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Physiological Effects of Gentian Violet on Broiler Chickens
ENVIRONMENT AND HEALTH Physiological Effects of Gentian Violet on Broiler Chickens R. G. STEWART, R. D. WYATT, G. M. LANZA, H. M. EDWARDS, and M. D. RUFF 1
Department of Poultry Science, University of Georgia, Athens, Georgia 30602 (Received for publication January 11, 1979)
INTRODUCTION Problems associated with mycotoxin contamination of animal feeds is a persistent problem in almost all phases of animal production. As a result of mycotoxin contamination of feedstuffs and the realization that mycotoxin concentrations may increase after feed manufacture, a variety of antifungal preparations have been used as feed additives in recent years. The expectation is that these preparations will retard or inhibit mold growth and subsequent mycotoxin formation. This, in turn, would lessen the economic losses caused by mycotoxicoses and reduce the many problems caused by mycotoxins interacting with other environmental stressors. Preparations containing gentian violet as the active ingredient were at one time used on a large scale within the poultry industry. During this time of widespread use, numerous field reports emerged suggesting improved growth rates, improvements in feed efficiency, increased livability, and improved carcass quality associated with the use of dietary gentian violet even in the absence of mold growth and mycotoxin contamination in the rations. A beneficial effect of gentian violet on such production parameters has not been documented; however, the existence of such an effect appears to
1 Present address: United States Department of Agriculture, Animal Parasitology Institute, ARS, Beltsville, MD 20705.
warrant consideration. For example, gentian violet exhibits antibacterial properties, specifically against gram positive organisms, an activity similar to that of penicillin. The fungistatic properties of gentian violet have been recognized for many years, especially its activity against Candida albicans, an opportunistic pathogen to both man and animal. The anthelmintic properties of gentian violet also have been exploited. Preparations containing gentian violet as the sole active ingredient are currently available for treatment of internal bacterial, antifungal, and anthelmintic properties and perhaps yet unidentified traits of gentian violet could result in improved performance of commercial poultry. The purpose of this investigation was to evaluate gentian violet for growth promoting and pharmacological activities.
MATERIALS AND METHODS In all experiments, day-old male chicks (Cobb) were obtained from a commercial hatchery and housed in electrically heated batteries under continuous lighting with feed and water available ad libitum. In Experiment 1, four dietary levels (0, 16, 32, and 64 /Ug/g) of USP grade crystal violet were attained by adding the pure compound to commercial broiler-starter ration free of medication. Sixteen groups of 10 birds each with 4 replicates per treatment were fed for four weeks at which time various blood and tissue analyses were 234
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ABSTRACT The effects of dietary gentian violet upon certain physiological parameters of broiler chickens were studied. Gentian violet exhibited no effect upon growth rates or feed conversion ratios at dietary levels of 16, 32, or 64 jug/g. In vitro intestinal absorption of methionine and glucose was also unaltered. Dietary gentian violet significantly increased hemoglobin concentration without an effect on packed cell volume. Furthermore, commercial gentian violet containing preparations, when incorporated into the diet, resulted in increased intestinal absorption of Fe 5 ' but this increase was dependent upon type of inert carrier used. Dietary gentian violet alleviated some of the growth suppression caused by dietary aflatoxin; however, no effect was observed on plasma pigmentation. These data suggest that dietary gentian violet possesses effects other than those for which it has been traditionally employed. 1980 Poultry Science 59:234-239
GENTIAN VIOLET AND BROILER CHICKENS
Experiment 2 was similar to Experiment 1 except the dietary treatments included the control group (basal ration) and two groups receiving the basal ration containing 16 fig of gentian violet/g of diet. The gentian violet was supplied by using two commercial products,
one with a calcium carbonate carrier (Marshall Mineral, Marshall, TX) and the other employing a corn cob fraction (Dan-Mar Enterprises, Commerce, GA) as the carrier. Each treatment consisted of 3 replicates of 3 birds each. The birds were fed each ration for 7 days at which time all birds were dosed by crop intubation with 1.0 ml of an aqueous solution containing .2 yc/ml of F e 5 9 . The initial absorption Fe was determined as described by Edwards (1966). The administration of the diets containing gentian violet was continued throughout a 12-day post-dosing period. Experiment 3 was of a 2 X 4 factorial design for the presence and absence of gentian violet supplied as Dye-Gen® (Dan-Mar Enterprises, Commerce, GA) at a level of 16 Mg/g of diet and the presence and absence of aflatoxin with aflatoxin levels of 1.25, 2.5, and 5.0 yitg/g of diet. The aflatoxin used was produced and added to the basal diet as described by Smith and Hamilton (1970). Each treatment consisted of 3 replicates of 10 birds each. The birds were fed each diet from day old to 3 weeks of age. During the experiment mortality, body weights and feed conversion ratios were determined weekly. At the end of the experiment a blood sample was taken via cardiac puncture and the plasma carotenoid concentration determined by the method of Wilson (1956) as modified by
Rufietal. (1974). All data were statistically analyzed by an analysis of variance (Bruning and Kintz, 1968). Statements of significance are based on P< .05. RESULTS
The addition of crystal violet to the diet of young broiler chicks had no significant effect on the 4-week growth rate (Table 1). In a similar fashion there was no pronounced effect on the weekly feed conversion ratios from day old through 4 weeks (Table 2). A significant effect on the feed conversion ratio was observed during the first week of the experiment with all levels of crystal violet; however, the magnitude of the improvement was slight and was not evident in subsequent weeks. Analysis of certain blood components revealed that dietary crystal violet had no effect upon the concentrations of total plasma protein, plasma glucose, or PCV (Table 3). Crystal violet at all dietary levels resulted in a significant increase in the hemoglobin concentration; however, the increase was only about 4% (Table 3).
