Delayed Reproductive Development Resulting from Aflatoxicosis in Juvenile Japanese Quail12

Delayed Reproductive Development Resulting from Aflatoxicosis in Juvenile Japanese Quail1'2 J. A. DOERR and M. A. OTTINGER Department of Poultry Science, University of Maryland, College Park, Maryland 20742 (Received for publication November 29, 1979)

1980 Poultry Science 59:1995-2001 INTRODUCTION

Aflatoxin is the trivial name given to a group of highly toxic metabolites produced by the mold Aspergillus flavus when growing in feedstuffs (Ciegler and Lillehoj, 1968). Aflatoxin has a pronounced effect on livers in chickens (Smith and Hamilton, 1970), resulting in reduction of serum proteins (Tung et al, 1975) and inhibition of lipid metabolism (Donaldson et al, 1972; Tung et al, 1972). Additionally, there have been some reports of the effects of aflatoxin on reproduction parameters. Kratzer et al. (1969) reported that feeding more than 2.5 Hg aflatoxin per gram of diet to White Leghorn hens did not alter egg production but did reduce hatchability. However, other workers have reported significant decreases in egg production (Sims et al, 1970; Garlich et al, 1973; Huff et al, 1975). Minimum egg production has been shown to occur 7 days follow-

1 Scientific Article No. A2682. Contribution No. 5727 of the Maryland Agricultural Experiment Station (Department of Poultry Science). 2 A preliminary report of a portion of this paper was presented at the 68th Annual Meeting of the Poultry Science Association, Gainesville, FL, 1979.

ing withdrawal of aflatoxin and to continue to be depressed 18 days after cessation of treatment (Garlich et al, 1973). During aflatoxicosis in Japanese quail, egg production, egg weight, and hatchability were depressed (Sawhney et al, 1973). There have been relatively few reports on aflatoxicosis and reproduction in males. Wyatt et al. (1973) found adverse effects on body weight, liver, pancreas, serum protein, and lipid but not serum cholesterol in broiler breeder males fed 20 ppm aflatoxin for 4 weeks. Briggs et al. (1974) studied semen from breeder males during aflatoxicosis. They observed no effect on sperm count, semen volume, or DNA or RNA of semen. Similarly, no histopathology was found in testes from males receiving an acutely toxic dose of the mycotoxin (Briggs et al, 1974). The reports cited described the response of sexually competent, adult birds to dietary aflatoxin. It seemed appropriate, therefore, to investigate aflatoxicosis in juveniles prior to and during sexual maturation. MATERIALS AND METHODS

Progeny from the University's colony of randombred, white egg-laying Japanese quail

1995

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ABSTRACT Aflatoxicosis was induced in young Japanese quail. In the first experiment five replicates of 30 birds per treatment were fed a soy-corn basal ration containing 0, 5, or 10 jug aflatoxin per gram of feed from 1 to 3 weeks of age. At 3 weeks, the animals were sacrificed and measurements taken. In the second experiment, 0 or 10 ng aflatoxin per gram of diet were fed from either 1 to 3 weeks of age or 2 to 4 weeks of age. At 3 weeks of age body weights were significantly (P<.05) reduced and relative liver weights were significantly (P<.05) increased. Testicular weights relative to body weight were depressed by up to 50%. Ovary wet weights, but not relative weights, were reduced. Testicular development (weight) was impaired through 6 weeks of age. Ovarian development, determined both by weight and number of developing follicles, was delayed as long as 3 weeks following withdrawal of aflatoxin from the diet. Radioimmunoassay for circulating androgens revealed that aflatoxin suppressed both the onset of production and the final concentration of male hormone. The data demonstrate that aflatoxin can exert a deleterious inhibition of sexual development in quail with subsequent impairment of reproductive capacity. These findings raise the implication of potential reproductive failure in economically important species such as broiler breeders. (Key words: aflatoxicosis, Japanese quail, reproduction, sexual maturation, testosterone)

