In vitro Inoculation of Fresh or Frozen Rumen Fluid Distinguishes Contrasting Microbial Communities and Fermentation Induced by Increasing Forage to Concentrate Ratio

Abstract

In vitro rumen batch culture is a technology to simulate rumen fermentation by inoculating microorganisms from rumen fluids. Although inocula (INO) are commonly derived from fresh rumen fluids, frozen rumen fluids are also employed for the advantages of storing, transporting, and preserving rumen microorganisms. The effects of frozen INO on microbial fermentation and community may be interfered with by substrate type, which has not been reported. This study was designed to test whether rumen fluid treatments (i.e., fresh and frozen) could interact with incubated substrates. A complete block design with fractional arrangement treatment was used to investigate the effects of INO (fresh or frozen rumen fluids) and concentrate-to-forage ratios (C/F, 1:4 or 1:1) on rumen fermentation and microbial community. The effects of increasing C/F were typical, including increased dry matter (DM) degradation and total volatile fatty acids (VFA) concentration (P < 0.001), and decreased acetate to propionate ratio (P = 0.01) and bacterial diversity of richness and evenness (P ≤ 0.005) with especially higher fermentative bacteria such as genus Rikenellaceae_RC, F082, Prevotella, Bacteroidales_BS11, Muribaculaceaege, and Christensenellaceae_R-7 (P ≤ 0.04). Although frozen INO decreased (P < 0.001) DM degradation and altered rumen fermentation with lower (P ≤ 0.01) acetate to propionate ratio and molar proportion of butyrate than fresh INO, typical effects of C/F were independent of INO, as indicated by insignificant INO × C/F interaction on substrate degradation, VFA profile and bacterial community (P ≥ 0.20). In summary, the effect of C/F on fermentation and bacterial diversity is not interfered with by INO type, and frozen INO can be used to distinguish the effect of starch content.

Prokaryote Composition and Structure of Rumen Fluid before and after In Vitro Rumen Fermentation

“…We found similarities in the overall bacterial community estimated by the Bray-Curtis index between two fluid types. Ma et al [26] observed similar results when fresh and frozen rumen fluids were studied for microbial community profiling. It was expected that the DNA of the inactive or dead microorganisms were retained in the fermentation flask in our research.…”

“…The source of rumen fluid and replication of the in vivo conditions in in vitro fermentation are the key components to overcome the challenging steps involved in in vitro rumen fermentation. The source of rumen fluid can be from live animals fed production [24] or maintenance rations [7], rumen fluid from slaughterhouses [25], or frozen rumen fluid [26,27]. Henderson et al [28] reported that the core microbiome in vivo is conserved across geographical regions no matter the species or feed; however, the relative abundances of the microbes vary with the diet and the ruminant species.…”

Forages and pastures symposium: an update on in vitro and in situ experimental techniques for approximation of ruminal fiber degradation

“…In agreement, the frozen treatment without a cryoprotectant seemed to offer the most favorable solution for rank-ordering in the absence of fresh rumen fluid ( Garcia et al, 2021 ). Ma et al (2022 ) compared IV fermentations with fresh and frozen ruminal fluid and the DM digestion was 4 percentage units less for frozen than fresh inocula; however, when the concentrate: forage ratio was 1:4 the DM digestion was 10 percent units less than a 1:1 ratio. Gas production was reduced when frozen inocula were used through the rank order of 1:4 and 1:1 concentrate: forage remained the same.…”