Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/142—Amino acids; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/105—Aliphatic or alicyclic compounds
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/111—Aromatic compounds
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/116—Heterocyclic compounds
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
  • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/205—Amine addition salts of organic acids; Inner quaternary ammonium salts, e.g. betaine, carnitine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/28—Compounds containing heavy metals
  • A61K31/285—Arsenic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine

Definitions

• compositions for Administration to Animals With
• This invention relates to industrial diets for domestic food animals, feed additives and methods for treating animals infected with coccidia.
• Coccidiosis is a common disease in domestic food animals, caused by protozoa belonging to the Eimeria family. Coccidiosis is found worldwide, and its economical impact, particularly on poultry farming, is huge. In the U.S. poultry industry alone, coccidiosis causes losses of 200-250 million dollars yearly. World-wide, coccidiosis is estimated to cause one third of all disease and mortality losses in the poultry industry (Trends in Veterinary Research and Development, part 6, Anti-coccidials- Lloyd-Evans, L.P.M. (ed.), PJB Publications Ltd., 1991). Since the 1950s, several dietary drugs (coccidiostats) have been developed to treat coccidiosis, but with only moderate success.
• coccidiostats have been: 1) rapidly developing resistance of the parasites; 2) adverse effects on host animals; and 3) risk for residuals or quality defects in the consumer products. While the universal use of coccidiostats has decreased serious mortality outbreaks, subclinical effects of coccidiosis on animals markedly decrease productivity (Jeng & Edgar, Highlights of Agricultural Research - Alabama, Agricultural Experiment Station, v.28, p.6 (1981)). In chickens, typical clinical signs of coccidiosis include ill-thrift, rapid loss of weight, diarrhea, and dysentery.
• coccidiosis causes severe disturbances in the acid-base, ionic and osmotic balance of the gut, and decreases nutrient absorption (Ruff, Georgia Coccidiosis Conference, Nov. 19- 21 (1986) pp. 169-183; Gwyther et al., Coccidia and Intestinal Coccidiomorphs, Vth International Conf., October 17-20 (1989) pp. 279-284).
• Many coccidiostats in common use kill parasites by breaking down their ionic and osmotic regulation.
• Betaine is an osmoprotectant. It increases the osmotic strength of cells without adversely affecting enzyme activity, and it protects enzymes from ionic or temperature inactivation (Nash et al. , A st. J. Plant Physiol. 9:47-57 (1982); Yancey et al, Science 224:1064-1069 (1982); Rudolph et al.,
• betaine might be useful in alleviating the undesirable side effects associated with the use of coccidiostats or that, betaine and coccidiostats might act synergistically to improve the commercial performance of domestic food animals suffering from coccidiosis.
• the present invention is directed to feeds for domestic food animals which contain a combination of betaine and coccidiostat. This combination has been found to be especially effective at reducing the mortality and improving the overall commercial performance of chicks infected with coccidia.
• Coccidiostats used may be of either the chemical or ionophore type.
• Preferred ionophore coccidiostats are salinomycin (tradename “Bio-Cox”) and lasalocid (tradename Avatec).
• the preferred chemical coccidiostat is halofuginone hydrobromide (tradename "Stenerol”).
• betaine is present in feed at a concentration of between 0.5 and 2.0 kg/ton of dry feed.
• the most preferred feeds are those used for chickens.
• the invention is also directed to dietary additives which contain a premixed combination of betaine and coccidiostat. Such additives can either be included in the feed given to animals or administered separately from such feed.
• the invention is directed to a method for reducing the mortality of animals, especially chickens, infected with coccidia by administering betaine to such animals.
• the betaine may either be administered alone or, more preferably, in combination with coccidiostat.
• the preferred ionophore coccidiostats for use in the method are salinomycin and lasalocid.
• the preferred chemical coccidiostat is halofuginone hydrobromide.
• FIGURE 1 Figure 1 shows the percent mortality for chicks grown for 45 days on unsupplemented diets, diets supplemented with 44 ppm of Bio- CoxTM (salinomycin), and or on diets with 66 ppm Bio-Cox. The effect of each diet was examined both with and without added betaine. It can be seen that betaine addition reduced chick mortality for each of the diets studied.
