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Cultural Methods and Some Characteristics of Some of the More Numerous Groups of Bacteria in the Bovine Rumen
... To determine how much acid was produced from d-xylose and cellobiose, bromocresol green was added. According to Bryant and Burkey [16], a glass electrode pH meter was applied to measure the generation of acid from glucose. ...
... The detection of starch hydrolysis was done by adding Gram's iodine solution to the cultures after a week when the color change occurred. Bryant and Burkey described how to use this technique [16]. ...
... A rubber stopper holding nutrient gelatin medium was used to inject a 48-h-old inoculum of test bacteria into the serum container using an insulin syringe. After cultures were incubated for a week, Bryant and Burkey tested their capacity to liquefy gelatin [16]. ...
Objective
To isolate and characterize cellulolytic rumen bacteria from the rumen of Sahiwal cattle using rumen bacterial inoculum to increase the nutritional value of rice bran used as broiler feed.
Materials and Methods
The ruminal liquid was kept at an optimal pH of 6.9 and a redox potential of less than −300 mV while being incubated anaerobically at 39°C in a medium containing rumen fluid glucose cellobiose agar. By using the Hungate technique, the organisms were detected based on their morphological, physiological, biochemical, and molecular testing.
Results
The findings revealed that the isolated Ruminococcus albus, and Ruminococcus flavifaciens were obligate anaerobic, generally Gram-positive, nonmotile cocci or rod, single or pair, occasionally short chain, producing yellow pigment when grown on cellulose, and having a clear zone around the colonies. Both isolate fermented sugars such as cellobiose, glucose, and lactose, as well as decomposed xylan. The results also showed that the isolates recognized as Ruminococcus spp., a cellulolytic rumen bacterium, were catalase-negative, indole-negative, and gelatin liquefaction-positive.
Conclusion
Isolation and characterization of Ruminococcus spp. may be helpful for Bangladesh in reducing the cost of producing poultry feed and circumventing restrictions on rice bran use. We can also develop more efficient and long-lasting plans to enhance poultry performance and feed efficiency, as well as increase the nutritional value of rice bran used as broiler feed, by understanding how various Ruminococcus spp. function in this process.
... 6 and no. 17 were received in a lyophilized condition and were reconstituted in rumen fluid-glucose-cellobiose agar (RGCA) medium (9) and grown anaerobically in the presence of carbon dioxide at 37 C. The strain designated as CHR was isolated in Toronto from fresh rumen juice. ...
... Later the stock cultures were replaced by the same strains, obtained from M. P. Bryant, University of Illinois, Urbana. Both sets of strains were received as stabs in RGCA rumen medium stubs (9). ...
... Isolation Methods for Selenomonads Rumen selenomonads were isolated from fresh rumen juice, obtained from a slaughter house (Canada Packers, Ltd., Toronto), by the roll tube method of Hungate (38) but using RGCA medium (9). ...
... The cultures were grown according to the method of Bryant and Burkey (1953). The initial tests for ability to incorporate sulfate were performed on broth A, which contained inorganic salts, glucose, cellobiose, and 40 per cent centrifuged sterile rumen liquid. ...
... The initial tests for ability to incorporate sulfate were performed on broth A, which contained inorganic salts, glucose, cellobiose, and 40 per cent centrifuged sterile rumen liquid. This broth differs from the one described by Bryant and Burkey (1953) only in that the sulfate salts were replaced by equivalent weights of their chlorides. Sufficient magnesium sulfate was included to give a final sulfate-sulfur concentration of about 0.03 mg per ml and an additional 0.03 mg sulfur per ml was present as cysteine. ...
... The radioactive sulfate used in this trial was essentially carrier-free. Nine tubes in each series (methionine and no-methionine) were inoculated with a straight needle from a 3-day old culture of L. multiparus on rumen fluid-glucose-cellobiose agar (Bryant and Burkey, 1953) and incubated at 39 C. One tube was removed from each series after incubation for 18 hr. Four tubes were removed after 25 hr and the remaining four tubes in each series were 363 on December 19, 2020 by guest http://aem.asm.org/ ...