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determined. Weekly body weights and feed conversion ratios were computed. At four weeks plasma was collected by drawing 3.0 ml of blood via cardiac puncture into silicon treated tubes (100 X 13 mm) containing .3 ml of .18 M sodium citrate. An additional blood sample was taken from the brachial vein for packed cell volume (PCV) and hemoglobin determination. Hemoglobin was determined according to the method of Sunderman et al. (1953), plasma glucose by the method of Dubrowski (1962), total plasma protein according to Wooten (1964), and PCV by the microhematocrit method. All birds were then killed by cervical dislocation and a segment of the duodenum approximately 3.0 cm in length was removed and placed in a cold (4 C) Krebs-Henseleit buffered saline (KHS, pH 7.2) which had been bubbled with 5% C 0 2 : 9 5 % 0 2 prior to use. The mesenteries were stripped and the intestine opened. Tissue punches, 7 mm in diameter, were removed from immediately below the bile and pancreatic duct using a No. 3 cork borer. Single punches from three different birds were pooled in a beaker. Three replicates, each containing tissue from three different birds, were utilized at each substrate and drug level. The intestinal tissue was held in cold (4 C) KHS containing 5 mM glucose as an energy source prior to incubation. The tissue was then transferred to beakers containing 9 ml of KHS and preincubated for 10 min at 40.5 ± .5 C. At zero time 1 ml of KHS containing 10 times the desired concentration of C 14 -L-methionine or C 14 -glucose was added to the beaker. Tissues were incubated for 10 min. The reaction was stopped by transferring the tissue to cold KHS and rinsing through three successive changes of cold buffer. The labeled substrate was extracted from the tissue with 70% ethanol. An aliquot of the extract was added to PCS scintillation fluid (Phase Combining System, Amersham/Searle) and the radioactivity measured using a Packard Tri-Carb Scintillation Counter. After extraction, the tissue was dried at 95 C and weighed. Results were expressed as mM of substrate absorbed/g weight dry tissue/10 min incubation.
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236
STEWART ET AL. TABLE 1. The effect of dietary crystal violet on the body weight of young broiler chickens (Experiment 1)
Crystal (mg/kg)
0
0 16 32 64
38 38 39 39
Weekly body weights (g) a 2
1 ±1 ±1 ±1 ±1
263 ± 264+ 255 ± 258+
119 ± 2 115 ± 3 115 + 1 117± 3
4 5 3 10
4
3 463 465 458 463
± ± ± ±
10 10 5 11
717 701 675 703
± ± ± ±
23 15 7 16
The in vitro intestinal uptake of methionine (Table 4) was not altered by any level of dietary crystal violet. At a substrate concentration of .5 mM and above and at a dietary level of crystal violet of 32 /ig/g there was a consistent stimulatory effect on glucose uptake; however, at no substrate concentration was there a significant response. Absorption of Fe 5 9 in response to two products containing gentian violet (Table 5) indicates that gentian violet on a corn cob fraction as the carrier will result in a significant increase in Fe absorption of about 50%. There was no significant effect on Fe absorption with gentian violet on the calcium carbonate carrier. The response of body weight of chicks to dietary aflatoxin and aflatoxin and gentian violet in combination is demonstrated in Table 6. The body weight of birds receiving aflatoxin alone responded as expected based upon findings of others (Smith and Hamilton, 1970). Feeding gentian violet alone did not result in an improvement in body weight (Tables 1 and 6);
when a dietary concentration of gentian violet of 16.0 fig/g was fed in combination with 2.50 Mg/g of dietary aflatoxin, the expected growth depression caused by the aflatoxin was partially alleviated. However, the feeding of 16.0 /zg/g of gentian violet in combination with 5.00 /ig/g of aflatoxin did not result in an alleviation of the body weight depression expected from the aflatoxin alone. Some field observations indicated that the use of dietary gentian violet enhanced carcass pigmentation of broilers. In this present investigation, neither dietary gentian violet alone (16 Mg/g) nor this same level of gentian violet in combination with dietary aflatoxin at levels up to 5.0 jug/g enhanced plasma pigmentation or prevented the decrease in plasma pigmentation associated with aflatoxicosis (Table 7).
