Effects of Roxarsone on Pigmentation and Coccidiosis in Broilers LARRY M. KOWALSKI 1 AND W . MALCOLM R E I D
Department of Poultry Science, University of Georgia, Athens, Georgia 30602 (Received for publication January 6, 1975)
POULTRY SCIENCE 54: 1544-1549, 1975
INTRODUCTION
R
OXARSONE (3 nitro-4-hydroxyphenylarsonic acid)2 as a feed additive has frequently been tested for effects on pigmentation, feed conversion and weight gains. Results reported in the literature are conflicting or equivocal. Some of the confusion appears to be due to lack of differentiation between growth stimulation and the anticoccidial activity properties ascribed to the product. Although roxarsone was discovered in an anticoccidial screening program (Morehouse and Mayfield, 1946), further testing of the compound also revealed growth-
1. Present address, Animal Industry Department, Tri-County Technical College, Pendleton, South Carolina 29670. 2. Marketed by Salsbury Laboratories, Charles City, Iowa, under the trade name 3-Nitro®. This product was originally approved (March 21, 1944) as an anticoccidial to market under the trade name Ren-O-Sal®.
stimulating properties when given at about one quarter the level required for anticoccidial activity (Morehouse, 1949). In a literature review Marusich et al. (1973) indicated that nine out of 37 studies reported that arsenicals did not increase growth rate, 12 out of 22 failed to show improved feed conversion, and six out of 14 could not substantiate greater pigmentation. Some of these tests were made in wire-floored batteries where coccidial infection was probably absent while others were made in floor-pens where exposure was probable. Most of these publications ignore any observations on presence or absence of coccidiosis and relationships to the anticoccidial effects of the drug. Roxarsone was cleared as a feed additive by the Food and Drug Administration (F.D.A.) in 1951. Indications for use included improvement of growth, feed efficiency, pigmentation and for prevention of coccidiosis. In 1971 the approval "for prevention of coccidiosis" was withdrawn following re-
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ABSTRACT Roxarsone (0.005%) medicated chickens inoculated at seven weeks of age with Eimeria maxima oocysts had significantly higher pigment levels in blood plasma than did unmedicated controls. Unmedicated E. maxima infected birds had significantly lower pigment levels compared to uninoculated controls. Uninoculated roxarsone medicated controls showed no increase in pigment levels over uninoculated unmedicated birds. This experimental design demonstrates that the beneficial effect of roxarsone medication is due to anticoccidial action rather than an increase in pigmentation after feeding the yellow-colored roxarsone. Since uninoculated birds gained more rapidly if roxarsone (0.025%) was added to the diet, the drug also appears to stimulate growth as well as to protect against coccidial infection. In two floor-pen experiments roxarsone demonstrated anticoccidial activity as measured by higher pigment scores than unmedicated infected controls. The coccidial infection was induced after seeding the litter with oocysts from birds infected with E. acervulina, E. brunetti, E. hagani, E. maxima, E. mivati, E. necatrix, E. praecox, and E. tenella. Pigmentation levels were significantly higher in plasma and skin of roxarsone medicated chickens in two experiments and in shanks in one experiment. Similar protection against depigmentation was demonstrated by adding roxarsone to feed medicated with nequinate. These results involving addition of roxarsone may explain some equivocal reports on pigmentation, weight gains or feed conversion. Some previous investigators appear to have used an inadequate experimental design by omitting use of unmedicated uninoculated controls or to have made no attempt to monitor for presence or absence of coccidiosis.
ROXARSONE, PIGMENTATION AND COCCIDIOSIS
MATERIALS AND METHODS In separate battery and floor-pen experiments broiler chicks (Cobb's) were fed ad libitum, a broiler starter ration for the first four weeks and a finisher ration for the final four weeks. The basal diet was the University of Georgia open-formula, broiler-starter ration (Dr. H. L. Fuller, Department of Poultry Science). Laboratory Experiment. Male chicks were maintained in electrically heated wire-floored battery brooders for the first four weeks and in finisher batteries for the remaining four weeks. Three 10-bird replications per treatment were randomized as to pen position at one day of age and started on medicated or unmedicated feed. Treatment groups included: 1. uninoculated, unmedicated controls; 2. inoculated, unmedicated controls; 3. uninoculated, roxarsone medicated (0.0025%); 4. inoculated, roxarsone medicated (0.0025%); 5. uninoculated, roxarsone medicated
(0.005%); and 6. inoculated, roxarsone medicated (0.005%). Inoculated birds were individually given approximately 200,000 E. maxima oocysts per os at seven weeks of age. This dosage was selected to produce weight depression and light mortality after completion of an assay trial. Floor-pen Pigmentation Experiments. Two experiments were conducted in an open-type commercial poultry house using 20 pens with concrete floors each measuring approximately 4.8 square meters. All pens were cleaned without attempting to sterilize prior to the experiment. Approximately 10 cm. of pine shavings litter were placed on the floor. Treatment groups were replicated five times and randomized in pen position with 25 male and 25 female chicks introduced per pen at one day of age. The experimental design was adopted to test the anticoccidial nequinate with and without roxarsone. Treatment groups included: 1. unmedicated controls; 2. (0.005%) roxarsone medicated; 3. (0.002%) nequinate 3 ; and 4. (0.002%) nequinate + (0.005%) roxarsone. To establish exposure to coccidial infection, oocysts-seeding was used (Edgar, 1958; Reid et al, 1969; and Mitchell and Scoggins, 1970). Five seeder birds were inoculated at one day of age with 10 x the recommended dosage of Coccivac D 4 and placed in suspended cages placed near the center of each pen when the principal birds were one day of age. Seeder birds were maintained in the suspended cages for three weeks. To assist in sporulation of oocysts the heat from two infra-red bulbs provided for brood-
3. Ayerst Research Laboratories, Chazy, New York; nequinate is marketed under the trade name Statyl®. 4. Coccivac D®, Sterwin Laboratories, Inc., is certified to contain live oocysts of Eimeria necattix, E. tenella, E. maxima, E. acervulina, E. brunetti, E. mivati, E. praecox, and E. hagani.
