Rumen Fluid Sampling and Analysis

Referring to the method described by Guo et al. [20 (link)], 50 mL of rumen fluid was collected from each sheep using a gastric tube rumen sampler prior to feeding, which was subsequently packaged in frozen storage tubes in liquid nitrogen for rapid freezing, and finally sent to the laboratory using liquid nitrogen to be stored at −80 °C for subsequent analysis. Acetic acid (AA), propionic acid (PA), isobutyric acid (IBA), butyric acid (BA), isovaleric acid (IVA), and valeric acid (VA) were determined by gas chromatograph (GC–7890B, Agilent Technologies), and the specific method used referred to the study of Liu [39 (link)] et al. Subsequently, the total volatile fatty acids (TVFAs), acetic acid/propionic acid (A/P), and VFA molar proportion (acetic acid (AAR), propionic acid (PAR), isobutyric acid (IBAR), butyric acid (BAR), isovaleric acid (IVAR), and valeric acid (VAR)) were calculated. Meanwhile, rumen fluid pHs were determined immediately using a portable pH meter (PHBJ–260F; INESA Scientific Instruments Co., LTD, Shanghai, China). Further, we evaluated microcrystalline cellulose (MCC), β–glucosidase (β–Glu), xylanase, lipase, amylase, carboxymethyl cellulose (CMC), and pepsin in rumen fluid with reference to the detailed instructions of the kit (Shanghai Kexing Biotechnology Co., Ltd., Shanghai, China).