In this meta-analysis, 17 rumen epithelial 16S rRNA gene Illumina MiSeq amplicon sequencing data sets were analyzed to identify a core rumen epithelial microbiota and core rumen epithelial OTUs shared between the different studies included. Sequences were quality-filtered and screened for chimeric sequences before performing closed-reference 97% OTU clustering, and de novo 97% OTU clustering. Closed-reference OTU clustering identified the core rumen epithelial OTUs, defined as any OTU present in ≥ 80% of the samples, while the de novo data was randomly subsampled to 10,000 reads per sample to generate phylum- and genus-level distributions and beta diversity metrics. 57 core rumen epithelial OTUs were identified including metabolically important taxa such as Ruminococcus, Butyrivibrio, and other Lachnospiraceae, as well as sulfate-reducing bacteria Desulfobulbus and Desulfovibrio. Two Betaproteobacteria OTUs (Neisseriaceae and Burkholderiaceae) were core rumen epithelial OTUs, in contrast to rumen content where previous literature indicates they are rarely found. Two core OTUs were identified as the methanogenic archaea Methanobrevibacter and Methanomethylophilaceae. These core OTUs are consistently present across the many variables between studies which include different host species, geographic region, diet, age, farm management practice, time of year, hypervariable region sequenced, and more. When considering only cattle samples, the number of core rumen epithelial OTUs expands to 147, highlighting the increased similarity within host species despite geographical location and other variables. De novo OTU clustering revealed highly similar rumen epithelial communities, predominated by Firmicutes, Bacteroidetes, and Proteobacteria at the phylum level which comprised 79.7% of subsampled sequences. The 15 most abundant genera represented an average of 54.5% of sequences in each individual study. These abundant taxa broadly overlap with the core rumen epithelial OTUs, with the exception of Prevotellaceae which were abundant, but not identified within the core OTUs. Our results describe the core and abundant bacteria found in the rumen epithelial environment and will serve as a basis to better understand the composition and function of rumen epithelial communities.

Forty sows (PIC Camborough 1050) from a single farm were randomly selected at 112 days of gestation to evaluate if gut bacteria transverse the blood system of the sow to deposit gut microbiota into colostrum for piglet gut inoculation via the entero-mammary pathway. Fourteen first-parity gilts and twenty third-parity sows were used for the study. At the time of farrowing, colostrum, fecal samples and blood samples were collected to evaluate the presence of bacteria in each sample. Colostrum and blood samples were processed via centrifugation to separate the immune cell fraction. Total deoxyribonucleic acid (DNA) was extracted from fecal, colostrum and white blood cell fractions. 16S ribosomal ribonucleic acid (rRNA) gene amplicon sequencing was conducted at the Iowa State University DNA Facility (Ames, IA) to further characterize the bacterial and archaeal taxa present within each sample. Data were analyzed using Mothur and using R v4.0.3 (R Core Team, 2020). The experimental unit was the sow. Tables were generated to demonstrate the relative abundances of bacteria and archaea present in each type of sample and also identify organisms differentially abundant between sample types. Firmicutes was the most abundant phylum in colostrum and fecal samples and Tenericutes had the greatest abundance in blood comparative to other phyla. Further evaluation of the classification of bacteria present demonstrated that a few genera of bacteria are present in all three samples. Clostridum_sensu_stricto 1 was present in high relative abundance in colostrum and in moderate abundance in the feces while also being present within the blood. Other genera present in all three sample types includes Ruminococcus and Mycoplasma. In conclusion, the data suggest that there are bacteria present in all three locations of the sow at the time of farrowing and that first parity sows have different microbial populations than third parity sows.

