Classifications

• • 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
  • A61K31/19—Carboxylic acids, e.g. valproic acid
• • 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
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
• • 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
  • A23K50/50—Feeding-stuffs specially adapted for particular animals for rodents
• • 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
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
• • 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
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
• • 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
  • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
  • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
  • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

• the present invention generally relates to compositions comprising abscisic acid, and/or salts, and derivatives thereof, and methods of their preparation and administration for uses with livestock and in aquaculture.
• Abscisic acid is a naturally occurring plant hormone that is well known to be involved in plant response to stress. The chemistry and physiology of abscisic acid and its analogs is described by Milborrow, Ann. Rev. Plant Physiol. 1974, 25, 259-307. The naturally occurring enantiomeric form of abscisic acid is (S)-(+)-abscisic acid. The stereochemistry of the side chain of the major part of naturally occurring abscisic acid is 2-cis-,4-trans-, since that is the isomer that is produced biosynthetically by all green plants and some microorganisms.
• Abscisic acid is a carboxylic acid, and thus in a medium having an acidic pH, it is protonated in its neutral undissociated form. This uncharged, undissociated form is more lipophilic than a salt of abscisic acid is in its dissociated form of abscisic acid that is present at higher pH (Blumenfeld and Bukovac 1972, Planta 107: 261-268).
• abscisic acid is used in agriculture and horticulture on or around crops and plants for various purposes, such as improving stress tolerance, slowing the growth rate, adjusting flowering phase, and other purposes. Abscisic acid has also been reported to possess insect inhibition qualities. See U.S. Pat. Nos. 4,434,180 and 4,209,530. Others have reported potential medicinal properties of abscisic acid, for example U.S. Patent Application No. 2006/0292215 discloses methods of using abscisic acid for anti-cancer purposes, and International Application No. WO 2007/042983 discloses anti-inflammatory activity of abscisic acid. All toxicology studies conducted to this point indicate abscisic acid, is a safe, nontoxic substance.
• abscisic acid, and/or salts, derivatives and analogs thereof have health and feed benefits for livestock and aquaculture.
• the present invention is generally directed to compositions comprising abscisic acid, and/or salts, and derivatives thereof (collectively referred to as “ABA” herein), of which (S)-(+)-abscisic acid is one enantiomer (hereinafter referred to as “S-ABA”), and methods of their use to maintain the health of livestock and fish comprising administering to livestock or fish a effective amount of ABA.
• ABA abscisic acid, and/or salts, and derivatives thereof
• the present invention is also directed to methods of increasing feed efficiency comprising administering to livestock or fish an effective amount of ABA.
• Another embodiment of the present invention is directed to methods of increasing weight gain of livestock and fish comprising administering to an animal an effective amount of ABA.
• Another embodiment of the present invention is directed to methods of decreasing the time to market of livestock and fish comprising administering to livestock or fish an effective amount of ABA.
• a different embodiment of the present invention is directed to methods of increasing the meat quantity of livestock and fish comprising administering to livestock or fish an effective amount of ABA.
• the present invention is directed to methods of increasing immunological function comprising administering to livestock or fish in need thereof an effective amount of ABA. This effect is supported by an increase in digestive tract weight, suggesting better overall animal health.
• the present invention is directed to methods for reducing or eliminating the use of antibiotics in feed comprising administering to an livestock or fish an effective amount of ABA.
• compositions of the present invention generally comprise ABA.
• Other components which enhance the biological activity of the ABA may optionally be included.
• the present invention is specifically directed to compositions comprising ABA, of which (S)-(+)-abscisic acid is one enantiomer, and methods of their use to treat various animal ailments, as well be used as feed for animals and animal breeders.
• ABS-ABA is the preferred compound of the compositions and uses herein and has the structure as follows:
• compositions and methods of the inventions encompass all isomeric forms of the described abscisic acids, their racemic mixtures, enol forms, solvated and unsolvated forms, analogs, prodrugs, derivatives, including but not limited to esters and ethers, and pharmaceutically acceptable salts
• suitable salts include inorganic salts such as the ammonium, lithium, sodium, potassium, magnesium, and potassium salts and organic amine salts such as the triethanolamine, dimethanolamine and ethanolamine salts.