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evaluation by a committee on veterinary drugs appointed by the National Research Council. Other more potent drugs were available by this time and the early experiments on anticoccidial activity had not been completed using the "4-pen" experimental design (Roe and Collins, 1943). This procedure, which is now generally required for approval, includes infected and uninfected, medicated treatment groups as well as unmedicated uninoculated and unmedicated inoculated controls. Such a 4-pen design revealed anticoccidial activity of roxarsone against Eimeria brunetti (Kowalski and Reid, 1972). A similar laboratory experiment with roxarsone and E. maxima inoculation has been conducted. These results together with pigmentation studies in floor-pen experiments where coccidiosis was induced and monitored in the presence or absence of roxarsone are herein reported.
1545
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L. M. KOWALSKI AND W. M. REID
ing was continued for eight weeks. Moisture content of the litter was determined frequently and increased when necessary to approximately 30 percent by adding water.
Visual Pigmentation Scores. Visual shank scores were made on 15 randomly selected birds per treatment prior to processing (Battery Experiment), 50 birds per treatment prior to processing (Floor-pen Experiment 1), and 20 processed birds per treatment (Floor-pen Experiment 2). Additional skin scores were made on these same birds from the laboratory experiment and from 20 birds (10 males and 10 females) per treatment from the two floorpen experiments. Birds were processed using simulated commercial procedures with a subscalding temperature (57° C. to 59" C ) . After processing, birds were chilled approximately 24 hours in slushed ice. The Ralston Purina Poultry Skin Guide was used as a basis for scoring the intensity of pigmentation. Shank scores were recorded from 0 to 13 according to the scale. For skin, scale readings were grouped as follows: 0 = score 0; 2 to 4 = score 1; 5 to 7 = 2; 8 to 10 = 3; 11 to 12 = 4; 13 = 5. Birds ranging throughout the scale were first selected as standards. Experimental data were analyzed using the analysis of variance and Duncan's multiple range test (Duncan, 1955). RESULTS Laboratory
Experiment.
Plasma
pigment
Skin pigmentation seven days PI with E. maxima was 22 percent lower in score than in uninoculated controls. In similar comparisons inoculated birds showed 24 percent reduction in pigment scores with 0.0025% roxarsone and 20 percent with 0.005% roxarsone. These differences were not significant. Shank scores of unmedicated, 0.0025% and 0.005% roxarsone medicated chicks showed respectively 9, 3 and 9 percent reductions in pigmentation scores 7 days PI with E. maxima compared with corresponding uninoculated groups. Differences were not significant. Weight gains of roxarsone-medicated (0.0025%) uninoculated birds were significantly greater than unmedicated controls. Such a difference indicates a growth promoting rather than anticoccidial activity since no coccidia were present. Gains of neither the 0.005% uninoculated roxarsone-medicated compared to uninoculated unmedicated controls nor of inoculated roxarsone-medicated birds at either level compared to unmedicated controls were significant. Numerical differences in favor of medicated birds suggest some growth promoting activity (0.005%) with uninoculated and some anticoccidial
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Plasma Pigment Determinations. Approximately two milliliters of blood were taken from each of 10 to 15 eight-week-old birds per treatment by cardiac puncture (Stephens, 1965; and Stephens et al., 1967). Plasma from five blood samples per treatment was pooled. The light transmittance of the pooled plasma was measured at 500 m\x. using a Bausch and Lomb Model 300 spectrophotometer and results converted to optical density (OD) or the amount of pigment present.
from unmedicated birds inoculated with Eimeria maxima showed a 42 percent decrease seven days postinoculation (PI) from that of uninoculated unmedicated birds (Table 1). These differences were highly significant (P s 0.01%). A 41 percent reduction of pigment levels also occurred in E. maxima inoculated birds fed on 0.0025% roxarsone and a 28 percent reduction when medicated with 0.005% roxarsone. However, depigmentation was significantly less at the higher level of roxarsone than in the lower level and in the unmedicated inoculated controls. Since uninoculated birds fed on roxarsone had no change in the degree of pigmentation when compared with uninoculated unmedicated controls, the beneficial effect in inoculated birds must be due to anticoccidial action.