The products of rumen microbial fermentations are considered essential for animal growth and performance. Changes in these microbial communities can have major effects on animal growth and performance. Saccharin-based artificial sweeteners can be included in livestock diets to increase palatability and encourage feed intake. Despite the importance of the rumen microbial fermentation, little or no research is available regarding how saccharin-based artificial sweeteners affect rumen content and rumen epithelial microbial communities. The aim of this study was to identify changes in both the rumen content and rumen epithelial microbial communities in response to the supplementation of Sucram®, a sodium-saccharin-based sweetener (Pancosma S.A./ADM Groups, Rolle, Switzerland) during standard, non-stress conditions using 16SrRNA gene amplicon sequencing.The rumen epithelial and rumen content microbiota of five Holstein-Friesian milking dairy cattle were compared before (baseline, BL) and after a 28-day supplementation of Sucram®. Illumina MiSeq-based 16S rRNA gene sequencing was conducted, and community analysis revealed significant changes in the abundance of specific phylotypes when comparing BL to Sucram® experimental groups. Sucram® did not have a significant effect on overall rumen microbial community structure between experimental groups. Statistically significant changes in microbial community composition following Sucram® supplementation were observed most consistently across a number of bacterial taxa in the rumen epithelium, while fewer changes were seen in the rumen content. Predicted genomic potentials of several significantly different OTUs were mined for genes related to feed efficiency and saccharin degradation. Operational taxonomic units (OTUs) classified as Prevotella and Sharpea were significantly (p<0.05) increased in samples supplemented with Sucram®, whereas a reduction in abundance was seen for OTUs classified as Treponema, Leptospiraceae, Ruminococcus and methanogenic archaea. This is the first study to report an effect of Sucram® on ruminant microbial communities, suggesting possible beneficial impacts of Sucram® on animal health and performance that may extend beyond increasing feed palatability.

The effect of a saccharin-based artificial sweetener was tested on animal performance measures and on the microbial communities associated with the rumen content and with the rumen epithelium during heat stress. Ten cannulated Holstein-Friesian milking dairy cattle were supplemented with 2 grams of saccharin-based sweetener per day, top-dressed into individual feeders for a 7-day adaptation period followed by a 14-day heat stress period. A control group of ten additional cows subjected to the same environmental conditions but not supplemented with sweetener were included for comparison. 16S rRNA gene amplicon sequencing was performed on rumen content and rumen epithelium samples from all animals, and comparisons of rumen content microbiota and rumen epithelial microbiota were made between supplemented and control populations. Supplementation of the saccharin-based sweetener did not affect the rumen content microbiota, but differences in the rumen epithelial microbiota beta-diversity (PERMANOVA, P = 0.003, R 2 = 0.12) and alpha-diversity (Chao species richness, P = 0.06 and Shannon diversity, P = 0.034) were detected between the supplemented and control experimental groups. Despite the changes detected in the microbial community, animal performance metrics including feed intake, milk yield, and short-chain fatty acid (acetic, propionic, and butyric acid) concentrations were not different between experimental groups. Thus, under the conditions applied, supplementation with a saccharin-based sweetener does not appear to affect animal performance under heat stress. Additionally, we detected differences in the rumen epithelial microbiota due to heat stress when comparing initial, pre-stressed microbial communities to the communities after heat stress. Importantly, the changes occurring in the rumen epithelial microbiota may have implications on barrier integrity, oxygen scavenging, and urease activity. This research adds insight into the impact of saccharin-based sweeteners on the rumen microbiota and the responsivity of the rumen epithelial microbiota to different stimuli, providing novel hypotheses for future research.

The objective was to determine the impact of feeding MCE on ruminal and intestinal morphology and microbiota composition of calves. A total of 10 male and 10 female crossbred (dairy × beef) calves (6 d of age) were assigned randomly to control (CTL; n = 10) or MCE-supplemented (TRT; n = 10) groups. The MCE was fed in the milk replacer and top-dressed on the calf starter during pre-weaning (6 to 49 d) and post-weaning (50 to 95 d) periods, respectively. Calves were slaughtered at 95 d to collect rumen and intestinal samples to determine volatile fatty acid (VFA) profile, mucosal morphology, and microbiota composition. The effects of MCE were analyzed by accounting for the sex and breed effects. Feeding MCE increased rumen papillae length (p = 0.010) and intestinal villus height: crypt depth (p < 0.030) compared to CTL but did not affect rumen VFA profile. The TRT had a negligible impact on microbial community composition in both the rumen and the jejunum. In conclusion, feeding MCE from birth through weaning can improve ruminal and small intestinal mucosa development of calves despite the negligible microbiota composition changes observed post-weaning.

