Eucalyptus oil

Acyclovir was purchased from Acros Organics, Morris Plains, NJ, USA. Tea tree oil, garlic oil, peppermint oil, sandalwood oil, anise oil, oregano oil, chamomile oil, lemon palm oil, thyme oil, and eucalyptus oil were procured from Sigma Aldrich, St. Louis, MO, USA. Oleic acid, PEG 200^®, PEG 400^®, Labrasol^® Propylene Glycol^® and Transcutol^®, Tween80^®, span80^®, tween60^®, span60^®, tween20^®, span20^®, hydroxyl propyl cellulose, and silicone were purchased from Gattefosse (Saint Priest Cedex, France). The chemicals used in the study consisted of analytical grade.

The CA was gifted by Ranbaxy (Mohali, India). Tween 20^®, ethyl oleate, eucalyptus oil, propylene glycol, polyethylene 400 (PEG 400), castor oil, dimethyl sulfoxide ≥99.5% (DMSO), thiazolyl blue tetrazolium bromide 98% (MTT) and methylcellulose were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tween 80^®, soybean oil and olive oil were purchased from R&M Chemicals Ltd. (Essex, UK). Capmul MCM EP was obtained from the Abitec Corporation (Janesville, WI, USA). Peceol and Maisine 35-1 were obtained from Gattefosse (Saint-Priest, France). Cremophor EL^® was obtained from BASF (Ludwigshafen, Germany). The dialysis membrane (MWCO 12,000 g/mole) was obtained from Sigma-Aldrich Sdn Bhd, Petaling Jaya, Malaysia. The Caco-2 cell line was bought from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Dulbecco’s modified eagle’s medium (DMEM) was purchased from GE Healthcare Life Sciences (Logan, UT, USA). Trypsin 0.25% was purchased from GE Healthcare Life Sciences (Logan, UT, USA). The penicillin-streptomycin solution (100×) was obtained from Biowest (Nuaillé, France). Fetal bovine serum (FBS) and Dulbecco’s phosphate-buffered saline (DPBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The passive lysis buffer (5×) was purchased from Promega (Madison, WI, USA).

Four blends of feed additives comprising graded doses (5, 10, 15, and 25 μL) of eucalyptus oil and a fixed quantity (15 μL) of aqueous extract of mulethi (Glycyrrhiza glabra root; commonly known as licorice or sweet wood) roots were prepared and tested for their suitability to modulate in vitro rumen fermentation for reducing ammonia-nitrogen and methane production without impeding feed digestion. The blends were prepared based on a series of preliminary in vitro rumen fermentation experiments with graded doses of eucalyptus oil and aqueous extract of mulethi roots individually (24 ). The eucalyptus oil (80% purity) used for in vitro and in vivo rumen fermentation studies were purchased from Sigma-Aldrich Ltd. (Bangalore, India). eucalyptus oil (60% purity) used for the in vivo feeding trial on growing buffaloes was purchased from Central Drug House, Delhi, India. Roots of mulethi were purchased from a local market at Hisar, Haryana, India were dried and ground to prepare extract (233.6 g/ L of distilled water) for in vitro studies.