Abstract:
A process of feeding animals and poultry a complete feed consisting only of (i) between 0.00077 and 0.005 percent by weight, based on the weight of the feed, of gentian violet and (ii) the remainder medically inert ingredients, such inert ingredients not having a sparing effect for poultry and animals from the toxic and/or lethal effects of aflatoxin. The gentian violet has the effect of sparing animals and poultry from the toxic effects of aflatoxins in feeds. The inert ingredients include all of the inert ingredients normally in a complete or basal animal or poultry feed.

Description:
This application is a continuation-in-part application of applicant&#39;s copending application Ser. No. 625,693, now abandoned, filed on Oct. 24, 1975, which in turn was a continuation-in-part of applicant&#39;s then co-pending application Ser. No. 595,876, now abandoned, filed July 14, 1975, which in turn is a continuation-in-part of two others of applicant&#39;s applications which were co-pending therewith, Ser. No. 454,000, filed Mar. 22, 1974, now U.S. Pat. No. 3,915,637, and application Ser. No. 342,290, filed Mar. 16, 1973, now U.S. Pat. No. 3,916,027 
    
    
     BACKGROUND OF THIS INVENTION 
     1. Field of This Invention 
     This invention relates to animal and poultry feed which contains a medicinal agent that is a selective fungicidal mold inhibitor of Candida albicans. This invention also relates to the use of such feed to spare animals and poultry from the toxic effects of aflatoxins and other mycotoxins in feed. 
     2. Art Section 
     It is known that a given strain of mold may gain or lose its ability to produce toxins without known reason. Several reasons for this phenomenon have been advanced, namely, change of substrate, genetics, stage of growth, accumulation and metabolism of the toxin, and actual stability of the toxin. 
     Facial eczema of sheep in New Zealand has been caused by Pithomyces chartarus -- the toxin caused liver damage and skin disorders. A malady in horses, cattle, and poultry has been caused by a toxin from a mold called Stachybotrys atra. A field outbreak in Georgia of mold toxicosis in swine resulted from corn left in the field -- the toxic molds were Aspergillus flavus and Penicillium rubrum. A massive outbreak of toxicity from moldy ground nut meal (peanut) occurred in England -- the toxin producing mold was a strain of Aspergillus flavus. The common name applied to the disease is &#34;Turkey X&#34; disease, and the poisonous material has been designated as &#34;Aflatoxin.&#34; Aflatoxin has carcinogenic properties. Hemorrhagic disease in chicks occurred -- toxins from certain molds will cause lesions indistinguishable from field hemorrhagic disease. Bovine hyperkeratosis has been caused by Aspergillus clavatus. A toxicosis from moldy feed at Texas Agricultural Experimental Station was caused by unidentified molds. The mold brought on acute lysine deficiency. Arginine was similarly affected. These amino acids alleviated most of the problem in poults. 
     Forgacs wrote in 1954 that aflatoxin was a feed mold problem. 
     Aflatoxicosis is recognized as a toxic disease problem occurring as a result of fungus growth in feeds and feedstuffs. Even if the fungus is killed in the feedstuffs the aflatoxin that has been produced by the growing fungus remains in the feed as a poison which then produces the symptoms of aflatoxicosis when the basal feed is ingested by poultry. There is a need for an effective active ingredient to be added to the inert ingredients of the basal feed to effect a reduction or &#34;sparing effect&#34; in the consuming poultry from the poisonous affects of the aflatoxin-containing feed. Such a sparing effect is obviously not produced by the ingredients which normally comprise a basal poultry feed, hence such ingredients are &#34;inert&#34; re the aflatoxicosis problem. 
     Aflatoxin, produced by Aspergillus flavus, causes Turkey X disease. The duck is most susceptible, followed by the turkey and chicken in that order. 
     Fungal growth can occur in ground grains having as little as 12 percent moisture. The heat and moisture given off by fungal growth activity encourages more fungal growth. Mold growth often occurs which causes certain molds to produce mycotoxins. Several of these mycotoxins are quite toxic and present a serious problem to animals and poultry by death, loss of weight or lessened weight gain, etc. Chronic mycotoxicoses from the long term low intake of mycotoxins often cause reduced growth rate and appetite, among other things. Recovery is slow even when switched to feeds free of mycotoxins. Another danger is that aflatoxin has been demonstrated to be a carcinogen. Mycotoxins, and aflatoxin in particular, are difficult to remove from feedstuffs. Heat is ineffective; U.V. irradiation and solvent extraction are effective, but quite economically unfeasible. Certain acids, bases and other compounds have been used to neutralize aflatoxin in feed, but most of such compounds appear to have been unsatisfactory for a number of reasons. 
     BROAD DESCRIPTION OF THIS INVENTION 
     An object of this invention is to provide a process for sparing animals and/or poultry from the toxic and/or lethal effects of mycotoxins, such as aflatoxins, in feed. Another object of this invention is to provide a composition for animals and/or poultry which has the property of sparing animals and/or poultry from the toxic and/or lethal effects of mycotoxins, such as aflatoxins, in feeds. Other objects and advantages are set out herein or are obvious herefrom to one ordinarily skilled in the art. 
     Such objects and inventions are achieved by the process and composition of this invention. 
     This invention involves a method for sparing an animal or poultry from the toxic and/or effects of a mycotoxin, such as an aflatoxin, in an animal or poultry feed. The method includes feeding a complete feed to the animal or poultry, the complete feed being comprised of (i) gentian violet and (ii) the remainder medically inert ingredients. The gentian violet spares the animal or poultry from the toxic and/or lethal effects of mycotoxin in the feed as a contaminant. the gentian violet is present in the feed in an amount between 0.00077 and 0.005 percent by weight, based on the weight of the complete feed. The medically inert ingredients do not have a sparing effect for poultry and animals from the toxic and/or lethal effects of mycotoxins. 
     The composition of this invention is preferably formulated by adding a premix concentrate to the feed. The premix contains between 0.1 and 10 percent by weight of gentian violet and the remainder medically inert ingredients. Enough of the premix concentrate to the other ingredients of the feed is used to obtain the recited amount of gentian violet in the complete feed. Other methods of preparing the complete feed can be used, for example, the gentian violet, by itself, can be directly admixed to the feed. 
     Preferably the feed contains between 0.00155 and 0.00232 percent by weight of gentian violet, based on the weight of the complete poultry feed. 
     This invention is quite useful for feeds that are contaminated with as much as 10 p.p.m. or more of mycotoxin, such as aflatoxin. 
     The sparing effect of gentian violet appears to be a linear function of the concentration of the aflatoxin in the feed. 
     The weight depression of animals or poultry eating mycotoxin-contaminated animal or poultry feeds which contain gentian violet at the disclosed levels is significantly less than for animals or poultry eating such mycotoxin-contaminated feed which does not contain gentian violet. 
     The levels of gentian violet used in this invention are low enough not to damage the consuming animal or poultry and not to produce an unacceptable residue in food from the animal or poultry. The level of residue is extremely low, particularly when a short withdrawal period is used before slaughter. 
     Gentian violet is essentially non-absorbable and stable at the pH range of the digestive tract of animals and poultry. 
     Other substituted benzophenone azoanilide dyes or rosaniline base dyes can be used in place of the gentian violet. Such are also known as triaminotritane dyes related to rosaniline. The --N(CH 3 ) 2  groups of gentian violet can be replaced by other auxochrome groups such as --OH, --NH 2 , --N(C 2  H 5 ) 2 , etc. The other various substituents, and the substituents on gentian violet, can be located at any position on the three benzyl rings. The other dyes must have at least one amino chloride group and/or substituted amino chloride group. 
     Examples of such other substituted benzophenone azoanilide dyes are: pentamethylpararosaniline chloride; tetramethylpararosaniline chloride; methyl green or light green, which is the methyl chloride addition product of gentian violet; ethyl green, which is the ethyl chloride addition product of gentian violet; sulfonic acids of the rosanilines, such as water blue and patent blue; phenylated rosanilines, such as triphenyl fuchsin, aniline blue, diphenylamine blue and spirit blue; pararosaniline; mixtures of pararosaniline with its methyl homologs, such as fuchsins and magentas. More broadly, any triphenylmethane dye that contains an amino group or substituted amino group can be used in place of gentian violet. Examples of such triphenylmethane dyes are the malachite green series of dyes, such as malachite green. 
     Most of such aniline dyes are rather toxic, but can be readily used if they are buffered by any generally non-toxic conventional buffering system to reduce their toxicity. The buffering system can be one composed from alkali and acid salts of phosphoric acid, acetic acid, carbonic acid and/or citric acid. Usually the buffer salts are the monobasic and tribasic salts thereof. 
     For the best effects the feed composition of this invention is fed to poultry, hogs or cattle. 
     Any complete poultry feed or basal poultry feed can be used. It can contain, for example, ground yellow corn, soybean oil meal, steamed bone meal, ground limestone, iodized salt, manganese sulfate, Vitamin A oil, dry Vitamin D-3, riboflavin, Vitamin B-12 and niacin. It can also contain, for example, fish meal and meat meal. 
     Any complete animal feed or basal animal feed can be used; it can contain, for example, any of the following ingredients: iodized salt, dry Vitamin D-3, riboflavin, Vitamin B-12, niacin, meat meal, D calcium pantothenate, cracked or milled grains such as corn, wheat, oats, barley and the like, dried molasses, dried sorghum, soybean meal, cottonseed meal, peanut meal, fish meal, essential amino acids, such as lysine, peptides and polypeptides containing essential amino acids, casein, soya bean protein, vitamins such as Vitamins A, D, E and K, mineral nutrients such as sodium chloride, ferrous salts, magnesium sulfate and calcium salts, proteins, buffers, dextrose, sucrose, lactose, maltose, corn syrup solids, hydrolized cereal solids, hay, etc. 
     The following is a typical or standard vitamin premix that is used in a complete poultry feed: 
     
