Ankom 220 fiber analyzer

The concentrate feed used in this in vitro study was the same as that provided to the rumen fluid donor animals, which consisted of 800 g of barley, 175 g of soybean meal, and 25 g of mineral and vitamin supplements per kg of dry matter (DM). This ensured that the rumen microbiota used in the in vitro gas production assay were acclimated to this specific feed, following the guidelines for in vitro gas production techniques [23 (link)]. The feed was ground using a Retsch mill (Retsch ZM200, Retsch GmbH, Haan, Germany), sieved through a 1 mm sieve, and stored in a dark glass bottle in a dry and cool place until the analysis of its chemical composition and in vitro rumen fermentation. The DM (AOAC 930.15), crude protein (CP, AOAC 954.01), and ash content (AOAC 942.05) were determined according to the methods of the Association of Official Analytical Chemists [24 ]. Neutral detergent fiber and acid detergent fiber were analyzed using an ANKOM²²⁰ fiber analyzer (ANKOM Technology, Macedon, NY, USA) following the methods outlined by Van Soest et al. [25 (link)]. The results of the chemical composition of the concentrate are presented in Table 1.

Forage samples were weighed, cut, and dried at 60 °C to approximately 90% dry matter (DM). They were then ground to pass a 1-mm screen for chemical analyses and a 2-mm screen for in vitro digestibility (IVD) tests. DM, ash, and crude protein (CP) were determined according to standard methods described by the Association of Official Analytical Chemists (AOAC 2000 ) (methods 930.15, 942.05, and 976.05, respectively). Additionally, ash-free neutral detergent fiber (NDFom) was determined using sodium sulfite in the neutral detergent (ND) following the method of Goeríng and Van Soest (1970 ) with an ANKOM 220 Fiber Analyzer (Ankom Technology Corporation).
In vitro dry matter digestibility (IVDMD) and organic matter digestibility (IVOMD) were determined using the two-stage pepsin-cellulose method (Pepcel) (Aufrere 1982 ). Digestible energy (DE) was estimated as 0.0185 × IVOMD (NRC 1988 ).

Representative samples of silages, concentrate, total mixed ration (TMR) and refusals were taken weekly and frozen at a temperature −25 °C. Thawed samples were dried at 60 °C in Binder dryers, and then ground in a mill (ZM 200, Retsch, Haan, Germany) to a 1 mm particle size. The proximate chemical composition of all feeds was determined by standard methods [25 ]. The content of DM was corrected according to the Porter and Murray method [26 (link)]. Silage samples were also assayed for pH with a pH-meter (HI 8314, Hanna Instruments, Woonsocket, RI, USA) and lactic acid by high-performance liquid chromatography (HPLC SHIMADZU) in a MetaCarb 67H P/N 5244 column (Varian, Palo Alto, CA, USA) and 0.0025 M sulfuric acid as the mobile phase, according to the manufacturer’s protocol. VFAs concentrations were determined using a gas chromatographer Varian 450-GC coupled with a flame ionization detector (FID) and a 25-meter-long capillary column CP-FFAP (the internal diameter was 0.53 mm and the thickness of the coating film was 1.0 μm). The contents of NDF, assayed with heat-stable amylase and expressed exclusive of residual ash, acid detergent fiber (ADF), expressed exclusive of residual ash, and acid detergent lignin (ADL) was determined by the method proposed by Van Soest et al. [27 (link)], using an ANKOM220 fiber analyzer (ANKOM Technology Corp., Macedon, NY, USA). The content of ammoniacal nitrogen (N-NH₃) were determined by direct distillation in a 2100 Kjeltec Distillation unit (FOSS Analytical A/S, Hillerød, Denmark) after increasing the pH of the samples by adding MgO. N protein was determined with the help of trichloroacetic acid (TCA) [28 (link)]. The content of nonprotein nitrogen (NPN) was determined as N total – N protein. In order to estimate the nutritional value of each silage, its in vitro DM digestibility (IVDMD) was determined by keeping a silage sample in a Daisy II incubator (ANKOM Technology Corp., Macedon, NY, USA) for 48 h with the ruminal fluid, after which extraction in neutral detergent solution was carried out according to the method proposed by Kowalski et al. [29 (link)]. The nutritional value of the silage was finally estimated with the help of the WINWAR software [30 ]. The chemical composition of the silages is specified in Table 2. The fatty acid composition was determined by gas chromatography (GC) on a VARIAN CP-3800 chromatograph (Varian Analytical Instruments 2700 Mitchell Drive, Walnut Creek, CA 94598-1675, USA) equipped with a flame ionization detector (FID). Fatty Acid Methyl Esters (FAMEs) were prepared by the Peisker’s method, as modified by Żegarska et al. [31 ]. The FAMEs were separated using a CP Sil 88 (0.20 μm) capillary column 50 m long and 0.25 mm in diameter. The column temperature was 180 °C. Injector and detector temperatures were both 250 °C. Helium carrier gas flow was 1.2 mL/min at a split ratio of 1:50. The composition of fatty acids in the silages is presented in Table 3.
Apparent total-tract digestibility of DM, CP, NDF, ADF was measured after 20 d of each period, using acid-insoluble ash (AIA) as an internal marker [32 (link)]. For 5 days, samples of feeds, refusals and feces were collected and frozen at a temperature of −25 °C. Fecal samples weighing approximately 100 g each were collected for all each cow from all excreta evacuated between 7:00 a.m. and 2:00 p.m. This schedule provided five representative samples of feces for each animal. After thawing, all samples from 5 days from a cow were mixed and blended, and then a laboratory sample was taken for determination of N total with the Kjeldahl method. The remaining mass was dried at 60 °C for 72 h, ground to pass a 1-mm screen, and stored for other chemical analyses. Digestibility was calculated from the intake and concentrations of AIA, as well as these components in the diets fed, refusals and in feces, according to this equation:
where AIAd = AIA concentration in the diet actually consumed, AIAf = AIA concentration in the feces, nutrientf = concentration of the nutrient in the feces, and nutrientd = concentration of the nutrient in the diet actually consumed [33 (link),34 (link)].