1996

DOERR AND OTTINGER

Aflatoxin was produced by growing Aspergillus parasiticus NRRL 2999 on rice according to the method of Shotwell et al. (1966) with the temperature modifications described by West et al. (1973). The fermented rice was steamed, dried, and ground to a fine powder which was analyzed spectrophotometrically for its aflatoxin content by the method of Nabney and Nesbitt (1965) as modified by Wiseman et

TABLE 1. Composition of soy-corn starter mash for quail Ingredient

% of Diet

Soybean meal Corn Mineral mix a Fat Vitamin mix^ Choline DL-Methionine

45.00 44.82 6.10 3.36 .50 .12 .10

Supplied the following amounts per kilogram of diet: calcium, 12.6 g; phosphorus, 8.12 g; magnesium, 616 mg; manganese, 91 mg; zinc, 67.8 mg; iron, 32 mg; iodine, 5.93 mg; copper, 3.98 mg; selenium, •2mg. Supplied the following amounts per kilogram of diet: niacin, 100 mg; Ca pantothenate, 40 mg; dla-tocopherol acetate, 50 mg; thiamine, 8 mg; riboflavin, 8 mg; pyridoxine, 8 mg; vitamin A, 3,000 IU; folic acid, 3 mg; vitamin K, 1 mg; biotin, 300 Mg: vitamin Bi2, 20 jug; vitamin D 3 , 800 IU.

al. (1967). Aflatoxin was identified and confirmed by the chromatographic method of Pons et al. (1966). Weighed amounts of the rice powder were added to the basal ration (Table 1) and thoroughly blended. Blood collection was either by cardiac puncture or by venous puncture of the brachial vein, with subsequent transfer of whole blood to clean glass tubes. Following clotting of the blood, serum was separated by centrifugation at 1900 X g at 4 C for 20 min. Sera were stored in individual glass vials at —40 C. Serum testosterone concentrations were measured by double antibody radioimmunoassay (RIA) (Micromedic Systems, Horsham, PA) as described by Ottinger and Brinkley (1978). The RIA was validated in quail serum. The sensitivity of the RIA was 7 pg, and parallelism was shown for serial dilutions of quail sera (Ottinger and Doerr, 1980). Body weights were measured weekly and organ weights were measured at sacrifice. Excised ovaries were fixed in 10% neutral buffered formalin, and follicles > .5 mm were counted in fixed ovaries using a 4X dissecting scope. Data were submitted to analysis of variance and significant means partitioned by the method of least significant differences, (Snedecor and Cochran, 1967). RESULTS

The growth response of Japanese quail fed dietary aflatoxin for 14 days is shown in Figure 1. Body weights at 2 and 3 weeks of age were depressed significantly (P<.05) by both treatment levels of aflatoxin. The weight depression at the highest dose of aflatoxin was 25% at 14 days and 28% at 21 days. For the strain of quail used, the apparent threshold for growth inhibition was less than 5 ppm administered for 7 days. In chickens aflatoxin produces an increase in the lipid content of liver resulting in an enlarged, yellow, friable liver (Smith and Hamilton, 1970). Liver weight and appearance were thus taken as indicators of acute toxicity in quail. Table 2 shows the response of quail liver to aflatoxin. Relative to body weight, liver weight was significantly (P<.05) elevated by both dose levels at 3 weeks of age. Among birds receiving 10 ppm aflatoxin, the increase in liver weight was greater than 60% of that measured in control birds. The livers in both aflatoxin groups exhibited tan to yellow coloration that was absent in the control birds.

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were obtained at hatch and placed in electrically heated, wire floored brooders under continuous lighting for 7 days. Feed and water were available ad libitum. The feed consisted of a soy-corn starter mash from which all medications were omitted (Table 1). This basal ration provided 2863 kcal metabolizable energy and 27% protein per kilogram. At 7 days of age, the birds were randomly assigned to treatments. In the first experiment, five pens of 30 birds per pen received one of three treatment levels of dietary aflatoxin, 0, 5, or 10 /ig/g of diet, to 21 days of age when the experiment was terminated. In the second experiment five pens of 30 birds per treatment received either 0 or 10 /ig/g aflatoxin in the diet for 2 weeks beginning at either 7 days of age (Treatment A) or 14 days of age (Treatment B). Following withdrawal of aflatoxin, quail were maintained on the basal ration until 5 weeks of age, at which time remaining quail were transferred to pedigree cages and provided a commercial layer ration.