• the organic amine salt is the triethanolamine salt.
• the organic amine salt is the dimethylethanolamine salt.
• the organic amine salt is the ethanolamine salt.
• salts are not limiting as other salts may also be suitable for use in the present invention.
• One presently preferred salt is the ammonium salt.
• Other preferred salts are the sodium and potassium salts.
• the salts may be prepared by contacting the acid form with a sufficient amount of the desired base to produce a salt in the conventional manner.
• the free acid forms may be regenerated by treating the salt with a suitable dilute aqueous acid solution such as dilute aqueous sulfuric, hydrochloric or phosphoric acid.
• the free acid forms differ from their respective salt forms somewhat in certain physical properties, such as their solubilities in polar solvents, but the salts are equivalent to their respective free acid forms for purposes of the invention. (See, for example S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharm. Sci., 66: 1-19 (1977) which is incorporated herein by reference).
• livestock and fish refer to any livestock or fish, or offspring of livestock or fish, who is receiving treatment, is in need of treatment, is taking or receiving treatment for prevention purposes, and/or is being administered the composition.
• offspring refers to progeny or descendants of livestock or fish, and includes born progeny, fetuses and embryos.
• Livestock shall include poultry, including chickens, which includes boilers and layers and male and female breeding stock, geese, duck, turkey, pheasant, cornish hens, swine, cattle, which includes beef and dairy production, sheep, and goats.
• “Fish” shall include catfish, carp, tilapia, trout, crayfish, shrimp, lobster, crab, aquatic mammals, salmon, and white fish.
• administering includes any means for introducing the ABA of the invention and other therapeutic agents, into the body, preferably into the systemic circulation.
• examples include but are not limited to oral, including feed and/or drinking water, buccal, sublingual, pulmonary, ophthalmic, transdermal, transmucosal, as well as subcutaneous, intraperitoneal, intravenous, intramuscular injection, transplacental transfer and lactation.
• treating and “treatment” have a commonly understood meaning of administration of a remedy to livestock or fish, or the parent of livestock or fish, who has or is suspected of having a disease or a condition, and refer to reversing, alleviating, inhibiting, or slowing the progress of the disease, disorder, or condition to which such terms apply, or one or more symptoms of such disease, disorder, or condition, or preventing or decreasing the chances of a disease, condition, disorder or outcome from occurring, or to increase effects of a specified physiological response or health benefit.
• preventing and prevention refer to prophylactic use to reduce the likelihood of a disease, disorder, or condition to which such term applies, or one or more symptoms of such disease, disorder, or condition. It is not necessary to achieve a 100% likelihood of prevention; it is sufficient to achieve at least a partial effect of reducing the risk of acquiring such disease, disorder, or condition.
• composition includes a product comprising ABA (and in the specified amounts, if indicated), including products with exogenous or up-regulated ABA, as well as any product which results, directly or indirectly, from combination of ABA with specified ingredients in the specified amounts.
• the term “effective amount” means an amount of a compound that, when administered to livestock or fish for treating a disease, condition or attaining a desired result, is sufficient to effect such treatment for the disease or desired result.
• the “effective amount” will vary depending on the compound, the disease state being treated or health benefit desired, the severity or the disease treated, the result desired, the age and relative health of the livestock or fish, the route and form of administration, the judgment of the attending medical or veterinary practitioner, breeder, trainer, or person attending or caring for the livestock or fish, and other factors.
• the amount of ABA that is “effective” will vary from composition to composition, depending on the particular use, the particular ABA, salts, derivatives and analogs thereof, and the like. Thus, it is not always possible to specify an exact “effective amount.” However, an appropriate “effective amount” in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
• Method of Ton refers to 2,205 pounds or 1,000 kg.
• Ton also known as a “short ton”, refers to 2,000 pounds or 907 kg.
• compositions of the present invention may be incorporated with compound feed, or commercial pelleted food produced in a feed mill and fed to domestic livestock and fish.
• the compositions of the present invention may be incorporated with fodder, or food given to domestic livestock including plants cut and carried to them.