• FIGURE 2 Chicks grown for 45 days on unsupplemented feeds or on feeds supplemented with 44 ppm coccidiostat and 66 ppm coccidiostat were necropsied and the severity of gut lesions ranked on a scale of 0 to 4. It can be seen that, in all cases, supplementation of diets with betaine led to a reduced severity of gut lesions.
• FIGURE 3 Figure 3 shows the end weights of chicks grown for 45 days on each of the three different types of diets described above. In each case, supplementation with betaine resulted in chicks with an increased end weight.
• FIGURE 4 The feed conversion efficiency of chickens grown on each of the three different types of diets was determined and the results are shown in Figure 4. In each instance, betaine resulted in chicks with improved efficiencies.
• FIGURE 5 Coccidia infected chicks were gown on diets containing either adequate methionine or on diets low in methionine was examined. All chicks received 66 ppm of salinomycin. The figure shows that the addition of betaine reduced the severity of gut lesions regardless of the level of methionine.
• FIGURE 6 Figure 6 shows the effect of betaine addition on the mortality of ?c ⁇ ' ⁇ # ⁇ -infected chicks. At a dose of 0.75 kg/ton, betaine reduced the mortality of chicks fed diets containing either a low or an adequate amount of methionine.
• FIGURE 7 Figure 7 shows that betaine increases the end weight of coccidia-infected chicks in diets which are both low in methionine or which contain adequate methionine
• FIGURE 8 Figure 8 shows that betaine improves feed conversion efficiency in chicks infected with coccidia regardless of whether the chicks are grown on feed low in methionine or with adequate methionine.
• starter diet A "starter diet” is the diet fed to chickens during the first 21 days of life.
• grower diet The term “grower diet” refers to the diet fed to chickens 21 to 40 days of age.
• Finisher diet The term “finisher diet” refers to the diet fed to chickens 40 to 49 days of age.
• Betaine also called “glycine betaine”, is defined chemically as l-Carboxy-N,N,N-trimethylmethanaminium hydroxide inner salt.
• Feed conversion efficiency is the ratio of the amount of weight gained by an animal divided by the amount of feed consumed by the animal. For example, a feed with an efficiency of
• Feed conversion ratio is the ratio of the amount of feed consumed by an animal divided by the amount of weight gained by the animal.
• Corn-sov feed “Corn-soy feed” is feed mainly comprised of yellow corn, soybean meal and soy oil.
• Significant As used herein the term “significant” means statistically significant. Thus, a statement that "treated chicks had significantly reduced mortality relative to untreated chicks” means that P ⁇ 0.05 using standard statistical analyses.
• Mortality Mortality is defined as the number of chicks within a treatment group that die during the course of an experiment. Typically mortality is expressed as a percentage and determined by dividing the number of chicks that die by the total number of chicks at the start of the experiment and then multiplying by 100.
• Feedstuffs are defined as those commonly used ingredients such as yellow corn or soybean meal which are combined to formulate the diet of chicks. •
• Basal diet is defined as the diet to be fed to chicks prior to supplementation with either methionine or betaine.
• domestic Food Animal is defined as any domestic animal that is consumed as a source of protein in the diet of humans or other animals.
• Typical domestic animals include: bovine animals(e.g. cattle); ovine animals (e.g. sheep); swine (e.g. pigs); fowl (e.g. chickens and turkeys); rabbit and the like.
• commercial performance refers to the extent to which animals raised under a specific set of conditions are commercially desirable as food animals. For the purposes of this invention, there are 4 parameters which determine commercial performance: mortality; end weight; feed conversion efficiency; and severity of gut lesions. An improvement in any one of these parameters results in an improved commercial performance provided that the remaining parameters either remain unchanged or also improve. 14.
• Premix When used as a noun, “premix” refers to a composition of two or more components which have been combined for a particular use. For example, a premix of the components betaine and coccidiostat could be added to chicken feed to produce a mixture with a desired final concentration without the necessity of making further adjustments in the concentration of either betaine or coccidiostat. 15.
• Ionophore coccidiostats are anti- coccidial agents which, by virtue of their molecular shape and structure, can act as ion transporters across biological membranes. All other anti-coccidial agents are referred to herein as "chemical coccidiostats. "
• Example 1 describes experiments in which chicks were inoculated with coccidia and then fed diets supplemented with betaine, coccidiostat (salinomycin) or a combination of betaine and coccidiostat. Chicks raised on the supplemented diets were compared with inoculated chicks fed unsupplemented diets and with chicks not inoculated with coccidia at all.
