Larval Development Test

The procedure used was a modification of the technique described previously.¹⁹ The experiment was conducted in plastic cups of 20 mL. The collected eggs were incubated at 27°C for 24 hours. An aliquot of 1 mL, containing 95–125 first-stage larvae (L₁) of H. contortus, was mixed with 5 g of feces collected from a sheep free of nematode eggs, and various serial concentrations of each plant extract. Serial concentrations of 50, 25, 12, 5, 6.25, 3.125 and 1.562 mg/mL of each plant were made in distilled water to make a total volume of 7 mL together with water containing L₁ and volume of egg-free feces. Ivermectin 1% (10 mg/mL) dissolved in 5% DMSO at concentrations of 0.5, 0.25, 0.12, 0.625, 0.3 12, 0.15, and 0.078 mg/mL was used as positive control while untreated eggs in 5% DMSO was used as negative control. There were three replicates for each extract concentration and control. The plates were further incubated for 5 days (for a total of 7 days), and further development was stopped by the addition of one drop of Lugol’s iodine solution: all L₁ and L₃ larvae in each well were counted under dissecting microscope at 40× magnification.