Influence of rumen contents’ processing method on microbial populations in the fluid and subsequent in vitro fermentation of substrates of variable composition

• Three methods of processing ruminal contents to obtain ruminal fluid were compared.
• Stomaching increased the cellulolytic bacteria in the fluid, but decreased protozoa.
• There were no differences between filtering through cheesecloth or 100-μm cloth.
• Most 24-h in vitro fermentation parameters were not influenced by processing method.

The in vitro batch culture technique is being increasingly used to study rumen fermentation, but the results are affected by several factors, being the source of the inoculum one of the most relevant. This work was conducted to assess the effects of different processing methods of ruminal contents on microbial populations in the obtained fluid, and its influence on fermentation parameters when the fluid was used as inoculum for in vitro incubations. Rumen contents were obtained from four rumen-fistulated sheep fed a 2:1 alfalfa hay:concentrate diet and subjected to the following treatments: SQ: squeezed through four layers of cheesecloth; FL: SQ treatment and further filtration through a 100-μm nylon cloth; STO: blended for three min at 230 rev min−1 in a Stomacher® and further filtrations as in SQ. Microbial populations’ abundance and bacterial diversity in the ruminal fluids were analysed by quantitative PCR (qPCR) and automated ribosomal intergenic spacer analysis (ARISA), respectively. Three forages (alfalfa hay, grass hay and barley straw) were incubated in vitro, either alone or mixed with concentrate (1:1), using each of the ruminal fluids as inoculum. There were no differences between SQ and FL methods in any of the microbial populations analysed, but STO increased the relative abundance of Fibrobacter succinogenes and Ruminococcus albus (P < 0.05) and decreased (P < 0.05) the concentration of protozoal DNA compared with SQ. Bacterial diversity was not affected (P > 0.05) by the processing methods. There were no interactions (P > 0.05) between the processing method and the characteristics of the substrates for any of the fermentation parameters analysed. Compared with SQ, the STO method resulted in greater (P < 0.05) methane production and ammonia-N concentrations in 8 h incubations. After 24 h of incubation, the use of STO inoculum increased (P < 0.05) methane production and dry matter degradability compared with SQ, with no differences in the rest of the parameters. No differences between SQ and FL methods were detected in any parameter. The results show that stomaching the rumen contents prior to inoculation of in vitro cultures modified some microbial populations, but had only subtle effects on fermentation parameters.