• the compositions of the present invention may be incorporated with forage, or growing plants eaten by domestic livestock. It is within a skill in the art to formulate the compositions with foodstuffs for oral consumption.
• compositions of the present invention may be formulated in a liquid composition for oral consumption. It is within a skill in the art to formulate the compositions in liquid compositions for oral consumption.
• compositions of the present invention may be added to water used for livestock or fish production.
• composition of the present invention is added to an aquaculture system used to grow fish, aquatic mammals, or crustaceans.
• An embodiment of the present invention is directed to methods of reducing mortality of livestock and fish comprising administering to livestock or fish an effective amount of ABA.
• Another varying embodiment of the present invention is directed to methods of increasing weight gain of livestock and fish comprising administering to livestock and fish an effective amount of ABA.
• Another embodiment of the present invention is directed to methods of decreasing the time to market of livestock and fish comprising administering to livestock and fish an effective amount of ABA. It is also contemplated that a different embodiment of the present invention is directed to methods of increasing the meat quantity of livestock and fish comprising administering to livestock and fish an effective amount of ABA.
• An embodiment of the present invention is directed to methods of increasing immunological function in livestock and fish comprising administering to livestock or fish in need thereof an effective amount of ABA.
• a preferred range of an effective amount of ABA for the various methods is from about 1 gram/MT of feed to about 2000 grams/MT of feed.
• a more preferred range of ABA is from about 5 grams/MT of feed to about 500 grams/MT of feed.
• a person having ordinary skill in the art would be able to adjust these ranges for various uses like aquaculture applications without undue experimentation.
• Animal feed for feeding poultry includes protein, fat, fiber, calcium, and phosphorous.
• a preferred feed would include corn and/or wheat, soybean meal, fat, animal by-product, meat and bone meal, and vitamins and minerals.
• premix When used in poultry feed, ABA can be initially mixed into a premix.
• premix is intended to mean a feed composition that is prepared as an initial mix containing the active ingredient and, for example, a carrier, and is then blended into the final feed.
• the premix is blended with about one metric ton (MT) of conventional feed, with the result that the necessary dosage requirements of the ABA are provided to the animals.
• MT metric ton
• the premix of the invention preferably comprises about 1 to 2000, and more preferably, about 5 to 500, grams of ABA.
• Carriers for use in a premix are well known by those having skill in the art, and appropriate concentrations can be readily determined.
• the ABA may be added to the carrier as a dry powder or as a liquid solution or suspension.
• the ABA When added as a liquid, the ABA may be dissolved or suspended in a liquid with stirring at room temperature.
• a liquid may be water or a suitable solvent or another product used for animal feed that is already in liquid form. Because of ABA's liquid solubility characteristics, it may form a suspension.
• a predetermined amount of ABA is then added to the conventional premix, and will not overly wet it. Once the premix is prepared, the premix is then added to the final feed, preferably at a rate in the range of one quarter of a pound (lb.) to five pounds (lbs.) of premix to one metric ton (MT) of feed, to supply daily requirements of the ABA for the poultry.
• the ABA solid or solution or liquid suspension can be added directly to the premix material, and then mixed.
• Mixing can be accomplished by any known means, such as by a standard horizontal or vertical blender. Mixing time will again vary depending upon the particular ingredients of the premix, and can take as long as is necessary to assure that the ingredients are thoroughly mixed.
• the premix is then incorporated into the feed to be fed to the poultry.
• ABA is blended with the carrier to form the premix, and the premix is directly blended into the final feed. While there is no evidence that use of the higher amounts would cause any toxicity problems in treated poultry, it will affect cost considerations. Because the premix generally will be added to one-ton charges of feed (as is common in the industry), the correspondence between one gram of ABA added to the premix yields about 1 ppm concentration of ABA in the feed. Thus, 5 grams of ABA added to one pound of premix, which in turn is added to one metric ton (MT) of feed, yields about a 5 ppm effective ABA concentration.
• MT metric ton
• ABA can be mixed with a premix material prior to incorporation into the finished feed
• the appropriate amount of ABA may be directly blended into or sprayed upon the feed.
