1636
P. C. MILLER AND M. L. SUNDE
Poultry Sci. 40: 708-716. Waldroup, P. W., B. L. Damron and R. H. Harms, 1966. The effect of low protein and high fiber grower diets on the performance of broiler pullets. Poultry Sci. 45: 393-402. Wolf, J. D., E. W. Gleaves, L. V. Tonkinson, R. H. Thayer and R. D. Morrison, 1969. Dietary protein, energy and volume in pullet grower diets as related to growing and laying performance. Poultry Sci. 48: 559-574. Wright, C. F., B. L. Damron, P. W. Waldroup and R. H. Harms, 1968. The performance of laying hens fed normal and low protein diets between 8-18 weeks of age. Poultry Sci. 47: 635-638. Yates, J. D., and P. J. Schaible, 1963. Skip-feeding and energy level of the ration for developing Leghorn type pullets. Feedstuffs, 35(46): 18.
Toxicity of Dietary Lead in Japanese Quail12 G. W. MORGAN, F. W. EDENS, P. THAXTON AND C. R. PARKHURST
Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27607 (Received for publication January 21, 1975)
ABSTRACT The toxicity of dietary lead in Japanese quail was investigated. The data indicated that dietary lead, in the form of lead acetate, was toxic to young quail at the level of 500 p.p.m. and this toxicity was evidenced by an inhibition of normal growth and by anemia. The anemic state in the lead toxic quail was more readily detected by reduced blood hemoglobin concentrations than by packed cell volumes. In addition, the data suggested that lead interfered with normal sexual development in the males. Lead at levels as high as 1000 p.p.m. did not prevent normal primary antibody responses to sheep erythrocytes. POULTRY SCIENCE 54: 1636-1642, 1975
INTRODUCTION
T eral
injection of rats with inorganic lead caused
HE effects of toxic levels of lead in mammals are diverse and represent sevfunctional
systemic
alterations.
However, the toxic effects of lead containing compounds in avian species have not been fully explored and are poorly understood at best. Yamamoto et al. (1974) reported that
mobilization of bone calcium and a concomitant increase in calcium content of the liver. Lead is known to potentiate the toxic effects of endotoxin in chicks (Truscott, 1970) and in mammals (Selye et al., 1966; Cook et
al,
1974). However, the mechanism of this effect remains obscure. Koller and Kovacic (1974) reported that mice exposed to lead had a reduced number of splenic antibody plaque
1. Paper number 4561 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, NC 27607. 2. A preliminary report of this paper was presented to the 72nd Annual Meeting of the SAAS, New Orleans, LA.
forming cells. Hilderbrand et al. (1973) discussed the possibility of hazardous effects of lead on sexuality and reproductive function in the rat. In addition, Goldberg (1972) reviewed the role of lead poisoning in heme
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 7, 2015
of photoperiodism and rearing period feed restriction on the performance of five Leghorn strains. Poultry Sci. 46: 1056-1072. Ringrose, R. C , 1958. Restricted feeding of growing pullets. Ag. Exp. Sta. Bull. 456, Univ. of New Hampshire, Durham, N.H. Strain, J. H., R. S. Gowe, R. D. Crawford, A. T. Hill, S. B. Slenand W. F. Mountain, 1965. Restricted feeding of growing pullets: 1. The effect on the performance traits of egg production stock. Poultry Sci. 44: 701-726. Sunde, M. L., W. W. Cravens, H. R. Bird and J. G. Halpin, 1954. The effect of complete and incomplete growing diets on subsequent performance of the laying hen. Poultry Sci. 33: 779-784. Turk, D. E., W. G. Hoekstra, H. R. Bird and M. L. Sunde, 1961. The effect of dietary protein and energy levels on the growth of replacement pullets.
L E A D TOXICITY IN QUAIL
biosynthesis. It is obvious that the effects of toxic levels of lead are diverse and represent no known central mechanistic lesion. This study was intended to provide a much needed model for future investigations concerning the toxic effects which lead may cause in all animal species. Dietary lead was fed to Japanese quail during the early juvenile period and growth, hematological changes, immunological responsiveness and the development of various major body organs were the criteria studied.
