5.3.3. Venom Activities Using Animal Models

Hemorrhagic and myotoxic activities were carried out as previously described [43] using male Swiss mice (18–20 g) under the approval of the Butantan Institute Ethics Committee on Animal Use (Protocol Number: 13710-14). Briefly, for hemorrhagic activity, samples containing 10 μg of each venom pool, diluted in 50 μL of PBS, were injected intradermally into the dorsal skin of mice. After 3 h, the animals were euthanized in a CO2 chamber, the dorsal skin was removed, and the hemorrhagic halos were measured. Groups of 5 animals were tested, and control group animals were injected with PBS only or *B. jararaca* venom (10 μg). Results represent the mean ± SE of the area of hemorrhagic spots (cm2) from mice tested in at least 2 independent experiments. The myotoxic activity was assayed using 50 μg of venom pools in 30 μL of PBS, injected intramuscularly into the gastrocnemius muscle in Swiss mice. After 3 h, the animals were bled via ophthalmic plexus and the sera were assayed for creatine-kinase activity with a commercial kit CK-UV (Bioclin), according to the manufacturer's instructions. Groups of 5 animals were tested, and animals from the control groups were injected with PBS only or *B. jararacussu* venom (50 μg). Results represent the mean ± SE of the CK activity in mice sera (U/mL) from mice tested in at least 2 independent experiments.

The lethality induced by the venoms was tested in murine (Swiss mice 18–20 g body weight) and avian (three days old Bovan chicks 20–30 g body weight) models with protocols approved by the Animal Ethical Use Committees of the Instituto Butantan (CEUAIB n◦ 13710-14). Groups of 6 mice or chicks were injected intraperitoneally with a single dose (200 μg) of venom samples, diluted in 500 μL of PBS. Following injection, the times of death for each group were recorded hourly until the sixth hour, and then at 12, 24, and 48 h after inoculation of the samples. After 48 h, the surviving animals were euthanized, using a CO2 chamber for the mice and injection of sodium pentobarbital overdose for the chicks. The data obtained were graphed on a survival curve plot. The tests were undertaken in two independent experiments.