AFLATOXIN AND REPRODUCTION 70r

• CONTROL • AFLATOXIN (5.0)ig/g) •AFLATOXIN ( I O ) i g / g )

H X e> ui

35-

>o o m

< u 2

_L 7

14 (days)

FIG. 1. Effect of graded levels of dietary aflatoxin on body weight of Japanese quail. Data points with vertical bars are the means of 5 groups of 30 birds ± SE.

Since the gonads of quail suffering aflatoxicosis seemed smaller and less developed by visual inspection, these organs were excised and weighed (Table 2). T h e r e was a significant ( P < . 0 5 ) r e d u c t i o n in testes weight relative to b o d y weight. T h e response of this organ was essentially linear in t h a t the effect was inversely p r o p o r t i o n a l to increasing levels of aflatoxin. Depression of testicular growth was on the order of 29% and 5 3 % of control values for t h e 5 and l O p p m aflatoxin t r e a t m e n t s , respectively. Similar investigation of ovaries revealed t h a t there was a significant ( P < . 0 5 ) depression in ovary wet weight; however, when analyzed relative to b o d y weight, n o effect on this organ could be d e m o n s t r a t e d . T h e foregoing data from 3-week-old quail suggested acute intoxication during a 14 day

exposure to dietary aflatoxin with severe consequences to the testes b u t n o t the ovaries. Therefore, a second trial was begun to e x a m i n e the male gonadal response in greater detail and t o confirm t h e a p p a r e n t lack of response b y t h e ovaries. Body weights of male Japanese quail (Table 3) were depressed in a highly significant ( P < . 0 1 ) m a n n e r by a 2 week feeding of aflatoxin (10 p p m ) initiated at either 7 ( T r e a t m e n t A) or 14 days of age ( T r e a t m e n t B). In t h e T r e a t m e n t A g r o u p , b o d y weights were 3 5 % lower than control at week 3 and 30% at week 4 ; T r e a t m e n t B evoked a less severe depression. At week 3 these birds weighed 29% less than control, while at week 4 b o d y weight was depressed by 24%. Seven days after withdrawal of aflatoxin, this group was still 15% behind its c o n t r o l . Using b o d y weight as the criterion, males recovered from aflatoxicosis at week 5 for T r e a t m e n t A and at week 6 for T r e a t m e n t B. T h e rate of testicular d e v e l o p m e n t during aflatoxicosis was assessed by testes weight. Weight segregates of testes are given in Table 4. A t 3 weeks of age all groups were i m m a t u r e , 100% of the testes pairs weighing less t h a n 100 mg. A m o n g control males d e v e l o p m e n t was observed at week 4 with a high percentage of testes pairs exceeding 1.0 g. By week 6 all controls surpassed 2.0 g testicular weight. Aflatoxin T r e a t m e n t A inhibited the rate of development. A t 5 weeks of age (2 weeks after withdrawal of the m y c o t o x i n diet from this g r o u p ) , the majority of testes were still less t h a n 1.0 g in weight. Recall t h a t b o d y weights in this group were equivalent to c o n t r o l weights at week 5 (Table 3). T r e a t m e n t A birds required 4 to 5 weeks after cessation of toxin administration t o achieve a 2.0 g testicular weight. With aflatoxin T r e a t m e n t B, the suppression of gonadal d e v e l o p m e n t was m o r e p r o n o u n c e d .

TABLE 2. Effect of dietary aflatoxin on some organ weights in three-week old Japanese quail

Aflatoxin —(Mg/g) — 0 5 10

Relative liver weight 3

Relative testes weight 3

-(g/100 g ) -

-(mg/100 g ) -

2.23 ± .10 3.15** ± .05 3.71** + .16

55.5 ±4.0 40.1* ±6.8 26.8** +4.5

Relative ovary weight 3 (mg/100 g) 58.7 ± 8.5 43.1 ±4.0 47.0 ± 2.7

Mean organ weight ± SE per 100 g body weight for 5 groups of 15 birds. Superscripts designate values which differ significantly (*P<.05; **P<.01) from control.