The aim of this pilot study was to identify changes in both the rumen content and rumen epithelial microbiota in response to the supplementation of Sucram ® , a sodium-saccharin-based sweetener (Pancosma S.A./ADM Groups, Rolle, Switzerland). Rumen microbial communities are essential for animal growth and performance, and changes in these communities can have major effects on these parameters. Little or no research is available regarding how saccharin-based artificial sweeteners, fed to cattle in attempts to increase palatability and encourage feed intake, affect rumen microbial communities. The rumen epithelium and rumen content microbiota of five lactating Holstein-Friesian dairy cattle were compared before (baseline, BL) and after a 28-day supplementation of Sucram ® using Illumina MiSeq-based 16S rRNA gene sequencing. After supplementation of Sucram ® , significant changes in the abundance of specific taxa were detected: an increase in Prevotella and Sharpea species, a decrease in Treponema , Leptospiraceae , Ruminococcus and methanogenic archaea (p<0.05), but Sucram® did not affect the overall rumen microbial community structure. This is the first study to report an effect of Sucram ® on ruminant microbial communities.

Forty sows (PIC Camborough 1050) from a single farm were randomly selected at 112 d of gestation to evaluate if gut bacteria transverse the blood system of the sow to deposit gut microbiota into colostrum for piglet gut inoculation via the entero-mammary pathway. Fourteen first-parity gilts and twenty third-parity sows were used for the study. At the time of farrowing, colostrum, fecal samples and blood samples were collected to evaluate the presence of bacteria in each sample. Colostrum and blood samples were processed via centrifugation to separate the immune cell fraction. Total deoxyribonucleic acid (DNA) was extracted from fecal, colostrum and white blood cell fractions. 16S ribosomal ribonucleic acid (rRNA) gene amplicon sequencing was conducted at the Iowa State University DNA Facility (Ames, IA) to further characterize the bacterial and archaeal taxa present within each sample. Data were analyzed using Mothur and using R v4.0.3 (R Core Team, 2020). The experimental unit was the female. Tables were generated to demonstrate the relative abundances of bacteria and archaea present in each type of sample and also identify organisms differentially abundant between sample types. Firmicutes was the most abundant phylum in colostrum and fecal samples and Tenericutes had the greatest abundance in blood comparative to other phyla. Further evaluation of the classification of bacteria present demonstrated that a few genera of bacteria are present in all 3 samples. Clostridum_sensu_stricto 1 was present in high relative abundance in colostrum and in moderate abundance in the feces while also being present within the blood. Other genera present in all 3 sample types includes Ruminococcus and Mycoplasma. In conclusion, the data suggests that there are bacteria present in all 3 locations of the sow at the time of farrowing and that gilts have different microbial populations than sows.

Sub-Acute Ruminal Acidosis (SARA) is a metabolic disorder in dairy cattle characterized by a lowered ruminal pH, as a result of high grain diets. Dairy cows experiencing SARA show decreased milk production and impaired health; thus, SARA has major economic impacts. SARA has been associated with changes in the rumen epithelial microbiota. Previously, we performed meta-transcriptome sequencing on rumen epithelial biopsy samples from 3 fistulated Holstein cows before (baseline) and after switching to high concentrate feed to induce SARA to analyze the gene expression of the rumen epithelial microbiota. In this previous analysis, 1,607 features were detected using the KEGG Ontology reference database, including a wide variety of metabolic genes, as well as stress response, motility, and other functions. However, only 3 features were differentially expressed between baseline and SARA conditions using this approach. Here, we present a re-analysis of the meta-transcriptomics data using an improved bioinformatics pipeline to reveal additional genetic diversity and differentially expressed genes. We performed a de novo transcriptome co-assembly with Trinity, mapped reads to the assembled contigs, used RSEM to determine read counts, and DESEQ2 for detection of differentially expressed genes (Q &lt; 0.05). Features were annotated using EggNOG, providing taxonomic predictions and GO, COG, and KEGG-based functional prediction. Using this method, a total of 76,861 transcripts were assembled, and 4,935 genes were differentially expressed between baseline and SARA conditions, representing a substantial improvement over the previous analysis. Our preliminary analysis indicates high expression levels of central metabolism and housekeeping genes, as well as oxidative stress response genes, and outer membrane proteins among rumen epithelial bacteria. Differentially expressed archaeal genes were primarily upregulated under SARA conditions and included genes involved in methanogenesis. Further analyses are ongoing and will provide insight into gene expression of the rumen epithelial microbiota, and their association with SARA.