Ability of Selenomonas ruminantium,Veillonella parvula,and Wolinella succinogenes to Reduce Nitrate and Nitrite with Special Reference to the Suppression of Ruminal Methanogenesis

When mixed ruminal microbes were grown in a medium containing ground hay and concentrate, cell numbers of nitrate-reducing bacteria, Veillonella parvula and Wolinella succinogenes, drastically decreased as estimated by competitive PCR. However, decrease in their numbers was prevented by the addition of nitrate, suggesting that energy acquisition by electron transport phosphorylation (ETP) coupled with nitrate and/or nitrite reduction is important for the survival of these bacteria in the rumen. On the other hand, the number of Selenomonas ruminantium increased, and addition of nitrate did not affect the number, suggesting that the numbers of nitrate-reducing strains of S. ruminantium are low. A nitrate-reducing strain of S. ruminantium subsp. lactilytica was found to have the ETP system coupled with both nitrate and nitrite reduction. V. parvula, W. succinogenes, S. ruminantium, and Streptococcus bovis were more tolerant to nitrite toxicity than other representative ruminal bacteria. Methanogens were particularly sensitive to nitrite. W. succinogenes reduced nitrate and nitrite at higher rates than V. parvula and the subsp.lactilytica , but growth of W. succinogenes on nitrate and H₂ was slower than the growth of V. parvula and the subsp. lactilytica. Methane production by unidentified methanogen mixture was markedly reduced by the coexistence of W. succinogenes, V. parvula or the subsp. lactilytica in the presence of nitrate and H₂. W. succinogenes was shown to be most effective to augment nitrate and nitrite reduction, and to reduce methanogenesis.     

Effects of Dietary Linseed Oil and Propionate Precursors on Ruminal Microbial Community,Composition, and Diversity in Yanbian Yellow Cattle

The rumen microbial ecosystem is a complex system where rumen fermentation processes involve interactions among microorganisms. There are important relationships between diet and the ruminal bacterial composition. Thus, we investigated the ruminal fermentation characteristics and compared ruminal bacterial communities using tag amplicon pyrosequencing analysis in Yanbian yellow steers, which were fed linseed oil (LO) and propionate precursors. We used eight ruminally cannulated Yanbian yellow steers (510 ± 5.8 kg) in a replicated 4 × 4 Latin square design with four dietary treatments. Steers were fed a basal diet that comprised 80% concentrate and 20% rice straw (DM basis, CON). The CON diet was supplemented with LO at 4%. The LO diet was also supplemented with 2% dl-malate or 2% fumarate as ruminal precursors of propionate. Dietary supplementation with LO and propionate precursors increased ruminal pH, total volatile fatty acid concentrations, and the molar proportion of propionate. The most abundant bacterial operational taxonomic units in the rumen were related to dietary treatments. Bacteroidetes dominated the ruminal bacterial community and the genus Prevotella was highly represented when steers were fed LO plus propionate precursors. However, with the CON and LO diet plus malate or fumarate, Firmicutes was the most abundant phylum and the genus Ruminococcus was predominant. In summary, supplementing the diets of ruminants with a moderate level of LO plus propionate precursors modified the ruminal fermentation pattern. The most positive responses to LO and propionate precursors supplementation were in the phyla Bacteriodetes and Firmicutes, and in the genus Ruminococcus and Prevotella. Thus, diets containing LO plus malate or fumarate have significant effects on the composition of the rumen microbial community.     

Effect of protein supplementation on ruminal parameters and microbial community fingerprint of Nellore steers fed tropical forages