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AGE

21

1997

1998

DOERR AND OTTINGER TABLE 3. Effect of dietary aflatoxin on body weight of Male Japanese quail Aflatoxin treatment Control

Age (weeks)

41.9 b * 59.3 b * 85.5 a 98.4 a 100.6 a

64.2 a ± 2.1 85.3 a ± 2.0 92.4 a ± 4.7 99.0 a ± 4.0 107.6 a ± 2.0

3 4 5 6

45.9 b * 62.lb* 78.5 b 94.1 a 105.6 a

± 2.9 ±6.7 ± 2.7 + 1.6 ± 3.9

± ± ± ± ±

3.8 4.9 6.3 2.9 .8

a.b Values are the mean body weights (g) of 5 birds ± SE. Values in a row with different superscripts differ significantly (P<.05, 'denotes P<.01). Aflatoxin (10 Mg/g) fed week 1 to 3.

2

Aflatoxin (10 Mg/g) fed week 2 to 4.

This group exhibited a delay in testicular m a t u r a t i o n even w h e n compared t o T r e a t m e n t A. Additionally, at 8 weeks of age (4 weeks following toxin administration and 2 weeks after recovery of normal b o d y weight), only 50% of the birds had reached the 2.0 g threshold. Figure 2 shows serum testosterone concentrations during aflatoxicosis. There was a signifi-

TABLE 4. Effect of aflatoxin on percent testicular development in Japanese quail Testes weight range

B2

100 3

100

100

20 80

40 60

1.0 2.0

40 60

<.l .1 1.0 >2.0

1.0 2.0

40 60

<.l .1 1.0 >2.0

1.0 2.0

. 1

1.0 >2.0

1.0 >2.0

•

60 40

UJ


£i CO

^

3.0-

5 75

75

100

75

25

100

00

50 50

2.0

Aflatoxin (10 Mg/g) fed week 1 to 3. Aflatoxin (10 Mg/g) fed week 2 to 4.

3

30 70

6.0r TERON

<.l <.l

1

A1

"(g)-

(weeks) 3

Aflatoxin treatment Control

Values are percent of birds per treatment.

3

SER

Age

cant ( P < . 0 5 ) depression of this h o r m o n e by both toxin t r e a t m e n t s through 4 weeks of age. By week 5, t h e T r e a t m e n t A group testosterone c o n c e n t r a t i o n s , although numerically lower, were statistically equivalent to the controls. Those birds receiving the B t r e a t m e n t , however, had n o t achieved control level concentrations by 6 weeks of age. During t h e 2 weeks following withdrawal of aflatoxin this group exhibited testosterone levels depressed by m o r e than 50%. Table 5 gives b o d y weights of female quail during aflatoxicosis. Both aflatoxin t r e a t m e n t s p r o d u c e d significant ( P < . 0 5 ) growth inhibition

4 AGE

5

6

(weeks)

FIG. 2. Effect of dietary aflatoxin on serum testosterone. Data points with vertical bars are the mean of 5 males ± SE.

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1

AFLATOXIN AND REPRODUCTION

1999

TABLE 5. Effect of dietary aflatoxin on body weight of female Japanese quail Aflatoxin t r e a t m e n t Age

A1

Control

B2

(weeks) 3

62.1a±2.1

4 5 6 8

82.2a±3.1 106.7a±6.2 127.0 a ± 5.0 119.7a±5.1

52.7b±

5 2 . 5 b ± 1.2

.7

68.7b±3.1 92.1b±1.7 106.7 b ± 9.0 111.0a±3.6

70.3 b 86.0 C 99.5b 121.7 a

± 2.4 ± 3.3 + 2.6 + 3.6

' ' Values are the mean body weights (g) of 5 birds ± SE. Values in a row with different superscripts differ significantly (P<.05). ' Aflatoxin (10 Mg/g) fed week 1 to 3. 2

Aflatoxin (10 Mg/g) fed week 2 to 4.

