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. 2020 Dec 23:11:618032.
doi: 10.3389/fmicb.2020.618032. eCollection 2020.

Comparison of Two Sampling Techniques for Evaluating Ruminal Fermentation and Microbiota in the Planktonic Phase of Rumen Digesta in Dairy Cows

Camila Flavia de Assis Lage  1 , Susanna Elizabeth Räisänen  1 , Audino Melgar  1 , Krum Nedelkov  1 , Xianjiang Chen  1 , Joonpyo Oh  1 , Molly Elizabeth Fetter  1 , Nagaraju Indugu  2 , Joseph Samuel Bender  2 , Bonnie Vecchiarelli  2 , Meagan Leslie Hennessy  2 , Dipti Pitta  2 , Alexander Nikolov Hristov  1
Affiliations

Comparison of Two Sampling Techniques for Evaluating Ruminal Fermentation and Microbiota in the Planktonic Phase of Rumen Digesta in Dairy Cows

Camila Flavia de Assis Lage et al. Front Microbiol. .

Abstract

The objective of this experiment was to compare ruminal fluid samples collected through rumen cannula (RC) or using an oral stomach tube (ST) for measurement of ruminal fermentation and microbiota variables. Six ruminally cannulated lactating Holstein cows fed a standard diet were used in the study. Rumen samples were collected at 0, 2, 4, 6, 8, and 12 h after the morning feeding on two consecutive days using both RC and ST techniques. Samples were filtered through two layers of cheesecloth and the filtered ruminal fluid was used for further analysis. Compared with RC, ST samples had 7% greater pH; however, the pattern in pH change after feeding was similar between sampling methods. Total volatile fatty acids (VFA), acetate and propionate concentrations in ruminal fluid were on average 23% lower for ST compared with RC. There were no differences between RC and ST in VFA molar proportions (except for isobutyrate), ammonia and dissolved hydrogen (dH2) concentrations, or total protozoa counts, and there were no interactions between sampling technique and time of sampling. Bacterial ASV richness was higher in ST compared with RC samples; however, no differences were observed for Shannon diversity. Based on Permanova analysis, bacterial community composition was influenced by sampling method and there was an interaction between sampling method and time of sampling. A core microbiota comprised of Prevotella, S24-7, unclassified Bacteroidales and unclassified Clostridiales, Butyrivibrio, unclassified Lachnospiraceae, unclassified Ruminococcaceae, Ruminococcus, and Sharpea was present in both ST and RC samples, although their relative abundance varied and was influenced by an interaction between sampling time and sampling method. Overall, our results suggest that ruminal fluid samples collected using ST (at 180 to 200 cm depth) are not representative of rumen pH, absolute values of VFA concentrations, or bacterial communities >2 h post-feeding when compared to samples of ruminal fluid collected using RC. However, ST can be a feasible sampling technique if the purpose is to study molar proportions of VFA, protozoa counts, dH2, and ammonia concentrations.

Keywords: non-invasive sampling techniques; rumen cannula; rumen fermentation; rumen microbiome; stomach tube; stomach tube method.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Effect of sampling techniques (rumen cannula vs stomach tube) on pH in lactating dairy cows (means ± SE; n = 66). Overall sampling method effect, P < 0.001; time of sampling effect, P < 0.001; sampling method × time of sampling interaction, P = 0.66.
FIGURE 2
FIGURE 2
Effect of sampling techniques (rumen cannula vs stomach tube) on total volatile fatty acid (VFA) concentration (mM) in lactating dairy cows (means ± SE; n = 69). Overall sampling method effect, P < 0.001; time of sampling effect, P < 0.001; sampling method × time of sampling interaction, P = 0.44.
FIGURE 3
FIGURE 3
(A–C) Comparison of alpha diversity of liquid fraction from samples collected using ruminal cannula (RC) and stomach tube (ST) at different sampling times relative to feeding [before feeding (hr00); 2 h after feeding (hr02), 4 h after feeding (hr04); 6 h after feeding (hr06); 8 h after feeding (hr08); and 12 h after feeding (hr12)] for (A) Species richness and (B) Shannon diversity (C) Faith’s phylogenetic diversity.
FIGURE 4
FIGURE 4
Comparison of bacterial community composition of liquid fraction from samples collected using stomach tube (ST) and ruminal cannula (RC) at different sampling time [before feeding (hr00); 2 h after feeding (hr02), 4 h after feeding (hr04); 6 h after feeding (hr06); 8 h after feeding (hr08) and 12 h after feeding (hr12)]. The principal coordinate (PC) plots show weighted pairwise UniFrac distances between samples.
See this image and copyright information in PMC

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