Unsexed Japanese quail chicks from the North Carolina State University colony were used in this study. At hatching the quail were placed in heated metal batteries. The brooding temperature was maintained at approximately 35° C. for the first week and weekly thereafter for the next four weeks it was reduced by 5°. Continuous 24 hour fluorescent lighting was provided during the first week. From one week of age until each experiment was terminated the lighting scheme consisted of one 14 hour photoperiod and 10 hours of darkness per 24 hour period. The experimental diets consisted of a nutritionally complete quail starter diet (N.C.S. S-l, 28% protein; Gehle and Briggs, 1973) to which lead, in the form of lead acetate, was added in varying quantities. In all cases the amount of lead added was corrected for the acetate content and the concentrations of lead (fig./gm. of feed) was expressed as parts per million. Feed and water were supplied ad libitum. All bleedings and injections were made intravenously via the brachial veins. Hemoglobin determinations were made by the oxyhemoglobin method (Sunderman et al., 1953) and packed cell volumes were determined by a microhematocrit method (Schalm, 1965). The quail were immunized with single injections of 0.5 ml. of a 15% saline suspension
of washed sheep red blood cells (SRBC). Blood samples for subsequent serum antibody analysis by a microhemagglutination method (Witlin, 1967) were collected, allowed to clot at room temperature, refrigerated at 4° C. for a minimum of six hours and then centrifuged at 500 x g for 45 minutes. The resulting serum samples were decanted into glass vials and stored frozen at -20° C. until antibody analyses were performed. Immediately prior to antibody titration the serum samples were thawed at room temperature and the complement activity was destroyed by heating in a water bath at 56° C. for 30 minutes. Immediately following the last bleeding the quail were killed by cervical dislocation and the bursa, spleen, heart, liver, adrenals and testes were excised and weighed. Relative organ weights were calculated and expressed as mg. organ weight per gm. body weight. In Trial 1 five experimental groups consisting of 30 quail per group were fed diets containing 0, 1,10, 100, or 1000 p.p.m. added lead beginning on day 6 of the trial. All groups received the basal diet during the first five days. At two weeks of age and at two week intervals thereafter through six weeks of age, body weights, packed cell volumes, and hemoglobin concentrations were determined on 20 quail which were randomly selected from each group. At five weeks of age 10 male quail from each group were immunized with SRBC. Blood samples for subsequent antibody titration were collected seven days post-immunization. At six weeks of age 10 randomly selected males from each group were sacrificed and the aforementioned organs were removed and weighed. In Trial 2 five experimental groups consisting of 30 quail each were fed diets containing 0, 10, 100, 500, or 1000 p.p.m. added lead. All groups were maintained on their respective diets for the full five week duration of the trial. Body weights, packed cell volumes, and hemoglobin concentrations (on 25,20 and
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 7, 2015
MATERIALS AND METHODS
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G. W. MORGAN, F. W. EDENS, P. THAXTON AND C. R. PARKHURST
RESULTS
evident when these groups were compared to the controls. As was the case with growth, blood hemoglobin concentrations were not significantly altered by 100 p.p.m. or lower levels of lead. The data depicting the effects of lead on packed cell volumes are presented in Figure 4. Again, only the data of Trial 2, which are representative of both trials, are presented. There were no significant differences among treatment groups during the first three weeks of the experiment and significantly (P < .01) reduced packed cell volumes at four and five weeks of age were observed only in the 1000 p.p.m. lead group. The relative organ weights of the quail of Trials 1 and 2 are presented in Tables 1 and
The effects of dietary lead acetate on the body weights of quail in Trials 1 and 2 are presented graphically in Figures 1 and 2, respectively. Lead acetate added to the diet of young quail resulted in toxicity at the level of 1000 p.p.m. of lead in Trial 1 and at 500 and 1000 p.p.m. of lead in Trial 2, as evidenced by a continuous significant inhibition of growth. This inhibition was observed initially at two weeks of age and persisted throughout the duration of the experiments. The body weights of the birds in Trial 1 which received 1000 p.p.m. and those in Trial 2 which received either 500 or 1000 p.p.m. added lead were significantly (P < .01) lower than those of the controls (0 p.p.m.). Additionally, it should be noted that the body weights of birds that received 1, 10, or 100 p.p.m. lead did not differ significantly from the controls at any time throughout the duration of the trials. The effects of dietary lead on the blood hemoglobin concentrations are illustrated in Figure 3. The results obtained in Trials 1 and 2 were similar, and the data of Trial 2 are presented as representative of both trials. Hemoglobin concentrations were reduced significantly (P < .01) by both 500 and 1000 p.p.m. of added lead. This was
AGE (weeks)
FIG. 1. Effect of lead on the body weight of the Japanese quail of Trial 1. Each point represents the mean of 20 observations. Vertical lines in this graph as well as the following graphs represent standard errors of the means (S.E.M.). O = 0 p.p.m.; • = 1 p.p.m.; A = 10 p.p.m.; + = 100 p.p.m.; * = 1000 p.p.m. added dietary lead.
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20 randomly selected quail, respectively) were determined for each of the five groups at 2, 3, 4, and 5 weeks of age. At four weeks of age the birds were immunized with SRBC and blood samples for subsequent antibody determinations were collected at 4 days postimmunization. Ten males from each group were sacrificed at five weeks of age and the relative organ weights were calculated. The data were analyzed by analysis of variance and when the F-ratio indicated significant treatment effects, the treatment means were compared by the multiple range test of Duncan (1955).
1639
LEAD TOXICITY IN QUAIL
45
r
4 AGE (weeks)
FIG. 2. Effect of lead on the body weight of the Japanese quail of Trial 2. Each point represents the mean of 25 observations. In this and all subsequent graphs O = 0 p.p.m.; • = 10 p.p.m.; A = 100 p.p.m.; + = 500 p.p.m.; * = 1000 p.p.m. added dietary lead.
I2r
3
4 AGE (weeks)
FIG. 3. Effect of lead on blood hemoglobin in the Japanese quail of Trial 2. Each point represents the mean of 20 observations.
4 AGE (weeks)
FIG. 4. Effect of lead on the packed cell volume of the Japanese quail of Trial 2. Each point represents the mean of 20 observations.
2, respectively. The relative weights of the bursa, spleen, liver and heart were not altered significantly in either trial by any of the dose levels of lead. However, the data of Trial 1 indicate that at six weeks of age the testes weights were reduced in the quail which received 1000 p.p.m. of lead, though they did not differ significantly from those of the controls (Table 1). The quail which received lOOOp.p.m. of lead exhibited a wider variation in testes size than those of the other four groups. Certain quail in this group possessed normal sized testes while others had near adolescent sized testes. In view of this individual discrepancy in testicular maturation time the decision was made to terminate Trial 2 when the quail reached five weeks of age. Thus in Trial 2 the effects of lead were determined at an earlier stage of testicular development. At five weeks of age (Table 2) testes size was reduced significantly (P < .05) in quail which were fed 1000 p.p.m. of lead. The reduction in testes size was relatively uniform throughout this treatment
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3
3
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G. W. MORGAN, F. W. EDENS, P. THAXTON AND C. R. PARKHURST
TABLE 1.—Mean relative organ weights (mg./gm. of body weight) of 6 week-old male Japanese quail of Trial 1 which received dietary lead acetate12-4 Lead (p.p.m.) 0 1 10 100 1000
Organ Spleen .300 .332 .429 .409 .3%
± ± ± ± ±
.033a .026a .048a .052a .033a
Adrenal 3
Bursa .650 .837 .792 .701 .824
± ± ± ± ±
.063a .087a .087a .059a .119a
.128 . 152 .120 . 156 .161
± .012a ±.018a ± . 0 1 la ±.025a ± .024a
Heart 10.79 ± .17a 11.26±.30a 10.52 ± . 2 5 a 11.26 ± .53a 11.95 ± .58a
Liver 22.57 21.99 22.68 26.30 27.28
± ± ± ± ±
1.11a 1.32a 1.37a 1.17a 1.57a
Testes 21.30 25.40 21.79 22.00 15.39
± ± ± ± ±
1.57a,b 1.84a 1.23a,b 1.32a,b 3.65b
1 Mean ± standard error of the mean. 2 Each mean represents 10 observations. 3 Both adrenals. 4
Means not followed by the same small letter differ significantly at P < .01 (Duncan, 1955).