In tropical regions, protein supplementation is a common practice in dairy and beef farming. However, the effect of highly degradable protein in ruminal fermentation and microbial community composition has not yet been investigated in a systematic manner. In this work, we aimed to investigate the impact of casein supplementation on volatile fatty acids (VFA) production, specific activity of deamination (SAD), ammonia concentration and bacterial and archaeal community composition. The experimental design was a 4×4 Latin square balanced for residual effects, with four animals (average initial weight of 280±10 kg) and four experimental periods, each with duration of 29 days. The diet comprised Tifton 85 (Cynodon sp.) hay with an average CP content of 9.8%, on a dry matter basis. Animals received basal forage (control) or infusions of pure casein (230 g) administered direct into the rumen, abomasum or divided (50 : 50 ratio) in the rumen/abomasum. There was no differences (P>0.05) in ruminal pH and microbial protein concentration between supplemented v. non-supplemented animals. However, in steers receiving ruminal infusion of casein the SAD and ruminal ammonia concentration increased 33% and 76%, respectively, compared with the control. The total concentration of VFA increased (P<0.05) in steers receiving rumen infusion of casein. SAD and the microbial protein concentration did not vary significantly among treatments during the feeding cycle, but mean SAD values were greater in steers supplemented in the rumen and rumen/abomasum. Ruminal ammonia concentration was positively correlated with SAD in animals receiving ruminal infusion of casein. Polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) analysis revealed low similarity between treatments, animals and time of sample collection. Richness analysis and determination of the Shannon–Wiener index indicated no differences (P>0.05) in species richness and diversity of γ-proteobacteria, firmicutes and archaea between non-supplemented Nellore steers and steers receiving casein supplementation in the rumen. However, species richness and the Shannon–Wiener index were lower (P<0.05) for the phylum bacteroidetes in steers supplemented with casein in the rumen compared with non-supplemented animals. Venn diagrams indicated that the number of unique bands varied considerably among individual animals and was usually higher in number for non-supplemented steers compared with supplemented animals. These results add new knowledge about the effects of ruminal and postruminal protein supplementation on metabolic activities of rumen microbes and the composition of bacterial and archaeal communities in the rumen of steers.     

Trace mineral source influences digestion,ruminal fermentation,and ruminal copper,zinc, and manganese distribution in steers fed a diet suitable for lactating dairy cows

High solubility of certain trace minerals (TM) in the rumen can alter nutrient digestibility and fermentation. The objectives of the present studies were to determine the effects of TM source on 1) nutrient digestibility and ruminal fermentation, 2) concentrations of soluble Cu, Zn, and Mn in the rumen following a pulse dose of TM, and 3) Cu, Zn, and Mn binding strength on ruminal digesta using dialysis against a chelating agent in steers fed a diet formulated to meet the requirements of a high producing dairy cow. Twelve Angus steers fitted with ruminal cannulae were adapted to a diet balanced with nutrient concentrations similar to a diet for a high producing lactating dairy cow for 21 d. Steers were then randomly assigned to dietary treatments consisting of 10 mg Cu, 40 mg Mn, and 60 mg Zn/kg DM from either sulfate (STM), hydroxychloride (HTM) or complexed trace minerals (CTM). The experimental design did not include a negative control (no supplemental Cu, Mn, or Zn) because the basal diet did not meet the National Research Council requirement for Cu and Zn. Copper, Mn, and Zn are also generally supplemented to lactating dairy cow diets at concentrations approximating those supplied in the present study. Following a 14-d adaptation period, total fecal output was collected for 5-d. Following the fecal collection period, rumen fluid was collected for Volatile fatty acid (VFA) parameters. On the following day, the same diet was provided for 14 d, without supplemental Cu, Zn, and Mn. This period served as a wash-out period. A pulse dose of 100, 400, and 600 mg of Cu, Zn, Mn, respectively, from either STM, HTM, or CTM, was administered via ruminal cannulae to the steers on day 15. Over a 24-h period ruminal samples were obtained every 2-h. Following centrifugation, the supernatant was analyzed for Cu, Mn, and Zn. Ruminal solid digesta samples from times 0, 12, and 24 h after bolus dosing were exposed to dialysis against Tris-EDTA. Digestibility of NDF and ADF were lesser in STM vs. HTM and vs. CTM supplemented steers. Steers receiving HTM and CTM had greater total VFA concentrations than STM, and molar proportions of individual VFA were not affected by treatment. Ruminal soluble Cu and Zn concentrations were greater post dosing in STM and CTM supplemented steers at 2, 4, and 6 h for Cu and 4, 6, 8, 10 and 12 h for Zn when compared to HTM supplemented steers. The release of Cu and Zn from ruminal solid digesta following dialysis against Tris-EDTA at 12 and 24 h postdosing was greater for steers receiving HTM compared to those receiving STM or CTM. Results indicate trace mineral source impacts: 1) how tightly bound Cu and Zn are to ruminal solid digesta; 2) fiber digestion; 3) and ruminal total VFA concentrations.     

