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,Meagan Leslie Hennessy,Dipti Pitta,Molly Elizabeth Fetter,Alexander Nikolov Hristov,Nagaraju Indugu,Joseph Samuel Bender,Bonnie Vecchiarelli,Krum Nedelkov,Audino Melgar,Xianjiang Chen,Joonpyo Oh,Susanna Elizabeth Räisänen

doi:10.3389/fmicb.2020.618032

Camila Flavia De Assis Lage, Meagan Leslie Hennessy + Show 11 more

Open Access

https://doi.org/10.3389/fmicb.2020.618032

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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.

Highlights

Sampling and analysis of ruminal fluid are important tools in ruminant nutrition research and in addition to studies focused on ruminal fermentation, there is an increasing interest in understanding ruminal microbiota
Ruminal fluid collected through stomach tubing (ST) had a pH on average 0.47 points greater (P < 0.001, Table 1) than ruminal fluid collected through Rumen cannulation (RC), and the difference between the two sampling techniques persisted throughout the course of sampling (Figure 1)
No differences in molar proportions of volatile fatty acids (VFA) were observed between the two sampling techniques, except for a slightly greater (P = 0.03) proportion of isobutyrate in ruminal fluid collected via ST in comparison to RC

Summary

INTRODUCTION

Sampling and analysis of ruminal fluid are important tools in ruminant nutrition research and in addition to studies focused on ruminal fermentation, there is an increasing interest in understanding ruminal microbiota. Ramos-Morales et al (2014) compared samplings using ST as an alternative to RC and observed similarities in rumen microbiota between techniques These authors, reported differences in microorganism distribution pre- and post-feeding. Stomach tubing recovers samples mostly from the planktonic phase with small finely digested particles and does not permit sampling from different sites within the rumen, and samples acquired using this method may have a different distribution of microbial communities compared to rumen contents collected via cannula (Firkins and Yu, 2015). The objective of this study was to compare rumen fermentation parameters and ruminal microbiota composition between RC and ST techniques and to investigate if time post-feeding can affect differences between the two techniques. We hypothesized that distribution of ruminal microbiota and fermentation patterns would differ between sampling techniques and these differences may be affected by sampling time post-feeding

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