... Rumen content samples were collected, bacteria were enumerated, and strains were isolated using the methods and rumen fluid-glucose-cellobiose agar (RGCA) roll tubes as described by Bryant and Burkey (1953), except that sugars added to the isolation medium included glucose, maltose, and cellobiose each at a 0.1 per cent (w/v) final concentration. After isolation strains were stored as RGCA slant cultures in a Dry Ice box. ...
... The ammonia test medium used to determine ammonia production of 75 previously described strains of ruminal bacteria contained mineral solutions 1 and 2 of Bryant and Burkey (1953) with (NH4)2SO4 deleted; resazurin, 0.0001 per cent (w/v); Trypticase, 1.5 per cent (w/v); a solution of the sodium salts of certain volatile fatty acids (acetate, n-valerate, isovalerate, DL-a-methyl-n-butyrate, and isobutyrate at concentrations of 420, 60, 30, 30, and 30 /.LM per ml, respectively), 1 per cent (v/v); clarified rumen fluid, 5 per cent (v/v); glucose, maltose, and cellobiose, 0.066 per cent (w/v) of each; cysteine HCl H20, 0.05 per cent (w/v); Na2CO3, 0.4 per cent (w/v); and a gaseous phase of carbon dioxide. The medium was prepared in a manner similar to that of Bryant and Burkey (1953) with cysteine, Na2CO3, and rumen fluid added as sterile solutions equilibrated with carbon dioxide after other ingredients of the medium were autoclaved. ...
... The ammonia test medium used to determine ammonia production of 75 previously described strains of ruminal bacteria contained mineral solutions 1 and 2 of Bryant and Burkey (1953) with (NH4)2SO4 deleted; resazurin, 0.0001 per cent (w/v); Trypticase, 1.5 per cent (w/v); a solution of the sodium salts of certain volatile fatty acids (acetate, n-valerate, isovalerate, DL-a-methyl-n-butyrate, and isobutyrate at concentrations of 420, 60, 30, 30, and 30 /.LM per ml, respectively), 1 per cent (v/v); clarified rumen fluid, 5 per cent (v/v); glucose, maltose, and cellobiose, 0.066 per cent (w/v) of each; cysteine HCl H20, 0.05 per cent (w/v); Na2CO3, 0.4 per cent (w/v); and a gaseous phase of carbon dioxide. The medium was prepared in a manner similar to that of Bryant and Burkey (1953) with cysteine, Na2CO3, and rumen fluid added as sterile solutions equilibrated with carbon dioxide after other ingredients of the medium were autoclaved. The clarified rumen fluid was prepared by centrifugation (12,500 X g for 10 min) of rumen fluid obtained by filtration through cheese cloth of rumen contents obtained from animal B at 11 a.m. ...
... Prevotella strains, detected as OTU218, were isolated from the rumen of two cows maintained at the National Institute of Livestock and Grassland Science, according to previously described procedures (Shinkai et al., 2022). Briefly, rumen fluid was collected using a rumen catheter, sieved through three layers of surgical gauge, and diluted serially (10-fold) with an anaerobic dilution solution (Bryant and Burkey, 1953). Subsequently, diluted rumen bacteria were inoculated into modified YTR agar medium (Bryant and Burkey, 1953) to form roll tubes with a gas-tight Hungate anaerobic tube. ...
... Briefly, rumen fluid was collected using a rumen catheter, sieved through three layers of surgical gauge, and diluted serially (10-fold) with an anaerobic dilution solution (Bryant and Burkey, 1953). Subsequently, diluted rumen bacteria were inoculated into modified YTR agar medium (Bryant and Burkey, 1953) to form roll tubes with a gas-tight Hungate anaerobic tube. The constituents of the modified YTR agar medium (per liter) include yeast extract, 1.2 g (Oxoid); Bacto peptone, 2 g (Difco); mineral solution I, 75 mL; mineral solution II, 75 mL; clarified rumen fluid, 300 mL; glucose, 1.5 g; cellobiose, 1.5 g; resazurin (0.1%), 10 mL; hemin solution (0.5 g /L), 10 mL; NaHCO3 (8%), 50 mL; L-cysteine hydrochloride, 3 g; bacto agar, 1.2 g (Difco); distilled water, 500 mL. ...