Effects of feed withdrawal duration on animal behaviour,rumen microbiota and blood chemistry in feedlot cattle: implications for rumen acidosis

Feed withdrawal (FW) is a frequent issue in open outdoor feedlot systems, where unexpected circumstances can limit the animals’ access to food. The relationship among fasting period, animal behaviour during feed reintroduction (FR) and acidosis occurrence has not been completely elucidated. Twenty steers fitted with rumen catheters were fed a high-concentrate diet (concentrate : forage ratio 85 : 15) and were challenged by a protocol of FW followed by FR. The animals were randomly assigned to one of the four treatments: FW for 12 h (T12), 24 h (T24), 36 h (T36) or no FW (control group) followed by FR. The steers’ behaviour, ruminal chemistry, structure of the ruminal microbial community, blood enzymes and metabolites and ruminal acidosis status were assessed. Animal behaviour was affected by the FW–FR challenge ( P < 0.05). Steers from the T12, T24 and T36 treatments showed a higher ingestion rate and a lower frequency of rumination. Although all animals were suspected to have sub-acute ruminal acidosis (SARA) prior to treatment, a severe case of transient SARA arose after FR in the T12, T24 and T36 groups. The ruminal pH remained below the threshold adopted for SARA diagnosis ( pH value = 5.6) for more than three consecutive hours (24, 7 and 19 h in the T12, T24 and T36 treatments, respectively). The FW–FR challenge did not induce clinical acute ruminal acidosis even though steers from the T36 treatment presented ruminal pH values that were consistent with this metabolic disorder (pH threshold for acute acidosis = 5.2). Total mixed ration reintroduction after the withdrawal period reactivated ruminal fermentation as reflected by changes in the fermentation end-products. Ruminal lactic acid accumulation in steers from the T24 and T36 treatments probably led to the reduction of pH in these groups. Both the FW and the FR phases may have altered the structure of the ruminal microbiota community. Whereas fibrolytic bacterial groups decreased relative abundance in the restricted animals, both lactic acid producer and utiliser bacterial groups increased ( P < 0.05). The results demonstrated a synchronisation between Streptococcus (lactate producer) and Megasphaera (lactate utiliser), as the relative abundance of both groups increased, suggesting that bacterial resilience may be central for preventing the onset of metabolic disturbances such as ruminal acidosis. A long-FW period (36 h) produced rumen pH reductions well below and lactic acid concentration increased well above the accepted thresholds for acute acidosis without any perceptible clinical signs.     

Impact of adding nitrate or increasing the lipid content of two contrasting diets on blood methaemoglobin and performance of two breeds of finishing beef steers

Adding nitrate to the diet or increasing the concentration of dietary lipid are effective strategies for reducing enteric methane emissions. This study investigated their effect on health and performance of finishing beef cattle. The experiment was a two×two×three factorial design comprising two breeds (CHX, crossbred Charolais; LU, Luing); two basal diets consisting of (g/kg dry matter (DM), forage to concentrate ratios) 520 : 480 (Mixed) or 84 : 916 (Concentrate); and three treatments: (i) control with rapeseed meal as the main protein source replaced with either (ii) calcium nitrate (18 g nitrate/kg diet DM) or (iii) rapeseed cake (RSC, increasing acid hydrolysed ether extract from 25 to 48 g/kg diet DM). Steers (n=84) were allocated to each of the six basal diet×treatments in equal numbers of each breed with feed offered ad libitum. Blood methaemoglobin (MetHb) concentrations (marker for nitrate poisoning) were monitored throughout the study in steers receiving nitrate. After dietary adaptation over 28 days, individual animal intake, performance and feed efficiency were recorded for a test period of 56 days. Blood MetHb concentrations were low and similar up to 14 g nitrate/kg diet DM but increased when nitrate increased to 18 g nitrate/kg diet DM (P<0.001). An interaction between basal diet and day (P<0.001) indicated that MetHb% was consistently greater in Concentrate – than Mixed-fed steers at 18 g nitrate/kg diet DM. Maximum individual MetHb% was 15.4% (of total Hb), which is lower than considered clinically significant (30%). MetHb concentrations for individual steers remained consistent across time. Concentrate-fed steers were more efficient (lower residual feed intake (RFI) values) than Mixed-fed steers (P<0.01), with lower dry matter intake (DMI) (kg/day) (P<0.001) and similar average daily gain (ADG). CHX steers were more efficient (lower RFI; P<0.01) than LU steers with greater ADG (P<0.01), lower DMI (/kg BW; P<0.01) and lower fat depth (P<0.001). ADG, BW or DMI did not differ across dietary treatments (P>0.05). Neither basal diet nor treatment affected carcass quality (P>0.05), but CHX steers achieved a greater killing out proportion (P<0.001) than LU steers. Thus, adding nitrate to the diet or increasing the level of dietary lipid through the use of cold-pressed RSC, did not adversely affect health or performance of finishing beef steers when used within the diets studied.     

