Quantification of the hemolysis associated with use of T-61^Ras a euthanasia agent in rabbits–a comparison with Euthanyl^R(pentobarbital sodium) and the impact on serum hexosaminidase measurements

Abstract

Albino rabbits (2-2.3 kg) were euthanized with T-61R (N-[2-(m-methoxyphenyl)-2-ethylbutyl-(1)-]-gamma-hydroxybutyram ide, 200 mg/mL; 4,4'-methylene-bis-(cyclohexyltrimethylammonium iodide), 50 mg/mL; tetracaine hydrochloride, 5 mg/mL; 0.2-1.0 mL/kg, i.v.) or EuthanylR (pentobarbital sodium, 100-240 mg/kg, i.v.) at 15:00 h, and samples of arteriovenous blood or venous blood were collected. The time to loss of consciousness was dose dependent for T-61, ranging from 7 to 1 s or less. Serum obtained after T-61 euthanasia, but not after Euthanyl, was routinely contaminated with measurable hemoglobin levels (up to an estimated 3 mg/mL). The extent of hemolysis increased with increasing T-61 doses. The activity of hexosaminidase in the serum (determined by hydrolysis of a methylumbelliferyl substrate at pH 4.6) was up to 80% lower in sera obtained after the use of T-61 compared with sera obtained after the use of Euthanyl or without euthanasia drugs. The reduction in measurable activity appears to be due to interference, by hemoglobin, in the fluorescence measurements of the methylumbelliferol reaction product (with threshold effects being detected at 0.1 mg/mL). Standardization of serum volumes used in these types of enzyme assays and reporting on hemoglobin levels is thus recommended.

Assessment of the Apparent Intra- and Inter-Sample Variability in the Collagen Fibril Diameter in the Posterior Corneal Stroma of Rabbits

“…or T-61 (0.5 ml/kg, i.v. ), between 14.30 and 15.30 h [7] . Routinely, one eye was enucleated and used in physiological studies, while the other was prepared for use in electron-microscopic studies.…”

Factors That Can Influence Animal Research

“…The euthanasia agent, T-61, causes intravascular hemolysis which interferes with serum hexosaminidase measurements and artifactual damage of the pulmonary parenchyma characterized by congestion, edema, and endothelial necrosis, as well as endothelial swelling of the renal glomerular tufts (Doughty and Stuart, 1995;Port et al, 1978;Prien et al, 1988). The pulmonary architecture is extremely sensitive to effects of a variety of euthanasia techniques (Feldman and Gupta, 1976).…”

Averaged Spacing and 2-D Organization of Collagen Fibrils in the Posterior Cornea of the Rabbit Eye Assessed by Transmission Electron Microscopy

“…or T-61 R (0.5 ml/kg, i.v. ), between 14.30 and 15.30 h. 18 One eye was immediately prepared for use in these electron microscopy studies, while the other was removed and used in corneal physiology studies.…”