Lead 9 ^ (p.p.m.) Spleen Bursa Adrenal3-4 Heart Liver Testes5 _ 0 .374 ± .022a .790 ± .103a .0263 ± .0029a 12.05 ± .28a 22.08 ± .67a 4.43 ± 1.60a,b 10 .388 ± .029a .989 ± .120a .0308 ± .0020a,b 11.00 ± .49a 24.52+ .98a 5.39 ± 1.06a 100 .397 ± .032a 1.010 ± .106a .0335 ± .0030a,b 11.68 ± .43a 24.05 ± .81a 2.23 ± .76b,c 500 .401 ± .026a .772 ± .060a .0504 ± .0082b,c 11.89 ± .51a 24.93 ± 1.13a 3.78 ± .69b 1000 .444 ± .031a .798 ± .084a .0559 ± .0080c 11.31 ± 1.33a 24.62 ± .76a .87 ± .12c 'Mean ± standard error of the mean. 2 Each mean represents 10 observations. 3 Means not followed by the same small letter differ significantly at P < .01 (Duncan, 1955). 4 Right adrenal only. 5 Means not followed by the same small letter differ significantly at P < .05 (Duncan, 1955).
group. Doses of lead lower than 1000 p.p.m. did not result in significantly reduced testes weights. In Trial 2 the relative adrenal weights were increased significantly (P < .01) by both 500 and 1000 p.p.m. of lead (Table 2). It should be noted that the same trend of adrenal hypertrophy occurred in the quail of Trial 1 which received 1000 p.p.m. of lead; however, significant increases were not found (Table 1). Doses of 100 p.p.m. of lead or lower did not result in significant changes in relative adrenal size in either trial. As indicated in Table 3, all of the quail which received lead demonstrated a normal ability to express a primary humoral immune response following antigenic challenge with SRBC. Significant treatment differences in anti-SRBC hemagglutinin titers were not
TABLE 3.—Anti-SRBC hemagglutinin levels (log2) of Japanese quail which received dietary lead' Lead (p.p.m.) 0 1
Trial l 2 4.5 ± .4
Trial 23 4.8 ± .2
4.1 ± .2
—
10 3.8 ± .3 4.7 100 3.7 ± .4 4.8 500 — 4.5 1000 3.3 ± .4 4.5 1 Mean ± standard error of the mean. 2 Each mean represents 10 observations. 3 Each mean represents 20 observations.
± ± ± ±
.2 .3 .3 .3
noted among any of the treatment groups in either trial. DISCUSSION The results of this study indicate that dietary lead in the form of lead acetate when administered at levels of 500 or 1000 p.p.m.
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 7, 2015
TABLE 2.—Mean relative organ weights (mg./gm. of body weight) of 5 week-old male Japanese quail of Trial 2 which received dietary lead'-2
L E A D TOXICITY IN QUAIL
Lead at the levels employed in this study did not prove to be immunosuppressive. Koller and Kovacic (1974) reported that oral administration of lead acetate at 13.75 p.p.m., 137.5 p.p.m. and 1,375 p.p.m. to mice in the drinking water resulted in significantly reduced numbers of splenic antibody plaque forming cells to sheep erythrocytes. These workers indicated however, that the secondary immune response was more susceptible to lead than the primary immune response. Whether quail are less susceptible to immune injury by lead than mice, or whether lead is simply more toxic when administered in the drinking water than in the feed, or both are as yet unanswered questions. However, our data indicated that constant exposure of Japanese quail to dietary lead resulted in
inhibition of growth and reduced hemoglobin levels at lead doses which were not suppressive to the primary immune response. In Trial 1, when the testes were removed for weight analysis at 6 weeks, it appeared that certain of the quail which were fed the highest level (1000 p.p.m.) of lead had severely retarded testicular development, while others in this same group had testes which approximated normal size. Since Japanese quail under lighting schemes similar to the one utilized in this study (Tanaka et al., 1965) normally reach sexual maturity at about six weeks of age, this finding suggested that a more pronounced effect might be noted at an earlier stage of sexual development. The results obtained in Trial 2 in which testes weights were measured at 5 weeks, supported this concept and suggested that lead at the level of 1000 p.p.m. delayed the development of normal testes size. An alternate interpretation is that since the quail in Trial 1 did not begin receiving lead until six days of age the body lead burden was not elevated sufficiently to prevent normal testicular development in all of the quail. In this regard it would be necessary to assume that there are individual variations in tolerance to the body burden of lead. It can also be noted that adrenal size was increased significantly in the quail which received 500 or 1000 p.p.m. lead at five weeks, but not at six weeks of age. To speculate on a possible role for lead in the adrenal-testes interrelationship would be premature at this time. However, Hilderbrand et al. (1973) noted the possibility of hazardous effects of lead on sexuality and reproductive function in sexually mature rats which received lead orally. The results of this study indicate that lead is toxic when administered in the diet to Japanese quail and that the primary manifestations of lead toxicity in young quail include growth inhibition and anemia. The effects of lead on testicular development in quail needs further clarification. Obviously,
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 7, 2015
for a period of 5 weeks interfered with normal growth in the Japanese quail. Additionally, it is concluded that the inhibition of normal growth is a primary characteristic of lead toxicity in the Japanese quail. Our data also indicate that the quail which received the growth inhibitory levels of lead experienced an anemia. Goldberg (1972) reviewed the relationship of toxic levels of lead to anemia in mammals. He indicated that anemia is a primary symptom of lead toxicity. The anemic state in quail was detected more readily by hemoglobin analysis than by packed cell volumes. This indicates that either the anemic state was caused by a reduced hemoglobin content of normal size cells or that fewer, but larger erythroid cells were present in the circulation. The mechanism by which lead causes anemia; whether it is by increasing hemolysis (Hasan et al., 1967), interfering with hemoglobin synthesis at the enzyme level (Kao and Forbes, 1973), or by reacting with hemoglobin directly (Barltrop and Smith, 1972) remains unknown. However, Barltrop and Smith (1972) presented evidence suggesting that free sulphydryl groups are not essential to the formation of the lead-hemoglobin complex.
