. Biol. 363. Chem. 252:3245-3251. Kato, R., K. Onoda, and Y. Omari, 1969. Effect of Zlatkis, A., B. Azk, and A. J. Boyle, 1953. A new aflatoxin B, on the incorporation of "C-acetate method for the direct determination of serum into cholesterol by rat liver. Experientia 25:1026. cholesterol. J. Lab. Clin. Med. 41:486-487. occur after 2 weeks of age in young chicks may be secondary effects to the primary effect of protein synthesis inhibition, since plasma protein and cholesterol values were significantly depressed soon after aflatoxin administration, while body weight and PCV depressions occurred later, possibly resulting from the accumulative effect of severe hepatic dysfunction. Although plasma proteins and cholesterol are both depressed at later ages, the severity of the decreases are not as dramatic as observed at early ages. Therefore, secondary effects such as body weight and PCV appear to return to normal, while primary biochemical disruption, though lessened, remains significantly affected.