Ruminal stability of terpenoids with anthelmintic activity on Haemonchus contortus

Abstract

Haemonchus contortus is one of the most pathogenic gastrointestinal nematodes from small ruminants. Indiscriminate application of synthetic anthelmintic has selected resistant nematodes. Several molecules, such as terpenoids, have perspectives to become as new tool to control parasites. However, it is necessary to evaluate the stability of these compounds in the ruminal environment to assure that the effective concentrations achieve the target parasites. The aim of this study was to evaluate the in vitro ruminal metabolism of carvacrol and thymol and their efficacy on H. contortus. The activity of terpenoids was assessed by the egg hatch test. Carvacrol and thymol were serially diluted in tween 80 (1%) from 2,500 µg/mL to 75 µg/mL. Four replicates were performed for each concentration. The eggs were incubated for 48 hours at 27 ºC and the first stage larvae were counted. The experiment was repeated three times. For ruminal metabolism, carvacrol and thymol (800 µg/mL) were added to 2 mL of ruminal content collected by an esophageal tube from sheep. Inactive ruminal content by boiling was used as control. The incubations were carried out by quadriplicate in a shaking water bath at 37 °C under anaerobic conditions for 30, 120 and 240 hours. Control samples of boiled ruminal content were prepared and incubated under the same conditions. An aliquot of samples was thoroughly mixed with acetonitrile, for 10 minutes and then centrifugated. The supernatant was injected to the HPLC with UV detection. Carvacrol and thymol showed low IC50 values in the egg hatch test (186 and 187.0 µg/mL), respectively). Carvacrol and thymol were very stable in active ruminal content. The carvacrol concentrations were 91.0% (30 minutes), 87.3% (120 minutes) and 84.0% (240 minutes) of the concentrations recovered in the control samples (inactive ruminal content). Thymol recovery was 90.6% 94.1% and 95.1% of the compound concentrations measured in the control samples at the same incubation times. Therefore, carvacrol and thymol were effective on the eggs of H. contortus and metabolic stable in active ruminal content showing a potential pharmacological tool against gastrointestinal nematodes.