1641
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G. W. MORGAN, F. W. EDENS, P. THAXTON AND C. R. PARKHURST
this is of considerable interest not only to those engaged in experimentation with birds but to those involved with lead exposure in man and other mammals. Experiments are in progress which should further clarify the relationship of lead to reproductive function and behavior. ACKNOWLEDGEMENTS
REFERENCES Barltrop, D., and A. Smith, 1972. Lead binding to human haemoglobin. Experientia, 28: 76-77. Cook, J. A., E. A. Marconi and N. R. DiLuzio, 1974. Lead, cadmium, endotoxin interaction: Effect on mortality and hepatic function. Toxicol. Appl. Pharmacol. 28: 292-302. Duncan, D. B., 1955. Multiple range and multiple " F " tests. Biometrics, 11: 1-42. Gehle, M. H., and D. M. Briggs, 1973. Comparison of starter and grower rations on Bobwhite quail performance. Poultry Sci. 52: 2031. Goldberg, A., 1972. Lead poisoning and heme biosynthesis. Brit. J. Haematol. 23: 521-524. Hasan, J., V. Wikho and S. Hernberg, 1967. Deficient red cell membrane (Na+ + K + )—ATPase in lead
NEWS AND NOTES (Continued from page 1623) Lowest Pounds of Feed Per Pound of Eggs Produced—2.19 lbs.—Carey Farms (Carey Nick 300), Marion, Ohio. Heaviest Average Egg Weight—27.3 oz.—Parks Poultry Farm (Sil-Go-Links), Altoona, Pennsylvania. Highest Percent Large and Extra Large Eggs— 91.1%—Harco Farms, Division of Arbor Acre Farms (Harco Sex Link), South Easton, Massachusetts. Highest Albumen Quality—86.5 Haugh units—Carey Farms (Carey Nick 310), Marion, Ohio.
Lowest Percent of Blood Spots 1 /8 Inch or More— 0.5%—Parks Poultry Farm (Keystone B-l), Altoona, Pennsylvania; Shaver Poultry Breeding Farms, Ltd. (Starcross 288), Cambridge, Ontario, Canada; and Colonial Poultry Farms, Inc. (TrueLine 365B), Pleasant Hill, Missouri. Lowest Percent of Blood Spots Less Than 1 /8 Inch— 0.7%—Carey Farms (Carey Nick 310), Marion, Ohio. Lowest Percent of Meat Spots Less Than 1/8 Inch— 0.1%—Colonial Poultry Farms, Inc. (True-Line 365B), Pleasant Hill, Missouri.
(Continued on page 1646)
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 7, 2015
Appreciation is extended to Mrs. Bonnie Goodwin, Mrs. Jeannine Gilbert, Mrs. Grace Brockman and Mrs. Frances Penny for excellent technical assistance.
poisoning. Arch. Environ. Health, 14: 313-318. Hilderbrand, D. C , R. Der, W. T. Griffin and M. S. Fahim, 1973. Effect of lead acetate on reproduction. Am. J. Obstet. Gynecol. 115: 1058-1065. Kao, R. L. C , and R. M. Forbes, 1973. Effects of lead on heme-synthesizing enzymes and urinary delta aminolevulinic acid in the rat. Proc. Soc. Exp. Biol. Med. 143: 234-237. Koller, L. D., and S. Kovacic, 1974. Decreased antibody formation in mice exposed to lead. Nature, 250: 148-150. Schalm, O. W., 1965. Veterinary Hematology, Lea and Febiger, Philadelphia, PA, p. 84-87. Selye, H., B. Tuchweber and L. Bertok, 1966. Effect of lead acetate on susceptibility of rats to bacterial endotoxins. J. Bacterid. 91: 884-890. Sunderman, F. W., R. P. MacFate, D. A. McFadyen, G. F. Stevenson and B. E. Copeland, 1953. Symposium on clinical hemoglobinometry. Am. J. Clin. Path. 23: 519-598. Tanaka, K„ F. B. Mather, W. O. Wilson and L. Z. McFarland, 1965. Effect of photoperiods on early growth of gonads and on potency of gonadotropins of the anterior pituitary in coturnix. Poultry Sci. 44: 662-665. Truscott, R. B., 1970. Endotoxin studies in chicks: Effect of lead acetate. Can. J. Comp. Med. 34: 134-137. Witlin, B., 1967. Detection of antibodies by microtitration techniques. Mycopathologia et Mycologia Applicata, 33: 241-257. Yamamoto, T., M. Yamaguchi and Y. Suketa, 1974. Calcium accumulation in liver and calcium mobilization in bone of lead-poisoned rats. Toxicol. Appl. Pharmacol. 27: 204-205.
