Adaptive responses in short-chain fatty acid absorption, gene expression, and bacterial community of the bovine rumen epithelium recovered from a continuous or transient high-grain feeding

Abstract

The feeding of high-grain diets to dairy cows commonly results in lowered pH and ruminal dysbiosis, characterized by changes in absorption dynamics of short-chain fatty acids (SCFA) across the reticuloruminal wall, epithelial function, and the epithelial bacteria community structure. Therefore, the present study evaluated the effect of high-grain feeding persistence on the absorption kinetics of reticuloruminal SCFA, gene expression in the rumen epithelium, and the associated shifts in the epithelial bacteria in cows recovering from either a long-term continuous high-grain feeding model or a long-term transient high-grain feeding model. In a crossover study design, 8 nonlactating Holstein cows were fed 60% concentrate either continuously for 4 wk (continuous) or with a 1-wk break in the second week of the high-grain feeding (transient). After the high-grain feeding, all animals were fed a diet of 100% forage (recovery) for an additional 8 wk. Rumen papilla biopsies and SCFA absorption measurements were taken at the start of the trial (baseline), after the 4-wk high-grain feeding (49 d), after 2-wk recovery forage feeding (63 d), and after 8-wk recovery forage (105 d). Absorption of SCFA was determined in vivo using the washed and isolated reticulorumen technique. Rumen papillae biopsies were used for adherent bacterial DNA and host RNA extraction. The epithelial bacteria were determined using Illumina MiSeq (Microsynth AG, Balgach, Switzerland) sequencing of the 16S rRNA gene. No significant effects of the high-grain feeding model were seen for bacterial diversity. However, bacterial diversity increased with time spent in the recovery forage feeding period regardless of feeding model. The relative abundance of Acidobacteria phyla and Acetivibrio spp. increased when animals were fed a transient high-grain feeding model. A trend toward increased CLDN4 expression was observed in the continuous model. Furthermore, there were interactions between feeding model and sampling day for gene targets CD14, DRA, NHE2, NHE3, and MCT2. When comparing length of recovery, in the continuous model increased relative absorption of SCFA was sustained at 63 d but dropped to baseline measurements at 105 d. A similar pattern was found with the transient model but it did not reach significance. The only gene target that was found to significantly correlate to relative absorption of SCFA was DRA (correlation coefficient ≤ −0.41). Whereas, genera Alkalibaculum, Anaerorhabdus, Coprococcus, and Dethiobacter all showed positive correlations to gene targets for pH regulation (NHE2 and NHE3) and SCFA uptake (MCT1) but negative correlations to SCFA absorption. We conclude that while the rumen absorption and epithelial bacteria were able to recover to baseline levels after 8 wk of forage feeding, the time needed for re-establishment of homeostasis in host gene expression is longer, especially when high-grain feeding is interrupted.

Feed Additives Differentially Impact the Epimural Microbiota and Host Epithelial Gene Expression of the Bovine Rumen Fed Diets Rich in Concentrates

“…We noted that the feeding of these feed additives reduced rumen and fecal associated toxins including biogenic amines and lipopolysaccharide (Humer et al, 2018). However, previous research has also shown that despite the changes of the digesta associated microbiota, the epithelial-associated microbe population can remain stable (Petri et al, 2013), and have also been shown to be correlated to rumen papillae gene expression associated with short-chain fatty acids (SCFA) absorption and pH regulation (Petri et al, 2019). However, the impact of feed additives on epithelial microbiota, host physiology, and the associations between the host and microbes to rumen endotoxin levels under SARA conditions remains largely unknown.…”

“…For RNA isolation, the method previously described by Petri et al (2019) was used. Briefly, 20 mg of papillae from each animal were combined with Lysis buffer (RNease Mini QIAcube kit, Qiagen, Hilden, Germany) and autoclaved ceramic beads (0.6 g; 1.4 mm; VWR), and homogenized 6.5 ms-1 for 30 s in a FastPrep-24 instrument (MP Biomedicals, Santa Ana, CA, United States).…”

“…The mean RNA integrity (RIN) value for all samples was 7.9 ± 0.67, with two samples having RIN values below 7.0 (5.6 and 6.2), and the other samples ranging between 7.0 and 9.2. Complementary DNA (cDNA) was synthesized (High Capacity cDNA RT kit, Life Technologies Limited, Vienna, Austria) from 2 µg RNA in duplicate using a 2-step PCR program (Mastercycler nexus, Eppendorf, Hamburg, Germany) according to the previously published method (Petri et al, 2019), using an incubation at 25 • C for 10 min, reverse transcription at 37 • C for 120 min, and a final heating step at 85 • for 5 min. Reverse transcription controls (1 µl × 1 µl) were included as a control for residual DNA contamination.…”

“…For gene expression analysis, qPCR reactions were conducted using the following thermal program: at 95 • C for 5 min, and 40 cycles of 95 • C for 10s melting, 60 • C for 30 s annealing, and 72 • C for 30 s final elongation (Mx3000P thermocycler, Agilent Technologies). Melting curve analysis was completed to determine primer specificity (Petri et al, 2019). The primers used for the RT-qPCR are listed in the Table 1, including their sequences and efficiencies.…”

“…As reference genes beta actin (ACTB), glyceraldehyde−3−phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1), ornithine decarboxylase antizyme 1 (OAZ1), and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) were considered. The amplicons of all primers were verified with PrimerBLAST 5 , dissociation curves were used to test for efficiency and specificity (Neubauer et al, 2019;Petri et al, 2019) and an additional dissociation stage was performed to verify the presence of a single PCR product. All reactions were run in duplicate and repeated when the cycle difference was more than 0.5 cycles between duplicates.…”