         ______________________________________                 gm/100 lb.______________________________________Manganese sulfate (feed grade)                   11.4Vitamin A oil (10,000 I.U./gm)                   22.7Dry Vitamin D-3 (1,6550 I.C.U./gm)                   22.7Riboflavin              0.15Vitamin B-12 (3 mg. lb.)                   45.4Niacin                  1.0______________________________________ 
    
     An exemplary cattle feed is 73 percent rolled shelled corn, 20 percent ground corn cobs, and a supplement containing soybean meal, alfalfa meal, cane molasses, urea, salt, dicalcium phosphate, Vitamin A concentrate and Vitamin D concentrate. 
     The greatest mycotoxin effect sparing is achieved in young, maturing animals or poultry, although excellent mycotoxin effect sparing is achieved in grown mature animals or poultry. 
     An important feature of this invention is that there is little or no residue of the gentian violet in the tissue of the animals fed the compositions of this invention--this is apparently so due, in part, to the levels of gentian violet used in the compositions of this invention. This means that there is no toxicological danger to animals or humans from ingestion of the edible tissues of animals which have been fed the compositions of this invention. 
     Applicant&#39;s disclosed concentration ranges in the animal and poultry feeds are important re efficacy, i.e., mycotoxin effect sparing in poultry or animals. If amounts lower than applicant&#39;s disclosed ranges are used, there is little or no efficacy in aflatoxin effect sparing. If an amount of gentian violet is used which is higher than applicant&#39;s disclosed range (in feed), then toxic effects in the poultry can be encountered. Obviously, the lower the amount of gentian violet used, the less chance of toxic effect. The toxic effect can kill the poultry or cause a decrease in the weight gain of the poultry, which represents a serious economic loss to commercial poultry raisers. The residue build up is a potential health hazard when such higher levels are used. 
     DETAILED DESCRIPTION OF THE INVENTION 
     As used herein, the term &#34;poultry&#34; means domestic fowls including chickens, ducks, turkeys, geese, etc. 
     As used herein, the term &#34;animal&#34; includes domestic pigs, other swine, cattle, sheep, goats, fish, such as trout, rabbits, etc. 
     As used herein, the term &#34;gentian violet&#34; is hexamethylpararosaniline chloride or a mixture of at least 96 percent of hexamethylpararosaniline chloride with minor portions of pentamethylpararosaniline chloride and/or tetramethylpararosaniline chloride, and preferably meeting all specifications of quality for U.S.P. grade as listed in the United States Pharmacopiea XIV. This assures that none of the deadly heavy metals are left as a residue in animal or poultry tissue that is to be consumed by man. 
     Gentian violet is a dark green powder or greenish, glistening pieces with a metallic luster. Gentian violet is soluble in water, chloroform and ethanol. Other names for gentian violet are methylrosaniline chloride and crystal violet. 
     Gentian violet, both as a contaminant in water and as a residue in poultry manure, is biodegradable in the presence of the ultra-violet rays of sunlight. 
     Toxicosis is a pathological condition caused by the action of a toxin. Toxin is a colloidal proteinaceous poisonous substance that is a specific product of the metabolic activities of a living organism. 
     A large number of toxins, broadly classed as mycotoxins, are produced by a variety of molds, fungi and yeast. These toxins have been proven to interfere with normal function of poultry and animals even to producing death. Prior to this invention, all efforts to overcome the harmful effects of these mycotoxins have proven fruitless. 
     The discovery of this invention is that when gentian violet is administered in the feed or drinking water at the proper dosage level, the gentian violet will exert a sparing effect from the mycotoxins to alleviate their harmful effect to poultry or other animals. Some of the mycotoxin producers are: Aspergillus sp., Fusarium sp., Alternaria sp., Penicillium sp., Mucor sp., Cladosporium sp. and Gibberella sp. 
     Examples of specific molds which cause mycotoxicoses in animals and poultry are: Aspergillus chevalieri, A. clavatus, A. flavus, A. fumigatus, A. glaucus, Paecilomyces varioti, Penicillium citrinum, P. purpuro-genum, P. rubrum, a species of Alternaria. Mycotoxin F-2 is produced by Fusarium graminearium, F. moniliforme and F. roseum; F-3 and F-5-3 are produced by F. graminearum; aflatoxin is produced by Aspergillus flavus, A. parasiticus, A. ruber, A. wenti, A. oryzae, A. niger, A. ostianus, A. ochraceus, Penicillium puberulum, P. variable, P. citrinum, P. frequentanus, and Rhizopus sp.; ochratoxin is produced by A. ochraceus; rubitoxin is produced by P. rubrum; ergot is produced by Claviceps purpurea; alimentary toxic aleukia is produced by F. sporotrichiodes and F. tricinctum; islandotoxin is produced by Pencillium islandicum; slaframine is produced by Rhizoctonia leguminicola; ipomeamarone is produced by Ceratosystic fimbriata; patulin is produced by P. urticae; maltoryzine is produced by A. oryzae; gliotoxin is produced by P. terlikowskii and Tricoderma viride; sporidesmin is produced by Pithomyces chartarum; chetomin is produced by Chetomium cochliodes; furocoumarins is produced by Sclerotinia sclerotiorum; kojic acid is produced by A. tamarii and A. oryzae; citreo-viridin is produced by P. citreo-viride; citrinin is produced by P. citrinum and A. terrus; and rugulosin is produced by P. rugulosum. 
     Mycotoxins (especially aflatoxins) are carcinogenic in or cause cancer in man, and so the presence of mycotoxins in the digestive trace (hence, possibly the flesh) of animals or poultry slaughtered for human consumption raises the specter of such a problem in the consuming person. 
     Aflatoxins are a type of mycotoxin and are produced primarily by certain strains of a group of fungi called Aspergillus flavus. 
     Aflatoxin is soluble in various lipid solvents. 
     Aflatoxin B-1 is the most toxic of the known mycotoxins, so the sparing effect of this invention has been primarily shown with such aflatoxin. It has been reported that 1 or 2 p.p.m., or less, of aflatoxin B-1 in feed is lethal to animals and poultry. (Pages 2, 3 and 6 of Wilcox, et al., ibid., sets out examples in various animals and poultry and discusses some F.D.A. levels in this field. As little as 0.4 p.p.b. in fish food has triggered the development of hepatic tumors in trout.) This invention has a significant sparing effect on poultry as high as 10 p.p.m. of aflatoxin in feed fed to poultry over many weeks. 
     Aflatoxin B. (C 17  H 12  O 6  : mol wt 312.27; C, 65.38%; H, 3.87%; 0 30.74%) is the toxic metabolite of the fungus Aspergillus flavus Link ex Fries and is the causative principle of turkey &#34;X&#34; disease. It has the structure: ##STR1## Aflatoxin B: crystals mp of 268° to 269°; exhibits blue fluoroescence; [a] D  -558° (in chloroform); absorption max in ethanol: 223, 265, 362 mu (25,600, 13,400, 21,800); and LD 50  orally in white pekin ducklings: 28.2 ug. 
     Aflatoxin G (C 17  H 12  O 7  ; mol wt 328.27; C, 62.20%; H, 3.68%; 0, 34.12%) is the toxic metabolite of the fungus Aspergillus flacus Link ex Fries. It has the structure: ##STR2## Aflatoxin G: crystals, mp of 244° to 246°; exhibits yellow-green fluorescence; [a] D  -556° (in chloroform) absorption max in ethanol: 243, 257, 264, 362 mu (11,500, 9,900, 10,000, 16,100); LD 50  orally in white pekin ducklings 90 ug. 
     Staphylococcal infections are common in poultry. Staphylococccus bacteria are quite common in animal populations, however certain factors promote the invasion of the bacteria resulting in infections. Some mycotoxins have been shown to induce lesions of the skin and mucous membranes or cause damages to the intestinal lining. In either case, entry of staphylococcus may be promoted. See Poultry, Feedstuffs, June 9, 1985, p. 18. The use of gentian violet (in the amount of this invention) in poultry feed contaminated with mycotoxins eliminates or greatly lessens the effects of the mycotoxins, hence the staphylococcal infections due to mycotoxin contaminated poultry feeds are eliminated or greatly lessened. 
     The mere inclusion of gentian violet in a feed is not very effective as a mold inhibitor. The gentian violet particles are not in contact with every feed particle. The inclusion of equal amounts of gentian violet particles and feed particles would not really produce a good mold inhibitor. 
     The term inert ingredients as regards the animal or poultry feed, includes the normal feed ingredients but excludes materials which have a therapeutic or pharmacological activity. The term, more specifically, is medically-inert ingredients, and excludes any agent besides gentian violet which has a sparing effect for poultry and animals from the toxic and/or lethal effects of mycotoxins, such as, aflatoxins. 
     A vitamin mixture can be used as the carrier for the gentian violet in the premix used in this invention. 
     This invention is not intended for use in humans. 
     Gentian violet is not effective against aflatoxin production in feeds, i.e., it is not a mold inhibitor in feeds. Various chemicals, in gaseous or liquid form, have been applied to feeds to neutralize aflatoxin in such feeds. There are many problems with such chemicals. Gentian violet is used in a solid form, so it does not operate in that manner in the feed. Gentian violet, within the scope of this invention is present in feed which is consumed by the animal or poultry -- the gentian violet spares the animal or poultry from the toxic effects of the aflatoxin in the consumed feed. 
     Herein all parts, ratios, proportions and percentages are on a weight basis, unless otherwise stated or obvious to one ordinarily skilled in the art. 
     The following examples illustrate this invention. 
    