P. C. MILLER AND M. L. SUNDE
Poultry Sci. 40: 708-716. Waldroup, P. W., B. L. Damron and R. H. Harms, 1966. The effect of low protein and high fiber grower diets on the performance of broiler pullets. Poultry Sci. 45: 393-402. Wolf, J. D., E. W. Gleaves, L. V. Tonkinson, R. H. Thayer and R. D. Morrison, 1969. Dietary protein, energy and volume in pullet grower diets as related to growing and laying performance. Poultry Sci. 48: 559-574. Wright, C. F., B. L. Damron, P. W. Waldroup and R. H. Harms, 1968. The performance of laying hens fed normal and low protein diets between 8-18 weeks of age. Poultry Sci. 47: 635-638. Yates, J. D., and P. J. Schaible, 1963. Skip-feeding and energy level of the ration for developing Leghorn type pullets. Feedstuffs, 35(46): 18.
Toxicity of Dietary Lead in Japanese Quail12 G. W. MORGAN, F. W. EDENS, P. THAXTON AND C. R. PARKHURST
Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27607 (Received for publication January 21, 1975)
ABSTRACT The toxicity of dietary lead in Japanese quail was investigated. The data indicated that dietary lead, in the form of lead acetate, was toxic to young quail at the level of 500 p.p.m. and this toxicity was evidenced by an inhibition of normal growth and by anemia. The anemic state in the lead toxic quail was more readily detected by reduced blood hemoglobin concentrations than by packed cell volumes. In addition, the data suggested that lead interfered with normal sexual development in the males. Lead at levels as high as 1000 p.p.m. did not prevent normal primary antibody responses to sheep erythrocytes. POULTRY SCIENCE 54: 1636-1642, 1975
INTRODUCTION
T eral
injection of rats with inorganic lead caused
HE effects of toxic levels of lead in mammals are diverse and represent sevfunctional
systemic
alterations.
However, the toxic effects of lead containing compounds in avian species have not been fully explored and are poorly understood at best. Yamamoto et al. (1974) reported that
mobilization of bone calcium and a concomitant increase in calcium content of the liver. Lead is known to potentiate the toxic effects of endotoxin in chicks (Truscott, 1970) and in mammals (Selye et al., 1966; Cook et
al,
1974). However, the mechanism of this effect remains obscure. Koller and Kovacic (1974) reported that mice exposed to lead had a reduced number of splenic antibody plaque
1. Paper number 4561 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, NC 27607. 2. A preliminary report of this paper was presented to the 72nd Annual Meeting of the SAAS, New Orleans, LA.
forming cells. Hilderbrand et al. (1973) discussed the possibility of hazardous effects of lead on sexuality and reproductive function in the rat. In addition, Goldberg (1972) reviewed the role of lead poisoning in heme
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 7, 2015
of photoperiodism and rearing period feed restriction on the performance of five Leghorn strains. Poultry Sci. 46: 1056-1072. Ringrose, R. C , 1958. Restricted feeding of growing pullets. Ag. Exp. Sta. Bull. 456, Univ. of New Hampshire, Durham, N.H. Strain, J. H., R. S. Gowe, R. D. Crawford, A. T. Hill, S. B. Slenand W. F. Mountain, 1965. Restricted feeding of growing pullets: 1. The effect on the performance traits of egg production stock. Poultry Sci. 44: 701-726. Sunde, M. L., W. W. Cravens, H. R. Bird and J. G. Halpin, 1954. The effect of complete and incomplete growing diets on subsequent performance of the laying hen. Poultry Sci. 33: 779-784. Turk, D. E., W. G. Hoekstra, H. R. Bird and M. L. Sunde, 1961. The effect of dietary protein and energy levels on the growth of replacement pullets.
L E A D TOXICITY IN QUAIL
biosynthesis. It is obvious that the effects of toxic levels of lead are diverse and represent no known central mechanistic lesion. This study was intended to provide a much needed model for future investigations concerning the toxic effects which lead may cause in all animal species. Dietary lead was fed to Japanese quail during the early juvenile period and growth, hematological changes, immunological responsiveness and the development of various major body organs were the criteria studied.