1547
ROXARSONE, PIGMENTATION AND COCCIDIOSIS
TABLE 1.—Pigmentation as affected by roxarsone and Eimeria maxima inoculation in broilers (battery experiment)*
Treatment Uninoculated unmedicated controls Inoculated unmedicated controls
Uninoculated, 0.005% roxarsone Inoculated, 0.005% roxarsone
Skin pigmentation % reducMean tion score
0.89c
6.8abc
0.52a
41.6
0.91c 0.54a 0.90c 0.66b
22.1
5.3c
reduction
3.1a
24.0
5.7bc
3.1a
20.0
3.1a
95.5c
8.8
69.1
369.7a 3.1
115.5c
68.8
315.8ab
3.4a
6.9abc
Weights Average gain Reduc(gms., tion wk. 7-8) (%) 309.1b
3.2a
8.5a 27.7
Mean score 3.4a
7.5ab 40.6
Shank pigmentation2
8.8
136.6c
56.7
'Values with the same small letter in a column are nonsignificant (P < 0.05). 2 Shank scores ranged from 0-5 with 5 being the most deeply pigmented; skin scores ranged from 0-13 with 13 being the most deeply pigmented. TABLE 2.—Pigmentation as affected by presence or absence of roxarsone in floor-pen reared broilers exposed to coccidiosis by seeding \ Shank pigmentation2 Skini pigmentation2 mean scores mean scores Female Average Male Female Average Male Female Average 2.0b 2.3c 7.6b 2.6b 6.7b 0.85b 0.95bc 8.4a 3.0a 2.7a 9.4a 3.3a 8.1a 1.14a 10.6a 1.14a 2.7b 2.4ab 7.9b 3.1a 6.8b 8.9a 0.80b 0.92c
Plasma Pigment (OD) Experiment I
2
treatment Unmeditated controls Roxarsone 0.005% Nequinate 0.002% Roxarsone 0.005% + nequinate 0.002% Unmedicated controls Roxarsone 0.005% Nequinate 0.002% Roxarsone 0.005% + nequinate 0.002%
Male 1.05a 1.14b 1.04a 1.13b 0.83a 1.01a 0.87a
0.88b 0.77a 0.94a 0.82a
1.00b 0.80c 0.98a 0.84bc
9.4a 6.0b 9.9a 7.6ab
0.90a
0.92a
0.91ab 9.7a
8.5a 7.6ab 9.6a 6.0b
9.0a 6.8b 9.8a 6.8b
3.3a 2.0a 2.7a 2.4a
2.7a 2.1a 2.0a 1.9a
3.0a 2.1b 2.4ab 2.2b
8.2a
9.0a
3.0a
2.5a
2.8a
•For any column values bearing the same small letter are nonsignificant (P s 0.05). 2 Skin scores ranged from 0-13 with 13 being the most deeply pigmented; shank scores ranged from 0-5 with 5 being the most deeply pigmented.
activity with inoculated birds. Floor-pen Experiments. In both experiments male, female and average plasma pigment scores were numerically greater in roxarsone medicated birds than in unmedicated controls (Table 2). These differences were significant in males, females, and pen averages in experiment 1 and in pen averages in experiment 2. Similarly, higher scores were recorded in birds medicated with roxarsone
plus nequinate than in nequinate medicated alone. These differences were significant with the males and the averages in experiment 1, but not in experiment 2. Skin Pigmentation. Scores in both experiments with males, females and averages were numerically higher in roxarsone medicated than in unmedicated controls. These differences were significant in pen averages in both experiments, in the females in Experi-
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Uninoculated, 0.0025% roxarsone Inoculated, 0.0025% roxarsone
Plasma pigment Mean Decrease (OD) (%)
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L. M. KOWALSKI AND W. M. REID
ment 1, and the males in Experiment 2. Similar higher pigment scores which were significant in both females and pen averages occurred if roxarsone was added to nequinate. With the exception of the unmedicated controls in Experiment 2, all other treatment groups showed higher pigment scores in males than in females.
DISCUSSION Results of the "4-pen" laboratory experiment indicate that roxarsone at 0.005 percent provided protection against E. maxima and prevented depigmentation of the birds. This benefit must have been due to anticoccidial activity rather than direct absorption of the yellow-colored roxarsone since no change in degree of pigmentation occurred in uninoculated controls. That E. maxima infection results in depigmentation has been demonstrated in this battery experiment and confirms findings of others (Mitchell et at, 1961, Bletner et al., 1966, Marusich et al., 1972, Ruff et al., 197'4). This "4-pen" design does not appear to have been used previously with roxarsone except by Kowalski and Reid (1972) who demonstrated efficacy against E. brunetti. Most previously conducted experiments with roxarsone show insufficient data on presence or absence of coccidiosis to evaluate effects of anticoccidial activity on pigmentation. Absence of coccidia would explain the results of Marusich et al. (1969, 1973) who found no improvement in pigmentation in
Results of this study indicate that the anticoccidial activity of roxarsone improves pigmentation by preventing depigmentation which accompanies infection with E. maxima or other species of coccidia. In uninfected chickens roxarsone showed growth promotion but no pigmentation properties. Evaluation experiments attempting to measure ef-
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Shank Pigmentation. Roxarsone medicated birds (males, females and averages) scored significantly higher in shank pigmentation than unmedicated controls in Experiment 1. Similar but nonsignificant trends appear in males in Experiment 2. Addition of roxarsone to nequinate medicated feed resulted in significant increases in average pigment scores in both experiments.