    
     EXAMPLE 1 
     160 day-old chicks were randomly divided into sixteen groups of 10 chicks each. The chicks were hatch run and were all from the same breeder flocks. All of the chicks were wing banded and debeaked at one day of age. All of the chicks were MD vaccinated on day one and were NC-IB vaccinated during the experiment. 
     The feed was prepared by preparing 1,600 pounds of a 24 percent protein broiler mash. The 1,600 pounds of feed were divided into sixteen equal batches of 100 lbs. each. The sixteen groups of chicks received gentian violet and/or aflatoxin B-1 in the amounts set out in the following table: 
     
                       TABLE 1______________________________________Checkerboard experimental design for groups ofchicks to receive gentian violet (0, 7, 21 or 35 ppm)and B-1 aflatoxin (0, 1, 4 or 10 ppm) in their feed.Gentian Violet (ppm)  0       7         21        35______________________________________B-1    0     Gp 1.sup.(a)                  Gp 2    Gp 3    Gp 4Afla-  1     Gp 5      Gp 6    Gp 7    Gp 8toxin.sup.(b)  4     Gp 9      Gp 10   Gp 11   Gp 12  10    Gp 13     Gp 14   Gp 15   Gp 16______________________________________ Notes: .sup.(a) 10 chicks per group .sup.(b) 916.7 mg B-1 aflaxtoxin plus 9.167 gm feed equal stock toxin at 100 ppm 
    
     Vigen premix contained 1.55 percent of gentian violet, 1 percent of white mineral oil, 1.65 percent of Micro-Cel E, and 91.8 percent of corn cob fractions and 4 percent distilled water and was manufactured by A.H.P., Inc., of Gainesville, Ga. Enough VIGEN premix was used to achieve the desired gentian violet level desired in each of the sixteen feed batches. A sample (1 lb.) for each feed batch (lot) was set aside for future testing purposes. Feed remaining at termination of the trial (day 20) was weighed to determine the feed consumed by each group of birds. 
     The test was conducted for 20 days. The parameters measured during the test were: (1) weight gains -- all of the groups vs. the controls -- by pen at the conclusion of the trial; and (2) the sparing effect of gentian violet from the toxicity of B-1 aflatoxin. 
     Analysis of the chick weights at 20 days of age was done utilizing an analysis of variance (one-way with equal and unequal replications). The principal object in performing the analysis of variance was to be able to compare the mean weights by the method of least significant differences. (For this reason, whether the analysis of variance was significant or not is of no consequence in this study). 
     During the test, continued observation was made of mortality, feed consumption, diarrhea, etc. On the tenth day after the start of the test, the chickens were vaccinated NC-IB by treatment Broilerbron spray. The chickens were weighed on a day of age, 7, 14 and 20 days of age. The chicken weights (at 7, 14 and 20 days of age) are given in the following table: 
     