Unsexed Japanese quail chicks from the North Carolina State University colony were used in this study. At hatching the quail were placed in heated metal batteries. The brooding temperature was maintained at approximately 35° C. for the first week and weekly thereafter for the next four weeks it was reduced by 5°. Continuous 24 hour fluorescent lighting was provided during the first week. From one week of age until each experiment was terminated the lighting scheme consisted of one 14 hour photoperiod and 10 hours of darkness per 24 hour period. The experimental diets consisted of a nutritionally complete quail starter diet (N.C.S. S-l, 28% protein; Gehle and Briggs, 1973) to which lead, in the form of lead acetate, was added in varying quantities. In all cases the amount of lead added was corrected for the acetate content and the concentrations of lead (fig./gm. of feed) was expressed as parts per million. Feed and water were supplied ad libitum. All bleedings and injections were made intravenously via the brachial veins. Hemoglobin determinations were made by the oxyhemoglobin method (Sunderman et al., 1953) and packed cell volumes were determined by a microhematocrit method (Schalm, 1965). The quail were immunized with single injections of 0.5 ml. of a 15% saline suspension
of washed sheep red blood cells (SRBC). Blood samples for subsequent serum antibody analysis by a microhemagglutination method (Witlin, 1967) were collected, allowed to clot at room temperature, refrigerated at 4° C. for a minimum of six hours and then centrifuged at 500 x g for 45 minutes. The resulting serum samples were decanted into glass vials and stored frozen at -20° C. until antibody analyses were performed. Immediately prior to antibody titration the serum samples were thawed at room temperature and the complement activity was destroyed by heating in a water bath at 56° C. for 30 minutes. Immediately following the last bleeding the quail were killed by cervical dislocation and the bursa, spleen, heart, liver, adrenals and testes were excised and weighed. Relative organ weights were calculated and expressed as mg. organ weight per gm. body weight. In Trial 1 five experimental groups consisting of 30 quail per group were fed diets containing 0, 1,10, 100, or 1000 p.p.m. added lead beginning on day 6 of the trial. All groups received the basal diet during the first five days. At two weeks of age and at two week intervals thereafter through six weeks of age, body weights, packed cell volumes, and hemoglobin concentrations were determined on 20 quail which were randomly selected from each group. At five weeks of age 10 male quail from each group were immunized with SRBC. Blood samples for subsequent antibody titration were collected seven days post-immunization. At six weeks of age 10 randomly selected males from each group were sacrificed and the aforementioned organs were removed and weighed. In Trial 2 five experimental groups consisting of 30 quail each were fed diets containing 0, 10, 100, 500, or 1000 p.p.m. added lead. All groups were maintained on their respective diets for the full five week duration of the trial. Body weights, packed cell volumes, and hemoglobin concentrations (on 25,20 and
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 7, 2015
MATERIALS AND METHODS
1637
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G. W. MORGAN, F. W. EDENS, P. THAXTON AND C. R. PARKHURST
RESULTS
evident when these groups were compared to the controls. As was the case with growth, blood hemoglobin concentrations were not significantly altered by 100 p.p.m. or lower levels of lead. The data depicting the effects of lead on packed cell volumes are presented in Figure 4. Again, only the data of Trial 2, which are representative of both trials, are presented. There were no significant differences among treatment groups during the first three weeks of the experiment and significantly (P < .01) reduced packed cell volumes at four and five weeks of age were observed only in the 1000 p.p.m. lead group. The relative organ weights of the quail of Trials 1 and 2 are presented in Tables 1 and
The effects of dietary lead acetate on the body weights of quail in Trials 1 and 2 are presented graphically in Figures 1 and 2, respectively. Lead acetate added to the diet of young quail resulted in toxicity at the level of 1000 p.p.m. of lead in Trial 1 and at 500 and 1000 p.p.m. of lead in Trial 2, as evidenced by a continuous significant inhibition of growth. This inhibition was observed initially at two weeks of age and persisted throughout the duration of the experiments. The body weights of the birds in Trial 1 which received 1000 p.p.m. and those in Trial 2 which received either 500 or 1000 p.p.m. added lead were significantly (P < .01) lower than those of the controls (0 p.p.m.). Additionally, it should be noted that the body weights of birds that received 1, 10, or 100 p.p.m. lead did not differ significantly from the controls at any time throughout the duration of the trials. The effects of dietary lead on the blood hemoglobin concentrations are illustrated in Figure 3. The results obtained in Trials 1 and 2 were similar, and the data of Trial 2 are presented as representative of both trials. Hemoglobin concentrations were reduced significantly (P < .01) by both 500 and 1000 p.p.m. of added lead. This was
AGE (weeks)
FIG. 1. Effect of lead on the body weight of the Japanese quail of Trial 1. Each point represents the mean of 20 observations. Vertical lines in this graph as well as the following graphs represent standard errors of the means (S.E.M.). O = 0 p.p.m.; • = 1 p.p.m.; A = 10 p.p.m.; + = 100 p.p.m.; * = 1000 p.p.m. added dietary lead.
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20 randomly selected quail, respectively) were determined for each of the five groups at 2, 3, 4, and 5 weeks of age. At four weeks of age the birds were immunized with SRBC and blood samples for subsequent antibody determinations were collected at 4 days postimmunization. Ten males from each group were sacrificed at five weeks of age and the relative organ weights were calculated. The data were analyzed by analysis of variance and when the F-ratio indicated significant treatment effects, the treatment means were compared by the multiple range test of Duncan (1955).
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LEAD TOXICITY IN QUAIL
45
r
4 AGE (weeks)
FIG. 2. Effect of lead on the body weight of the Japanese quail of Trial 2. Each point represents the mean of 25 observations. In this and all subsequent graphs O = 0 p.p.m.; • = 10 p.p.m.; A = 100 p.p.m.; + = 500 p.p.m.; * = 1000 p.p.m. added dietary lead.
I2r
3
4 AGE (weeks)
FIG. 3. Effect of lead on blood hemoglobin in the Japanese quail of Trial 2. Each point represents the mean of 20 observations.
4 AGE (weeks)
FIG. 4. Effect of lead on the packed cell volume of the Japanese quail of Trial 2. Each point represents the mean of 20 observations.
2, respectively. The relative weights of the bursa, spleen, liver and heart were not altered significantly in either trial by any of the dose levels of lead. However, the data of Trial 1 indicate that at six weeks of age the testes weights were reduced in the quail which received 1000 p.p.m. of lead, though they did not differ significantly from those of the controls (Table 1). The quail which received lOOOp.p.m. of lead exhibited a wider variation in testes size than those of the other four groups. Certain quail in this group possessed normal sized testes while others had near adolescent sized testes. In view of this individual discrepancy in testicular maturation time the decision was made to terminate Trial 2 when the quail reached five weeks of age. Thus in Trial 2 the effects of lead were determined at an earlier stage of testicular development. At five weeks of age (Table 2) testes size was reduced significantly (P < .05) in quail which were fed 1000 p.p.m. of lead. The reduction in testes size was relatively uniform throughout this treatment
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G. W. MORGAN, F. W. EDENS, P. THAXTON AND C. R. PARKHURST
TABLE 1.—Mean relative organ weights (mg./gm. of body weight) of 6 week-old male Japanese quail of Trial 1 which received dietary lead acetate12-4 Lead (p.p.m.) 0 1 10 100 1000
Organ Spleen .300 .332 .429 .409 .3%
± ± ± ± ±
.033a .026a .048a .052a .033a
Adrenal 3
Bursa .650 .837 .792 .701 .824
± ± ± ± ±
.063a .087a .087a .059a .119a
.128 . 152 .120 . 156 .161
± .012a ±.018a ± . 0 1 la ±.025a ± .024a
Heart 10.79 ± .17a 11.26±.30a 10.52 ± . 2 5 a 11.26 ± .53a 11.95 ± .58a
Liver 22.57 21.99 22.68 26.30 27.28
± ± ± ± ±
1.11a 1.32a 1.37a 1.17a 1.57a
Testes 21.30 25.40 21.79 22.00 15.39
± ± ± ± ±
1.57a,b 1.84a 1.23a,b 1.32a,b 3.65b
1 Mean ± standard error of the mean. 2 Each mean represents 10 observations. 3 Both adrenals. 4
Means not followed by the same small letter differ significantly at P < .01 (Duncan, 1955).