birds fed roxarsone in battery experiments. In our battery experiment a growth promoting activity of roxarsone was demonstrated by feeding to uninfected controls. Since weight gain is also greatly reduced in inoculated unmedicated birds compared to uninoculated, anticoccidial activity of roxarsone against specific coccidial species could also permit growth at the normal rate. In our floor-pen experiments addition of roxarsone to the feed with or without nequinate produced higher pigment scores than in unmedicated controls in measurements taken from plasma, skin and shanks. More depigmentation of skin and shanks in the floor-pen than the battery experiment may have been due to earlier exposure (2-6 weeks compared to 7 weeks) to coccidiosis. These results follow the observation of Craig (1974, personal communication, A. W. Perdue and Company, Salsbury, Md.) who indicated that lower pigment readings were detected first in plasma, second in the skin, and finally at a later time in the shanks. Herrick et al. (1970) observed that depigmentation occurs more rapidly in the skin than in the shanks. Monitoring of the floor-pen birds for lesions at three weeks of age demonstrated active coccidiosis (Kowalski, 1974), but since eight species had been introduced there is no way to tell which species caused greatest reduction in pigment levels. Ruff et al. (1974) have shown that the six principal species all cause decreases in plasma pigment. Maximum decreases were calculated as follows: E. tenella, 50%; E. mivati, 62%; E. necatrix, 67%; E. acervulina, 68%; E. maxima, 69%; and E. brunetti, 74%.
ROXARSONE, PIGMENTATION AND COCCIDIOSIS
fects of roxarsone on pigmentation, growthpromotion or feed-conversion must be considered incomplete if no attempt is made to measure presence or absence of coccidial exposure. Since unplanned exposure to coccidial oocysts is to be expected in experiments involving floor-pen management, efficacy and growth-promotion studies of anticoccidials should also include a "4-pen" design conducted in batteries.
The assistance of Mrs. Joyce Johnson during these experiments is greatly appreciated. Inoculum containing oocysts was provided through the courtesy of Sterwin Laboratories (Opelika, Alabama). The coccidial strains were originally isolated by Dr. S. A. Edgar (Auburn University, Auburn, Alabama). REFERENCES Bletner, J. K., R. P. Mitchell, Jr. and R. L. Tugwell, 1966. The effect of Eimeria maxima on broiler pigmentation. Poultry Sci. 45: 689-694. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Edgar, S. A., 1958. Problems in the control of coccidiosis. Proc. Semi-Annual Nutr. Council, Amer. Feed Manuf. Assoc, p. 19-26. Herrick, G. M., J. L. Fry and R. H. Harms, 1970. Repletion and depletion of xanthophyll in the skin and shanks of broilers. Poultry Sci. 49: 1396-1397. Kowalski, L. M., and W. M. Reid, 1972. Roxarsone: Efficacy against Eimeria brunetti infections in chickens. Poultry Sci. 51: 1586-1589. Kowalski, L. M., 1974. Some effects of roxarsone on coccidial infections in the domestic fowl. Ph.D. Dissertation, University of Georgia, Athens. Marusich, W. L., E. Ogrinz, M. Brand and M. Mitro-
vie, 1969. Safety and compatibility of sulfadimethoxine potentiated mixture (Ro 5-0013), a new broad spectrum coccidiostat-antibacterial, in chickens. Poultry Sci. 48: 217-222. Marusich, W. L., E. F. Ogrinz, P. R. Brown and M. Mitrovic, 1973. Effect of roxarsone and canthaxanthin on broiler pigmentation. Br. Poultry Sci. 14: 23-30. Marusich, W. L., E. Schildknecht, E. F. Ogrinz, P. R. Brown and M. Mitrovic, 1972. Effect of coccidiosis on pigmentation in broilers. Br. Poultry Sci. 13: 577-585. Mitchell, R. P., Jr., J. K. Bletner and R. L. Tugwell, 1961. Factors affecting the xanthophyll pigment in chicks. Poultry Sci. 40: 1432. Mitchell, G. A., and R. W. Scoggins, 1970. Avian Eimeria infection technique: suspended seeder cage. Exp. Parasitol. 28: 87-89. Morehouse, N. F., 1949. Accelerated growth in chickens and turkeys produced by 3-nitro-4-hydroxyphenylarsonic acid. Poultry Sci. 28: 375-384. Morehouse, N. F., and O. J. Mayfield, 1946. The effect of some aryl arsonic acids on experimental coccidiosis infection in chickens. J. Parasitol. 32: 20-24. Reid, W. M., R. N. Brewer, J. Johnson, E. M. Taylor, K. S. Hegde and L. M. Kowalski, 1969. Evaluation of techniques used in studies on efficacy of anticoccidial drugs in chickens. Am. J. Vet. Res. 30: 447-459. Roe, G. C , and J. H. Collins, 1943. A method of testing coccidiosis remedies in poultry. Proc. 47th Annual Meeting U.S. Live Stock Sanitary Assoc, p. 178-183. Ruff, M. D., W. M. Reid and J. K. Johnson, 1974. Lowered blood carotenoid levels in chickens infected with coccidia. Poultry Sci. 53: 1801-1809. Stephens, J. F., 1965. Some physiological effects of coccidiosis caused by Eimeria necatrix in the chicken. J. Parasitol. 51:331-335. Stephens, J. F., L. M. Kowalski and W. J. Borst, 1967. Some physiological effects of coccidiosis caused by Eimeria maxima in young chickens. J. Parasitol. 53: 176-179.
NEWS AND NOTES (Continued from page 1543) Science, North Carolina State University, and First Vice President of the Poultry Science Association, Inc., presented Harold E. Ford, Executive Director of Se.P. & E.A. with the Honorary Membership scroll
in the Association. He was elected at the annual meeting at West Virginia University in August. J. W. Marshall was the recipient of the Workhorse of the Year Award.