                       Table 2______________________________________Weight of Chickens     Wt. at   Wt. at     Wt. at     7 days,  14 days,   20 days,     gm.      gm.        gm.______________________________________Group No. 1Chicken No. 1        111        251        4682            108        234        3953            126        249        3954            105        288        3965            90         211        4596            111        281        4027            118        262        3678            111        255        4209            111        248        43310           111        253        395Group No. 2Chicken No. 1        126        264        4182            123        255        3653            133        278        4444            108        288        3375            98         277        4166            94         247        4327            125        270        4558            105        205        4389            114        177        42010           113        271        334Group No. 3Chicken No. 1        122        252        4072            105        288        4293            120        243        4164            114        237        3845            116        276        4096            100        252        4197            98         238        3968            127        271        4149            109        226        39710           114        259        417Group No. 4Chicken No. 1        120        303        4192            105        269        4853            127        242        4224            110        247        4125            124        222        4386            109        277        4077            116        269        3628            108        276        4039            95         255        33410           101        203        286Group No. 5Chicken No. 1        114        272        4362            114        259        4413            109        259        3734            115        275        4375            117        268        4136            130        306        3847            112        258        4178            102        230        4279            121        247        50110           119        275        471Group No. 6Chicken No. 1        119        242        4352            98         256        3873            118        248        3954            111        274        4385            118        224        3986            98         240        3597            118        239        4098            110        243        4239            108        273        39310           100        265        429Group No. 7Chicken No. 1        87         237        3982            116        226        4073            109        258        3924            91         259        3965            114        278        3876            103        254        3687            107        257        4088            108        292        3959            117        243        37510           101        238        402Group No. 8Chicken No. 1        100        255        3732            102        267        3873            104        269        3994            105        235        4445            101        258        4376            112        242        3797            104        233        4588            109        255        3169            99         241        40110           115        201        410Group No. 9Chicken No. 1        99         202        2852            106        198        3143            105        202        2064            94         188        3055            107        179        1876            76         213        2987            99         141        2468            78         165        2629            88         125        23510           88         102        --Group No. 10Chicken No. 1        105        208        2942            105        212        2983            116        259        3974            90         230        3925            88         218        3296            116        205        2927            112        235        2218            104        146        2099            103        165        32310           112        239        329Group No. 11Chicken No. 1        114        275        2912            121        261        3443            112        256        4044            116        259        4005            105        218        2516            114        247        2857            109        191        3628            100        166        3379            90         185        39310           81         --         428Group No. 12Chicken No. 1        88         218        3442            121        230        3243            122        191        2524            113        228        3475            122        193        3036            108        189        2737            97         163        2908            113        132        3669            99         216        29810           114        157        197Group No. 13Chicken No. 1        86         182        2082            85         193        1163            93         164        2354            103        144        2805            97         121        2196            104        141        2207            78         153        1048            88         136        1989            97         77         18910           113        --         --Group No. 14Chicken No. 1        101        167        2492            97         178        2443            94         142        2324            94         178        2925            89         189        1966            75         137        2117            102        158        1898            98         138        2599            94         129        18010           96         --         --Group No. 15Chicken No. 1        104        210        2482            77         239        2543            97         164        3114            116        188        2715            103        161        2296            71         189        2327            100        162        2418            87         157        --9            98         84         --10           --         --         --Group No. 16Chicken No. 1        90         158        2432            102        128        2503            90         193        1984            88         147        2545            91         147        2246            89         134        1807            96         129        2178            69         170        1779            83         146        18210           105        105        230______________________________________ 
    
     To address the question of the sparing effect of gentian violet in aflatoxin intoxication, four comparisons were made: 
     (1) The mean weight of the chicks receiving gentian violet but no B-1 aflatoxin. 
     These results and analysis are presented in Table 3. In this instance no significant difference was demonstrated between the mean weights of the chicks receiving either 0, 7, 21 or 35 ppm of genian violet in their feed. 
     
                       TABLE 3______________________________________Control Groups. The weights of four groups ofchicks (10 chicks per group) fed either 0, 7,21 or 35 ppm gentian violet in their feed at 20 daysof age.                    Chicken No.                    In The  Gentian Violet in The Feed                    Particular    0 ppm   7 ppm   21 ppm                          35 ppm                                Group______________________________________weight,  468     418     407   419   1gm.      395     365     429   485   2    395     444     416   422   3    396     337     384   412   4    459     416     409   438   5    402     432     419   407   6    367     455     396   362   7    420     438     414   403   8    433     420     397   334   9    395     334     417   286   10    .sup.-x=413              405.9   408.8                            396.8    n=10     10      10    10ANOVASource ofVariation   df        SS        MS      F______________________________________Treatments   4-1=3     1414.2750 471.4250                               0.2993 (NS)Error   36        56688.1   1574.6694______________________________________Total   39        58102.375lsd (0.5) = t .05 S.sup.- d = 2.0336 × 17.7463 = 36.0889Difference Between Mean Weightsgentian violet (ppm)35       7           21          0______________________________________396.8    405.9       408.8       413                            .sup.(a)______________________________________ .sup.(a) = any mean underscored by the same line is not significantly different at the 5% level of probability.    (2) The mean weights of chicks receiving B-1 aflatoxin at the 1 ppm level and gentian violet. 
    
     These results and analysis are given in Table 4. The mean weight of the birds receiving no gentian violet (weighing 430 gms) was not significantly different than the birds receiving gentian violet at the 7 ppm level (406.6). On the other hand gentian violet fed at either 7, 35 or 21 ppm did not result in any significant differences in the mean weights of the groups in question. 
     
                       TABLE 4______________________________________Aflatoxin (1ppm). The weights of four groups ofchicks (10 per group) all fed B-1 aflatoxin(1 ppm in feed) and either 0, 7, 21 or 35 ppmgentian violet in their feed at 20 days of age.                    Chicken No.                    in the  Gentian Violet in the Feed                    particular    0 ppm   7 ppm   21 ppm                          35 ppm                                group______________________________________weight,  436     435     398   373   1gm.      441     387     407   387   2    373     395     392   399   3    437     438     396   444   4    413     398     387   437   5    384     359     368   379   6    417     409     408   458   7    427     423     395   316   8    501     393     375   401   9    471     429     402   410   10    .sup.-x=430              406.6   392.8                            400.4    n=10     10      10    10ANOVASource ofVariation   df        SS        MS      F______________________________________Treatments    4-1=3    7735.5    2578.5  2.6474 (NS)Error   36        35062.4   973.9555______________________________________Total   40-1=39   42797.9lsd (0.05) = t 0.05 s.sup.-d = 2.0336 × 13.9567 = 28.3823Difference Between Mean WeightsGentian Violet (ppm)21       35          7           0______________________________________392.8    400.4       406.6       430                            .sup.(a)______________________________________ .sup.(a) = any mean underscored by the same line is not significantly different at the 5% level of probability 
    
     (3) The mean weights of the chicks receiving B-1 aflatoxin at the 4 ppm level and gentian violet. 
     These results and analysis are given in Table 5. The mean weight of the birds receiving gentian violet at the 21 ppm level was significantly heavier (P&gt;0.05) than the birds not receiving gentian violet (349.5 gms vs. 259.4 gms). In turn, there was no significant differences in the mean weights of the birds receiving gentian violet. 
     
                       TABLE 5______________________________________Aflatoxin (4ppm). The weights of four groups ofchicks (10 per group) all fed B-1 aflatoxin(4 ppm in feed) and either 0, 7, 21 or 35 ppmgentian violet in their feed at 20 days of age.                    Chicken No.                    in theGentian Violet in the Feed                    particular   0 ppm    7 ppm   21 ppm                          35 ppm                                group______________________________________weight, 285      294     291   344   1gm.     314      298     344   324   2   206      397     404   252   3   305      392     400   347   4   187      329     251   303   5   298      292     285   273   6   246      221     362   290   7   262      209     337   366   8   235      323     393   298   9   --       329     428   197   10.sup.-x=259.8     308.4    349.5   299.4n=9      10       10      10ANOVASource ofVariation   df        SS        MS      F______________________________________Treatments    4-1=3     38630.5803                       12876.8601                               4.387 *Error   35        104114.8556                       2974.7101______________________________________Total   39-1=38   142745.4359lsd (0.05) = t 0.05 (35) S.sup.-d           = 2.2205 × 25.0591 = 55.6437.sup.(a)           = 2.2205 × 24.3914 = 54.1611Gentian Violet (ppm)0        35          7           21______________________________________259.4    299.4       308.4       349.5                            .sup.(b)______________________________________ .sup.(a) = for any comparison involving 9 replicates (Gentian Violet 0 ppm) .sup.(b) = any mean underscored by the same line is not significantly different at the 5% level of probability 
    
     (4) The mean weights of the chicks receiving B-1 aflatoxin at the 10 ppm level and gentian violet. 
     These results and analysis are presented in Table 6. Again, the mean weight of the group of chicks receiving gentian violet at the 21 ppm level was significantly larger than the group not receiving gentian violet (P&gt;0.05). There were no significant differences in the mean weights of the chicks receiving gentian violet at any level. 
     