Lead 9 ^ (p.p.m.) Spleen Bursa Adrenal3-4 Heart Liver Testes5 _ 0 .374 ± .022a .790 ± .103a .0263 ± .0029a 12.05 ± .28a 22.08 ± .67a 4.43 ± 1.60a,b 10 .388 ± .029a .989 ± .120a .0308 ± .0020a,b 11.00 ± .49a 24.52+ .98a 5.39 ± 1.06a 100 .397 ± .032a 1.010 ± .106a .0335 ± .0030a,b 11.68 ± .43a 24.05 ± .81a 2.23 ± .76b,c 500 .401 ± .026a .772 ± .060a .0504 ± .0082b,c 11.89 ± .51a 24.93 ± 1.13a 3.78 ± .69b 1000 .444 ± .031a .798 ± .084a .0559 ± .0080c 11.31 ± 1.33a 24.62 ± .76a .87 ± .12c 'Mean ± standard error of the mean. 2 Each mean represents 10 observations. 3 Means not followed by the same small letter differ significantly at P < .01 (Duncan, 1955). 4 Right adrenal only. 5 Means not followed by the same small letter differ significantly at P < .05 (Duncan, 1955).
group. Doses of lead lower than 1000 p.p.m. did not result in significantly reduced testes weights. In Trial 2 the relative adrenal weights were increased significantly (P < .01) by both 500 and 1000 p.p.m. of lead (Table 2). It should be noted that the same trend of adrenal hypertrophy occurred in the quail of Trial 1 which received 1000 p.p.m. of lead; however, significant increases were not found (Table 1). Doses of 100 p.p.m. of lead or lower did not result in significant changes in relative adrenal size in either trial. As indicated in Table 3, all of the quail which received lead demonstrated a normal ability to express a primary humoral immune response following antigenic challenge with SRBC. Significant treatment differences in anti-SRBC hemagglutinin titers were not
TABLE 3.—Anti-SRBC hemagglutinin levels (log2) of Japanese quail which received dietary lead' Lead (p.p.m.) 0 1
Trial l 2 4.5 ± .4
Trial 23 4.8 ± .2
4.1 ± .2
—
10 3.8 ± .3 4.7 100 3.7 ± .4 4.8 500 — 4.5 1000 3.3 ± .4 4.5 1 Mean ± standard error of the mean. 2 Each mean represents 10 observations. 3 Each mean represents 20 observations.
± ± ± ±
.2 .3 .3 .3
noted among any of the treatment groups in either trial. DISCUSSION The results of this study indicate that dietary lead in the form of lead acetate when administered at levels of 500 or 1000 p.p.m.
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TABLE 2.—Mean relative organ weights (mg./gm. of body weight) of 5 week-old male Japanese quail of Trial 2 which received dietary lead'-2
L E A D TOXICITY IN QUAIL
Lead at the levels employed in this study did not prove to be immunosuppressive. Koller and Kovacic (1974) reported that oral administration of lead acetate at 13.75 p.p.m., 137.5 p.p.m. and 1,375 p.p.m. to mice in the drinking water resulted in significantly reduced numbers of splenic antibody plaque forming cells to sheep erythrocytes. These workers indicated however, that the secondary immune response was more susceptible to lead than the primary immune response. Whether quail are less susceptible to immune injury by lead than mice, or whether lead is simply more toxic when administered in the drinking water than in the feed, or both are as yet unanswered questions. However, our data indicated that constant exposure of Japanese quail to dietary lead resulted in
inhibition of growth and reduced hemoglobin levels at lead doses which were not suppressive to the primary immune response. In Trial 1, when the testes were removed for weight analysis at 6 weeks, it appeared that certain of the quail which were fed the highest level (1000 p.p.m.) of lead had severely retarded testicular development, while others in this same group had testes which approximated normal size. Since Japanese quail under lighting schemes similar to the one utilized in this study (Tanaka et al., 1965) normally reach sexual maturity at about six weeks of age, this finding suggested that a more pronounced effect might be noted at an earlier stage of sexual development. The results obtained in Trial 2 in which testes weights were measured at 5 weeks, supported this concept and suggested that lead at the level of 1000 p.p.m. delayed the development of normal testes size. An alternate interpretation is that since the quail in Trial 1 did not begin receiving lead until six days of age the body lead burden was not elevated sufficiently to prevent normal testicular development in all of the quail. In this regard it would be necessary to assume that there are individual variations in tolerance to the body burden of lead. It can also be noted that adrenal size was increased significantly in the quail which received 500 or 1000 p.p.m. lead at five weeks, but not at six weeks of age. To speculate on a possible role for lead in the adrenal-testes interrelationship would be premature at this time. However, Hilderbrand et al. (1973) noted the possibility of hazardous effects of lead on sexuality and reproductive function in sexually mature rats which received lead orally. The results of this study indicate that lead is toxic when administered in the diet to Japanese quail and that the primary manifestations of lead toxicity in young quail include growth inhibition and anemia. The effects of lead on testicular development in quail needs further clarification. Obviously,
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for a period of 5 weeks interfered with normal growth in the Japanese quail. Additionally, it is concluded that the inhibition of normal growth is a primary characteristic of lead toxicity in the Japanese quail. Our data also indicate that the quail which received the growth inhibitory levels of lead experienced an anemia. Goldberg (1972) reviewed the relationship of toxic levels of lead to anemia in mammals. He indicated that anemia is a primary symptom of lead toxicity. The anemic state in quail was detected more readily by hemoglobin analysis than by packed cell volumes. This indicates that either the anemic state was caused by a reduced hemoglobin content of normal size cells or that fewer, but larger erythroid cells were present in the circulation. The mechanism by which lead causes anemia; whether it is by increasing hemolysis (Hasan et al., 1967), interfering with hemoglobin synthesis at the enzyme level (Kao and Forbes, 1973), or by reacting with hemoglobin directly (Barltrop and Smith, 1972) remains unknown. However, Barltrop and Smith (1972) presented evidence suggesting that free sulphydryl groups are not essential to the formation of the lead-hemoglobin complex.