• chicks fed a diet containing a combination of betaine and salinomycin for 21 days had significantly higher body weights and significantly fewer gut lesions than chicks fed a diet containing an equivalent concentration of either betaine or salinomycin alone.
• betaine alone nor coccidiostat alone were able to completely offset the effects of coccidia infection on feed conversion efficiency, when administered together, they produced an efficiency which was comparable to that of the noninoculated controls.
• Mortality in 21 day old chicks fed diets containing 44 ppm of salinomycin was significantly reduced relative to the mortality of infected chicks fed either an unsupplemented diet or a diet supplemented with betaine alone. When betaine and 44 ppm coccidiostat were used together, mortality was reduced to a level which was not significantly higher than the mortality of the noninoculated chicks.
• chicks receiving feed containing both betaine and coccidiostat had a mortality significantly lower than chicks receiving feed with only one of the agents and that the mortality of the chicks administered both agents was reduced to the point where it was no longer significantly higher than that of the noninoculated controls.
• the body weight and feed conversion efficiency of chicks on diets with betaine and 66 pm of coccidiostat were not significantly different from the control group, whereas chicks receiving only one of the agents had significantly lower body weights and a significantly higher feed conversion efficiency.
• Example 2 The results shown in Example 2 indicate that the positive effects of betaine and coccidiostat on the commercial performance of chicks infected with coccidia do not depend upon the methionine content of the diet. Thus, the anti-coccidial effect of betaine is independent of its ability to substitute for methionine. The results suggest that the protective effect of betaine is probably related to its ability to offset the detrimental effects of coccidiosis on nutrient absorption. Supporting this hypothesis is the finding that infected chicks fed diets with betaine have less severe gut lesions than chickens fed either unsupplemented diets or diets supplemented with coccidiostat alone
• Example 3 demonstrates that the synergistic improvement in commercial performance evidenced by the combination of betaine and salinomycin is maintained both with other types of ionophore coccidiostats (lasalocid) as well as with chemical coccidiostats (halofuginone hydrobromide) .
• coccidiostats suitable for use in combination with betaine are shown in Table 1.
• the appropriate dosage of coccidiostat for administration to animals is the dosage recommended by the U.S. Food and Drug Administration (see FDA 1994 Feed Additive Compendium. U.S. Food and Drug Administration, 1994) and betaine is present at a concentration of between 0.5 and 2.0 kg/ton dry feed.
• Coccidiostats that have been approved in Europe include the following (product license numbers are shown in parentheses): amprolmix (PL 0025/4008); avatec premix (PL 0031/4011); carbigran premix (PL 0006/4075); clopidol (PL 3405/4017); clopidol 250 (PL 3405/4025); coyden 25 (PL 0621/4001); cycostat 66 (PL 0095/4000); cygro premix (PL 0095/4042); deccox pure (PL 8327/4038); deccox poultry premix (PL 0012/4052); deccox sheep premix (PL 8327/4066); dinitolmide (PL 10101/4000); dinormix SR 25 (PL 0109/4000); DOT (dinitolmide (PL 0109/4002); elancoban G200 (PL 0006/4047); monensin 200 (PL 3405/4006); lerbek (
• the European-approved coccidiostats may also be used to practice the claimed invention.
• concentration of coccidiostat used should be equivalent to that recommended by the manufacturer and betaine should be present at a concentration of between 0.5 and 2.0 kg/ton dry feed.
• Feeds for Chickens The present invention is directed to feeds for chickens formulated to contain both coccidiostat and betaine.
• a basal diet for chicks is first formulated using any of a variety of routine feedstuffs such as corn, soy, wheat and barley (see AFMA Feed Ingredient Guide, published by the American Feed Manufacturer's Association, Arlington, VA., U.S.A.; H. Patrick et al. , Poultry: Feeds & Nutrition. Second Edition, AVI Publishing Co. Inc., Westport, Conn., chapter 37, (1980)). All mixing and other preparation of feeds takes place using routine procedures well-known in the art (see e.g., H.
