Body Temperature Drop as a Humane Endpoint in Snake Venom-Lethality Neutralization Tests

Abstract

Snake venom neutralization potency tests are required for quality control assessment by manufacturers and regulatory authorities. These assays require the use of large numbers of mice that manifest severe signs associated with pain and distress and long periods of suffering. Despite this, many animals make a full recovery; therefore, the observation of clinical signs as a predictor of animal death is highly subjective and could affect the accuracy of the results. The use of a more objective parameter such as body temperature measurement could help establish a humane endpoint that would contribute to significantly reducing the suffering of large numbers of animals. We determined the temperature drop in BALB/c mice exposed to the mixtures of Bothrops asper or Lachesis stenophrys venom and a polyvalent antivenom by using an infrared thermometer. Our data show that, based on the temperature change from baseline, it is possible to predict which animals will survive during the first 3 h after inoculation. The data provided in this study may contribute to future reductions in animal suffering, in concordance with general trends in the use of laboratory animals for the quality control of biologicals.

Keywords: antivenom; refinement; temperature; venom.

Figure 1

Kaplan–Meier survival analysis of (A) B. asper and (B) L. stenophrys venoms stratified into five groups (controls and 2, 3, 4.5, and 6.75 mg venom/mL antivenom) shows a significantly better overall survival for groups receiving lower venom/antivenom dosages. Lethality rates were not different between venoms.

Figure 2

Temperature differences between surviving and non-surviving mice. Groups of five mice were inoculated with mixtures of a fixed dose of B. asper (A) or L. stenophrys (B) venoms (4xLD₅₀) and variable doses of antivenom (2, 3, 4.5, and 6.75 mg venom/mL antivenom). Body temperature was recorded before inoculation and every hour for 3 h. Graphs represent mean ± 95% CI of temperature from surviving (n = 40 for B. asper; n = 39 for L. stenophrys) and non-surviving (n = 35 for B. asper; n = 35 for L. stenophrys) mice. Animals that did not survive had significantly lower temperatures at all time points (except at 0 h) relative to animals that survived, (** p < 0.01; **** p < 0.0001).