The Type of Forage Substrate Preparation Included as Substrate in a RUSITEC System Affects the Ruminal Microbiota and Fermentation Characteristics

Abstract

In vitro fermentation systems such as the rumen simulation technique (RUSITEC) are frequently used to assess dietary manipulations in livestock, thereby limiting the use of live animals. Despite being in use for nearly 40 years, improvements are continually sought in these systems to better reflect and mimic natural processes in ruminants. The aim of this study was to evaluate the effect of forage preparation, i.e., frozen minced (FM) and freeze-dried and ground (FDG), on the ruminal microbiota and on fermentation characteristics when included as a substrate in a RUSITEC system. A completely randomized design experiment was performed over a 15-day period, with 7 days of adaptation and an 8-day experimental period. Fermentation parameters (total gas, CH4, and volatile fatty acid production) were analyzed on a daily basis over the experimental period and the archaeal and bacterial microbiota (liquid-associated microbes [LAM] and solid-associated microbes [SAM] was assessed at 0, 5, 10, and 15 days using high-throughput sequencing of the 16S rRNA gene. Results from this study suggested a tendency (P = 0.09) of FM treatment to increase daily CH4 (mg/d) production by 16.7% when compared with FDG treatment. Of the major volatile fatty acids (acetate, propionate, and butyrate), only butyrate production was greater (P = 0.01) with FM treatment compared with FDG substrate. The archaeal and bacterial diversity and richness did not differ between the forage preparations, although feed particle size of the forage had a significant effect on microbial community structure in the SAM and LAM samples. The Bacteroidetes phylum was more relatively abundant in the FM substrate treatment, while Proteobacteria was enriched in the FDG treatment. At the genus-level, Butyrivibrio, Prevotella, and Roseburia were enriched in the FM substrate treatment and Campylobacter and Lactobacillus in the FDG substrate treatment. Evidence from this study suggests that forage preparation affects CH4 production, butyrate production, and the structure of the rumen microbiota during in vitro fermentation.

The application of rumen simulation technique (RUSITEC) for studying dynamics of the bacterial community and metabolome in rumen fluid and the effects of a challenge with Clostridium perfringens

“…To distinguish whether the observed shifts are linked to the diet change between animal and RUSITEC or directly related to the in vitro system further studies are required comparing samples from donor animals and the RUSITEC receiving the same diet. Here, the overall bacterial community structure within the current RUSITEC experiment did not exhibit statistically significant changes along the time line, which is in contrast to the study by Duarte et al [ 11 ]. In our study, the putative loss of lowly abundant phylotypes is reflected by a shift in the unweighted UniFrac analysis, whereas no clear clustering of sampling days could be detected in the weighted UniFrac analysis.…”

“…Bacteroidetes and Firmicutes were the predominant phyla detected in the RUSITEC samples, which is in agreement with the studies by Duarte et al [ 11 , 38 ], most in vivo studies [ 39 – 42 ] and the native rumen fluid samples of the two donor cows. The high overlap of OTUs found in the RUSITEC samples and in the native rumen fluid samples indicates a high degree of stability of rumen fluid bacteria in the RUSITEC system.…”

“…Therefore, several fingerprint methods and qPCR have been intensively used for comparing bacterial communities within the RUSITEC system [ 28 , 36 , 37 ]. Belanche et al [ 9 , 10 ] were the first, who used next generation sequencing methods to evaluate the impact of dietary supplementation in the RUSITEC system and Duarte and colleagues [ 11 ] detected a shift in the microbiome between day 5 and 10 of a RUSITEC trial by using next generation sequencing. Our study investigated the stability of the bacterial community structure as well as its metabolome in the RUSITEC system along the time line in shorter intervals then in the latter studies.…”

“…Belanche and colleagues were the first, who used next generation sequencing methods to evaluate the impact of dietary supplementation in the RUSITEC system [ 9 , 10 ]. Recently, Duarte and colleagues [ 11 ] reported an effect of the sampling day on the liquid-associated microbiota in the RUSITEC using Illumina sequencing.…”

Fatty acid profiling in animal feeds and related food matrixes using a fast GC/MS method and in situ derivatization

“…In turn, the type of substrate modifies the rumen itself and its fermentation characteristics (Duarte et al, 2017). Microorganisms such as bacteria, fungi, and protozoa can degrade complex structures (e.g., forages) and free utilizable nutrients (NRC, 2001;FEDNA, 2009;Duarte et al, 2017). After rumen-mediated lipolysis (where metabolisms of long-chained fatty acids and hydrogenation of unsaturated fatty acids take place), fat biohydrogenation takes place, a process responsible for the concentration and proportion of fatty acids in tissue and milk (Woods and Fearon, 2009;Castillo et al, 2013).…”