Diversity of the Intestinal Bacteria of Cattle Fed on Diets with Different Doses of Gelatinized Starch-Urea

Gelatinized starch-urea (Starea, SU) is an effective and economical source of urea for ruminants. Here we assessed the influence of dietary supplementation with gelatinized starch-urea on the diversity of intestinal bacteria in finishing cattle. Fifty steers were randomly allotted to five treatments with diets supplemented with different doses of Starea [0 % (SU0), 8 % (SU8), 16 % (SU16), 24 % (SU24), and 32 % (SU32) of urea-N in total nitrogen]. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes was used to examine the effect of dietary supplementation of Starea on intestinal bacterial flora. Shannon–Weaver and Simpson diversity indices consistently showed the lowest bacterial diversity in the SU0 treatment. Increasing doses of Starea increased the diversity up to SU24 after which, diversity decreased. Cluster analysis of 16S rRNA gene DGGE profiles indicates that the intestinal bacterial communities associated with cattle that were not supplemented with Starea in feed differed in composition and structure from those supplemented with Starea. The amount of Starea supplemented in cattle diets influenced the abundance of several key species affiliated with Lachnospiraceae, Ruminococcaceae, Peptostreptococcaceae, Comamonadaceae and Moraxellaceae. These results suggest that Starea influences the composition and structure of intestinal bacteria which may play a role in promoting ruminant health and production performance.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0526-8) contains supplementary material, which is available to authorized users.     

Campylobacter fetus Induced Proinflammatory Response in Bovine Endometrial Epithelial Cells

Campylobacter fetus subsp. fetus is the causal agent of sporadic abortion in bovines and infertility that produces economic losses in livestock. In many infectious diseases, the immune response has an important role in limiting the invasion and proliferation of bacterial pathogens. Innate immune sensing of microorganisms is mediated by pattern-recognition receptors (PRRs) that identify pathogen-associated molecular patterns (PAMPs) and induces the secretion of several proinflammatory cytokines, like IL-1β, TNF-α, and IL-8. In this study, the expression of IL-1β, TNF-α, IL-8, and IFN-γ in bovine endometrial epithelial cells infected with C. fetus and Salmonella Typhimurium (a bacterial invasion control) was analyzed. The results showed that expression levels of IL-1β and IL-8 were high at the beginning of the infection and decreased throughout the intracellular period. Unlike in this same assay, the expression levels of IFN-γ increased through time and reached the highest peak at 4 hours post infection. In cells infected with S. Typhimurium, the results showed that IL8 expression levels were highly induced by infection but not IFN-γ. In cells infected with S. Typhimurium or C. fetus subsp. fetus, the results showed that TNF-α expression did not show any change during infection. A cytoskeleton inhibition assay was performed to determine if cytokine expression was modified by C. fetus subsp. fetus intracellular invasion. IL-1β and IL-8 expression were downregulated when an intracellular invasion was avoided. The results obtained in this study suggest that bovine endometrial epithelial cells could recognize C. fetus subsp. fetus resulting in early proinflammatory response.     

ARISA analysis of ruminal bacterial community dynamics in lactating dairy cows during the feeding cycle

The bovine rumen undergoes substantial changes in environmental conditions during the animal's feeding cycle, but the effects of these changes on microbial populations have not been examined systematically. Two dairy cows fed a mixed forage/concentrate ration at 12 h intervals over 4 feeding cycles displayed substantial changes in ruminal pH and volatile fatty acid (VFA) concentrations. Automated ribosomal intergenic spacer analysis (ARISA) of solid- and liquid-associated bacterial populations in samples collected at 2, 4, 6, 9, and 12 h after feeding revealed a high degree of bacterial diversity. A total of 155 different amplicon lengths (ALs) were detected across all 83 samples, and 11–74 detected per sample. A substantial proportion (11%) of the ALs was detected in one cow but not in the other. The proportions of ALs that were detected only in the liquid phase or the solid phase were 13.5% and 1.9%, respectively. Correspondence analysis indicated that bacterial community composition differed between cows and between solid or liquid phases, but overall the solid-associated population displayed less change in composition within and across feeding cycles. The data support the notion that cows fed the same diets can have substantial differences in bacterial community composition, and that the solids-associated (biofilm) communities display greater stability than do associated planktonic communities.     

