Samodha C. Fernando

Rumen Bacterial Community Composition in Holstein and Jersey Cows Is Different under Same Dietary Condition and Is Not Affected by Sampling Method

The rumen microbial community in dairy cows plays a critical role in efficient milk production. However, there is a lack of data comparing the composition of the rumen bacterial community of the main dairy breeds. This study utilizes 16S rRNA gene sequencing to describe the rumen bacterial community composition in Holstein and Jersey cows fed the same diet by sampling the rumen microbiota via the rumen cannula (Holstein cows) or esophageal tubing (both Holstein and Jersey cows). After collection of the rumen sample via esophageal tubing, particles attached to the strainer were added to the sample to ensure representative sampling of both the liquid and solid fraction of the rumen contents. Alpha diversity metrics, Chao1 and observed OTUs estimates, displayed higher (P = 0.02) bacterial richness in Holstein compared to Jersey cows and no difference (P > 0.70) in bacterial community richness due to sampling method. The principal coordinate analysis displayed distinct clustering of bacterial communities by breed suggesting that Holstein and Jersey cows harbor different rumen bacterial communities. Family level classification of most abundant (>1%) differential OTUs displayed that OTUs from the bacterial families Lachnospiraceae and p-2534-18B5 to be predominant in Holstein cows compared to Jersey cows. Additionally, OTUs belonging to family Prevotellaceae were differentially abundant in the two breeds. Overall, the results from this study suggest that the bacterial community between Holstein and Jersey cows differ and that esophageal tubing with collection of feed particles associated with the strainer provides a representative rumen sample similar to a sample collected via the rumen cannula. Thus, in future studies esophageal tubing with addition of retained particles can be used to collect rumen samples reducing the cost of cannulation and increasing the number of animals used in microbiome investigations, thus increasing the statistical power of rumen microbial community evaluations.

In vivo determination of rumen undegradable protein of dried distillers grains with solubles and evaluation of duodenal microbial crude protein flow

The objectives of this trial were to determine the rumen undegradable protein (RUP) of dried distillers grains with solubles (DDGS), to compare the estimates of duodenal bacterial CP (BCP) flow using diaminopumelic acid (DAPA) or DNA as bacterial markers, and to estimate duodenal protozoal CP (PCP) and yeast CP (YCP) flow when DDGS are fed. Three crossbred steers fitted with ruminal and double L-shaped duodenal cannulae (average BW 780 ± 137 kg) were used in a 3 treatment, 6 period crossover design. Animals were housed in individual free stalls and fed twice daily at 0700 and 1900 h. Diets (DM basis) were 1) CONTROL, which is 0% DDGS but with 19.5% corn bran, 20% sorghum silage, 60% brome hay, 0.5% trace minerals, and 0.25% urea, 2) LOW DDGS, which is inclusion of 9.75% DDGS replacing equal percentage of corn bran, and 3) HIGH DDGS, which is inclusion of 19.5% DDGS completely replacing corn bran. Duodenal BCP flow was estimated using DAPA and DNA as bacterial markers. In addition, duodenal PCP and YCP flow were estimated using DNA markers. The value of DDGS RUP as a percent of CP was determined to be 63.0 ± 0.64%. Estimates of duodenal BCP flow using DAPA were 473, 393, and 357 ± 78 g/d (P = 0.09) for CONTROL, LOW DDGS, and HIGH DDGS, respectively. Estimates of duodenal BCP flow using DNA were 479, 397, and 368 ± 74 g/d (P = 0.14), respectively. Average BCP flow across treatments was unaffected (P = 0.71) by marker type and were 404 and 417 ± 83 g/d for DAPA and DNA markers, respectively. Estimates of duodenal PCP flow were 82, 80, and 78 ± 12 g/d (P = 0.64) for CONTROL, LOW DDGS, and HIGH DDGS, respectively. Estimates of duodenal YCP flow were 0.15, 1.94, and 4.80 ± 0.66 g/d (P < 0.01) for CONTROL, LOW DDGS, and HIGH DDGS, respectively. Duodenal BCP flow tended to decrease with DDGS inclusion, but estimates were not affected by marker type. In addition, DDGS did not affect duodenal PCP supply and provided small amounts of duodenal YCP. Overall, the value of DDGS RUP determined in this study will contribute to a better understanding of the effect of this coproduct in ruminant nutrition.

Loss of Sirt1 function improves intestinal anti-bacterial defense and protects from colitis-induced colorectal cancer.

Dysfunction of Paneth and goblet cells in the intestine contributes to inflammatory bowel disease (IBD) and colitis-associated colorectal cancer (CAC). Here, we report a role for the NAD+-dependent histone deacetylase SIRT1 in the control of anti-bacterial defense. Mice with an intestinal specific Sirt1 deficiency (Sirt1int−/−) have more Paneth and goblet cells with a consequent rearrangement of the gut microbiota. From a mechanistic point of view, the effects on mouse intestinal cell maturation are mediated by SIRT1-dependent changes in the acetylation status of SPDEF, a master regulator of Paneth and goblet cells. Our results suggest that targeting SIRT1 may be of interest in the management of IBD and CAC.