                       TABLE 6______________________________________Aflatoxin (10 ppm). The weights of four groupsof chicks (10 per group) all fed B-1 aflatoxin(10 ppm in feed) and either 0, 7, 21 or 35 ppmgentian violet in their feed at 20 days of age.______________________________________                     Chicken No.                     in theGentian Violet in the Feed                     particular   0 ppm     7 ppm   21 ppm                           35 ppm                                 group______________________________________weight, gm.   208       249     248   243   1   116       244     254   250   2   235       232     311   198   3   280       292     271   254   4   219       196     229   224   5   220       211     232   180   6   104       189     241   217   7   198       259     --    177   8   189       180     --    182   9   --        --      --    230    10   .sup.-x = 196.6             228     255.1 215.5   n = 9      9      7     10ANOVASource ofVariation   df         SS        MS      F______________________________________Treatments   4 - 1 = 3  14260.5920                        4753.5306                                3.0461 (NS)Error   31         48375.5795                        1560.5025Total   35 - 1 = 341sd(0.05) = t.05(31)Sd.sup.-         = 2.0399 × 18.1499 = 37.0240.sup.(a)         = 2.0399 × 19.6803 = 40.1458         = 2.0399 × 18.6210 = 37.9850         = 2.0399 × 19.9050 = 40.6042Difference Between Mean WeightsGentian Violet (ppm)0         35          7          21______________________________________196.6     215.5       228        255.1                        .sup.(b)______________________________________ .sup.(a) = for comparisons involving n&#39;s of 10 and 9, 10 and 7, 9 and 9, and 7, respectively .sup.(b) = any mean underscored by the same line is not significantly different at the 5% 
    
     The deleterious effects of aflatoxin is well illustrated by Tables 7, 8, 9 and 10. In this instance the effects of B-1 aflatoxin was observed within the groups of chicks receiving gentain violet at the 0, 7, 21 and 35 ppm dosage levels. In every instance there was no significant difference (P&lt;=0.05) in the mean weights of the groups of chicks receiving either 0 or 1 ppm B-1 aflatoxin. However, in every case the mean weights of the groups of chicks receiving 0 or 1 ppm aflatoxin was significantly larger than the groups of chicks receiving 4 ppm (P&gt;0.05). In turn, the mean weights of groups receiving 4 ppm was significantly heavier than the mean weights of the groups receiving 10 ppm. 
     
                       TABLE 7______________________________________Gentian Violet (0 ppm). The weights of four groups ofchicks (10 per group) all fed B-1 aflatoxinin their feed at either 0, 1, 4 or 10 ppm at20 days of age.______________________________________                     Chicken No.                     in theB-1 Aflatoxin In the Feed particular   0 ppm     1 ppm   4 ppm 10 ppm                                 group______________________________________weight, gm.   468       436     285   208   1   395       441     314   116   2   395       373     206   235   3   396       437     305   280   4   459       413     187   219   5   402       384     298   220   6   367       417     246   104   7   420       427     262   198   8   433       501     235   189   9   395       471     --    --    10   .sup.-x = 413             430     259.8 196.6   n = 10     10     9     9ANOVASource ofVariation   df         SS         MS      F______________________________________Treatment   4 - 1 = 3  373536.9853                         124512.3284                                 67.5533.sup.aError   =  34       62667.7779                          1843.1699______________________________________Total   38 - 1 = 37              436204.7632a = P &gt; 0.0051sd(0.05) = t.05(34)Sd.sup.-         = 2.0336 × 19.1998 = 39.0447.sup.(b)         = 2.0336 × 19.7254 = 40.1136.sup.(c)         = 2.0336 × 20.2374 = 41.1548.sup.(d)Difference in Mean WeightsB-1 Aflatoxin (ppm)10        4           0          1______________________________________196.6     259.8       413        430                            .sup.(e)______________________________________ .sup.(b) = for comparisons involving 10 replicates in each mean .sup.(c) = for comparisons involving 10 replicates in one mean and 9 in the other .sup.(d) = for comparisons involving 9 replicates in each mean .sup.(e) = any means underscored by the same line are not significantly different at the 5% level of probability 
    
     
                       TABLE 8______________________________________Gentian Violet (7 ppm). The weights of four groups ofchicks (10 per group) all fed gentian violet 7ppm (feed)and B-1 aflatoxin in their feed at either0, 1, 4 or 10 ppm at 20 days of age.______________________________________                     Chicken No.                     in theB-1 Aflatoxin In the Feed particular   0 ppm     1 ppm   4 ppm 10 ppm                                 group______________________________________weight, gm.   418       435     294   249   1   365       387     298   244   2   444       395     397   232   3   337       438     392   292   4   416       398     329   196   5   432       359     292   211   6   455       409     221   189   7   438       423     209   259   8   420       393     323   180   9   334       429     329   --    10______________________________________   .sup.-x = 405.9             406.6   308.4 228   n = 10    10      10     9Source ofVariation   df         SS         MS      F______________________________________Treatments   4 - 1 = 3  210,665.2744                         70,221.7581                                 36.0715.sup.aError   35         68,135.7     1946.7342Total   39 - 1 = 38              278,800.9744(a) = P &gt; 0.0051sd(0.05) = t.05(35)Sd.sup.-         = 2.0315 × 19.7318 = 40.0852.sup.(b)           2.0315 × 20.2720 = 41.1826.sup.(c) ##STR3## ##STR4## 
    
     
         Difference in Mean WeightsB-1 Aflatoxin (ppm)10        4           0          1______________________________________228       308.4       405.9      406.6                            .sup.(d) .sup.(d) =  any means underscored by the same line are not significantly different at the 5% level of probability 
    
     
                       TABLE 9______________________________________Gentian violet (21 ppm). The weights of four groups ofchicks (10 per group) all fed gentian violet 21 ppm (feed)and B-1 aflatoxin in their feed at either0, 1, 4 or 10 ppm at 20 days of age.                     Chicken No.                     in the  B-1-Aflatoxin      particular0 ppm    1 ppm     4 ppm      10 ppm                                group______________________________________weight, gm.407      398       291        248   1429      407       344        254   2416      392       404        311   3384      396       400        271   4409      387       251        229   5419      368       285        232   6396      408       362        241   7414      395       337        --    8397      375       393        --    9417      402       428        --    10______________________________________-x = 408.8    392.8     349.5      255.1n = 10   10        10         7______________________________________ANOVASource ofVariation    df        SS         MS      F______________________________________Treatments    4-1=3     112,626.1456                         37,542.0485                                 31.6441.sup.(a)Error    33         39,150.5572                          1,186.3805______________________________________Total    37-1=36   151,776.7028______________________________________               .sup.(a) =  P &gt; 0.005lsd(0.05) = t.05S-d = 2.0357 × 15.4037 = 31.3573.sup.(b)= 2.0357 × 16.9721 = 34.5501.sup.(c) ##STR5## ##STR6## 
    
     
         Difference in Mean WeightsB-1 aflatoxin (ppm)10        4           1           0255.1     349.5       392.8       408.8______________________________________.sup.(d) .sup.(d) = any means underscored by the same line are not significantly different at the 5% level of significance 
    