Characterization of the Core Rumen Microbiome in Cattle during Transition from Forage to Concentrate as Well as during and after an Acidotic Challenge

This study investigated the effect of diet and host on the rumen bacterial microbiome and the impact of an acidotic challenge on its composition. Using parallel pyrosequencing of the V3 hypervariable region of 16S rRNA gene, solid and liquid associated bacterial communities of 8 heifers were profiled. Heifers were exclusively fed forage, before being transitioned to a concentrate diet, subjected to an acidotic challenge and allowed to recover. Samples of rumen digesta were collected when heifers were fed forage, mixed forage, high grain, during challenge (4 h and 12 h) and recovery. A total of 560,994 high-quality bacterial sequences were obtained from the solid and liquid digesta. Using cluster analysis, prominent bacterial populations differed (P≤0.10) in solid and liquid fractions between forage and grain diets. Differences among hosts and diets were not revealed by DGGE, but real time qPCR showed that several bacteria taxon were impacted by changes in diet, with the exception of Streptococcus bovis. Analysis of the core rumen microbiome identified 32 OTU''s representing 10 distinct bacterial taxa including Bacteroidetes (32.8%), Firmicutes (43.2%) and Proteobacteria (14.3%). Diversity of OTUs was highest with forage with 38 unique OTUs identified as compared to only 11 with the high grain diet. Comparison of the microbial profiles of clincial vs. subclinical acidotic heifers found a increases in the relative abundances of Acetitomaculum, Lactobacillus, Prevotella, and Streptococcus. Increases in Streptococcus and Lactobacillus likely reflect the tolerance of these species to low pH and their ability to proliferate on surplus fermentable carbohydrate. The acetogen, Acetitomaculum may thereforeplay a role in the conversion of lactate to acetate in acidotic animals. Further profiling of the bacterial populations associated with subclinical and clinical acidosis could establish a microbial fingerprint for these disorders and provide insight into whether there are causative microbial populations that could potentially be therapeutically manipulated.     

Development of a miniaturized nitrate reduction test for the identification of oral bacteria

A miniaturized nitrate reduction test (MNRT) for oral bacteria was developed and its reliability compared with a conventional nitrate reduction test (CNRT). In the MNRT 100 μl aliquots of freshly grown heavy suspension of various oral bacterial species, in physiological saline, were added to equal volumes of 0.1% filter-sterilized KNO₃ solution in distilled water in wells of transparent plastic plates. Duplicate plates were incubated aerobically or anaerobically at 35°C for 12–15 h. At the end of the incubation period the test was performed by adding either a trace amount of a non-liquid reagent (mixture of l-(+)-tartaric acid, sulfanilic acid and 1-naphthylenediamine dihydrochloride, 10:1:1, wt/wt) or conventional liquid reagents A and B (sulfanilic acid and N,N-dimethyl-1-naphthylamine). In the conventional nitrate reduction test (CNRT), tubes of a basal anaerobic broth were inoculated with the same bacterial species used for MNRT, and the nitrate reduction tests performed after anaerobic incubation of the cultures for 4–6 days. Several hundred anaerobic and facultative bacterial isolates belonging to genera Veillonella, Bacteroides, Fusobacterium, Selenomonas, Actinomyces and Capnocytophaga were characterized by MNRT and CNRT. Analysis of the data showed that MNRT and CNRT systems were comparable. In the MNRT system Veillonella parvula and Selenomonas sputigena were capable of reducing nitrate only under anaerobic conditions. Actinomycetes reduced the nitrates under aerobic and anaerobic conditions, while all black-pigmented Bacteroides, Fusobacterium and Capnocytophaga species did not reduce nitrate. These findings suggest that the MNRT is reliable, rapid and may be conveniently used in clinical or research laboratories with a heavy microbiological work load.     