DISCUSSION In no instance was there an enhancement of the growth rate of broiler chicks associated with dietary crystal or gentian violet. These
TABLE 2. Effect of dietary crystal violet on feed conversion ratios of young broiler chicks (Experiment 1) Weekly feed conversion ratio (g feed consumed/g wt gained)
Crystal (Mg/g)
Oto 1
0 16 32 64
1.434 1.345 1.345 1.329
1 to 2 ± ± ± ±
.024 a .016 b .018 b ,020 b
1.567 1.571 1.627 1.687
+ + + ±
.041 .062 .065 .071
2 to 3
3 to 4
1.779 ± .073 1.759 +• .071 1.896 ± .070 1.717+ .087
2.036 ± 2.105 ± 2.119 ± 2.054 ±
Each value (mean ± standard error of the mean) represents the mean of 4 replicates of 10 birds each. These values differ significantly (P<.05) from the corresponding control group.
.093 .102 .086 .086
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Each value (mean ± standard error of the mean) is the average of 4 replicates of 10 birds each. There were no significant (P>.05) differences among means during any week.
GENTIAN VIOLET AND BROILER CHICKENS
237
TABLE 3. Effect of dietary crystal violet on blood components of young broiler chickens (Experiment 1) Crystal violet (Mg/g)
Plama protein (mg/100 ml)
Plasma glucose (mg/100 ml)
0 16 32 64
2705 ± 181a 2880 ± 186 2738 ± 205 2898± 126
282 ± 287 ± 274 ± 252 ±
Packed cell volume (%)
Hemglobin (g/100 ml) 7.00 ± .10 7.34 + .05b
19 25 14 12
29.7 ± 4.7 29.0 ± 3.2 28.1 ± 3.9 28.9 ± 3.0
7.31 ± .08 b 7.32 ± .09°
data confirm those of Stewart et al. (1977) where no improvement in growth rate was observed in broilers grown for 6 weeks under commercial-type conditions. The improvement in feed efficiency observed in this present study is of questionable significance since this effect was present only at one time (1 week) of the growing period and was not evident at later times. Likewise, Stewart et al. (1977) did not observe an improvement in feed efficiency due to dietary gentian violet. Of interest is the increase in the hemoglobin concentration in the blood associated with all levels of dietary gentian violet. Since the PCV was not increased, this suggests an increased
intestinal absorption of iron which, in turn, allowed for additional hemoglobin synthesis. This suspected facilitation of iron uptake was confirmed by determination of in vivo Fe 5 9 absorption (Table 5) and offers one possible explanation for the increase in hemoglobin. The difference between the two products in this ability to apparently increase iron uptake may lie in the inherent tendency of the gentian violet to be solubilized and dissociate from the carrier. In the case of the calcium carbonate carrier, the gentian violet is readily solubilized. This is evident in the degree of staining of the crop and proventriculus noted during routine necropsy of the birds at the termination of the
TABLE 4. Uptake of methionine and glucose by intestinal tissue of broiler chicks fed graded levels of crystal violet (Experiment 1)
Crystal
m M Substrate
(Mg/g)
.25
1.0
0 16 32 64
2.58 2.18 2.52 1.95
+ + ± +
.22b .07 .07 .11
4.26 4.09 3.25 3.95
± ± ± ±
0 16 32 64
3.72 3.28 3.42 2.87
+ + + +
.47 .14 .79 .34
5.97 6.43 8.96 4.29
± .22 ± .32 ± 1.12 ± .26
.5
2.5
5.0
10.0
Methionine .25 .53 .28 .58
7.78 6.13 6.20 5.33
± 1.23 ± .39 ± .35 ± .43
1 1 . 7 3 ± 1.15 1 1 . 6 0 + .68 11.28 ± 1 . 5 4 8.85 ± .64
15.69 14.33 12.04 12.91
± 2.32 ± .28 ± .16 ± .82
2 1 . 8 6 + 2.57 19.53 ± .90 15.60 ± 1.30 1 7 . 4 4 ± 1.41
30.40 33.10 35.00 31.19
± ± ± ±
40.16 47.70 45.01 36.36
Glucose 10.06 13.12 13.26 9.56
± .21 ± .90 ±1.31 ±1.68
21.10 27.13 31.89 21.78
±4.30 ±1.09 ±1.81 ±1.94
1.05 2.97 1.69 2.96
± ± ± ±
3.89 3.64 1.87 2.30
Crystal violet was added to the ration as a pulverized powder containing 93% total dye content (Fisher Scientific). Each value (mean ± standard error of the mean) represents mM substrate absorbed/gram dry tissue/10 minutes.