• the nutrient content of the basal diet may be determined using standard feedstuff analysis tables (see e.g., H. Patrick et al, Poultry: Feeds & Nutrition. Second Edition, AVI Publishing Co. Inc., Westport, Conn., pp. 438-449 (1980); H. Titus et al. , The Scientific Feeding of Chickens. Fifth Edition, The Interstate Publishers, Danville, 111., chapter 13 (1971)).
• Vitamins, minerals and other nutrients may be added to concentrations determined by turning to various available references (see e.g., Nutrient Requirements of Poultry. National Research Council, National Academy of Sciences, Washington, D.C. (1984)). These references are well-known, and the data provided is generally accepted by those skilled in the art.
• betaine and coccidiostat are added. These agents may either be added individually, or they may be added together as a premix.
• the final concentration of betaine in the feed should be between 0.5 and 2.0 kg/ton dry feed.
• the final concentration of coccidiostat will vary depending upon the particular type of coccidiostat used.
• the concentration recommended by the U.S. Food and Drug Administration should be suitable in all instances. Typical recommended inclusion rates for chick diets are: monensin: 100-120 ppm; salinomycin: 60 ppm; naracin: 70 ppm; and lasalocid: 90 ppm.
• the feed may be administered to chickens in any convenient manner. For example, it may be formed into pellets or administered as a powder. It may be given to all chicks as a preventative or it may only be given to chicks after they have been identified as being infected with coccidia.
• Coccidiosis occurs in a number of species of domestic food animals other than the chicken.
• Table 1 contains coccidiostats that have been approved by the Food and Drug Administration (FDA) for the treatment of various target animals (FDA 1994 Feed Additive Compendium. U.S. Food and Drug Administration, 1994).
• FDA Food and Drug Administration
• Cattle, sheep, swine and turkeys are all known to be susceptible to infection. These species are all treated with the same coccidiostats as chickens and are all subject to the same adverse side effects.
• the present invention encompasses feeds for these animals which contain both coccidiostat and betaine.
• a diet suitable for the particular domestic food animal being raised is formulated using standard feed tables.
• a standard diet for cattle may be formulated using the information provided by the Merck Veterinary Manual, sixth edition, pages 1104-1132 (1986).
• standard diets can be prepared for rabbits (pages 1210-12110); sheep (1211- 1221); swine (pages 1221-1230); and poultry (pages 1188-1210).
• coccidiostat and betaine are added.
• the concentration of coccidiostat should be that recommended by the Food and Drug Administration (FDA 1994 Feed Additive Compendium. U.S. Food and Drug Administration, 1994).
• the concentration of betaine which has been found to be successful in potentiating the effects of coccidiostat in chickens may be used initially in formulating feeds for other animals and adjusted up or down as experience dictates. Because of the synergistic effect of betaine, it may be possible to reduce the concentration of coccidiostat administered to animals and to still obtain commercially acceptable results. Thus, the use of betaine in combination with coccidiostat may reduce the cost of formulating an acceptable feed.
• the present invention encompasses other compositions containing these two agents which are administered to domestic food animals for the purpose of preventing the adverse effects of coccidiosis.
• a premix of betaine and coccidiostat may be administered either in the form of tablets, capsules or liquids. In all cases the concentration of betaine and coccidiostat should result in a dietary inclusion rate comparable to that for feeds formulated to contain these agents.
• the betaine/coccidiostat composition may be supplemented with additives to improve its flavor or to provide other dietary supplements or therapeutic agents needed by animals.
• Compounds containing betaine and coccidiostat for parenteral administration will be formulated according to known methods for preparing pharmaceutically useful compositions in which these agents are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation are described, for example, in Remington's Pharmaceutical Sciences (16th edition, A. Oslow, ed., Mack, Easton, PA 1980). The required dosage will depend upon the type of animal being treated and the type of coccidiostat being administered (FDA 1994 Feed Additive Compendium. U.S. Food and Drug Administration, 1994).
• Controlled delivery may be accomplished by selecting appropriate macromolecules such as polyesters, polyaminoacids, polypyrrolidone, ethylene vinylacetate, methylcellulose, carboxymethyl- cellulose or protamine sulfate and combining these according to well- established procedures in order to control release.
• the duration of action of coccidiostat and betaine may also be controlled by incorporating these agents into particles of polymeric materials such polyesters, polyaminoacids, hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers.