(Continued on page 1555)
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ACKNOWLEDGMENTS
1549
Department of Poultry Science, University of Georgia, Athens, Georgia 30602 (Received for publication January 6, 1975)
POULTRY SCIENCE 54: 1544-1549, 1975
INTRODUCTION
R
OXARSONE (3 nitro-4-hydroxyphenylarsonic acid)2 as a feed additive has frequently been tested for effects on pigmentation, feed conversion and weight gains. Results reported in the literature are conflicting or equivocal. Some of the confusion appears to be due to lack of differentiation between growth stimulation and the anticoccidial activity properties ascribed to the product. Although roxarsone was discovered in an anticoccidial screening program (Morehouse and Mayfield, 1946), further testing of the compound also revealed growth-
1. Present address, Animal Industry Department, Tri-County Technical College, Pendleton, South Carolina 29670. 2. Marketed by Salsbury Laboratories, Charles City, Iowa, under the trade name 3-Nitro®. This product was originally approved (March 21, 1944) as an anticoccidial to market under the trade name Ren-O-Sal®.
stimulating properties when given at about one quarter the level required for anticoccidial activity (Morehouse, 1949). In a literature review Marusich et al. (1973) indicated that nine out of 37 studies reported that arsenicals did not increase growth rate, 12 out of 22 failed to show improved feed conversion, and six out of 14 could not substantiate greater pigmentation. Some of these tests were made in wire-floored batteries where coccidial infection was probably absent while others were made in floor-pens where exposure was probable. Most of these publications ignore any observations on presence or absence of coccidiosis and relationships to the anticoccidial effects of the drug. Roxarsone was cleared as a feed additive by the Food and Drug Administration (F.D.A.) in 1951. Indications for use included improvement of growth, feed efficiency, pigmentation and for prevention of coccidiosis. In 1971 the approval "for prevention of coccidiosis" was withdrawn following re-
1544
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ABSTRACT Roxarsone (0.005%) medicated chickens inoculated at seven weeks of age with Eimeria maxima oocysts had significantly higher pigment levels in blood plasma than did unmedicated controls. Unmedicated E. maxima infected birds had significantly lower pigment levels compared to uninoculated controls. Uninoculated roxarsone medicated controls showed no increase in pigment levels over uninoculated unmedicated birds. This experimental design demonstrates that the beneficial effect of roxarsone medication is due to anticoccidial action rather than an increase in pigmentation after feeding the yellow-colored roxarsone. Since uninoculated birds gained more rapidly if roxarsone (0.025%) was added to the diet, the drug also appears to stimulate growth as well as to protect against coccidial infection. In two floor-pen experiments roxarsone demonstrated anticoccidial activity as measured by higher pigment scores than unmedicated infected controls. The coccidial infection was induced after seeding the litter with oocysts from birds infected with E. acervulina, E. brunetti, E. hagani, E. maxima, E. mivati, E. necatrix, E. praecox, and E. tenella. Pigmentation levels were significantly higher in plasma and skin of roxarsone medicated chickens in two experiments and in shanks in one experiment. Similar protection against depigmentation was demonstrated by adding roxarsone to feed medicated with nequinate. These results involving addition of roxarsone may explain some equivocal reports on pigmentation, weight gains or feed conversion. Some previous investigators appear to have used an inadequate experimental design by omitting use of unmedicated uninoculated controls or to have made no attempt to monitor for presence or absence of coccidiosis.
ROXARSONE, PIGMENTATION AND COCCIDIOSIS
MATERIALS AND METHODS In separate battery and floor-pen experiments broiler chicks (Cobb's) were fed ad libitum, a broiler starter ration for the first four weeks and a finisher ration for the final four weeks. The basal diet was the University of Georgia open-formula, broiler-starter ration (Dr. H. L. Fuller, Department of Poultry Science). Laboratory Experiment. Male chicks were maintained in electrically heated wire-floored battery brooders for the first four weeks and in finisher batteries for the remaining four weeks. Three 10-bird replications per treatment were randomized as to pen position at one day of age and started on medicated or unmedicated feed. Treatment groups included: 1. uninoculated, unmedicated controls; 2. inoculated, unmedicated controls; 3. uninoculated, roxarsone medicated (0.0025%); 4. inoculated, roxarsone medicated (0.0025%); 5. uninoculated, roxarsone medicated
(0.005%); and 6. inoculated, roxarsone medicated (0.005%). Inoculated birds were individually given approximately 200,000 E. maxima oocysts per os at seven weeks of age. This dosage was selected to produce weight depression and light mortality after completion of an assay trial. Floor-pen Pigmentation Experiments. Two experiments were conducted in an open-type commercial poultry house using 20 pens with concrete floors each measuring approximately 4.8 square meters. All pens were cleaned without attempting to sterilize prior to the experiment. Approximately 10 cm. of pine shavings litter were placed on the floor. Treatment groups were replicated five times and randomized in pen position with 25 male and 25 female chicks introduced per pen at one day of age. The experimental design was adopted to test the anticoccidial nequinate with and without roxarsone. Treatment groups included: 1. unmedicated controls; 2. (0.005%) roxarsone medicated; 3. (0.002%) nequinate 3 ; and 4. (0.002%) nequinate + (0.005%) roxarsone. To establish exposure to coccidial infection, oocysts-seeding was used (Edgar, 1958; Reid et al, 1969; and Mitchell and Scoggins, 1970). Five seeder birds were inoculated at one day of age with 10 x the recommended dosage of Coccivac D 4 and placed in suspended cages placed near the center of each pen when the principal birds were one day of age. Seeder birds were maintained in the suspended cages for three weeks. To assist in sporulation of oocysts the heat from two infra-red bulbs provided for brood-
3. Ayerst Research Laboratories, Chazy, New York; nequinate is marketed under the trade name Statyl®. 4. Coccivac D®, Sterwin Laboratories, Inc., is certified to contain live oocysts of Eimeria necattix, E. tenella, E. maxima, E. acervulina, E. brunetti, E. mivati, E. praecox, and E. hagani.
