Rumen Fluid Analysis in Lambs

At the end of the experiment, approximately 3 h after morning feeding, rumen fluid was collected from five randomly selected lambs from each treatment group using an oral stomach tube (Wuhan Anscitech Farming Technology Co., Ltd, Wuhan, China). Rumen pH was measured using a pH meter (DDSJ-318; Shanghai INESA Scientific Instrument Co., Ltd, Shanghai, China). The rumen fluid samples were subsequently filtered through four layers of sterile cheesecloth. For NH₃-N analysis, 5 mL of filtered rumen fluid was placed in a centrifuge tube containing 1 mL of H₂SO₄ (20 g/L). To determine the volatile fatty acid (VFA) concentration, another 5 mL of filtered rumen fluid was placed in a centrifuge tube containing 1 mL of metaphosphoric acid (250 g/L). The samples for NH₃-N and VFA analysis were stored at −20°C until analysis.
The rumen fluid VFA concentration was measured using gas chromatography (Agilent, GC7890, Santa Clara, CA, USA) according to Jeon et al [18 (link)]. The rumen NH₃-N concentration was determined using a colorimetric spectrophotometer (UV-759; Shanghai Automation Instrument Co., Ltd, Shanghai, China) based on AOAC [16 ].