T A B L E 6. Effect of aflatoxin on percent development in Japanese quail

Age

range

Control

ovaries exhibiting signs of development. In week 5, the ovaries of control birds developed rapidly while those in aflatoxin treated groups remained unchanged. Even at eight weeks (4 to 5 weeks following withdrawal of aflatoxin) only one half of the Treatment A group had achieved rapid ovarian development, and two-thirds of the B treatment group remained in the intermediate classification. Follicle counts (Fig. 3) confirmed the retarded ovarian development suggested by the previous data (Table 6). Follicles in the control birds began development at week 3 and accelerated rapidly through weeks 5 and 6. Both aflatoxin groups were severely repressed. Evidence of follicular maturation was not

ovarian

A1

B2

l7 LLI

(weeks)

(g) <1

100 3

100

<.l

100

100

? n o

_i LLI

ri

> > n \ Jhi O

<.l

.1 - 1.0 >1.0

100 40 60

<.l

1

LLI

_l

.1 - 1.0 >1.0

20 80

40 40 20

.1 - 1.0 >1.0

100

50 50

Aflatoxin (10 Mg/g) fed week 1 to 3.

2

Aflatoxin (10 Mg/g) fed week 2 to 4.

3

Values are percent of birds per treatment.

£

ir>

o

A\ _J ri _l 7

(> •—• LL.

OkAl^ tr 3

Le^4 AGE

"I 5

1 6

(Weeks)

FIG. 3. Effect of dietary aflatoxin on follicular development. Data points with vertical bars are the mean of 5 females ± SE.

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which continued for as long as 3 weeks following withdrawal of aflatoxin from the diet. Compared to males (Table 3), the body weights of females were not depressed as severely, but the depression continued for a longer time posttreatment. The weights of the B treatment group were equivalent to Treatment A at week 4 although the B group was still receiving the aflatoxin diet. Evaluation of ovarian response to aflatoxin is shown in Table 6. Again using arbitrary weight limits, ovaries were segregated into three categories. At weeks 3 and 4, there were no

2000

DOERR AND OTTINGER

found until week 5, and at the end of the observation period this parameter continued to be depressed in both groups by 70% or more. Additionally, progress of maturation was linear rather than exponential as was the case in the control group. DISCUSSION

These investigations clearly demonstrate that dietary aflatoxin can produce both acute and delayed effects on the developing reproductive system of juvenile quail. The severity of the response is linked to coincidence of challenge with the onset of puberty. The delayed responses to aflatoxin serve to reinforce earlier reports of diagnostic frustration when symptoms

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Acute aflatoxicosis was induced in young quail receiving graded levels of aflatoxin in the diet. Body weight and relative liver weight responses were similar to those seen in young chickens (Smith and Hamilton, 1970). A twofold reduction in relative testes weights in males occurred during acute aflatoxicosis; however, there was no equivalent manifestation in the females, suggesting that the ovaries were refractory to aflatoxin. This was a surprising result since other toxicological studies in laying birds have demonstrated severe effects of aflatoxin on egg production (Sims et al, 1970; Sawhney et al, 1973; Huff et al, 1975). The apparent discrimination of effect by sex was resolved in the second experiment. During a longitudinal study it was found that sexual development in either sex was impaired and that ovarian response was best determined during and after the normal period of maturation. In the case of males, testicular growth exhibited a lag of approximately 20 to 70% at any given time depending on the aflatoxin treatment imposed. Treatment A, which induced acute toxicity early in the life of the bird (1 to 3 weeks of age), delayed testicular maturation by more than 2 weeks; Treatment B (2 to 4 weeks of age) prolonged maturation by more than 4 weeks. Similarly, testosterone concentrations were depressed 1 week beyond the acute toxicity phase (Treatment A) and for at least 2 weeks more when Treatment B was given. Since the onset of rapid maturation of testes in the Japanese quail occurs 25 days posthatch,andperipheral testosterone concentrations are highly correlated with testicular weights (Ottinger and Brinkley, 1979), it would seem likely that the effect of an acute aflatoxin insult terminating on day 21 (Treatment A) represents generalized growth depression with relatively mild postinsult sequelae. By contrast, acute aflatoxicosis coincident with the onset and progress of testicular maturation and androgen synthesis (Treatment B) resulted not only in growth inhibition but apparently also in suppression of anabolic mechanisms in the

