2011 Annual Report
1a.Objectives (from AD-416)
The overall objective of this study is to evaluate effects of a slow-release formulation on the in vivo rates of gastrointestinal absorption of an organic acids that is an effective anti-bacterial agent in broiler chicks and rats. Specific objectives are:.
1)To determine the rates and sites of organic acid absorption with and without formulation on proprietary beads in broiler chicks and rats..
2)To determine the in vitro kinetics of organic acid release from formulated beads under various conditions (temperature, pH, mechanical grinding, etc.).
1b.Approach (from AD-416)
Animal Studies. A detailed study protocol will be written and agreed upon prior to initiation of studies using rats and broilers. Formulated and unformulated forms of two 14C-labeled organic acids will be gavage-dosed to broiler chicks and to rats. Animals will be placed into cages configured to allow the collection of respiratory gases and excreta. Respiratory gases will be collected at regular intervals from each animal until slaughter. Sets of chicks and rats will be humanely slaughtered 1, 2, 3, and 4 hours after gavage dosing. At slaughter blood; the upper gastrointestinal tract; the mid-gastrointestinal tract, the lower gastrointestinal tract; and remainder of the carcass will be collected. Excreta will also be collected. Gastrointestinal contents will be separated from each gastrointestinal fraction and each will be immediately frozen or processed prior to analysis for total 14C-radioactive residues. Total radioactive residues in respiratory gases will be analyzed by liquid scintillation counting of trapped gases and 14C-residues in collected tissues, intestinal fluids, and carcasses will be analyzed after conversion to [14C]-CO2 by combustion analysis. Data will be summarized and a final report will be provided.
Laboratory Studies. A detailed study protocol will be written and agreed upon prior to initiation of laboratory studies..
1)Release into water: 14C-Organic acid formulated onto beads will be weighed into vials, a known volume of water will be added to each vial, vials will be re-weighed, and subsequently placed in a rotary water bath at 23 'C with agitation at 60 rpm. Every 15 minutes for a 2 hour period an aliquot of the suspended material will be removed, weighed, and centrifuged to remove particulate matter. Volumes will be maintained by replacing volumes removed at each sampling time with water. Radioactive organic acid released by the formulated beads will be measured in the centrifugation supernatant by liquid scintillation counting. At the end of the 2- hour period, each parent vial will be weighed and radioactivity remaining in the beads will be determined after centrifugation by combustion analysis of aliquots of the pelleted beads..
2)Release into simulated gastrointestinal fluid. The general protocol described above for “release into water” will be replicated using sequential additions of simulated gastric and intestinal fluids. Radioactivity in the fluids will be analyzed by liquid scintillation counting and radioactivity remaining in beads will be quantified by combustion analysis.
Laboratory and live phase portions of studies have been completed. Upon the mutual consent of the cooperating parties, an additional broiler study was substituted for the rat study. In vitro dissipation studies indicated that the formulated organic acid is sparingly released into water with even lesser quantities released into simulated gastric fluid. Dissipation of organic acid into simulated intestinal fluid (pH 7.5) was greater than in water or simulated gastric fluid, but was still less than 20% of the total formulated activity. Formulation of organic acid into a slow-release product delayed loss of radioactivity through respiration, increased loss of radioactivity in excreta, and slowed the release of radioactivity into liver and carcass fractions of broiler chicks. Overall, the study strongly suggested that formulation of organic acids into slow release particles allowed greater gastrointestinal access to organic acid relative to unformulated product. Data are being reviewed and a manuscript has been drafted.