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J Anim Sci Biotechnol. 2016 Jun 14;7:35. doi: 10.1186/s40104-016-0094-8. eCollection 2016.

Methodological factors affecting gas and methane production during in vitro rumen fermentation evaluated by meta-analysis approach.

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Department of Comparative Biomedicine and Food Science (BCA), University of Padova, Viale dell'Università 16, 35020 Legnaro (PD), Italy.
Department of Agronomy, Food, Natural resources, Animals and Environment (DAFNAE), University of Padova, Viale dell'Università 16, 35020 Legnaro (PD), Italy.


Effects of some methodological factors on in vitro measures of gas production (GP, mL/g DM), CH4 production (mL/g DM) and proportion (% CH4 on total GP) were investigated by meta-analysis. These factors were considered: pressure in the GP equipment (0 = constant; 1 = increasing), incubation time (0 = 24; 1 = ≥ 48 h), time of rumen fluid collection (0 = before feeding; 1 = after feeding of donor animals), donor species of rumen fluid (0 = sheep; 1 = bovine), presence of N in the buffer solution (0 = presence; 1 = absence), and ratio between amount of buffered rumen fluid and feed sample (BRF/FS; 0 = ≤ 130 mL/g DM; 1 = 130-140 mL/g DM; 2 = ≥ 140 mL/g DM). The NDF content of feed sample incubated (NDF) was considered as a continuous variable. From an initial database of 105 papers, 58 were discarded because one of the above-mentioned factors was not stated. After discarding 17 papers, the final dataset comprised 30 papers (339 observations). A preliminary mixed model analysis was carried out on experimental data considering the study as random factor. Variables adjusted for study effect were analyzed using a backward stepwise analysis including the above-mentioned variables. The analysis showed that the extension of incubation time and reduction of NDF increased GP and CH4 values. Values of GP and CH4 also increased when rumen fluid was collected after feeding compared to before feeding (+26.4 and +9.0 mL/g DM, for GP and CH4), from bovine compared to sheep (+32.8 and +5.2 mL/g DM, for GP and CH4), and when the buffer solution did not contain N (+24.7 and +6.7 mL/g DM for GP and CH4). The increase of BRF/FS ratio enhanced GP and CH4 production (+7.7 and +3.3 mL/g DM per each class of increase, respectively). In vitro techniques for measuring GP and CH4 production are mostly used as screening methods, thus a full standardization of such techniques is not feasible. However, a greater harmonization of analytical procedures (i.e., a reduction in the number of available protocols) would be useful to facilitate comparison between results of different experiments.


Experimental factors; Gas production; In vitro rumen fermentation, Meta-analysis, Methane production

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