     
                       TABLE 10______________________________________Gentian violet (35 ppm).The weights of four groups of -chicks (10 per group) all fed gentianviolet 35 ppm(feed) and B-1 aflatoxin in their feed at either0, 1, 4 or 10 ppm at 20 days of age.______________________________________                     Chicken No.                     in the  B-1 Aflatoxin In the Feed                     particular0 ppm    1 ppm     4 ppm      10 ppm                               group______________________________________weight, gm.419      373       344        243   1485      387       324        250   2422      399       252        198   3412      444       347        254   4438      437       303        224   5407      379       273        180   6362      458       290        217   7403      316       366        177   8334      401       298        182   9286      410       197        230    10-x = 396.8    400.4     299.4      215.5n = 10   10        10         10______________________________________ANOVASource ofVariation    df        SS         MS      F______________________________________Treatment    4-1=3     234,494.0750                         78,164.6916                                 37.8792.sup.(a)Error    36        74,286.9    2,063.5250______________________________________Total    40-1=39______________________________________               .sup.(a) = P&gt;0.0051sd(0.05) = t.05(36)Sd = 2.0294 × 20.3151 = 41.2275 ##STR7##Difference in Mean WeightsB-1 Aflatoxin (ppm)10        4           0           1215.5     299.4       396.8       400.4______________________________________.sup.(b) .sup.(b) = any two means underscored by the same line are not significantly different at the 5% level of probability 
    
     When B-1 aflatoxin was fed at the 0 to 1 ppm level, the gentian violet did not appear to have any effect on the mean weights of chicks at 20 days of age. When B-1 aflatoxin was fed at the 4 ppm level, gentian violet began to show a sparing effect in that the mean weight of the group of chicks receiving 21 ppm gentian violet (349.5 gms) was significantly larger (P&gt;0.05) than the chicks receiving no gentian violet (259.4 gms). This is also true when B-1 aflatoxin was fed at the 10 ppm level. The reason that sparing effect was not evident until the B-1 aflatoxin is fed in 4 to 10 ppm is that these are the levels at which intoxication becomes manifest. 
     The sparing effect of gentian violet in the presence of intoxicating levels of B-1 aflatoxin apparently assumes a sigmoid curve. The dosage of 21 ppm appears on the linear part of this curve and appears to be the &#34;best&#34; dosage. The dosages of 7 and 35 ppm are at either extreme of the sigmoid curve. At this stage (20 days of age) no significant differences are in evidence between the mean wieghts of the 7, 21, and 35 ppm levels. However, a trend is evident at 20 days of age -- see Example 2 for the significant differences that developed as the test was continued. 
     The gentian violet was not toxic even at the 35 ppm level in the feed. 
     It can be concluded that groups of chicks receiving 21 ppm gentian violet were significantly heavier than the groups not receiving gentian violet when they were fed B-1 aflatoxin at intoxicating levels (4 or 10 ppm in the feed). 
     EXAMPLE 2 
     Example 1 was continued, in part, for an additional three weeks (for a test duration of six weeks). The groups held beyond three weeks of age were controls, 21 ppm of gentian violet, 4 ppm of aflatoxin and 21 ppm of gentian violet with 4 ppm of aflatoxin. All other groups were terminated since 1 ppm of aflatoxin gave no apparent effect and 10 ppm of aflatoxin produced excessive effects. The 21 ppm of gentian violet was selected on the basis of results at 3 weeks of age. 
     
                       TABLE 11______________________________________     wt. at   wt. at     wt. at     4 wk.,   5 wk.,     6 wk.,     gm.      gm.        gm.______________________________________Group No. 1Chicken No. 1       680        735        9642           590        744        10213           495        774        8724           604        812        10445           568        665        9926           630        646        10437           592        750        9598           575        770        8589           583        833        86410          616        868        996Ave wt. :   5933         759.7      961.3Group No. 9(4 ppm ofaflatoxin)Chicken No. 1       270        348        6972           420        400        5183           315        413        7824           391        512        5335           175        259        7046           430        570        7167           500        582        5278           400        552        9349           325        714        39810          --         --         --Ave. wt. :    358.4      483.3      645.4based on9 birds)Group No. 3(21 ppm ofgentian violet)Chicken No. 1       610        753        10502           643        795        10653           673        878        9944           572        800        10525           666        828        10046           630        834        10707           610        800        10318           613        825        9699           594        786        95510          --         --         --Ave wt. :     623.4      811.0     1021.1Group No. 11(4 ppm ofaflatoxin and21 ppm of gentianviolet)Chicken No. 1       566        670        7682           425        722        8033           423        487        7964           550        587        7735           376        592        7406           466        565        8877           387        636        7268           450        618        6489           --         --         --10          --         --         --Ave wt. :     455.4      609.6      767.6(based on 8birds)______________________________________ 
    
     By way of review, this example is an extension of Example 1 where the question was asked if gentian violet caused a sparing effect from the toxicity of B-1 aflatoxin in broiler chicks. In that experiment, which was terminated at 21 days of age, it was established that VIGEN did indeed cause a sparing effect. Further, it was established that the dose of aflatoxin delivered in the feed was critical in that 1 PPM had little effect on weight production and that 10 PPM was overwhelming. It was also observed that the best treatment dosage of gentian violet was about 21 PPM. 
     To determine if gentian violet continued to cause a sparing effect from the toxicity of aflatoxin, the Example 1 was continued until the broiler chickens were 6 weeks of age in selected groups. 
     This example is a continuation of Example 1 in four selected groups of broiler chickens. These groups are: 
     Group 1: Control. These birds were not given gentian violet or aflatoxin in their feed. 
     Group 3: 21 PPM of gentian violet. These birds were being given gentian violet at the rate of 21 PPM daily in their feed. 
     Group 9: 4 PPM B-1 of aflatoxin. These birds were being daily poisoned with aflatoxin being given at the dosage level of 4 PPM in their feed. 
     Group 11: 4 PPM B-1 of aflatoxin and 21 PPM of gentian violet. These birds were receiving both aflatoxin and gentian violet daily in their feed at the rates indicated. 
     Initially, at one-day of age, each group of birds contained 10 chicks each. There was normal chick attrition and at the start of this example, Group 1 had 10 chicks, Groups 3 and 9 had 9 chicks each, and Group 11 had 8 chicks. At the start of this trial, the birds were 3 weeks of age. The broiler chickens were weighed individually at 4, 5, and 6 weeks of age to the nearest gram. 
     The hypothesis proposed was that gentian violet does not have a sparing effect on broiler chicks when they are being poisoned with 4 PPM aflatoxin. Whether there is a sparing effect or not was judged on the basis of weight. 
     Analysis of the weight data was done utilizing an analysis of variance (one-way with unequal replication). The desired end was to compare the mean weights of the four groups of chicks by the method of least significant difference. 
     The weights of the broiler chickens at 4, 5 and 6 weeks of age are given in Tables 12, 13 and 14 respectively. Subsequent analysis of this data to compare the mean weight of each group to the mean weight of every other group is given in Table 15. 
     
                       Table 12______________________________________Individual Weights.sup.(a) (gms) of BirdsReceiving Either 4 PPM aflatoxin,21 PPM gentian violet, 4 PPM aflatoxinand 21 PPM gentian violet, or a Controlat 4 weeks of Age.                           4 PPM                 21 PPM    Aflafoxin &amp;      4 PPM      Gentian   21 PPMControl    Aflatoxin  Violet    Gentian Violet______________________________________680        270        610       566590        420        643       425495        315        673       423604        391        572       550568        175        666       376630        430        630       466592        500        610       387575        400        613       450583        325        594616n =  0      9          9         8mean = 593.3       358.4      623.4     455.4______________________________________ .sup.(a) Each group of birds initially contained 10 birds; 4 birds died for reasons not pertenant to the experiment. 
    