Downloaded from http://ps.oxfordjournals.org/ at California Institute of Technology on June 21, 2015
evaluation by a committee on veterinary drugs appointed by the National Research Council. Other more potent drugs were available by this time and the early experiments on anticoccidial activity had not been completed using the "4-pen" experimental design (Roe and Collins, 1943). This procedure, which is now generally required for approval, includes infected and uninfected, medicated treatment groups as well as unmedicated uninoculated and unmedicated inoculated controls. Such a 4-pen design revealed anticoccidial activity of roxarsone against Eimeria brunetti (Kowalski and Reid, 1972). A similar laboratory experiment with roxarsone and E. maxima inoculation has been conducted. These results together with pigmentation studies in floor-pen experiments where coccidiosis was induced and monitored in the presence or absence of roxarsone are herein reported.
1545
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L. M. KOWALSKI AND W. M. REID
ing was continued for eight weeks. Moisture content of the litter was determined frequently and increased when necessary to approximately 30 percent by adding water.
Visual Pigmentation Scores. Visual shank scores were made on 15 randomly selected birds per treatment prior to processing (Battery Experiment), 50 birds per treatment prior to processing (Floor-pen Experiment 1), and 20 processed birds per treatment (Floor-pen Experiment 2). Additional skin scores were made on these same birds from the laboratory experiment and from 20 birds (10 males and 10 females) per treatment from the two floorpen experiments. Birds were processed using simulated commercial procedures with a subscalding temperature (57° C. to 59" C ) . After processing, birds were chilled approximately 24 hours in slushed ice. The Ralston Purina Poultry Skin Guide was used as a basis for scoring the intensity of pigmentation. Shank scores were recorded from 0 to 13 according to the scale. For skin, scale readings were grouped as follows: 0 = score 0; 2 to 4 = score 1; 5 to 7 = 2; 8 to 10 = 3; 11 to 12 = 4; 13 = 5. Birds ranging throughout the scale were first selected as standards. Experimental data were analyzed using the analysis of variance and Duncan's multiple range test (Duncan, 1955). RESULTS Laboratory
Experiment.
Plasma
pigment
Skin pigmentation seven days PI with E. maxima was 22 percent lower in score than in uninoculated controls. In similar comparisons inoculated birds showed 24 percent reduction in pigment scores with 0.0025% roxarsone and 20 percent with 0.005% roxarsone. These differences were not significant. Shank scores of unmedicated, 0.0025% and 0.005% roxarsone medicated chicks showed respectively 9, 3 and 9 percent reductions in pigmentation scores 7 days PI with E. maxima compared with corresponding uninoculated groups. Differences were not significant. Weight gains of roxarsone-medicated (0.0025%) uninoculated birds were significantly greater than unmedicated controls. Such a difference indicates a growth promoting rather than anticoccidial activity since no coccidia were present. Gains of neither the 0.005% uninoculated roxarsone-medicated compared to uninoculated unmedicated controls nor of inoculated roxarsone-medicated birds at either level compared to unmedicated controls were significant. Numerical differences in favor of medicated birds suggest some growth promoting activity (0.005%) with uninoculated and some anticoccidial
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Plasma Pigment Determinations. Approximately two milliliters of blood were taken from each of 10 to 15 eight-week-old birds per treatment by cardiac puncture (Stephens, 1965; and Stephens et al., 1967). Plasma from five blood samples per treatment was pooled. The light transmittance of the pooled plasma was measured at 500 m\x. using a Bausch and Lomb Model 300 spectrophotometer and results converted to optical density (OD) or the amount of pigment present.
from unmedicated birds inoculated with Eimeria maxima showed a 42 percent decrease seven days postinoculation (PI) from that of uninoculated unmedicated birds (Table 1). These differences were highly significant (P s 0.01%). A 41 percent reduction of pigment levels also occurred in E. maxima inoculated birds fed on 0.0025% roxarsone and a 28 percent reduction when medicated with 0.005% roxarsone. However, depigmentation was significantly less at the higher level of roxarsone than in the lower level and in the unmedicated inoculated controls. Since uninoculated birds fed on roxarsone had no change in the degree of pigmentation when compared with uninoculated unmedicated controls, the beneficial effect in inoculated birds must be due to anticoccidial action.