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Each value ± standard error of the mean is the average of 4 replicates of 10 birds each. These values differ significantly (P<.05) from the corresponding control group.
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STEWART ET AL.
TABLE 5. Effect of two gentian violet products on FeS9 absorption of young broiler chicks (Experiment 2)
Gentian violet (Mg/g-carrier)
Fe s ' absorption (% of dose administered)
0 16-calcium carbonate 16-corn cob fraction
19.62 ± 1.03a 22.50 ± 1.49a 30.08 + 1.01 ^
experiments. With the corn cob fraction carrier, the gentian violet is solubilized slower, which will result in less upper gastrointestinal staining. It appears therefore, that more gentian violet would be associated with the corn cob carrier in the lower gastrointestinal tract (i.e., duodenal loop), and through enzymatic action or pH changes this gentian violet could be liberated for action on the intestinal absorptive surface. Estimations of in vitro intestinal uptake failed to reveal an enhancement of either methionine or glucose by crystal violet. This correlates with the findings of no change in plasma protein and glucose levels associated with crystal violet, however, caution must be taken in the interpretation of in vitro studies when the results are in support of an in vivo effect. Gentian violet did prevent some of the growth depression caused by dietary aflatoxin at a level of 2.50 /lg/g; however, at 5.00 /ig/g the addition of gentian violet did not alter the growth depression caused by dietary aflatoxin. Since it has been postulated that some of the
ACKNOWLEDGMENTS The authors wish to thank Ginger Smith and Fran Denman for their excellent technical assistance.
TABLE 6. Effect on body weight of feeding gentian violet and aflatoxin simultaneously to young broiler chicks (Experiment 3) violet (Mg/g) 0 16.0
0 443 ± 9 a 436 ± 11
1.25
Aflatoxin (Mg/g) 2.50
440 ± 25 434 ± 4
358± 8 402 + l l b
5.00 324 ± 8 314 ± 6
Each value (mean ± standard error of the mean) is the average 4 week body weight (g) of 4 replicates of 10 birds each. This value differs significantly from the group receiving the same level of aflatoxin and no gentian violet.
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Each value (mean ± standard error of the mean) represents the mean of 3 replicates of 3 birds each. The values differ significantly from the corresponding control group.
growth depression caused by dietary aflatoxin is the result of altered absorption, this alleviation of the growth depression could be due to enhanced absorption. In view of the effect of gentian violet on iron absorption, this hypothesis appears reasonable. The lowered plasma pigmentation during aflatoxicosis is also thought to be due to impaired intestinal uptake of the carotenoid pigments. The failure of gentian violet to restore the lowered plasma pigments and coupled with no significant effect on in vitro intestinal uptake of either methionine or glucose this suggests that the enhancement of absorption is specific and not a generalized effect for all nutrients. It can be concluded from this study that dietary gentian violet has no effect on the growth rate, feed conversion, or intestinal uptake or absorption of methionine, glucose or carotenoid pigments of broilers. There is a stimulatory effect upon iron uptake provided the gentian violet is on a corn cob fraction as the carrier. This increased iron uptake will result in a slight elevation in hemoglobin. Dietary gentian violet also appears to partially protect young broilers from the growth depression caused by dietary aflatoxin. It appears possible for gentian violet to result in an improvement in poultry health in view of these physiological activities. This could account for the numerous field reports indicating improvement in poultry production when dietary gentian violet is used.
GENTIAN VIOLET AND BROILER CHICKENS
239
TABLE 7. Effect of plasma pigment of simultaneous feeding of gentian violet and aflatoxin to young broiler chicks (Experiment 3) Gentian violet (Mg/g)
0
1.25
Aflatoxin (Mg/g) 2.50
5.00
0 16.0
8.10+ .23 a 8.72 ±1.23
7.91+ .15 8.69 ±1.59
4.82 ±.87 4.66 ± .34
4.38 ± .78 4.11 ± .31
a Each value (mean ± standard error of the mean) is the average plasma carotenoid concentration of 4 week old broiler chicks expressed as (3-carotene equivalents/ml.
REFERENCES Bruning, J. L., and B. L. Kintz, 1968. Computational handbook of statistics. Scott, Foresman Co., Glenview, IL. Dubrowski, K. M., 1962. An o-toluidine method for body-fluid glucose determination. Clin. Chem. 8:215-235. Edwards, H. M., Jr., 1966. The effect of protein source in the diet on Zn 65 absorption and excretion by chickens. Poultry Sci. 45:421—422. 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.
Smith, J. W., and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49:207-215. Stewart, R. G., R. D. Wyatt, and J. Kiker, 1977. Effect of commercial antifungal compounds on performance of broiler chickens. Poultry Sci. 56:1664-1666. Sunderman, F. W., R. P. MacFate, D. A. McFayden, G. F. Stevenson, and B. E. Copeland, 1953. Symposium on clinical hemoglobinometry. Amer. J. Clin. Pathol. 23:519-598. Wilson, W. O., 1956. Identifying non-laying chicken hens. Poultry Sci. 35:226-227. Wooten, I. D. P., 1964. Micro-analysis in medical biochemistry. Greene and Stratton, Inc., New York, NY.