Ruminal methane production is the main sink for metabolic hydrogen generated during rumen fermentation, and is a major contributor to greenhouse gas (GHG) emission. Individual ruminants exhibit varying methane production efficiency; therefore, understanding the microbial characteristics of low-methane-emitting animals could offer opportunities for mitigating enteric methane. Here, we investigated the association between rumen fermentation and rumen microbiota, focusing on methane production, and elucidated the physiological characteristics of bacteria found in low methane-producing cows. Thirteen Holstein cows in the late lactation stage were fed a corn silage-based total mixed ration (TMR), and feed digestion, milk production, rumen fermentation products, methane production, and rumen microbial composition were examined. Cows were classified into two ruminal fermentation groups using Principal component analysis: low and high methane-producing cows (36.9 vs. 43.2 L/DMI digested) with different ruminal short chain fatty acid ratio [(C2+C4)/C3] (3.54 vs. 5.03) and dry matter (DM) digestibility (67.7% vs. 65.3%). However, there were no significant differences in dry matter intake (DMI) and milk production between both groups. Additionally, there were differences in the abundance of OTUs assigned to uncultured Prevotella sp., Succinivibrio, and other 12 bacterial phylotypes between both groups. Specifically, a previously uncultured novel Prevotella sp. with lactate-producing phenotype was detected, with higher abundance in low methane-producing cows. These findings provide evidence that Prevotella may be associated with low methane and high propionate production. However, further research is required to improve the understanding of microbial relationships and metabolic processes involved in the mitigation of enteric methane.
... Efforts to cultivate these strictly O 2 -sensitive organisms resulted in the further modification of anaerobic culturing techniques and prepared the space for the cultivation of methanogens. The focus was on the cultivation of anaerobic bacteria from the rumen environment, such as cellulose and xylene-degrading bacteria [32,33]. A pioneer in the further development of anaerobic cultivation techniques was Hungate. ...
... Bryant preferred to inoculate the picked colonies into a slant medium rather than a liquid medium. The concentrated inoculum was found to be better adapted to the conditions in the new environment, as inoculation into liquid media is sometimes unsuccessful [32,81,98]. Applying this method, the microorganisms grow through the agar and form surface and subsurface colonies. ...
The cultivation and investigation of strictly anaerobic microorganisms belong to the fields of anaerobic microbial physiology, microbiology, and biotechnology. Anaerobic cultivation methods differ from classic microbiological techniques in several aspects. The requirement for special instruments, which are designed to prevent the contact of the specimen with air/molecular oxygen by different means of manipulation, makes this field more challenging for general research compared to working with aerobic microorganisms. Anaerobic microbiological methods are required for many purposes, such as for the isolation and characterization of new species and their physiological examination, as well as for anaerobic biotechnological applications or medical indications. This review presents the historical development of methods for the cultivation of strictly anaerobic microorganisms focusing on methanogenic archaea, anaerobic cultivation methods that are still widely used today, novel methods for anaerobic cultivation, and almost forgotten, but still relevant, techniques.
... The bacteria contained in the hydrolysis stage were Bacillus and Clostridium (Wirth et al. 2012). This was in accordance with (Bryant and Burkey 1976) that microbes that had a role in the hydrolysis process were Clostridium aceticum, Bacteriodes ruminicola, Bifidobacterium sp., E. coli, Enterobacter sp., Desulfurvibrio sp., Pseudomonas sp., Flavobacterium alkaligenes, and Aerobacter. Observations based on the genus found Lactobacillus and syntrophomonas bacteria, which showed the acidogenesis process. ...
... Observations based on the genus found Lactobacillus and syntrophomonas bacteria, which showed the acidogenesis process. Similarly, (Bryant and Burkey 1976) suggested that microbes that had a role in the acetogenesis were Syntrophobacter sp. and Pelotomaculum sp. ...
Increasing energy demand is not alongside the availability of limited fossil fuels. Alternative and renewable energy sources are not only an option to overcome energy problems but also essential to minimize global warming. Another critical and promising renewable energy source is biomass-derived from livestock feces. Beef cattle feces contain a microorganism consortium that can be used as a starter with coal media to form biogas. Indonesia recently developed coal waste processing into renewable energy, such as biogas. This study aimed to overview the ecological diversity of microbial consortium of beef cattle feces, lignite coal waste, and a combination of livestock and lignite coal waste under mesophilic conditions. This research is an explorative method, the data obtained were analyzed descriptively. The process of formation was carried out anaerobically on a bottle containing the rumen fluid medium. The fermentation process lasted 42 days at 39℃ of temperature. After that, the sample was electrophoresis, followed by next-generation sequencing (NGS) method. NGS data is processed with the MG-Rast website. This study demonstrates the ecological diversity of microbial consortium of beef cattle, lignite coal waste, and a combined consortium. The results showed ecological diversity in the form of taxonomy dominated by bacteria, eukaryotes, and archaea.
