JIA-2018-09

2084 TIAN Xing-zhou et al. Journal of Integrative Agriculture 2018, 17(9): 2082–2095 until measure. Lactic acid (LA), acetic acid (AA), propionic acid (PA), and butyric acid (BA) were determined using high performance liquid chromatography (HPLC; 1260 Infinity II LC, Agilent Technologies, Santa Clara, CA, USA) as per the methods of Kudo et al . (1987) and Song et al . (2012); ammonia nitrogen (NH 3 -N) was analyzed by the steam distillation method of Bremner and Keeney (1965). Moreover, the anthocyanin content in silage was extracted using 1% hydrochloric acid (HCl) dissolved in 95%methanol solution at 50°C for 24 h, and then the supernatant was collected and transferred into a 50-mL volumetric flask for the determination of anthocyanin composition by HPLC according to Hosoda et al . (2009) and Yang et al . (2009). The remaining samples were dried at 65°C in a vacuum oven for 72 h, then ground and passed through a 1-mm sieve. Dry matter (DM), crude protein (CP), and ash were measured according to the feed proximate analysis of the Association of Offcial Analytical Chemists (AOAC 1990). Neutral detergent fiber (NDF) was detected with sodium sulfite and a heat stable amylase, whereas acid detergent fiber (ADF) was measured by sequential analysis of the residual NDF by the methods of AOAC (1990) and Van Soest et al . (1991). Each sample was run in triplicate. Organic matter (OM) and hemicellulose were calculated using the following formulas, respectively: OM=100−Ash; Hemicellulose=NDF−ADF. Gross energy (GE) was analyzed using a Parr 6200 calorimeter (Moline, Illinois, USA). Water soluble carbohydrates (WSC) were assayed by a microplate reader (Spectracount TM , Packard Canberra, Meriden, CT, USA) after reaction with anthrone reagent (Sigma-Aldrich, Pcode: 101694154) as described by McDonald and Henderson (1964). Alternatively, dry matter intake (DMI), digestible dry matter (DDM), relative feed value (RFV), and net energy for lactation (NE L ) were predicted according to the following equations adapted from Lithourgidis et al . (2006): DMI=120/%NDF, DM basis; DDM=88.9− (0.779×%ADF, DM basis); RFV=%DDM×%DMI×0.775; and NE L =(1.044−0.0119×%ADF)×2.205 2.3. DPPH scavenging activity Antioxidant activity for the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity of the corn stover silage extract was determined spectrophotometrically according to Thaipong et al . (2006) and Zhang et al . (2018) using a stable free radical DPPH (Sigma-Aldrich, Pcode: 101845869) with a minor modification. Briefly, an aliquot of 2.00 mL of the appropriate dilution (1/5, 1/4, 1/3, 1/2, 1) of two corn stover silage extracts at 21 d was added to 2.00 mL of 0.1 mmol L –1 DPPH solution and then shaken vigorously. The OD value was detected at 517 nm using a microplate reader after incubation for 30 min in a 30°C water bath. The containers were wrapped in aluminum foil to ensure that the incubation process was carried out in the dark. Each sample was run in triplicate. The percentage of DPPH (%DPPH SC ) was calculated by the following formula: %DPPH SC =(Ac−As)×100/Ac Where, Ac is the absorbance of the control and As is the absorbance of the sample. The half maximal inhibitory concentration (IC 50 ) value was calculated by GraphPad Prism 5 software, which denotes the concentration of a substance required for 50% inhibition in vitro . 2.4. In vitro rumen fermentation Three healthy multiparous Saanen dairy goats, body weight (42.50±0.50) kg (mean±standard deviation) were used as ruminal fluid donors. The experimental animals were housed in clean individual pens with free access to water and were fed diets with a concentrate/roughage ratio of 50:50 (10.13% CP and 66.33% total digestible nutrient) according to the National Research Council (NRC 1981). Ration was offered in equal amounts twice daily at 08:00 and 17:00. Ruminal fluid was obtained from goats before morning feeding via the mouth using a vacuum pump into an Erlenmeyer flask. The ruminal fluid was immediately passed through four layers of cheesecloth and mixed in equal volume, and then transported to the laboratory. The culture fluid was prepared by mixing the ruminal fluid and a phosphate-bicarbonate buffer with carbon dioxide (CO 2 ) gas in a 39°C water bath as per Menke and Steingass (1988). A total of 100-mL glass gas-tight syringes (Kabuskiki Kaisha, Japan) were used as fermentation vessels; they were filled with 30-mL of mixture solution with 0.50 g of substrate in each syringe for 3, 6, 9, 12, 24, 48, 72, and 96 h incubation to estimate gas production (GP). The fermentation was stopped by submerging the syringe into ice-cold water, and then the pH of the rumen fluid was immediately measured using the portable pH meter. In the meantime, 20-mL of fermentation liquid and 5 mL of HCl (6 mol L –1 ) were kept in a container after being mixing together, then stored in a refrigerator at –20°C until the samples were analyzed for NH 3 -N and individual volatile fatty acid (VFA) content. The total VFA (TVFA) value was calculated from the following equation: TVFA=AA+PA+BA. Each sample had three replications and two control replications. Moreover, organic matter digestibility (OMD), metabolizable energy (ME), and effective degradability (ED) were calculated using the following formulas by Menke et al . (1979), Menke and Steingass (1988), and Eliman and Ørskov (1984), respectively: OMD (%)=0.986×GP (24 h)+0.0606×CP+11.03 (1) ME (MJ kg –1 )=–0.20+0.1410×OMD (2) ED (%)=a+bc/(k+c) (3)