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This value differs significantly from the group receiving the same level of aflatoxin and no gentian violet.
Department of Poultry Science, University of Georgia, Athens, Georgia 30602 (Received for publication January 11, 1979)
INTRODUCTION Problems associated with mycotoxin contamination of animal feeds is a persistent problem in almost all phases of animal production. As a result of mycotoxin contamination of feedstuffs and the realization that mycotoxin concentrations may increase after feed manufacture, a variety of antifungal preparations have been used as feed additives in recent years. The expectation is that these preparations will retard or inhibit mold growth and subsequent mycotoxin formation. This, in turn, would lessen the economic losses caused by mycotoxicoses and reduce the many problems caused by mycotoxins interacting with other environmental stressors. Preparations containing gentian violet as the active ingredient were at one time used on a large scale within the poultry industry. During this time of widespread use, numerous field reports emerged suggesting improved growth rates, improvements in feed efficiency, increased livability, and improved carcass quality associated with the use of dietary gentian violet even in the absence of mold growth and mycotoxin contamination in the rations. A beneficial effect of gentian violet on such production parameters has not been documented; however, the existence of such an effect appears to
1 Present address: United States Department of Agriculture, Animal Parasitology Institute, ARS, Beltsville, MD 20705.
warrant consideration. For example, gentian violet exhibits antibacterial properties, specifically against gram positive organisms, an activity similar to that of penicillin. The fungistatic properties of gentian violet have been recognized for many years, especially its activity against Candida albicans, an opportunistic pathogen to both man and animal. The anthelmintic properties of gentian violet also have been exploited. Preparations containing gentian violet as the sole active ingredient are currently available for treatment of internal bacterial, antifungal, and anthelmintic properties and perhaps yet unidentified traits of gentian violet could result in improved performance of commercial poultry. The purpose of this investigation was to evaluate gentian violet for growth promoting and pharmacological activities.
MATERIALS AND METHODS In all experiments, day-old male chicks (Cobb) were obtained from a commercial hatchery and housed in electrically heated batteries under continuous lighting with feed and water available ad libitum. In Experiment 1, four dietary levels (0, 16, 32, and 64 /Ug/g) of USP grade crystal violet were attained by adding the pure compound to commercial broiler-starter ration free of medication. Sixteen groups of 10 birds each with 4 replicates per treatment were fed for four weeks at which time various blood and tissue analyses were 234
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ABSTRACT The effects of dietary gentian violet upon certain physiological parameters of broiler chickens were studied. Gentian violet exhibited no effect upon growth rates or feed conversion ratios at dietary levels of 16, 32, or 64 jug/g. In vitro intestinal absorption of methionine and glucose was also unaltered. Dietary gentian violet significantly increased hemoglobin concentration without an effect on packed cell volume. Furthermore, commercial gentian violet containing preparations, when incorporated into the diet, resulted in increased intestinal absorption of Fe 5 ' but this increase was dependent upon type of inert carrier used. Dietary gentian violet alleviated some of the growth suppression caused by dietary aflatoxin; however, no effect was observed on plasma pigmentation. These data suggest that dietary gentian violet possesses effects other than those for which it has been traditionally employed. 1980 Poultry Science 59:234-239
GENTIAN VIOLET AND BROILER CHICKENS
Experiment 2 was similar to Experiment 1 except the dietary treatments included the control group (basal ration) and two groups receiving the basal ration containing 16 fig of gentian violet/g of diet. The gentian violet was supplied by using two commercial products,
one with a calcium carbonate carrier (Marshall Mineral, Marshall, TX) and the other employing a corn cob fraction (Dan-Mar Enterprises, Commerce, GA) as the carrier. Each treatment consisted of 3 replicates of 3 birds each. The birds were fed each ration for 7 days at which time all birds were dosed by crop intubation with 1.0 ml of an aqueous solution containing .2 yc/ml of F e 5 9 . The initial absorption Fe was determined as described by Edwards (1966). The administration of the diets containing gentian violet was continued throughout a 12-day post-dosing period. Experiment 3 was of a 2 X 4 factorial design for the presence and absence of gentian violet supplied as Dye-Gen® (Dan-Mar Enterprises, Commerce, GA) at a level of 16 Mg/g of diet and the presence and absence of aflatoxin with aflatoxin levels of 1.25, 2.5, and 5.0 yitg/g of diet. The aflatoxin used was produced and added to the basal diet as described by Smith and Hamilton (1970). Each treatment consisted of 3 replicates of 10 birds each. The birds were fed each diet from day old to 3 weeks of age. During the experiment mortality, body weights and feed conversion ratios were determined weekly. At the end of the experiment a blood sample was taken via cardiac puncture and the plasma carotenoid concentration determined by the method of Wilson (1956) as modified by
Rufietal. (1974). All data were statistically analyzed by an analysis of variance (Bruning and Kintz, 1968). Statements of significance are based on P< .05. RESULTS
The addition of crystal violet to the diet of young broiler chicks had no significant effect on the 4-week growth rate (Table 1). In a similar fashion there was no pronounced effect on the weekly feed conversion ratios from day old through 4 weeks (Table 2). A significant effect on the feed conversion ratio was observed during the first week of the experiment with all levels of crystal violet; however, the magnitude of the improvement was slight and was not evident in subsequent weeks. Analysis of certain blood components revealed that dietary crystal violet had no effect upon the concentrations of total plasma protein, plasma glucose, or PCV (Table 3). Crystal violet at all dietary levels resulted in a significant increase in the hemoglobin concentration; however, the increase was only about 4% (Table 3).