• the preferred additive range of ABA in finished feed, whether added directly or via a premix, is about 1 to 2000 grams per metric ton (MT) of feed; more preferably about 5 to 500 grams per metric ton (MT) of feed.
• Feed is conventionally prepared in a large bin or mixer in which the feed ingredients are added in descending weight order according to their prevalence in the ultimate feed mixture. Thus, cracked or ground grain would be the primary ingredient. Minor ingredients are then added. Micro-ingredients are added last. These include vitamins, drugs, growth promoters, antibiotics, and, in the present case, ABA. Thus, ABA can be one of the micro-ingredients and is added to the feed in the final blending step. The feed is blended for conventional time periods.
• the feed comprising the ABA is fed to livestock and fish in standard form, such as a mash, crumble or pellet, and at standard feed dosage ranges and rates.
• Another embodiment of the present invention is liquid compositions that can be prepared as either ready-to-use dilutions or dilutable concentrates.
• the embodiment of the present invention can be a solution containing from 0.5% to as much as 50% by weight of ABA.
• the dilutable concentrates can be diluted into water directly to a final application concentration or to any intermediate dilution, without risk of precipitation of the active ingredient.
• the aqueous formulations according to one embodiment of the present invention are inexpensive to manufacture, safe to handle and use, and the ABA active ingredient is stable under storage and shipping conditions.
• a person having ordinary skill in the art would be able to determine how to prepare the final aqueous solution concentration for direct application to animals without undue experimentation, without any chance of causing precipitation of the active ingredient, and without long and laborious stirring to bring the active ingredient into solution.
• Another embodiment of the present invention is an ABA water solution that serves as a drinking source of water for the livestock and fish.
• Such supplemented water solution could be prepared by dissolving dry powder ABA in drinking water or by using a liquid solution or suspension concentrate of ABA.
• the preferred additive range of ABA in drinking water is about 1 to 2000 parts per million (ppm) or about 1 to 2000 milligrams of ABA per liter of water; more preferably about 5 to 500 parts per million (ppm) or about 5 to 500 milligrams of ABA per liter of water.
• a person having ordinary skill in the art would be able to determine how to prepare the final aqueous solution for direct application to livestock and fish without undue experimentation, without any chance of causing precipitation of the active ingredient, and without long and laborious stirring to bring the active ingredient into solution.
• ABA can be applied directly onto animal feed once it has been prepared.
• the ABA can be applied directly to the finished feed.
• an aqueous solution of ABA is sprayed onto the finished feed in its final form, such as a pellet, prior to delivering the feed to the livestock and fish.
• mice Male Cobb 500 chickens were fed different levels of S-ABA supplemented into starter, grower, and finisher feed diets for their entire 42 day commercial life cycle. On day 43, samples of chickens (sometimes referred to as “birds”) from each treatment were processed.
• the starter, grower, and finisher basal diets represented standard commerical broiler diets.
• the starter and grower diets contained Salinomycin (a coccidiostat) at 40 grams/ton.
• the basal diets and final treatment diets were prepared using a 500 or 4,000 pound capacity vertical mixer or a 14,000 pound capacity horizontal mixer. After correcting for percent purity of S-ABA in the S-ABA dry powder, S-ABA was added to the basal diets at concentrations of 5, 50, or 500 ppm (4.6, 46, or 455 grams of S-ABA/ton).
• Control treatments included (1) commercial diet only and (2) commercial diet supplemented with antibiotics (bacitracin methylene disalicylate (BMD), and STAFAC® virginiamycin (hereinafter “Stafac”). STAFAC is a registered trademark of Phibro Animal Health Corporation). All feed diets were pelleted at ⁇ 65° C. or below.
• a 90-pen test facility was divided into 10 blocks containing 9 pens per block. Treatments were assigned to the pens using a complete randomized block design. Birds were assigned to the pens randomly. Specific treatment groups were as follows:
• Birds were vaccinated for Mareks at the hatchery. Birds were vaccinated upon receipt for the study (day 0) for Newcastle and Infectious Bronchitis via spray cabinet. No other vaccinations or treatments, except what is provided in the diets were administered during the study.
