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

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Abstract
Albino rabbits (2–2.3 kg) were euthanized with T-61R(N-[2-(m-methoxyphenyl)-2-ethylbutyl-(1)-]-γ-hydroxybutyramide, 200 mg/mL; 4,4′-methylene-bis-(cyclohexyltrimethylammonium iodide), 50 mg/mL; tetracaine hydrochloride, 5 mg/mL; 0.2–1.0 mL/kg, iv) or EuthanylR(pentobarbital sodium, 100–240 mg/kg, iv) 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 methylumbeiliferol 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.Key words: euthanasia, rabbits, T-61R, N-[2-(m-methoxyphenyl)-2-ethylbutyl-(1)-]-γ-hydroxybutyramide, 4,4′-methylene-bis-(cyclohexyltrimethylammonium iodide), tetracaine hydrochloride, EuthanylR, pentobarbital sodium, serum, hemoglobin, hexosaminidase.

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