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determined. Weekly body weights and feed conversion ratios were computed. At four weeks plasma was collected by drawing 3.0 ml of blood via cardiac puncture into silicon treated tubes (100 X 13 mm) containing .3 ml of .18 M sodium citrate. An additional blood sample was taken from the brachial vein for packed cell volume (PCV) and hemoglobin determination. Hemoglobin was determined according to the method of Sunderman et al. (1953), plasma glucose by the method of Dubrowski (1962), total plasma protein according to Wooten (1964), and PCV by the microhematocrit method. All birds were then killed by cervical dislocation and a segment of the duodenum approximately 3.0 cm in length was removed and placed in a cold (4 C) Krebs-Henseleit buffered saline (KHS, pH 7.2) which had been bubbled with 5% C 0 2 : 9 5 % 0 2 prior to use. The mesenteries were stripped and the intestine opened. Tissue punches, 7 mm in diameter, were removed from immediately below the bile and pancreatic duct using a No. 3 cork borer. Single punches from three different birds were pooled in a beaker. Three replicates, each containing tissue from three different birds, were utilized at each substrate and drug level. The intestinal tissue was held in cold (4 C) KHS containing 5 mM glucose as an energy source prior to incubation. The tissue was then transferred to beakers containing 9 ml of KHS and preincubated for 10 min at 40.5 ± .5 C. At zero time 1 ml of KHS containing 10 times the desired concentration of C 14 -L-methionine or C 14 -glucose was added to the beaker. Tissues were incubated for 10 min. The reaction was stopped by transferring the tissue to cold KHS and rinsing through three successive changes of cold buffer. The labeled substrate was extracted from the tissue with 70% ethanol. An aliquot of the extract was added to PCS scintillation fluid (Phase Combining System, Amersham/Searle) and the radioactivity measured using a Packard Tri-Carb Scintillation Counter. After extraction, the tissue was dried at 95 C and weighed. Results were expressed as mM of substrate absorbed/g weight dry tissue/10 min incubation.
235
236
STEWART ET AL. TABLE 1. The effect of dietary crystal violet on the body weight of young broiler chickens (Experiment 1)
Crystal (mg/kg)
0
0 16 32 64
38 38 39 39
Weekly body weights (g) a 2
1 ±1 ±1 ±1 ±1
263 ± 264+ 255 ± 258+
119 ± 2 115 ± 3 115 + 1 117± 3
4 5 3 10
4
3 463 465 458 463
± ± ± ±
10 10 5 11
717 701 675 703
± ± ± ±
23 15 7 16
The in vitro intestinal uptake of methionine (Table 4) was not altered by any level of dietary crystal violet. At a substrate concentration of .5 mM and above and at a dietary level of crystal violet of 32 /ig/g there was a consistent stimulatory effect on glucose uptake; however, at no substrate concentration was there a significant response. Absorption of Fe 5 9 in response to two products containing gentian violet (Table 5) indicates that gentian violet on a corn cob fraction as the carrier will result in a significant increase in Fe absorption of about 50%. There was no significant effect on Fe absorption with gentian violet on the calcium carbonate carrier. The response of body weight of chicks to dietary aflatoxin and aflatoxin and gentian violet in combination is demonstrated in Table 6. The body weight of birds receiving aflatoxin alone responded as expected based upon findings of others (Smith and Hamilton, 1970). Feeding gentian violet alone did not result in an improvement in body weight (Tables 1 and 6);
when a dietary concentration of gentian violet of 16.0 fig/g was fed in combination with 2.50 Mg/g of dietary aflatoxin, the expected growth depression caused by the aflatoxin was partially alleviated. However, the feeding of 16.0 /zg/g of gentian violet in combination with 5.00 /ig/g of aflatoxin did not result in an alleviation of the body weight depression expected from the aflatoxin alone. Some field observations indicated that the use of dietary gentian violet enhanced carcass pigmentation of broilers. In this present investigation, neither dietary gentian violet alone (16 Mg/g) nor this same level of gentian violet in combination with dietary aflatoxin at levels up to 5.0 jug/g enhanced plasma pigmentation or prevented the decrease in plasma pigmentation associated with aflatoxicosis (Table 7).
