D lactate assay kit

The reaction system for the serum concentration of DAO determination included 0.1 mL (4 μg) horseradish peroxidase solution (Sigma-Aldrich, St. Louis, USA), 3 mL PBS (0.2 M, pH 7.2), 0.1 mL O-dianisidine methanol solution (500 μg of O-dianisidine) (Sigma-Aldrich, St. Louis, USA), 0.5 mL sample, and 0.1 mL substrate solution (175 μg of cadaverine dihydrochloride) (Sigma-Aldrich, St. Louis, USA). The processed samples were incubated in an incubator chamber at 37°C for 30 min and measured at 436 nm by UV/visible spectrophotometer-UV-2450 (Shimadzu, Kyoto, Japan) [23 (link)]. Serum D-lactate was determined using a D-Lactate Assay Kit (BioVision, Mountain View, San Francisco, USA) in accordance with the manufacturer's instruction [24 (link)].

The permeability of the intestinal barrier was evaluated by detecting the levels of lipopolysaccharides (LPS) and D-lactate and the activity of diamine oxidase (DAO) in serum. Four samples from each group were randomly chosen for these tests. The LPS content was measured using the ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (GenScript, USA). The D-lactate level was determined by the colorimetric method according to the manufacturer’s protocol (D-lactate Assay Kit, BioVision, USA). The DAO activity was assessed by the reaction of cadaverine dihydrochloride (Sigma, USA) as described in our previous work [22 (link)].

A Glucose Uptake Assay Kit (Biovision, CA, USA) was used to measure the uptake of glucose. The production of lactate acid, ATP and pyruvic acid was examined by D-Lactate Assay Kit, ATP Colorimetric/Fluorometric Assay Kit and Pyruvate Colorimetric/Fluorometric Assay Kit (Biovision), respectively. The corresponding absorbances were detected using a microplate reader.

Rumen SCFA concentrations were determined using a gas chromatograph (HP6890N, Agilent Technologies, Wilmington, DE, United States) as described by Yang et al. (2012) (link). Rumen Ni concentration was determined in the supernatant of a 10 ml rumen fluid sample (centrifugation at 900 × g for 5 min) with the atomic absorption spectrometry fitted with a nickel hollow cathode lamp according to the method described by Rahnama and Najafi (2016) . Concentrations of L-lactate, D-lactate, and ammonia, and urease activity were determined by using the L-Lactate Assay Kit, D-Lactate Assay Kit, Ammonia Assay Kit, and Urease Activity Assay Kit (ab65331, ab83429, ab83360, and ab204697, Abcam Trading Co., Ltd., Shanghai, China), respectively, and a microplate reader (Infinite M200 PRO, Tecan Trading AG, Switzerland). SPSS V21.0 (SPSS Inc., Chicago, IL, United States) was applied for statistical comparison using the two-tailed t-test. Differences were considered significant when P < 0.05 in the two-tailed t-test.

D-lactate was measured by using the D-Lactate Assay Kit (Colorimetric) from Abcam (Cambridge, MA, USA), according to the manufacturer’s instructions.

Before performing d-lactate quantification, 1 ml of culture supernatants was deproteinized by precipitation with 10% (v/v) TCA 6N^{68 (link)}. The presence of d-lactate in supernatants was evaluated by using the colorimetric d-lactate Assay Kit (abcam) according to manufacturer’s instructions. Deproteinized BHIG was used as negative control.