Impact of feed efficiency and diet on adaptive variations in the bacterial community in the rumen fluid of cattle

Limited knowledge of the structure and activities of the ruminal bacterial community prevents the understanding of the effect of population dynamics on functional bacterial groups and on host productivity. This study aimed to identify particular bacteria associated with host feed efficiency in steers with differing diets and residual feed intake (RFI) using culture-independent methods: PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR analysis. PCR-DGGE profiles were generated from the ruminal fluid of 55 steers fed a low-energy-density diet and then switched to a high-energy-density diet. Bacterial profile comparisons by multivariate statistical analysis showed a trend only for RFI-related clusters on the high-energy diet. When steers (n = 19) belonging to the same RFI group under both diets were used to identify specific bacterial phylotypes related to feed efficiency traits, correlations were detected between dry matter intake, average daily gain, and copy numbers of the 16S rRNA gene of Succinivibrio sp. in low-RFI (efficient) steers, whereas correlations between Robinsoniella sp. and RFI (P < 0.05) were observed for high-RFI (inefficient) animals. Eubacterium sp. differed significantly (P < 0.05) between RFI groups that were only on the high-energy diet. Our work provides a comprehensive framework to understand how particular bacterial phylotypes contribute to differences in feed efficiency and ultimately influence host productivity, which may either depend on or be independent from diet factors.     

The effect of dietary addition of nitrate or increase in lipid concentrations,alone or in combination,on performance and methane emissions of beef cattle

Adding nitrate to or increasing the concentration of lipid in the diet are established strategies for reducing enteric methane (CH₄) emissions, but their effectiveness when used in combination has been largely unexplored. This study investigated the effect of dietary nitrate and increased lipid included alone or together on CH₄ emissions and performance traits of finishing beef cattle. The experiment was a 2×4 factorial design comprising two breeds (cross-bred Aberdeen Angus (AAx) and cross-bred Limousin (LIMx) steers) and four dietary treatments (each based on 550 g forage : 450 g concentrate/kg dry matter (DM)). The four dietary treatments were assigned according to a 2×2 factorial design where the control treatment contained rapeseed meal as the main protein source, which was replaced either with nitrate (21.5 g nitrate/kg DM); maize distillers dark grains (MDDG, which increased diet ether extract from 24 to 37 g/kg DM) or both nitrate and MDDG. Steers (n=20/dietary treatment) were allocated to each of the four treatments in equal numbers of each breed with feed offered ad libitum. After 28 days adaptation to dietary treatments, individual animal intake, performance and feed efficiency were recorded for 56 days. Thereafter, CH₄ emissions were measured over 13 weeks (six steers/week). Increasing dietary lipid did not adversely affect animal performance and showed no interactions with dietary nitrate. In contrast, addition of nitrate to diets resulted in poorer live-weight gain (P<0.01) and increased feed conversion ratio (P<0.05) compared with diets not containing nitrate. Daily CH₄ output was lower (P<0.001) on nitrate-containing diets but increasing dietary lipid resulted in only a non-significant reduction in CH₄. There were no interactions associated with CH₄ emissions between dietary nitrate and lipid. Cross-bred Aberdeen Angus steers achieved greater live-weight gains (P<0.01), but had greater DM intakes (P<0.001), greater fat depth (P<0.01) and poorer residual feed intakes (P<0.01) than LIMx steers. Cross-bred Aberdeen Angus steers had higher daily CH₄ outputs (P<0.001) but emitted less CH₄ per kilogram DM intake than LIMx steers (P<0.05). In conclusion, inclusion of nitrate reduced CH₄ emissions in growing beef cattle although the efficacy of nitrate was less than in previous work. When increased dietary lipid and nitrate inclusion were combined there was no evidence of an interaction between treatments and therefore combining different nutritional treatments to mitigate CH₄ emissions could be a useful means of achieving reductions in CH₄ while minimising any adverse effects.     

Isolation of Pseudobutyrivibrio ruminis and Pseudobutyrivibrio xylanivorans from rumen of Creole goats fed native forage diet

We isolated and identified functional groups of bacteria in the rumen of Creole goats involved in ruminal fermentation of native forage shrubs. The functional bacterial groups were evaluated by comparing the total viable, total anaerobic, cellulolytic, hemicellulolytic, and amylolytic bacterial counts in the samples taken from fistulated goats fed native forage diet (Atriplex lampa and Prosopis flexuosa). Alfalfa hay and corn were used as control diet. The roll tubes method increased the possibility of isolating and 16S rDNA gene sequencing allowed definitive identification of bacterial species involved in the ruminal fermentation. The starch and fiber contents of the diets influenced the number of total anaerobic bacteria and fibrolytic and amylolytic functional groups. Pseudobutyrivibrio ruminis and Pseudobutyrivibrio xylanivorans were the main species isolated and identified. The identification of bacterial strains involved in the rumen fermentation helps to explain the ability of these animals to digest fiber plant cell wall contained in native forage species.     