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this is of considerable interest not only to those engaged in experimentation with birds but to those involved with lead exposure in man and other mammals. Experiments are in progress which should further clarify the relationship of lead to reproductive function and behavior. ACKNOWLEDGEMENTS
REFERENCES Barltrop, D., and A. Smith, 1972. Lead binding to human haemoglobin. Experientia, 28: 76-77. Cook, J. A., E. A. Marconi and N. R. DiLuzio, 1974. Lead, cadmium, endotoxin interaction: Effect on mortality and hepatic function. Toxicol. Appl. Pharmacol. 28: 292-302. Duncan, D. B., 1955. Multiple range and multiple " F " tests. Biometrics, 11: 1-42. Gehle, M. H., and D. M. Briggs, 1973. Comparison of starter and grower rations on Bobwhite quail performance. Poultry Sci. 52: 2031. Goldberg, A., 1972. Lead poisoning and heme biosynthesis. Brit. J. Haematol. 23: 521-524. Hasan, J., V. Wikho and S. Hernberg, 1967. Deficient red cell membrane (Na+ + K + )—ATPase in lead
NEWS AND NOTES (Continued from page 1623) Lowest Pounds of Feed Per Pound of Eggs Produced—2.19 lbs.—Carey Farms (Carey Nick 300), Marion, Ohio. Heaviest Average Egg Weight—27.3 oz.—Parks Poultry Farm (Sil-Go-Links), Altoona, Pennsylvania. Highest Percent Large and Extra Large Eggs— 91.1%—Harco Farms, Division of Arbor Acre Farms (Harco Sex Link), South Easton, Massachusetts. Highest Albumen Quality—86.5 Haugh units—Carey Farms (Carey Nick 310), Marion, Ohio.
Lowest Percent of Blood Spots 1 /8 Inch or More— 0.5%—Parks Poultry Farm (Keystone B-l), Altoona, Pennsylvania; Shaver Poultry Breeding Farms, Ltd. (Starcross 288), Cambridge, Ontario, Canada; and Colonial Poultry Farms, Inc. (TrueLine 365B), Pleasant Hill, Missouri. Lowest Percent of Blood Spots Less Than 1 /8 Inch— 0.7%—Carey Farms (Carey Nick 310), Marion, Ohio. Lowest Percent of Meat Spots Less Than 1/8 Inch— 0.1%—Colonial Poultry Farms, Inc. (True-Line 365B), Pleasant Hill, Missouri.
(Continued on page 1646)
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Appreciation is extended to Mrs. Bonnie Goodwin, Mrs. Jeannine Gilbert, Mrs. Grace Brockman and Mrs. Frances Penny for excellent technical assistance.
poisoning. Arch. Environ. Health, 14: 313-318. Hilderbrand, D. C , R. Der, W. T. Griffin and M. S. Fahim, 1973. Effect of lead acetate on reproduction. Am. J. Obstet. Gynecol. 115: 1058-1065. Kao, R. L. C , and R. M. Forbes, 1973. Effects of lead on heme-synthesizing enzymes and urinary delta aminolevulinic acid in the rat. Proc. Soc. Exp. Biol. Med. 143: 234-237. Koller, L. D., and S. Kovacic, 1974. Decreased antibody formation in mice exposed to lead. Nature, 250: 148-150. Schalm, O. W., 1965. Veterinary Hematology, Lea and Febiger, Philadelphia, PA, p. 84-87. Selye, H., B. Tuchweber and L. Bertok, 1966. Effect of lead acetate on susceptibility of rats to bacterial endotoxins. J. Bacterid. 91: 884-890. Sunderman, F. W., R. P. MacFate, D. A. McFadyen, G. F. Stevenson and B. E. Copeland, 1953. Symposium on clinical hemoglobinometry. Am. J. Clin. Path. 23: 519-598. Tanaka, K„ F. B. Mather, W. O. Wilson and L. Z. McFarland, 1965. Effect of photoperiods on early growth of gonads and on potency of gonadotropins of the anterior pituitary in coturnix. Poultry Sci. 44: 662-665. Truscott, R. B., 1970. Endotoxin studies in chicks: Effect of lead acetate. Can. J. Comp. Med. 34: 134-137. Witlin, B., 1967. Detection of antibodies by microtitration techniques. Mycopathologia et Mycologia Applicata, 33: 241-257. Yamamoto, T., M. Yamaguchi and Y. Suketa, 1974. Calcium accumulation in liver and calcium mobilization in bone of lead-poisoned rats. Toxicol. Appl. Pharmacol. 27: 204-205.
