Embryonated eggs as an alternative to animals in the determination of median lethal dose (LD50) in bitis venom

Abstract

Determination of median lethal dose (LD50) is a vital tool adopted by the World Health Organization for pre-clinical assessment of products for use in the management of snakebite envenoming, a condition which is now included among the list of Neglected Tropical Diseases in 2017. The current trend in the determination of LD50 involves the use of laboratory animals, tens or even hundreds of animals are sacrificed to achieve this goal. This study aimed to find reliable alternatives to this sacrificing of laboratory animals for research purposes. This study investigated the comparative similarities or differences in results obtained from the use of laboratory animals and embryonated eggs in the determination of LD50 in snake venom research. The median lethal dose (LD50) was determined using female mice using the up and down method and Probit method as well as embryonated eggs. There was no statistical difference in the LD50 of the venom of Bitis arietans obtained by the up and down method and that of the conventional probit analysis (p≤0.05) (0.325 mg/kg [probit] and 0.351 mg/kg [up and down] respectively). There was also no statistical difference in the LD50 of the venom of Bitis arietans by the up and down method, conventional probit method, and by the use of embryonated eggs (p≤0.05) (0.325 mg/kg [probit], 0.351 mg/kg [up and down], and 0.392 mg/kg [embryonated eggs). The three methods used produced values of LD50 that were within the range reported on the Australian snake and venom database of 2007. The results suggest embryonated eggs can conveniently replace the use of laboratory animals in the determination of LD50 in snake venom research to ease the ethical challenges posed by excessive use of laboratory animals in snake venom research.