DISCUSSION In no instance was there an enhancement of the growth rate of broiler chicks associated with dietary crystal or gentian violet. These
TABLE 2. Effect of dietary crystal violet on feed conversion ratios of young broiler chicks (Experiment 1) Weekly feed conversion ratio (g feed consumed/g wt gained)
Crystal (Mg/g)
Oto 1
0 16 32 64
1.434 1.345 1.345 1.329
1 to 2 ± ± ± ±
.024 a .016 b .018 b ,020 b
1.567 1.571 1.627 1.687
+ + + ±
.041 .062 .065 .071
2 to 3
3 to 4
1.779 ± .073 1.759 +• .071 1.896 ± .070 1.717+ .087
2.036 ± 2.105 ± 2.119 ± 2.054 ±
Each value (mean ± standard error of the mean) represents the mean of 4 replicates of 10 birds each. These values differ significantly (P<.05) from the corresponding control group.
.093 .102 .086 .086
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Each value (mean ± standard error of the mean) is the average of 4 replicates of 10 birds each. There were no significant (P>.05) differences among means during any week.
GENTIAN VIOLET AND BROILER CHICKENS
237
TABLE 3. Effect of dietary crystal violet on blood components of young broiler chickens (Experiment 1) Crystal violet (Mg/g)
Plama protein (mg/100 ml)
Plasma glucose (mg/100 ml)
0 16 32 64
2705 ± 181a 2880 ± 186 2738 ± 205 2898± 126
282 ± 287 ± 274 ± 252 ±
Packed cell volume (%)
Hemglobin (g/100 ml) 7.00 ± .10 7.34 + .05b
19 25 14 12
29.7 ± 4.7 29.0 ± 3.2 28.1 ± 3.9 28.9 ± 3.0
7.31 ± .08 b 7.32 ± .09°
data confirm those of Stewart et al. (1977) where no improvement in growth rate was observed in broilers grown for 6 weeks under commercial-type conditions. The improvement in feed efficiency observed in this present study is of questionable significance since this effect was present only at one time (1 week) of the growing period and was not evident at later times. Likewise, Stewart et al. (1977) did not observe an improvement in feed efficiency due to dietary gentian violet. Of interest is the increase in the hemoglobin concentration in the blood associated with all levels of dietary gentian violet. Since the PCV was not increased, this suggests an increased
intestinal absorption of iron which, in turn, allowed for additional hemoglobin synthesis. This suspected facilitation of iron uptake was confirmed by determination of in vivo Fe 5 9 absorption (Table 5) and offers one possible explanation for the increase in hemoglobin. The difference between the two products in this ability to apparently increase iron uptake may lie in the inherent tendency of the gentian violet to be solubilized and dissociate from the carrier. In the case of the calcium carbonate carrier, the gentian violet is readily solubilized. This is evident in the degree of staining of the crop and proventriculus noted during routine necropsy of the birds at the termination of the
TABLE 4. Uptake of methionine and glucose by intestinal tissue of broiler chicks fed graded levels of crystal violet (Experiment 1)
Crystal
m M Substrate
(Mg/g)
.25
1.0
0 16 32 64
2.58 2.18 2.52 1.95
+ + ± +
.22b .07 .07 .11
4.26 4.09 3.25 3.95
± ± ± ±
0 16 32 64
3.72 3.28 3.42 2.87
+ + + +
.47 .14 .79 .34
5.97 6.43 8.96 4.29
± .22 ± .32 ± 1.12 ± .26
.5
2.5
5.0
10.0
Methionine .25 .53 .28 .58
7.78 6.13 6.20 5.33
± 1.23 ± .39 ± .35 ± .43
1 1 . 7 3 ± 1.15 1 1 . 6 0 + .68 11.28 ± 1 . 5 4 8.85 ± .64
15.69 14.33 12.04 12.91
± 2.32 ± .28 ± .16 ± .82
2 1 . 8 6 + 2.57 19.53 ± .90 15.60 ± 1.30 1 7 . 4 4 ± 1.41
30.40 33.10 35.00 31.19
± ± ± ±
40.16 47.70 45.01 36.36
Glucose 10.06 13.12 13.26 9.56
± .21 ± .90 ±1.31 ±1.68
21.10 27.13 31.89 21.78
±4.30 ±1.09 ±1.81 ±1.94
1.05 2.97 1.69 2.96
± ± ± ±
3.89 3.64 1.87 2.30
Crystal violet was added to the ration as a pulverized powder containing 93% total dye content (Fisher Scientific). Each value (mean ± standard error of the mean) represents mM substrate absorbed/gram dry tissue/10 minutes.