... The feed ingested by the ruminant animal undergoes extensive microbial digestion and fermentation in the cattle rumen, producing a range of short chain fatty acids (SCFAs) and energy for the host, and also releases methane, hydrogen, and carbon di-oxide to the atmosphere281 . This anaerobic environment is densely (>10 10 -10 11 cells/g of contents) populated by diverse and interdependent species of Bacteria, Protozoa, Fungi, Archaea and Viruses, which are involved in breakdown of complex carbohydrates and polymers from plants, hydrogen transfer, and inter-species transaction of fermentation products and of oligomers and monomers[282][283][284][285] . Among the major bacterial players are the phyla Bacteroidetes and Firmicutes, with an abundance of 21% and 12% of the bacterial population in the rumen, respectively280 . ...
... The representative community successfully captured most of the known metabolic interactions with the three functional guilds of microbes, as was evident from the flux values of the shared metabolites. The extent and directionalities of the major metabolic exchanges in the community are shown inFigure 4.1, which shows that the model was able to capture the known rumen microbiome behavior in terms of metabolite production, consumption, and inter-species transactions280,[282][283][284][285] . Complex plant carbohydrates and proteins are utilized by R. flavefaciens and P. ruminicola, and the short-chain fatty acids (SCFAs) are absorbed by the rumen epithelium.M. gottschalkii accepts hydrogen, formate, carbon di-oxide from the other two members, and releases methane to the atmosphere, similar to what was described in previous reports280,[288][289][290] . ...
Computations and modeling have emerged as indispensable tools that drive the process of understanding, discovery, and redesign of biological systems. With the accelerating pace of genome sequencing and annotation information generation, the development of computational pipelines for the rapid reconstruction of high-quality genome-scale metabolic networks has received significant attention. These models provide a rich tapestry for computational tools to quantitatively assess the metabolic phenotypes for various systems-level studies and to develop engineering interventions at the DNA, RNA, or enzymatic level by careful tuning in the biophysical modeling frameworks. in silico genome-scale metabolic modeling algorithms based on the concept of optimization, along with the incorporation of multi-level omics information, provides a diverse array of toolboxes for new discovery in the metabolism of living organisms (which includes single-cell microbes, plants, animals, and microbial ecosystems) and allows for the reprogramming of metabolism for desired output(s). Throughout my doctoral research, I used genome-scale metabolic models and omics-integrative analysis tools to study how microbes, plants, animal, and microbial ecosystems respond or adapt to diverse environmental cues, and how to leverage the knowledge gleaned from that to answer important biological questions. Each chapter in this dissertation will provide a detailed description of the methodology, results, and conclusions from one specific research project. The research works presented in this dissertation represent important foundational advance in Systems Biology and are crucial for sustainable development in food, pharmaceuticals and bioproduction of the future. Advisor: Rajib Saha
... Members of the genus require anaerobic conditions, a carbohydrate energy source such as cellulose, cellobiose, or xylan, and some require carbon dioxide even in complex media (Bryant and Burkey, 1953). It was early recognized that unknown and apparently unusual growth factors were required by some strains (Hungate 1950;Sijpesteijn, 1951). ...
... The strains of ruminococci studied were previously described and specifically identified (Bryant et al., 1958b, c). Stock cultures were grown in stabs of rumen fluid-glucose-cellobiose agar (RGCA) slant medium (Bryant and Burkey, 1953) and stored in a Dry Ice box with transfers at about 6-month intervals. ...
... Assay for ,1-isopropylmalate dehydrogenase. Cells of Ruminococcusflavefaciens, strain C94, were grown on a medium similar to the RGCA medium of Bryant and Burkey (11), except that glucose and agar were deleted. To maintain anaerobiosis, the medium was bubbled slowly with O2-free CO2 while it was cooled in ice water immediately after it was removed from the autoclave. ...