Ration formulations containing reduced-fat dried distillers grains with solubles and their effect on lactation performance, rumen fermentation, and intestinal flow of microbial nitrogen in Holstein cows

Sixteen multiparous lactating Holstein cows were used in 2 experiments to evaluate the effects of reduced-fat dried distillers grains with solubles (RFDG) on milk production, rumen fermentation, intestinal microbial N flow, and total-tract nutrient digestibility. In experiment 1, RFDG was fed at 0, 10, 20, or 30% of diet dry matter (DM) to 12 noncannulated Holstein cows (mean ± standard deviation: 89 ± 11 d in milk and 674 ± 68.2 kg of body weight) to determine effects on milk production. In experiment 2, the same diets were fed to 4 ruminally and duodenally cannulated Holstein cows (mean ± standard deviation: 112 ± 41 d in milk; 590 ± 61.14 kg of body weight) to evaluate the effects on rumen fermentation, intestinal flow of microbial N, and total-tract nutrient digestibility. In both experiments, cows were randomly assigned to 4 × 4 Latin squares over 21-d periods. Treatments (DM basis) were (1) control (0% RFDG), (2) 10% RFDG, (3) 20% RFDG, and (4) 30% RFDG. Feed intake and milk yield were recorded daily. In both experiments, milk samples were collected on d 19 to 21 of each period for analysis of milk components. In experiment 2, ruminal pH was measured; samples of rumen fluid, duodenal digesta, and feces were collected on d 18 to 21. Microbial N was estimated by using purines and DNA as microbial markers. Milk yield was not affected by treatment and averaged 34.0 ± 1.29 kg/d and 31.4 ± 2.81 kg/d in experiments 1 and 2, respectively. Percentage of milk protein tended to increase in experiment 1; estimates were 3.08, 3.18, 3.15, and 3.19 ± 0.06% when RFDG increased from 0 to 30% in the diets. However, milk protein concentration was not affected in experiment 2 and averaged 3.02 ± 0.07%. Percentage of milk fat was not affected and averaged 3.66 ± 0.05% and 3.25 ± 0.14% in experiments 1 and 2, respectively. Total ruminal volatile fatty acids and ammonia concentrations were not affected by treatment and averaged 135.18 ± 6.45 mM and 18.66 ± 2.32 mg/dL, respectively. Intestinal microbial N flow was not affected by treatment; however, purines yielded higher estimates of flow compared with DNA markers. When averaged across treatments, intestinal flow of microbial N was 303 and 218 ± 18 g of N/d, using purines and DNA as the markers. Dry matter, organic matter, neutral detergent fiber, and nonfiber carbohydrate digestibility tended to increase with increasing inclusion of RFDG. Results from these experiments indicate that dairy rations can be formulated to include up to 30% RFDG while maintaining lactation performance, volatile fatty acids concentration, and intestinal supply of microbial N.

Rumen bacterial communities can be acclimated faster to high concentrate diets than currently implemented feedlot programs

Recent studies have demonstrated RAMP®, a complete starter feed, to have beneficial effects for animal performance. However, how RAMP may elicit such responses is unknown. To understand if RAMP adaptation results in changes in the rumen bacterial community that can potentially affect animal performance, we investigated the dynamics of rumen bacterial community composition in corn‐adapted and RAMP‐adapted cattle.

Ruminal degradation and intestinal digestibility of protein and amino acids in high-protein feedstuffs commonly used in dairy diets

A study was conducted to determine the rumen degradation and intestinal digestibility of crude protein (CP) and AA, and AA composition of the rumen-undegradable protein (RUP) from 3 sources of blood meal (BM1, BM2, and BM3), canola meal (CM), low-fat distillers dried grains with solubles (LFDG), soybean meal (SBM), and expeller soybean meal (ESBM). Two Holstein cows fitted with ruminal and proximal duodenal cannulas were used for in situ incubation of 16h and for the mobile bag technique. To correct for bacterial contamination of the RUP, 2 methods were used: purines and DNA as bacterial markers. Ruminal degradations of CP were 85.3, 29.8, 40.7, 75.7, 76.9, 68.8, and 37.0 ± 3.93% for BM1, BM2, BM3, CM, LFDG, SBM, and ESBM, respectively. Ruminal degradation of both total essential AA and nonessential AA followed a similar pattern to that of CP across feedstuffs. Based on the ratio of AA concentration in the RUP to AA concentration in the original feedstuff, ruminal incubation decreased (ratio <1) the concentrations of His, Lys, and Trp, and increased (ratio >1) the concentrations of Ile and Met across feedstuffs. Compared with purines, the use of DNA as bacterial marker resulted in a higher estimate of bacterial CP contamination for CM and lower estimates for LFDG and ESBM. Intestinal digestibility of RUP could not be estimated for BM1, BM3, and SBM due to insufficient recovery of residue. For the remaining feedstuffs, intestinal digestibility of RUP was highest for ESBM, followed by BM2 and LFDG, and lowest for CM: 98.8, 87.9, 89.7, and 72.4 ± 1.40%, respectively. Intestinal absorbable dietary protein was higher for BM2 compared with CM and LFDG, at 61.7, 17.9, and 20.7 ± 2.73% CP, respectively. As prices fluctuate, intestinal absorbable protein or AA may be used as a tool to aid in the selection among feedstuffs with different protein quality.