Garlic skin induces shifts in the rumen microbiome and metabolome of fattening lambs

Garlic (Allium sativum L.) and its constituents have been shown to modify rumen fermentation and improve growth performance. Garlic skin, a by-product of garlic processing, contains similar bioactive components as garlic bulb. This study aimed to investigate the effects of garlic skin supplementation on growth performance, ruminal microbes, and metabolites in ruminants. Twelve Hu lambs were randomly assigned to receive a basal diet (CON) or a basal diet supplemented with 80 g/kg DM of garlic skin (GAS). The experiment lasted for 10 weeks, with the first 2 weeks serving as the adaptation period. The results revealed that the average daily gain and volatile fatty acid concentration were higher (P < 0.05) in lambs fed GAS than those in the CON group. Garlic skin supplementation did not significantly (P > 0.10) affect the α-diversity indices, including the Chao1 index, the abundance-based coverage estimator value, and the Shannon and Simpson indices. At the genus level, garlic skin supplementation altered the ruminal bacterial composition by increasing (P < 0.05) the relative abundances of Prevotella, Bulleidia, Howardella, and Methanosphaera and decreasing (P < 0.05) the abundance of Fretibacterium. Concentrations of 139 metabolites significantly differed (P < 0.05) between the GAS and the CON groups. Among them, substrates for rumen microbial protein synthesis were enriched in the GAS group. The pathways of pyrimidine metabolism, purine metabolism, and vitamin B6 metabolism were influenced (P < 0.05) by garlic skin supplementation. Integrated correlation analysis also provided a link between the significantly altered rumen microbiota and metabolites. Thus, supplementation of garlic skin improved the growth performance of lambs by modifying rumen fermentation through shifts in the rumen microbiome and metabolome.     

Interaction Between Nickel and Protein Source in the Ruminant

Eighty growing steers were used to determine the effect of nickel supplementation on performance and metabolic parameters of steers fed corn silage-based diets supplemented with different crude protein sources. Crude protein sources examined included: (1) soybean meal, (2) blood meal, (3) urea, and (4) blood meal-urea (two-thirds of supplemental nitrogen from blood meal and one-third from urea). The protein sources differed in ruminal degradability, nitrogen solubility, and nickel content. Nickel was added within each protein treatment to supply either 0 or 5 ppm of supplemental nickel. The experiment was 84 d in duration and rumen fluid and blood samples were collected on days 42 and 80. Average daily gain and feed efficiency were not affected by nickel supplementation. The addition of 5 ppm supplemental nickel greatly increased rumen bacterial urease activity regardless of protein source. When samples were collected prior to feeding on day 80, nickel increased serum urea nitrogen concentrations in steers fed urea, but decreased circulating urea concentrations in animals fed blood meal or the blood meal-urea combination.Ad libitum intake of trace mineral salt was greatly reduced in steers receiving 5 ppm supplemental nickel. The present study suggests that the source of protein may influence ruminant responses to dietary nickel.     

Methodology for concurrent determination of urea kinetics and the capture of recycled urea nitrogen by ruminal microbes in cattle

We measured the incorporation of recycled urea-nitrogen (N) by ruminal microbes, using five ruminally and duodenally fistulated steers (237 kg) fed low-quality grass hay (47 g crude protein/kg dry matter (DM)). Three received 1 kg/day of soybean meal (SBM) and two received no supplemental protein (control). The experiment was 15 days long. Background enrichments of 15N were measured on day 9 and continuous jugular infusion of 0.12 g/day [15N15N]urea began on day 10. Daily samples of urine, feces, ruminal bacteria and duodenal digesta from days 10 through 14 were used to determine plateaus in 15N enrichment. Duodenal and bacterial samples collected on day 15 were used to measure duodenal N flows. Bacterial N flow was calculated as duodenal N flow multiplied by duodenal 15N enrichment divided by bacterial 15N enrichment. Bacterial N from recycled urea-N was calculated as bacterial N flow multiplied by bacterial 15N enrichment divided by urinary urea 15N enrichment. Urinary enrichment of [15N15N]urea plateaued within 24 h, whereas 14N15N urea plateaued within 48 h of [15N15N]urea infusion. Bacteria reached a plateau in 15N enrichment within 24 h and duodenal samples within 48 h. Urea production was 17.6 g of urea-N/day for control and 78.0 g/day for SBM. Gut entry was 0.99 g of urea-N/g of urea-N produced for control and 0.87 g/g for SBM. Incorporation of recycled N into microbial N was 9.0 g of N/day for control and 23.0 g/day for SBM. Recycled urea-N accounted for 0.33 g of N/g of microbial N at the duodenum for control and 0.27 g/g for SBM. Our methods allowed measurement of incorporation of recycled urea-N into ruminal microbial N.     