     
                       Table 13______________________________________Individual Weights (gms) of Birds Receivingeither 4 PPM aflatoxin, 21 PPM gentian violet,4 PPM aflatoxin &amp; 21 PPM gentian violet, oracting as a Control at 5 Weeks of Age.                           4 PPM      4 PPM      Gentian   aflatoxin &amp;Control    aflatoxin  Violet    21 PPM Gentian______________________________________735        348        753       670744        400        795       722774        413        878       487812        512        800       587665        259        834       565646        570        800       636750        582        825       618770        552        786833        714        786868n =  10     9          9         8mean = 759.7       483.3      811.0     609.6______________________________________ 
    
     
                       Table 14______________________________________Individual Weights (gms) of Birds Receiving4 PPM aflatoxin, 21 PPM gentian violet, 4 PPM aflatoxin21 PPM gentian violet, or acting as a Controlat 6 Weeks of Age.                           4 PPM                 21 PPM    aflatoxin &amp;      4 PPM      Gentian   21 PPMControl    aflatoxin  Violet    Gentian Violet______________________________________964        697        1050      7681021       518        1065      303872        782         994      7961044       533        1052      773992        704        1004      7401043       716        1070      887959        527        1031      726858        934         969      648864        398         955996n =  10     9          9         8mean = 961.3        645.44     1021.11   767.62______________________________________ 
    
     
                       Table 15______________________________________Comparison of the mean weights of broilerchickens receiving in their feed either 4 PPMaflatoxin, 21 PPM gentian violet, 4 PPM aflatoxin &amp;21 PPM gentian violet, or a control by the method ofleast significant difference at 4, 5 and 6 Weeksof Age.Mean Weights of Group (gms)                                 21 PPM  4 PPM    4 PPM aflatoxin  Con- GentianAge    aflatoxin           &amp; 21 PPM Gentian Violet                            trol Violet______________________________________4 weeks  358.4    455.4            593.3                                 623.4                                 .sup.(a)5 weeks  483.3    609.6            759.7                                 811.06 weeks  645.4    767.6            961.3                                 1021.1______________________________________ .sup.(a) any mean underscored by the same line is not significantly different at the 5% level of probability 
    
     In every instance the comparison of the mean weight of the group receiving 4 PPM of aflatoxin to the group receiving 4 PPM of aflatoxin and 21 PPM of gentian violet demonstrated the group receiving gentian violet was significantly heavier (P&gt;0.05). For example: 
     
         ______________________________________            aflatoxin andaflatoxin   gentian violet      SignificantGroup       Group               orAge  Weight in gms            Weight in gms                        Difference                                Not______________________________________4    358.4       455.4        97.0   yes5    483.3       609.6       126.3   yes6    645.4       767.6       122.2   yes______________________________________ 
    
     At the three age levels examined in this study, the mean weights of the control group was not significantly different than the group of birds receiving 21 PPM of gentian violet in their feed. Both of these groups have mean weights significantly heavier than the birds receiving 4 PPM aflatoxin and 21 PPM of gentian violet. 
     Since the group of birds receiving 4 PPM of aflatoxin and 21 PPM of gentian violet weighed significantly more than the group of broiler chickens being poisoned with 4 PPM of aflatoxin, the hypothesis that gentian vilet does not cause a sparing effect in broiler chickens being poisoned with aflatoxin must be rejected. Instead, the alternate hypothesis that gentian violet causes a sparing effect in broiler chickens when they are being poisoned with aflatoxin has to be accepted. 
     At ages 4, 5 and 6 weeks, the group of birds receiving 21 PPM of gentian violet in their feed had mean weights 30.1, 51.3 and 59.8 gms heavier than their control counterparts. These differences, because of the small number of experimental units, are not significant. However, in this and other experiments, the trend for birds to gain weight faster than their control counterparts is patent. EXAMPLE 3 
     Example 1 was repeated, using the following parameters: 
     Secured 160 day-old broiler pullets 
     Divided into 16 groups of 10 each 
     Aflatoxin used at levels of 0, 1, 4 and 10 PPM 
     Gentian violet used at levels of 0, 7, 21 and 35 PPM 
     Broiler feed mixed to contain 24% protein 
     Prepared 70 pounds of feed at each level of gentian violet 
     Mixed 916.7 mg of B-1 Aflatoxin with 9,167 gm of feed to provide stock toxin at 100 PPM. 
     Added: 700 gm toxin stock to 6,300 gm feed - 10 PPM 
     280 gm toxin stock to 6,720 gm feed - 4 PPM 
     70 gm toxin stock to 6,930 gm feed - 1 PPM 
     These amounts added to feed with 0, 7, 21 and 35 PPM of gentian violet to provide the 16 combination of toxin and gentian violet. 
     Chicks were weighed at day-old, 7, 14 and 20 days of age with the results given on Table No. 16. 
     Hematocrits at 20 days were within the normal range in all groups with averages ranging from 28.9 to 36.5. There was no pattern to indicate any marked effect by level of either the aflatoxin or gentian violet. Blood clotting times (CT) were secured on all groups receiving 0 or 7 PPM of gentian violet. Untreated controls (0 toxin - 0 gentian violet) had average CT of 1.5 minutes. All others had longer CT with many over 5 minutes. However, there was marked variation both within and between groups and were too erratic to allow interpretation. The capillary tube method gives an indication of clotting time but a better method is needed before conclusive results can be obtained. It was generally observed, however, that the clotting times were quite long and a few birds bled to death from the wing vein puncture. 
     Feed conversions were calculated. The 4 and 10 PPM of aflatoxin had much poorer feed conversion and 1 PPM had little effect. Addition of gentian violet at 7 PPM improved feed conversion only with 1 PPM of aflatoxin. However, at 21 PPM of gentian violet and 4 PPM aflatoxin, there was marked improvement in feed conversion as well as weight gain. 
     Groups receiving 0 and 4 PPM of aflatoxin with 0, 7 and 21 PPM of gentian violet were being continued to 6 weeks of age. All other groups, 0 toxin - 35 of gentian violet, 4 toxin - 35 of gentian violet and 1 and 10 of toxin with 0, 7, 21 and 35 were necropsied. The 1 PPM of toxin produced slight subcutaneous hemorrhage with 0, 7 or 21 PPM of gentian violet but little or no liver or kidney changes. However, 10 PPM of aflatoxin caused considerable hemorrhage and marked liver and kidney changes with all levels (0, 7 and 21 PPM) of gentian violet. Changes were also evident at 4 and 10 PPM toxin with 35 PPM of gentian violet. 
     