• Water was provided ad libitum throughout the study via one automatic four-nipple drinker in each pen. Drinkers were checked twice daily to assure a constant and clean water supply to the birds.
• Feed was provided ad libitum throughout the study via one handing, ⁇ 17-inch diameter tube feeder per pen. Chick feeder trays were placed in each pen for approximately the first 4 days. Feed added and removed from pens was weighed and recorded from day 0 to day 42. Diet changes were conducted at the same time for all pens. The feeding period for each diet was as shown below:
• Birds were weighed by pen at 0, 7, 14, 21, 28, 35, and 42 days of age. The feed remaining in each pen was weighed and recorded on study day 0, 7, 14, 21, 28, 35, and 42 days of age. The feed intake during days 0-7, 0-14, 0-21, 0-28, 0-35, and 0-42 was calculated.
• Performance data was summarized by average weight per bird at each body weights measurement period.
• the average feed conversion was calculated for each respective body weight period using the total feed consumption in a pen divided by the total weight of surviving birds (i.e. days 0-7, 0-14, 0-21, etc.). Adjusted feed conversion was calculated using the total feed consumption in a pen divided by the total weight of surviving birds and weight of birds that died or were removed from that pen.
• TABLE 3 shows that S-ABA in the feed diet increases the weight of digestive tracts compared to the commercial feed diets (control 1 and control 2).
• Increased digestive tract weights may be directly related to better overall health. Further, data in TABLE 3 suggest that S-ABA may also reduce or eliminate the need for antibiotics in feed.
• TABLE 4 shows the economic benefit of improved health and growth of broiler chickens and increased feeding efficiency resulting from the addition of S-ABA into commercial feed.
• the starter, grower, and finisher basal diets represented standard commercial broiler diets.
• the starter and grower diets contained Salinomycin (a coccidiostat) at 40 grams/ton.
• the basal diets and final treatment diets were prepared using a 500 or 4,000 pound capacity vertical mixer or a 14,000 pound capacity horizontal mixer. After correcting for percent purity of S-ABA in the S-ABA dry powder, S-ABA was added to the basal diets at concentrations of 2.5, 5.0, or 7.5 ppm (2.3, 4.6, or 6.8 grams of S-ABA/ton).
• Control treatments included (1) commercial diet only and (2) commercial diet supplemented with antibiotics (bacitracin methylene disalicylate and Stafac). All feed diets were pelleted at ⁇ 65° C. or below.
• a 108-pen test facility was divided into 18 blocks containing 6 pens per block. Treatments were assigned to the pens using a complete randomized block design. Birds were assigned to the pens randomly. Specific treatment groups were as follows:
• Birds were vaccinated for Mareks at the hatchery. Birds were vaccinated upon receipt for the study (day 0) for Newcastle and Infectious Bronchitis via spray cabinet. No other vaccinations or treatments, except what is provided in the diets were administered during the study.
• Water was provided ad libitum throughout the study via one automatic four-nipple drinker in each pen. Drinkers were checked twice daily to assure a constant and clean water supply to the birds.
• Feed was provided ad libitum throughout the study via one handing, ⁇ 17-inch diameter tube feeder per pen. Chick feeder trays were placed in each pen for approximately the first 4 days. Feed added and removed from pens was weighed and recorded from day 0 to day 42. Diet changes were conducted at the same time for all pens. The feeding period for each diet was as shown below:
• Birds were weighed by pen at 0, 7, 14, 21, 28, 35, and 42 days of age. The feed remaining in each pen was weighed and recorded on study day 0, 7, 14, 21, 28, 35, and 42 days of age. The feed intake during days 0-7, 0-14, 0-21, 0-28, 0-35, and 0-42 was calculated.
• Performance data was summarized by average weight per bird at each body weights measurement period.
• the average feed conversion was calculated for each respective body weight period using the total feed consumption in a pen divided by the total weight of surviving birds (i.e. days 0-7, 0-14, 0-21, etc.). Adjusted feed conversion was calculated using the total feed consumption in a pen divided by the total weight of surviving birds and weight of birds that died or were removed from that pen.

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