... Cysteine-HCI and Na2CO3 solutions were added to the cooled medium and Na2S-9H2O (0.5 mg/ml, sterilized under N2) was added just prior to inoculation. Cells from 2 liters of medium were harvested by centrifugation after growth for 17 hr to optical density (OD) 0.39 (600 m, in 18-mm cuvettes) and were washed once with anaerobic dilution solution (11). ...
Certain anaerobic ruminal bacteria synthesize the leucine carbon skeleton by use of a pathway different from that described in other microorganisms. These organisms carboxylate the intact carbon skeleton of isovalerate, synthesizing leucine-2-C¹⁴ from isovalerate-1-C¹⁴. Strains of Bacteroides ruminicola and Peptostreptococcus elsdenii were like Ruminococcus flavefaciens in that they incorporated appreciable amounts of C¹⁴ from isovalerate-1-C¹⁴ into cellular protein and in that the only labeled amino acid found was leucine. The specific activity of β-isopropylmalate dehydrogenase in extracts from R. flavefaciens and from the mixed bacterial population from the rumen was very low as compared with the specific activity of this enzyme in extracts from Escherichia coli. This suggests that the pathway of leucine biosynthesis that operates in many aerobic and facultative microorganisms is not the major pathway in rumen bacteria. This was supported by the finding that after fermentation of whole rumen contents with acetate-2-C¹⁴, leucine from the bacterial cells had a specific activity lower than one would expect if acetate was incorporated directly into carbons 1 and 2 of leucine.
... B. fibrisolvens D1 T is Gram positive but stains Gram negative (2) and can ferment a wide range of carbohydrates, including xylan, pectin, and starch, with the production of H 2 , CO 2 , butyrate, formate, and lactate. D1 T was originally isolated from bovine rumen contents inoculated into 40% rumen fluid-glucose-cellobiose agar medium roll tubes (3). The available draft genome of D1 T (GCA_900129945) is the sole member of Butyrivibrio fibrisolvens species group in the Genome Taxonomy Database Toolkit (GTDB-Tk), while 10 other isolate genomes cluster as Butyrivibrio fibrisolvens_C (4,5). ...
Butyrivibrio are anaerobic bacteria and members of the family Lachnospiraceae with important roles in fiber digestion in both animals and humans. This report describes the complete genome of Butyrivibrio fibrisolvens type strain D1T (DSM 3071) consisting of a chromosome (CP146963), megaplasmid (pNP243), and small plasmid (pNP21).
... Ten-milliliter volumes of the C. jejuni-inoculated, sugar-amended Bolton broth were then distributed under a continuous flow of microaerobic gas mix (10 % CO 2 , 5 % O 2 , 85 % N 2 ) to presterilized 18 × 150 mm crimp-top culture tubes. Triplicate sets of the culture tubes were subsequently inoculated individually (0.2 mL) or jointly (0.1 mL each) with suspensions of the freshly collected gastrointestinal tract populations, previously serially 10-fold-diluted in anaerobic dilution solution [30] to 1:10,000, to compare effects of the bovine rumen, bovine fecal microorganisms or their combination. The freshly collected bovine rumen and fecal samples were each diluted to deplete endogenous substrate and to dilute to extinction the potential effect of wildtype Campylobacter that may have been present in the samples. ...
Infection with the foodborne pathogen Campylobacter is the leading bacterial cause of human foodborne illness in the United States. The objectives of this experiment were to test the hypothesis that mixed microbial populations from the bovine rumen may be better at excluding Campylobacter than populations from freshly voided feces and to explore potential reasons as to why the rumen may be a less favorable environment for Campylobacter than feces. In an initial experiment, C. jejuni cultures inoculated without or with freshly collected bovine rumen fluid, bovine feces or their combination were cultured micro-aerobically for 48 h. Results revealed that C. jejuni grew at similar growth rates during the first 6 h of incubation regardless of whether inoculated with the rumen or fecal contents, with rates ranging from 0.178 to 0.222 h−1. However, C. jejuni counts (log10 colony-forming units/mL) at the end of the 48 h incubation were lowest in cultures inoculated with rumen fluid (5.73 log10 CFUs/mL), intermediate in cultures inoculated with feces or both feces and rumen fluid (7.16 and 6.36 log10 CFUs/mL) and highest in pure culture controls that had not been inoculated with the rumen or fecal contents (8.32 log10 CFUs/mL). In follow-up experiments intended to examine the potential effects of hydrogen and hydrogen-consuming methanogens on C. jejuni, freshly collected bovine feces, suspended in anaerobic buffer, were incubated anaerobically under either a 100% carbon dioxide or 50:50 carbon dioxide/hydrogen gas mix. While C. jejuni viability decreased <1 log10 CFUs/mL during incubation of the fecal suspensions, this did not differ whether under low or high hydrogen accumulations or whether the suspensions were treated without or with the mechanistically distinct methanogen inhibitors, 5 mM nitrate, 0.05 mM 2-bromosulfonate or 0.001 mM monensin. These results suggest that little if any competition between C. jejuni and hydrogen-consuming methanogens exists in the bovine intestine based on fecal incubations.