Experimental Evaluation of Host Adaptation of Lactobacillus reuteri to Different Vertebrate Species

ABSTRACT The species Lactobacillus reuteri has diversified into host-specific lineages, implying a long-term association with different vertebrates. Strains from rodent lineages show specific adaptations to mice, but the processes underlying the evolution of L. reuteri in other hosts remain unknown. We administered three standardized inocula composed of strains from different host-confined lineages to mice, pigs, chickens, and humans. The ecological performance of each strain in the gastrointestinal tract of each host was determined by typing random colonies recovered from fecal samples collected over five consecutive days postadministration. Results revealed that rodent strains were predominant in mice, confirming previous findings of host adaptation. In chickens, poultry strains of the lineage VI (poultry VI) and human isolates from the same lineage (human VI) were recovered at the highest and second highest rates, respectively. Interestingly, human VI strains were virtually undetected in human feces. These findings, together with ancestral state reconstructions, indicate poultry VI and human VI strains share an evolutionary history with chickens. Genomic analysis revealed that poultry VI strains possess a large and variable accessory genome, whereas human VI strains display low genetic diversity and possess genes encoding antibiotic resistance and capsular polysaccharide synthesis, which might have allowed temporal colonization of humans. Experiments in pigs and humans did not provide evidence of host adaptation of L. reuteri to these hosts. Overall, our findings demonstrate host adaptation of L. reuteri to rodents and chickens, supporting a joint evolution of this bacterial species with several vertebrate hosts, although questions remain about its natural history in humans and pigs. IMPORTANCE Gut microbes are often hypothesized to have coevolved with their vertebrate hosts. However, the evidence is sparse and the evolutionary mechanisms have not been identified. We developed and applied an experimental approach to determine host adaptation of L. reuteri to different hosts. Our findings confirmed adaptation to rodents and provided evidence of adaptation to poultry, suggesting that L. reuteri evolved via natural selection in different hosts. By complementing phylogenetic analyses with experimental evidence, this study provides novel information about the mechanisms driving host-microbe coevolution with vertebrates and serve as a basis to inform the application of L. reuteri as a probiotic for different host species.

Effect of feeding dried distillers grains with solubles on ruminal biohydrogenation, intestinal fatty acid profile, and gut microbial diversity evaluated through DNA pyro-sequencing

The objectives of this study were to evaluate the effect of dried distillers grains with solubles (DDGS) on ruminal biohydrogenation and duodenal flow of fatty acids, and to evaluate effects on the ruminal and duodenal microbial community using Roche 454 pyro-sequencing. Three crossbred steers (average BW 780 ± 137 kg) fitted with ruminal and duodenal cannulae were used in a 3-diet, 6-period crossover design. Animals were housed in individual free stalls and fed twice daily at 0700 and 1900 h. Diets (DM basis) were 1) CONTROL, 19.5% corn bran, 20% sorghum silage, 60% brome hay, 0.5% trace minerals, and 0.25% urea, but no DDGS; 2) LOW DDGS, inclusion of 9.75% DDGS replacing equal percentage of corn bran; 3) HIGH DDGS, inclusion of 19.5% DDGS completely replacing corn bran. Feed ingredients and duodenal digesta samples were analyzed for fatty acid composition. The DNA was extracted from isolated mixed ruminal bacterial samples and from intestinal digesta samples. The V1-V3 region of the 16S rRNA gene was sequenced, and bacterial phylogenetic analysis was conducted. Data were analyzed using the MIXED procedure of SAS. Biohydrogenation of C18:1 increased (P < 0.01) with DDGS inclusion; means were 68.3, 75.6, and 79.3 ± 4.3% for CONTROL, LOW DDGS, and HIGH DDGS, respectively. In the same order, means of biohydrogenation of C18:2 (P < 0.05) were 84.1, 91.5, and 93.3 ± 3.4%. Duodenal flow of total fatty acids increased (P < 0.01) with DDGS inclusion; means were 134, 168, and 223 ± 33 g/d for CONTROL, LOW DDGS, and HIGH DDGS, respectively. In the same order, means of C18:0 flow (P < 0.01) were 51, 86, and 121 ± 18 g/d. DDGS did not affect the predominant bacterial phyla in the gut, which were Bacteroidetes (P = 0.62) and Firmicutes (P = 0.71). However, the phylum Fibrobacteres decreased (P < 0.01) when DDGS was fed with means of 5.5, 6.0 and 3.7 ± 0.6% for CONTROL, LOW DDGS, and HIGH DDGS, respectively. Fibrobacteres were lower (P < 0.01) in isolated ruminal bacterial samples compared to duodenal digesta samples with means of 0.1 and 10.1 ± 0.6%, respectively. Overall, the inclusion of DDGS in diets increased ruminal biohydrogenation of C18:1 and C18:2, which increased duodenal flow of C18:0. In addition, the bacterial community of the rumen clustered separately from that of the duodenum suggesting different bacterial diversity between isolated ruminal bacteria and duodenal digesta.