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ROXARSONE, PIGMENTATION AND COCCIDIOSIS
TABLE 1.—Pigmentation as affected by roxarsone and Eimeria maxima inoculation in broilers (battery experiment)*
Treatment Uninoculated unmedicated controls Inoculated unmedicated controls
Uninoculated, 0.005% roxarsone Inoculated, 0.005% roxarsone
Skin pigmentation % reducMean tion score
0.89c
6.8abc
0.52a
41.6
0.91c 0.54a 0.90c 0.66b
22.1
5.3c
reduction
3.1a
24.0
5.7bc
3.1a
20.0
3.1a
95.5c
8.8
69.1
369.7a 3.1
115.5c
68.8
315.8ab
3.4a
6.9abc
Weights Average gain Reduc(gms., tion wk. 7-8) (%) 309.1b
3.2a
8.5a 27.7
Mean score 3.4a
7.5ab 40.6
Shank pigmentation2
8.8
136.6c
56.7
'Values with the same small letter in a column are nonsignificant (P < 0.05). 2 Shank scores ranged from 0-5 with 5 being the most deeply pigmented; skin scores ranged from 0-13 with 13 being the most deeply pigmented. TABLE 2.—Pigmentation as affected by presence or absence of roxarsone in floor-pen reared broilers exposed to coccidiosis by seeding \ Shank pigmentation2 Skini pigmentation2 mean scores mean scores Female Average Male Female Average Male Female Average 2.0b 2.3c 7.6b 2.6b 6.7b 0.85b 0.95bc 8.4a 3.0a 2.7a 9.4a 3.3a 8.1a 1.14a 10.6a 1.14a 2.7b 2.4ab 7.9b 3.1a 6.8b 8.9a 0.80b 0.92c
Plasma Pigment (OD) Experiment I
2
treatment Unmeditated controls Roxarsone 0.005% Nequinate 0.002% Roxarsone 0.005% + nequinate 0.002% Unmedicated controls Roxarsone 0.005% Nequinate 0.002% Roxarsone 0.005% + nequinate 0.002%
Male 1.05a 1.14b 1.04a 1.13b 0.83a 1.01a 0.87a
0.88b 0.77a 0.94a 0.82a
1.00b 0.80c 0.98a 0.84bc
9.4a 6.0b 9.9a 7.6ab
0.90a
0.92a
0.91ab 9.7a
8.5a 7.6ab 9.6a 6.0b
9.0a 6.8b 9.8a 6.8b
3.3a 2.0a 2.7a 2.4a
2.7a 2.1a 2.0a 1.9a
3.0a 2.1b 2.4ab 2.2b
8.2a
9.0a
3.0a
2.5a
2.8a
•For any column values bearing the same small letter are nonsignificant (P s 0.05). 2 Skin scores ranged from 0-13 with 13 being the most deeply pigmented; shank scores ranged from 0-5 with 5 being the most deeply pigmented.
activity with inoculated birds. Floor-pen Experiments. In both experiments male, female and average plasma pigment scores were numerically greater in roxarsone medicated birds than in unmedicated controls (Table 2). These differences were significant in males, females, and pen averages in experiment 1 and in pen averages in experiment 2. Similarly, higher scores were recorded in birds medicated with roxarsone
plus nequinate than in nequinate medicated alone. These differences were significant with the males and the averages in experiment 1, but not in experiment 2. Skin Pigmentation. Scores in both experiments with males, females and averages were numerically higher in roxarsone medicated than in unmedicated controls. These differences were significant in pen averages in both experiments, in the females in Experi-
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Uninoculated, 0.0025% roxarsone Inoculated, 0.0025% roxarsone
Plasma pigment Mean Decrease (OD) (%)
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L. M. KOWALSKI AND W. M. REID
ment 1, and the males in Experiment 2. Similar higher pigment scores which were significant in both females and pen averages occurred if roxarsone was added to nequinate. With the exception of the unmedicated controls in Experiment 2, all other treatment groups showed higher pigment scores in males than in females.
DISCUSSION Results of the "4-pen" laboratory experiment indicate that roxarsone at 0.005 percent provided protection against E. maxima and prevented depigmentation of the birds. This benefit must have been due to anticoccidial activity rather than direct absorption of the yellow-colored roxarsone since no change in degree of pigmentation occurred in uninoculated controls. That E. maxima infection results in depigmentation has been demonstrated in this battery experiment and confirms findings of others (Mitchell et at, 1961, Bletner et al., 1966, Marusich et al., 1972, Ruff et al., 197'4). This "4-pen" design does not appear to have been used previously with roxarsone except by Kowalski and Reid (1972) who demonstrated efficacy against E. brunetti. Most previously conducted experiments with roxarsone show insufficient data on presence or absence of coccidiosis to evaluate effects of anticoccidial activity on pigmentation. Absence of coccidia would explain the results of Marusich et al. (1969, 1973) who found no improvement in pigmentation in
Results of this study indicate that the anticoccidial activity of roxarsone improves pigmentation by preventing depigmentation which accompanies infection with E. maxima or other species of coccidia. In uninfected chickens roxarsone showed growth promotion but no pigmentation properties. Evaluation experiments attempting to measure ef-
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Shank Pigmentation. Roxarsone medicated birds (males, females and averages) scored significantly higher in shank pigmentation than unmedicated controls in Experiment 1. Similar but nonsignificant trends appear in males in Experiment 2. Addition of roxarsone to nequinate medicated feed resulted in significant increases in average pigment scores in both experiments.
