Abstract In grazing systems, cattle produce a high molar ratio of acetate, resulting in greater methane production and greater energy loss via gas production. Thus, the objective of this study was to evaluate in vitro gas production and methane concentration using a mineral mixture containing 0.4% HMTBa in a Tifton hay-based substrate. The experiment was carried out under anaerobic conditions using ANKOM Gas Pressure Monitor bottles kept at 39 ºC containing artificial saliva, ruminal fluid, and two grams of a substrate from each treatment: (FE) Control, 1.75 g of Tifton hay and 0.25 g of mineral mixture; and (FM) 1.75 g of Tifton hay and 0.25 g of Suplemax mineral mixture (Suplemento Nutrição Animal) containing 0.4% of HMTBa. Duplicate substrates and duplicate blanks were fermented for each treatment in two runs, with a total of four repetitions per treatment. The accumulated pressure resulting from the fermentation was measured automatically over 48 hours using the Gas Monitor software connected to the ANKOM modules of each bottle. The conversion of pressure to gas volume was calculated as follows: Volume (mL) = 2.3932 + 3.4053 × pressure (R2 = 0.9146; n = 1.151). At the end of each run, a gas sample was collected from each bottle to measure methane concentration using gas chromatography. To obtain the methane production for every 100 mL of gas produced, the total volume of methane produced was multiplied by 100 and divided by the total gas production. Data were analyzed in a completely randomized design using analysis of variance (ANOVA) at α = 0.05. Gas production was not affected by the inclusion of HMTBa (P = 0.3938), with an average of 54.14 mL (Table 1). There was a reduction in methane production from 25.41 to 20.05 mL with the addition of HMTBa (P = 0.0034), representing a decrease of 21.09%. There was a tendency to reduce methane production for each 100 mL of gas produced from 46.01 to 37.86 mL (P = 0.0889). Although HMTBa reduces the molar proportion of propionate, it is considered to be one of the main hydrogen users. In environments with a high concentration of H2 (above 1 mmol) and the presence of an anti-methanogen, the most favored path for using hydrogen is acetogenesis, which reduces methane production. Thus, the use of a mineral mixture containing 0.4% HMTBa in a Tifton-hay-based substrate reduced methane production in an in vitro assay.
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