Obesity-Dependent Increases in Oocyte mRNAs Are Associated With Increases in Proinflammatory Signaling and Gut Microbial Abundance of Lachnospiraceae in Female Mice

RNAs stored in the metaphase II-arrested oocyte play important roles in successful embryonic development. Their abundance is defined by transcriptional activity during oocyte growth and selective degradation of transcripts during LH-induced oocyte maturation. Our previous studies demonstrated that mRNA abundance is increased in mature ovulated oocytes collected from obese humans and mice and therefore may contribute to reduced oocyte developmental competence associated with metabolic dysfunction. In the current study mouse models of diet-induced obesity were used to determine whether obesity-dependent increases in proinflammatory signaling regulate ovarian abundance of oocyte-specific mRNAs. The abundance of oocyte-specific Bnc1, Dppa3, and Pou5f1 mRNAs as well as markers of proinflammatory signaling were significantly increased in ovaries of obese compared with lean mice which were depleted of fully grown preovulatory follicles. Chromatin-immunoprecipitation analyses also demonstrated increased association of phosphorylated signal transducer and activator of transcription 3 with the Pou5f1 promoter in ovaries of obese mice suggesting that proinflammatory signaling regulates transcription of this gene in the oocyte. The cecum microbial content of lean and obese female mice was subsequently examined to identify potential relationships between microbial composition and proinflammatory signaling in the ovary. Multivariate Association with Linear Models identified significant positive correlations between cecum abundance of the bacterial family Lachnospiraceae and ovarian abundance of Tnfa as well as Dppa3, Bnc1, and Pou5f1 mRNAs. Together, these data suggest that diet-induced changes in gut microbial composition may be contributing to ovarian inflammation which in turn alters ovarian gene expression and ultimately contributes to obesity-dependent reduction in oocyte quality and development of infertility in obese patients.

Dietary energy drives the dynamic response of bovine rumen viral communities

BackgroundRumen microbes play a greater role in host energy acquisition than that of gut-associated microbes in monogastric animals. Although genome-enabled advancements are providing access to the vast diversity of uncultivated microbes, our understanding of variables shaping rumen microbial communities is in its infancy. Viruses have been shown to impact microbial populations through a myriad of processes, including cell lysis and reprogramming of host metabolism. However, little is known about the processes shaping the distribution of rumen viruses or how viruses may modulate microbial-driven processes in the rumen. To this end, we investigated how rumen bacterial and viral community structure and function responded in five steers fed four randomized dietary treatments in a crossover design.ResultsTotal digestible nutrients (TDN), a measure of dietary energy, best explained the variation in bacterial and viral communities. Additional ecological drivers of viral communities included dietary zinc content and microbial functional diversity. Using partial least squares regression, we demonstrate significant associations between the abundances of 267 viral populations and variables driving the variation in rumen viral communities. While rumen viruses were dynamic, 14 near ubiquitous viral populations were identified, suggesting the presence of a core rumen virome largely comprised of novel viruses. Moreover, analysis of virally encoded auxiliary metabolic genes (AMGs) indicates rumen viruses have glycosidic hydrolases to potentially augment the breakdown of complex carbohydrates to increase energy production. Other AMGs identified have a role in redirecting carbon to the pentose phosphate pathway and one carbon pools by folate to boost viral replication.ConclusionsWe demonstrate that rumen bacteria and viruses have differing responses and ecological drivers to dietary perturbation. Our results show that rumen viruses have implications for understanding the structuring of the previously identified core rumen microbiota and impacting microbial metabolism through a vast array of AMGs. AMGs in the rumen appear to have consequences for microbial metabolism that are largely in congruence with the current paradigm established in marine systems. This study provides a foundation for future hypotheses regarding the dynamics of viral-mediated processes in the rumen.