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Each value ± standard error of the mean is the average of 4 replicates of 10 birds each. These values differ significantly (P<.05) from the corresponding control group.
238
STEWART ET AL.
TABLE 5. Effect of two gentian violet products on FeS9 absorption of young broiler chicks (Experiment 2)
Gentian violet (Mg/g-carrier)
Fe s ' absorption (% of dose administered)
0 16-calcium carbonate 16-corn cob fraction
19.62 ± 1.03a 22.50 ± 1.49a 30.08 + 1.01 ^
experiments. With the corn cob fraction carrier, the gentian violet is solubilized slower, which will result in less upper gastrointestinal staining. It appears therefore, that more gentian violet would be associated with the corn cob carrier in the lower gastrointestinal tract (i.e., duodenal loop), and through enzymatic action or pH changes this gentian violet could be liberated for action on the intestinal absorptive surface. Estimations of in vitro intestinal uptake failed to reveal an enhancement of either methionine or glucose by crystal violet. This correlates with the findings of no change in plasma protein and glucose levels associated with crystal violet, however, caution must be taken in the interpretation of in vitro studies when the results are in support of an in vivo effect. Gentian violet did prevent some of the growth depression caused by dietary aflatoxin at a level of 2.50 /lg/g; however, at 5.00 /ig/g the addition of gentian violet did not alter the growth depression caused by dietary aflatoxin. Since it has been postulated that some of the
ACKNOWLEDGMENTS The authors wish to thank Ginger Smith and Fran Denman for their excellent technical assistance.
TABLE 6. Effect on body weight of feeding gentian violet and aflatoxin simultaneously to young broiler chicks (Experiment 3) violet (Mg/g) 0 16.0
0 443 ± 9 a 436 ± 11
1.25
Aflatoxin (Mg/g) 2.50
440 ± 25 434 ± 4
358± 8 402 + l l b
5.00 324 ± 8 314 ± 6
Each value (mean ± standard error of the mean) is the average 4 week body weight (g) of 4 replicates of 10 birds each. This value differs significantly from the group receiving the same level of aflatoxin and no gentian violet.
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Each value (mean ± standard error of the mean) represents the mean of 3 replicates of 3 birds each. The values differ significantly from the corresponding control group.
growth depression caused by dietary aflatoxin is the result of altered absorption, this alleviation of the growth depression could be due to enhanced absorption. In view of the effect of gentian violet on iron absorption, this hypothesis appears reasonable. The lowered plasma pigmentation during aflatoxicosis is also thought to be due to impaired intestinal uptake of the carotenoid pigments. The failure of gentian violet to restore the lowered plasma pigments and coupled with no significant effect on in vitro intestinal uptake of either methionine or glucose this suggests that the enhancement of absorption is specific and not a generalized effect for all nutrients. It can be concluded from this study that dietary gentian violet has no effect on the growth rate, feed conversion, or intestinal uptake or absorption of methionine, glucose or carotenoid pigments of broilers. There is a stimulatory effect upon iron uptake provided the gentian violet is on a corn cob fraction as the carrier. This increased iron uptake will result in a slight elevation in hemoglobin. Dietary gentian violet also appears to partially protect young broilers from the growth depression caused by dietary aflatoxin. It appears possible for gentian violet to result in an improvement in poultry health in view of these physiological activities. This could account for the numerous field reports indicating improvement in poultry production when dietary gentian violet is used.
GENTIAN VIOLET AND BROILER CHICKENS
239
TABLE 7. Effect of plasma pigment of simultaneous feeding of gentian violet and aflatoxin to young broiler chicks (Experiment 3) Gentian violet (Mg/g)
0
1.25
Aflatoxin (Mg/g) 2.50
5.00
0 16.0
8.10+ .23 a 8.72 ±1.23
7.91+ .15 8.69 ±1.59
4.82 ±.87 4.66 ± .34
4.38 ± .78 4.11 ± .31
a Each value (mean ± standard error of the mean) is the average plasma carotenoid concentration of 4 week old broiler chicks expressed as (3-carotene equivalents/ml.
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Smith, J. W., and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49:207-215. Stewart, R. G., R. D. Wyatt, and J. Kiker, 1977. Effect of commercial antifungal compounds on performance of broiler chickens. Poultry Sci. 56:1664-1666. Sunderman, F. W., R. P. MacFate, D. A. McFayden, G. F. Stevenson, and B. E. Copeland, 1953. Symposium on clinical hemoglobinometry. Amer. J. Clin. Pathol. 23:519-598. Wilson, W. O., 1956. Identifying non-laying chicken hens. Poultry Sci. 35:226-227. Wooten, I. D. P., 1964. Micro-analysis in medical biochemistry. Greene and Stratton, Inc., New York, NY.
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This value differs significantly from the group receiving the same level of aflatoxin and no gentian violet.