testes as evidenced by the prolonged, severe depression of serum testosterone (Fig. 2). The mechanism by which this effect occurs (i.e., interruption of androgen synthesis, depression of androgen precursors, synthesis of defective androgen, altered profile of androgen species, or other) remains to be determined. In these experiments, females seemed more sensitive to aflatoxin in terms of residual growth effects. Although males recovered normal body weight and growth rate by 2 weeks posttreatment, females still exhibited depressed body weights as late as 3 weeks after withdrawal of the aflatoxin challenge. Additionally, the ovary was revealed to be sensitive to aflatoxin although a measurable effect was not evoked before the 4 weeks of age. The ovary, which in Coturnix undergoes a rapid growth stage between 28 and 43 days of age (Fitzgerald, 1969), exhibited weight depression as late as 5 weeks after the birds were removed from aflatoxin treatment. Follicles constitute the major tissue reservoir in the quail ovary (Fitzgerald, 1969), and, therefore, follicular counts served to confirm the impression of retarded ovarian development derived from the weight data. Committment of follicles to maturation was quantitively impaired at week 4 by either aflatoxin treatment. Garlich et al (1973) demonstrated that during acute aflatoxicosis in laying hens, depressed serum constituents were preferentially channeled into developing eggs with the result that ova already committed to maturation proceed through full development. They also determined that initiation of maturation among uncommitted follicles is arrested. Our data are thus consistent with the response observed in chickens. However, in this study qualitative parameters of maturing and noncommitted follicles were not investigated. Although it has been reported that follicular maturation, as determined by lipid soluble dye accumulation, is evident in follicles of 2.0 mm diameter (Bacon and Koontz, 1971), the foregoing follicle data (Fig. 3) were based on a .5 mm limit which, we believe, provided greater sensitivity to the toxic effects of aflatoxin.

AFLATOXIN AND REPRODUCTION

occur subsequent to consumption of the responsible, mycotoxin contaminated feed (Garlich et ai, 1973; Hamilton, 1975, 1978). Finally, the data have ominous implications for breeder programs given the prospect of suboptimal reproductive performance among birds exposed at puberty to aflatoxin. ACKNOWLEDGMENTS

The authors wish to thank Cynthia Duchala and Stacey Zlotnik for their technical assistance. REFERENCES

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Bacon, W. L., and M. Koontz, 1971. Ovarian follicular growth and maturation in Coturnix quail. Poultry Sci. 50:233-236. Briggs, D. M., R. D. Wyatt, and P. B. Hamilton, 1974. The effect of dietary aflatoxin on semen characteristics of mature broiler breeder males. Poultry Sci. 53:2115-2119. Ciegler, A., and E. G. Lillehoj, 1968. Mycotoxins. Adv. Appl. Microbiol. 10:155-219. Donaldson, W. E., H. T. Tung, and P. B. Hamilton, 1972. Depression of fatty acid synthesis in chick liver (Gallus domesticus) by aflatoxin. Comp. Biochem. Physiol. 41B:843-847. Fitzgerald, T. A., 1969. The Coturnix quail. The Iowa State University Press, Ames, IA. Garlich, J. D., H. T. Tung, and P. B. Hamilton, 1973. The effects of term feeding on egg production and some plasma constituents of the laying hen. Poultry Sci. 52:2206-2211. Hamilton, P. B., 1975. Proof of mycotoxicoses being a field problem and a simple method for their control. Poultry Sci. 54:1706-1708. Hamilton, P. B., 1978. Fallacies in our understanding of mycotoxins. J. Food Protect. 41:404—408. Huff, W. E., R. D. Wyatt, and P. B. Hamilton, 1975. Effects of dietary aflatoxin on certain egg yolk parameters. Poultry Sci. 54:2014-2018. Kratzer, F. H., D. Bandy, M. Wiley, and A. N. Booth, 1969. Aflatoxin effects in poultry. Proc. Soc. Exp. Biol. Med. 131:1281-1284. Nabney, J., and B. F. Nesbitt, 1965. A spectrophotometric method of determining the aflatoxins. Analyst 90:155-160.

2001