                       Table 16______________________________________GENTIAN VIOLET WITH AFLATOXINChick Weights in Grams  Gentian Violet PPM in Feed      0      7        21       35______________________________________    0       39*       39     38     38            110      114    112    112            253      253    254    256            413      406    412    397    1        38       39     37     37Aflatoxin        115      110    115     95PPM in feed      265      248    254    192            430      407    393    400    4        38       38     39     38             94      105    106    110            172      212    229    192            260      308    350    299    10       38       37     38     37             94       94     95     91            146      157    173    146            197      205    255    216______________________________________ *Wt. in gms at day-old  7 days 14 days 20 days 
    
     EXAMPLE 4 
     Example 1 was repeated, except that lower aflatoxin levels and gentian violet were used at 7, 14 and 21 PPM. The sixteen groups of chicks received gentian violet and/or aflatoxin B-1 in the amounts set out in the following table: 
     
                       Table 17______________________________________Checkerboard experimental design for groups ofchicks to receive gentian violet (0, 7, 14 or 21 PPM)and B-1 aflatoxin (0, 1, 2 or 3 PPM) in their feed.Gentian Violet (PPM)   0      7         14        21______________________________________ 0      Gp 1.sup.(a)                  Gp 5    Gp 9    Gp 13 1      Gp 2      Gp 6    Gp 10   Gp 14B-1   2      Gp 3      Gp 7    Gp 11   Gp 15Afla-toxin 3      Gp 4      Gp 8    Gp 12   Gp 16______________________________________ Notes: .sup.(a) 15 chicks per group 
    
     The chicken weights are given in the following table: 
     
                       TABLE 18______________________________________WEIGHT OF CHICKENS     wt. at   wt. at     wt. at     7 days,  14 days,   21 days,     gm.      gm.        gm.______________________________________Group No. 1Chicken No. 1       121        257        4852           116        226        4233           119        247        3574           114        262        3655           109        229        3546           110        247        4287           119        294        3508           131        198        3779           112        268        39210          112        239        44211          114        230        45612           98        218        38813          105        288        32614          108        222        41315           92        195        334Group No. 2Chicken No. 1       111        255        3942           136        255        4833           118        232        4064           111        311        4695           113        247        4136           130        309        3497           103        281        3758           104        238        3609           108        268        40210          103        231        45811          136        230        35112          123        258        40013          116        237        36114          108        226        39715          124        256        380Group No. 3Chicken No. 1       131        241        4242           113        242        3923           104        241        4214           107        244        4735            81        212        3686           115        216        4177           106        244        4488           126        276        4099           121        219        36610          118        245        31811          102        223        33212          110        184        36913          126        223        35714          112        251        31615          116        199        332Group No. 4Chicken No. 1       115        210        3932           114        231        3723            49        186        3304            85        152        3965           112        253        3256           112        208        3717           112        224        2718           123        221        4179           113        198        36210          114        230        55511          104        188        33712          110        270        29313          125        206        33314           92        207        20515          --         --         --Group No. 5Chicken No. 1        81        227        4962            79        248        4033           120        212        4254           115        232        4435           123        258        3966           106        287        3007           102        261        3718           103        229        4239           122        215        41610          106        196        32811          112        228        36412          100        239        27713          134        238        38114          101        204        34215           94        160        396Group No. 6Chicken No. 1       124        260        4092           116        158        3843           109        267        4424           125        248        3895           114        263        4016           119        253        4217           120        260        3958           125        265        4139           101        239        29810           54        234        27411          125        264        30212          105        265        38713          121        149        40514           50        257        42915           55        163        407Group No. 7Chicken No. 1       110        253        4302           122        233        3453           106        238        3864           116        270        4665           120        230        3426           133        252        3927           124        250        4268           123        215        4289           124        268        46910          133        240        43111          113        255        30212           46        138        47013          130        248        36214          120        228        37115          --         --         --Group No. 8Chicken No. 1       103        237        3222           112        206        3813            91        234        3824            97        219        4015           107        246        2896            96        229        3727           112        195        2858           104        197        3639           102        235        31410          109        219        39811          119        240        35212          107        197        36613          113        172        36014          105        225        32115          --         --         --Group No. 9Chicken No. 1       143        272        4432           124        273        4663           107        277        4234           119        279        4335           123        259        4346           116        285        4537           131        263        4228           127        261        4409           144        260        38410          118        263        38811          125        271        38512          137        248        45413          114        235        42214          125        198        43615           98        234        334Group No. 10Chicken No. 1       116        275        3552           117        302        3923           126        274        3854           132        258        3715           116        251        3956           122        265        3827           126        236        3418           133        226        4309           119        264        38510          104        271        34811          127        267        41212          128        246        33113          134        264        36414          132        281        38315          105        223        413Group No. 11Chicken No. 1       107        284        4002           125        227        3553           117        273        4404           121        244        4165           115        242        3376           133        274        3727           137        243        3808           104        227        4269           113        250        42410          125        252        35711          131        254        43012          128        226        48113          114        224        34014          112        226        39415          119        249        349Group No. 12Chicken No. 1       116        238        3482           113        198        4383           107        233        3434           115        202        4025           121        240        3916           125        251        3547           115        213        3728           111        225        3289           104        220        28610          121        195        35611          102        221        32412          125        194        38013          108        229        36514          110        211        39515          118        222        318Group No. 13Chicken No. 1       121        226        3942           116        233        4313           118        236        4384           122        225        4165           125        230        4456           118        232        4437           137        222        4358           112        213        3929           113        241        43510          127        227        42711          116        212        41312          113        218        39013          127        228        40414          123        248        42215          108        216        415Group No. 14Chicken No. 1       116        305        4902           114        268        5003           110        256        4824            62        240        5065           118        283        3926           121        244        4327           122        253        4258           125        267        4239           114        292        45510          133        279        40011          138        273        44212          111        243        47713          127        154        30914          130        --         --15          --         --         --Group No. 15Chicken No. 1        32        281        4532           128        280        4393           116        277        3284           109        215        4105           132        273        4506           134        280        3487            49        244        3868           136        262        4479           138        247        41710          108        211        44411          116        214        40012          128        252        33713          117        146        29114          107        --         --15          111        --         --Group No. 16Chicken No. 1       118        272        4142            92        219        3693           116        145        3814           131        226        3745           126        231        2496           129        206        4497           105        268        3648           123        224        3539           109        219        32510          132        245        30611          114        232        33012          111        228        37913          115        219        35314          101        203        31815          107        202        328______________________________________ 
    
     The feed consumed by the birds is given in the following table: 
     
                       TABLE 19______________________________________Gms. Feed Consumed to 21 Days (No/Group)(Gm/Bird)Aflatoxin    PPM Gentian VioletPPM      0         7         14      21______________________________________0        9,000 (15)              8,300 (15)                        9,230 (15)                                8,970 (15)    (600)     (553)     (615)   (598)1        8,697 (15)              8,595 (15)                        9,005 (15)                                8,654 (13)    (580)     (573)     (600)   (666)2        8,460 (15)              8,230 (14)                        8,555 (15)                                8,030 (13)    (564)     (588)     (570)   (618)3        7,547 (14)              7,296 (14)                        7,823 (15)                                7,447 (15)    (539)     (521)     (522)   (496)______________________________________ 
    
     The average weight of each group of chicks over the test period is given in the following table: 
     
                       TABLE 20______________________________________Ave Weights-GramsAflatoxin   Age      PPM Gentian VioletPPM     Days     0        7      14     21______________________________________0       7        112.0    106.5  123.4  119.7   14       241.3    228.9  258.5  242.3   21       392.7    384.1  421.1  4201       7        116.3    104.2  122.5  117.2   14       255.6    236.3  260.2  237.2   21       399.9    383.7  379.1  4412       7        112.5    114.1  118.7  100.7   14       230.7    238.3  246.2  228.3   21       382.8    401.4  393.4  396.23       7        110.9    104.9  114.0  115.3   14       206.3    217.9  219.5  222.6   21       347.1    350.4  360.0  352.8______________________________________ 
    
     The sparing effect of gentian violet appears to be a linear function of the concentration of aflatoxin in the feed (administered or consumed). The groups receiving the gentian violet are significantly heavier than the groups not receiving gentian violet when they were fed B-1 aflatoxin. 
     EXAMPLE 5 
     Several rats were offered rat feed containing 1,000 PPM of gentian violet -- the rats refused to eat the feed. 
     Several dogs were offered dog feed containing 1,000 PPM of gentian violet -- the dogs refused to eat the feed. 
     This shows that animals will often refuse to eat feed containing high (toxic) levels of gentian violet.