• Various materials and methods for making and using microcapsules are disclosed in Remington's Pharmaceutical Sciences, (16th edition, A. Oslow, ed., Mack, Easton, PA 1980).
• the present invention is also directed to a method of treating domestic food animals infected with coccidia by administering a combination of betaine and coccidiostat.
• Tables 5 and 11 demonstrate that the administration of betaine and coccidiostat to c ⁇ c ⁇ -infected chicks significantly improves their commercial performance in terms of a diminished severity of gut lesions, reduced mortality, higher end weights and improved feed conversion efficiency.
• betaine and coccidiostat may be administered to animals either orally or parenterally as described above.
• betaine and coccidiostat are incorporated into the feed of animals.
• betaine should constitute between 0.5 and 2.0 kg/ton dry feed.
• salinomycin is used as the coccidiostat for treating chickens, it should be present at an inclusion rate of between 44 and 66 ppm.
• Other coccidiostats which may be used include monensin (100-200 ppm); naracin (70 ppm) and lasalocid (90 ppm), as well as the other coccidiostats shown in Table 1.
• Betaine may be purchased from commercial suppliers such as Finnsugar Bioproducts (sold under the tradename of "Betafin”). Suitable coccidiostats and their suppliers include the following: monensin: Elanco Products Ltd. (tradename “Elancoban” or “Romensin”); salinomycin: Hoechst (UK) Ltd. (tradename “Sacox”): narasin: Elanco Products Ltd. (tradename “Monteban”); and lasalocid: Roche (tradename "Avatec”). Additional coccidiostats that have been approved by the FDA are shown in Table 1. In species other than the chicken, the concentration of coccidiostat should be that recommended by the U.S. Food an Drug Administration (FDA 1994 Feed
• the initial concentration of betaine in the feed should be between 0.5 and 2.0 kg/ton dry feed. This may be adjusted either up or down as experience dictates. Other procedures for administering the combination of betaine and coccidiostat to animals are described above.
• mice 2,464 one-day-old male and 2,464 one-day-old female broiler chicks of commercial strain (Peterson x Arbor Acres) were randomly allotted to 56 floor pens on built-up litter. Chicks were grown to 45 days of age. There were seven treatment groups, each with eight replicates. The experimental design is shown in Table 4.
• Chicks in treatments 1 to 6 were inoculated at 14 days of age with a mixture of E. acervulina, E. maxima and E. tenella via drinking water. At 21 days of age, two males and two females from each pen were necropsied and scored for coccidiosis (0-4, 4 is most severe). Treatment 7 was a non-inoculated control, but chicks received a natural contaminant via the litter.
• Diets Corn-soy diets (starter and grower) were formulated to meet or exceed the nutritional requirements set forth in Nutrient Requirements of Poultry. National Research Council, National Academy of Sciences, Washington, D.C. (1984). Diet composition is shown in Table 2 and calculated analysis in Table 3. The diets were supplemented with betaine and a commercial coccidiostat, Bio-Cox, according to the experimental design (Table 4). Diets were supplied in crumble and pellet form ad libitum. A complete record of feed consumption was maintained.
• Chicks were grown to 47 days of age on a basal diet containing salinomycin and supplemented with methionine, betaine, or a combination of both. The experimental design is shown in Table 8. Each of the five treatments had eight replicates.
• Inoculation Chicks were inoculated at 15 days of age with a mixture of E. acervulina, E. maxima and E. tenella via drinking water.
• Diets Corn-soy diet (starter and grower) was formulated to meet or exceed the nutritional requirements set forth in NRC 1984, except for methionine, which was deficient in diets 1, 3 and 4. Diet composition is shown in Table 6 and calculated analysis in Table 7. The diets were supplemented with betaine and a commercial coccidiostat, salinomycin, according to the experimental design (Table 8). Diets were supplied in crumble and pellet form ad libitum. A complete record of feed consumption was maintained.
• Chicks in treatments 1, 2 and 5-10 were inoculated at 14 days of age with a mixture of E. acervulina, E. maxima and E. tenella via drinking water. At 21 days of age, 4 birds from each cage were necropsied and scored for coccidiosis (0-4, 4 is most severe). Treatments 3 and 4 were non-inoculated controls.
• Treatments marked with the same letter do not differ significantly with regard to the measured parameter.

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