DM, crude protein, crude fiber, ether extract, and crude ash were determined according to AOAC [85 ]. Chemicals used in the analysis of crude protein, lipids, and crude fiber were purchased from Avantor Performance Materials Poland S.A., Gliwice, Poland. To determine dry matter, samples were dried at 105 °C to constant weight. Crude protein (N × 6.25) was identified by the Kjeldahl method, using a Büchi Scrubber B414 unit and a Büchi 324 distillation set (Büchi Labortechnik AG, Flawil, Switzerland). Crude fat content was assigned using traditional Soxhlet extraction method with diethyl ether. Crude fiber was determined as the residue after sequential treatment with 1.25% H₂SO₄ and with 1.25% NaOH using an ANKOM²²⁰ Fiber Analyzer (ANKOM Technology, New York, NY, USA). Crude ash was measured by burning in a muffle furnace at 580 °C for 8 h. NFE content of each diet was calculated on the basis of the assessed chemical composition. The results are expressed in g per 100 g DM.
On the basis of identified chemical composition, ME (kcal/100 g DM) of the foods was calculated, according to the equation provided by the NRC [4 ], using Atwater factors.

The initial 5 days of the 10-day experimental period were allowed for pigs to adapt to the diets. Fecal samples were collected via grab sampling from d 6 to 7. On d 8 and d 10 of this experimental period, Whirl-Pak^® bags (NASCO, Fort Atkinson, WI, USA) containing 10 mL of 10% formic acid were attached to the T-cannulas to collect ileal digesta from 08:30 h to 16:30 h. The attached bags were inspected every 30 min and the filled bags were changed. Fecal and ileal digesta samples were stored at −20 °C immediately after collection. At the end of the collection, samples of fecal and ileal digesta from each pig were thawed and mixed evenly. Then, the fecal samples were dried at 65 °C for 72 h, and the ileal digesta samples were freeze-dried. Experimental diets, ingredients, fecal samples, and ileal digesta samples were ground to pass through a 0.5 mm screen before analysis.
Dry matter (DM) [20 (link)], crude protein (CP) [21 (link)], ether extract (EE; method 996.01; AOAC [22 ]), and ash (method 942.15; AOAC [22 ]) of all samples were analyzed. The neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents of samples were analyzed using an Ankom-220 fiber analyzer with filter bag techniques (F57) based on the procedure offered by the instrument company (method 6 and method 7, ANKOM Technology, Fairport, NY, USA) [23 (link)]. Total carbohydrate (CHO) content was calculated as the following equation: CHO = DM − (CP + EE + ash) [24 (link)]. Starch was determined using the Megazyme Total Starch Assay Procedure (Megazyme International Ireland Ltd., Wicklow, Ireland) based on thermostable α-amylase and amyloglucosidase. The content and composition of soluble non-starch polysaccharides (SNSP) and insoluble non-starch polysaccharides (INSP) were measured by gas chromatography-mass spectrometry (Agilent 7890A-5975C equipped with DB-225 capillary column (30 m × 0.25 mm × 0.25 μm), Agilent J&W Scientific, Santa Clara, CA, USA) according to the method described by Theander et al., [25 (link)] with minor modification in our research group. The content and composition of total non-starch polysaccharides (TNSP) were the sums of the INSP and SNSP contents. Gross energy (GE) was analyzed by an adiabatic bomb calorimeter (model 6400; Parr Instrument, Moline, IL, USA). The concentrations of AA in diets and ileal digesta samples were analyzed (method 982.30 E (a, b); AOAC [22 ]). The method of Fenton and Fenton was used to determine chromic oxide content of diets, ileal digesta, and fecal samples [26 (link)].