Influence of main dietary chemical constituents on the in vitro gas and methane production in diets for dairy cows

Incubation

The 7 diets were incubated in 4 repeated incubation runs, conducted in 4 successive wk. Two incubations were stopped at 24 h, whereas the other two were stopped after 48 h. The incubation times of 24 and 48 h were chosen as they are, respectively, the reference times for measuring in vitro GP [17] and in vitro degradability of NDF [18]. In each of the four incubation runs, we tested 7 diets?×?5 replications, plus 5 blanks (bottles containing only the buffered rumen fluid; 5 blanks/run), for a total of 160 bottles incubated. A commercial GP apparatus (Ankom^RF Gas Production System, Ankom Technology®, NY, USA) was used, consisting of 40 bottles equipped with pressure sensors (pressure range: from – 69 to 3,447 kPa; resolution: 0.27 kPa; accuracy: ± 0.1 % of measured value) and wireless connected to a computer. Each bottle (317 mL) was filled with 1.000?±?0.0010 g of diet, 100 mL of a buffer solution, and 50 mL of rumen fluid (headspace volume?=?167 mL), keeping the headspace of bottles flushed with CO₂.

The buffer solution was prepared according to [17], heated in a water bath at 39?±?0.4 °C and purged continuously with CO₂ for 30 min, to maintain anaerobic conditions. Rumen fluid was collected by an esophageal probe, as described by [19], 2 h before morning feeding from 3 dry Holstein-Friesian cows housed at the experimental farm of the University of Padova (Italy) and fed hay ad libitum and 2.5 kg/d of concentrates (0.5 kg of dry sugar beet pulp, 1 kg of corn grain, and 1 kg of sunflower meal). During the collection of rumen fluid, cows were handled according to the Italian law on animal care (Legislative Decree No. 26 of March 14, 2014). Rumen fluid was poured into thermal flasks preheated to 39?±?0.5 °C, immediately transferred to the laboratory, strained through 3 layers of cheesecloth, to eliminate feed particles, and mixed with buffer solution in a 1 to 2 ratio [17]. Operations were conducted under anaerobic conditions, by flushing with CO₂, and required less than 30 min to be completed. Bottles were placed in a ventilated oven at 39?±?0.4 °C and automatically vented at a fixed pressure (6.8 kPa), to avoid overpressure conditions and alterations of gas and CH₄ measures [20]. In vitro GP was monitored in continuous, using a setting of the GP system that allows to record pressure values every minute. Other in vitro parameters (rumen degradability, VFA and N-NH₃ concentrations, CH₄ production) were measured only at the end of incubation (at 24 or 48 h), to avoid opening of the oven during the incubation, with alteration of fermentation process.

At the end of incubations (24 or 48 h), two aliquots (5 mL) of fermentation fluid were collected from each bottle and stored at ?20 °C with 1 mL of metaphosphoric acid (25 %, w/v) to be later analyzed for ammonia N and volatile fatty acids (VFA). The content of ammonia N was measured using the FIAstar™ 5000 Analyzer (FOSS Analytical, Hilleroed, Denmark). The VFA profile was analyzed by GC with flame ionization detection (7820A GC system, Agilent Technologies, Milan, Italy) using a 30-m stainless steel column (JW DB-FFAP, Agilent Technologies, Milan, Italy) and H₂ as carrier gas (flow rate: 30 mL/min; isothermal oven temperature: 150 °C). Fermentation fluids were filtered into weighed crucibles (30 mL, Robu Glasfilter-Geräte GMBH®, Hattert, Germany) and analyzed for residual NDF using a Fibretech Analyzer (VELP® Scientifica, Milan, Italy).

At the end of each incubation (24 or 48 h), gas was collected with a 10-mL gas-tight syringe (Artsana S.p.A., Como, Italy) from the bottle headspace (HS). At each sampling, the syringe was flushed in order to collect a homogeneous sample, which was immediately transferred into a 9-mL vacuette (Greiner Bio-One GmbH, Kremsmunster, Austria). From each vacuette, an aliquot (10 ?L) of gas was sampled with a gas-tight syringe (1701 N, Hamilton, Bonaduz, Switzerland) and immediately analyzed for CH₄ concentration by GC with flame ionization detection (7820A GC system, Agilent Technologies, Milan, Italy) using a 15-m carbon layer column (GS-CarbonPLOT, Agilent Technologies, Milan, Italy) and H₂ as carrier gas (flow rate: 1.6 mL/min; isothermal oven temperature: 40 °C). An 11-point calibration curve was generated from eleven gas mixtures containing 2, 4, 8, 16, 24, 32, 60, 100, 140, 180, and 240 mL of CH₄/L (99.5 % pure, SAPIO s.r.l., Monza, Italy), respectively, and known volumes of air. Mixtures were prepared using the same graduated gas-tight syringe (1701 N, Hamilton). The calibration regression had R²??0.99.