Evolution of bacterial denitrification and denitrifier diversity

Little is known about the role of nitrate in evolution of bacterial energy-generating mechanisms. Denitrifying bacteria are commonly regarded to have evolved from nitrate-respiring bacteria. Some researchers regard denitrification to be the precursor of aerobic respiration; others feel the opposite is true. Currently recognized denitrifying bacteria such as Hyphomicrobium, Paracoccus, Pseudomonas and Thiobacillus form a very diverse group. However, inadequate testing procedures and uncertain taxonomic identification of many isolates may have overstated the number of genera with species capable of denitrification. Nitrate reductases are structurally similar among denitrifying bacteria, but distinct from the enzymes in other nitrate-reducing organisms. Denitryfying bacteria have one of two types of nitrite reductase, either a copper-containing enzyme or an enzyme containing a cytochrome cd moiety. Both types are distinct from other nitrate reductases. Organisms capable of dissimilatory nitrate reduction are widely distributed among eubacterial groups defined by 16S ribosomal RNA phylogeny. Indeed, nitrate reduction is an almost universal property of actinomycetes and enteric organisms. However, denitrification is restricted to genera within the purple photosynthetic group. Denitrification within the genus Pseudomonas is distributed in accordance with DNA and RNA homology complexes. Denitrifiers seem to have evolved from a common ancestor within the purple photosynthetic bacterial group, but not from a nitrate-reducing organism such as those found today. Although denitrification seems to have arisen at the same time as aerobic respiration, the evolutionary relationship between the two cannot be determined at this time.     

Analysis of the Rumen Bacterial Diversity under two Different Diet Conditions using Denaturing Gradient Gel Electrophoresis,Random Sequencing,and Statistical Ecology Approaches

Bacterial community structure and diversity in the rumen of steers in conditions of hay and corn diets was assessed by in vitro retrieval and analysis of the variable region (V3) of 16S rDNA. Two types of libraries were generated in this study: DGGE libraries, which further were analysed by excising, reamplification, and sequencing, and random shotgun sequence libraries. Phylogenetic and sequence similarity analyses of the resultant 68 clone sequences in DGGE libraries revealed the presence of 42 operational taxonomic units (OTUs) or phylotypes defined as having more than 97% of sequence similarity. One hundred and thirty four clone sequences in shotgun libraries were clustered into 72 phylotypes. The phylotype similarity, diversity, richness, and evenness in these libraries were estimated using a variety of diversity indices. In relation to diet, the corn-fed animals displayed more diverse and rich bacterial populations, which were mostly contributed by CFB-related phylotypes. Proteobacteria were also numerically prevalent on this diet (27%) but were represented by a few phylotypes thus diminishing the overall diversity and species richness values. On hay diet, the principal contributors to general diversity and species richness appeared to be low-G + C gram-positives. Although the ruminal Treponemaes were encountered only in hay-fed animals, their impact on species diversity on hay diet was low because of the limited number of phylotypes.     

Effect of Haylage and Monensin Supplementation on Ruminal Bacterial Communities of Feedlot Cattle

The objective of this study was to investigate the ruminal bacterial communities as affected by monensin, haylage, and their interaction of feedlot cattle fed 60 % dried distillers grains with solubles in a replicated 4 × 4 Latin square design. Pyrosequencing analysis of the V1–V3 region (about 500 bp) of 16S rRNA gene from the four dietary treatments (3 treatment plus one control diets) collectively revealed 51 genera of bacteria within 11 phyla. Firmicutes and Bacteroidetes were the first and the second most predominant phyla, respectively, irrespective of the dietary treatments. Monensin supplementation decreased the proportion of Gram-positive Firmicutes while increasing that of Gram-negative Bacteroidetes. However, the monensin supplementation did not reduce the proportion of all genera of Gram-positive bacteria placed within Firmicutes and lowered that of some genera of Gram-negative bacteria placed within Bacteroidetes. Haylage supplementation appeared to attenuate inhibition of monensin on some genera of bacteria. Factors other than monensin and haylage could affect ruminal bacterial communities.