birds fed roxarsone in battery experiments. In our battery experiment a growth promoting activity of roxarsone was demonstrated by feeding to uninfected controls. Since weight gain is also greatly reduced in inoculated unmedicated birds compared to uninoculated, anticoccidial activity of roxarsone against specific coccidial species could also permit growth at the normal rate. In our floor-pen experiments addition of roxarsone to the feed with or without nequinate produced higher pigment scores than in unmedicated controls in measurements taken from plasma, skin and shanks. More depigmentation of skin and shanks in the floor-pen than the battery experiment may have been due to earlier exposure (2-6 weeks compared to 7 weeks) to coccidiosis. These results follow the observation of Craig (1974, personal communication, A. W. Perdue and Company, Salsbury, Md.) who indicated that lower pigment readings were detected first in plasma, second in the skin, and finally at a later time in the shanks. Herrick et al. (1970) observed that depigmentation occurs more rapidly in the skin than in the shanks. Monitoring of the floor-pen birds for lesions at three weeks of age demonstrated active coccidiosis (Kowalski, 1974), but since eight species had been introduced there is no way to tell which species caused greatest reduction in pigment levels. Ruff et al. (1974) have shown that the six principal species all cause decreases in plasma pigment. Maximum decreases were calculated as follows: E. tenella, 50%; E. mivati, 62%; E. necatrix, 67%; E. acervulina, 68%; E. maxima, 69%; and E. brunetti, 74%.
ROXARSONE, PIGMENTATION AND COCCIDIOSIS
fects of roxarsone on pigmentation, growthpromotion or feed-conversion must be considered incomplete if no attempt is made to measure presence or absence of coccidial exposure. Since unplanned exposure to coccidial oocysts is to be expected in experiments involving floor-pen management, efficacy and growth-promotion studies of anticoccidials should also include a "4-pen" design conducted in batteries.
The assistance of Mrs. Joyce Johnson during these experiments is greatly appreciated. Inoculum containing oocysts was provided through the courtesy of Sterwin Laboratories (Opelika, Alabama). The coccidial strains were originally isolated by Dr. S. A. Edgar (Auburn University, Auburn, Alabama). REFERENCES Bletner, J. K., R. P. Mitchell, Jr. and R. L. Tugwell, 1966. The effect of Eimeria maxima on broiler pigmentation. Poultry Sci. 45: 689-694. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Edgar, S. A., 1958. Problems in the control of coccidiosis. Proc. Semi-Annual Nutr. Council, Amer. Feed Manuf. Assoc, p. 19-26. Herrick, G. M., J. L. Fry and R. H. Harms, 1970. Repletion and depletion of xanthophyll in the skin and shanks of broilers. Poultry Sci. 49: 1396-1397. Kowalski, L. M., and W. M. Reid, 1972. Roxarsone: Efficacy against Eimeria brunetti infections in chickens. Poultry Sci. 51: 1586-1589. Kowalski, L. M., 1974. Some effects of roxarsone on coccidial infections in the domestic fowl. Ph.D. Dissertation, University of Georgia, Athens. Marusich, W. L., E. Ogrinz, M. Brand and M. Mitro-
vie, 1969. Safety and compatibility of sulfadimethoxine potentiated mixture (Ro 5-0013), a new broad spectrum coccidiostat-antibacterial, in chickens. Poultry Sci. 48: 217-222. Marusich, W. L., E. F. Ogrinz, P. R. Brown and M. Mitrovic, 1973. Effect of roxarsone and canthaxanthin on broiler pigmentation. Br. Poultry Sci. 14: 23-30. Marusich, W. L., E. Schildknecht, E. F. Ogrinz, P. R. Brown and M. Mitrovic, 1972. Effect of coccidiosis on pigmentation in broilers. Br. Poultry Sci. 13: 577-585. Mitchell, R. P., Jr., J. K. Bletner and R. L. Tugwell, 1961. Factors affecting the xanthophyll pigment in chicks. Poultry Sci. 40: 1432. Mitchell, G. A., and R. W. Scoggins, 1970. Avian Eimeria infection technique: suspended seeder cage. Exp. Parasitol. 28: 87-89. Morehouse, N. F., 1949. Accelerated growth in chickens and turkeys produced by 3-nitro-4-hydroxyphenylarsonic acid. Poultry Sci. 28: 375-384. Morehouse, N. F., and O. J. Mayfield, 1946. The effect of some aryl arsonic acids on experimental coccidiosis infection in chickens. J. Parasitol. 32: 20-24. Reid, W. M., R. N. Brewer, J. Johnson, E. M. Taylor, K. S. Hegde and L. M. Kowalski, 1969. Evaluation of techniques used in studies on efficacy of anticoccidial drugs in chickens. Am. J. Vet. Res. 30: 447-459. Roe, G. C , and J. H. Collins, 1943. A method of testing coccidiosis remedies in poultry. Proc. 47th Annual Meeting U.S. Live Stock Sanitary Assoc, p. 178-183. Ruff, M. D., W. M. Reid and J. K. Johnson, 1974. Lowered blood carotenoid levels in chickens infected with coccidia. Poultry Sci. 53: 1801-1809. Stephens, J. F., 1965. Some physiological effects of coccidiosis caused by Eimeria necatrix in the chicken. J. Parasitol. 51:331-335. Stephens, J. F., L. M. Kowalski and W. J. Borst, 1967. Some physiological effects of coccidiosis caused by Eimeria maxima in young chickens. J. Parasitol. 53: 176-179.
NEWS AND NOTES (Continued from page 1543) Science, North Carolina State University, and First Vice President of the Poultry Science Association, Inc., presented Harold E. Ford, Executive Director of Se.P. & E.A. with the Honorary Membership scroll
in the Association. He was elected at the annual meeting at West Virginia University in August. J. W. Marshall was the recipient of the Workhorse of the Year Award.
(Continued on page 1555)
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ACKNOWLEDGMENTS
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