... Native strain isolation. Aliquots from each collected ruminal fluid were added into Petri dishes containing the culture medium proposed by Bryant and Burkey (1953) for cellulolytic bacteria isolation and physiological studies, changing the primary substrate according to the strains to be isolated (Table 1). In all inoculated Petri dishes, classic plate − casting processes were used for inoculation according to Stanier et al. (1986), and incubated at 39 (±1)°C for 24, 48, and 72 h in an anaerobic incubator (CO 2 incubator, model NU-5510, Nuaire, Plymouth, MN, USA). ...
Increasing the digestibility of agricultural residues is one goal of sustainable livestock production, and the role of rumen microbiome-feed interaction is critical for efficient digestion. This study was aimed at isolating and evaluating in vitro the effect on dry-matter digestibility (IVDMD), relative feed values (RFV), volatile fatty acid (VFA) synthesis, pH changes, and gas production (GP) by the inclusion of native rumen strains in mixtures of corn, sorghum, and oat agricultural residues with ruminal fluid. The results show that an inoculum of 1 × 10¹⁰ colony-forming unit per millilitre (CFU/mL) of the aero-tolerant native rumen strain Lysinibacillus fusiformis increases the IVDMD, RFV, and VFA values of all mixtures. During agricultural residue fermentation, the pH was stable, and propionic acid was the primary fatty acid synthesized, indicating increased energy availability for efficient cattle growth performance while limiting molecular hydrogen (H2) synthesis for conversion to methane (CH4). These results suggest that L. fusiformis could be used as a direct-feed microbial to promote sustainable livestock production. To the best of our knowledge, this is the first report to link the fermentation of fibrous agricultural residues in ruminal fluid inoculated with a fibrolytic native strain and digestibility in favour of increasing the efficiency of livestock production.
... Ileum contents were placed into sterile tubes and immediately used to examine the microbial population. After all microbial samples were subjected to anaerobic diluents of serial dilution (10 À4 −10 À6 ) and inoculated with sterile agar, the procedure of Bryant and Burkey (1953) was followed to perform microbial analysis. For each selective culture, a specific agar was used to grow and account for the total aerobic bacteria and specific colony type of coliforms, lactic acid bacteria, Escherichia coli, Salmonella spp. ...
The experiment was implemented to assess the influence of dietary supplementation of laying quails with creatine monohydrate (CrM), L-carnitine (CAR) and their mixture (CrMCAR) as antioxidants against oxidative stress (OS) induced by 2.5 ppm lead acetate (LA) in drinking water on productive, physiological and microbial aspects. In total, 400 laying quail females at 10 wk of age were divided into a randomized design with 5 groups and 4 replicates of 20 birds each. Birds were fed ad libitum with a balanced diet for 8 wk. The control group was kept under no-stress conditions and was given fresh water without any additives (G1). While birds in other groups were exposed to OS induced experimentally by 2.5 ppm LA in drinking water with no feed additive (G2) or supplemented with 500 mg/kg CrM (G3) or 500 mg/kg CAR (G4) or combination of 250 mg/kg each of CrM and CAR (CrMCAR, G5) to feed mixture.
Compared to G2, G5 demonstrated the reduction (P ≤ 0.05) of feed conversion ratio, feed intake, mortality and ileal total coliform, as well as serum and egg malondialdehyde and serum lipid hydroperoxide, uric acid, glucose, cholesterol, enzymatic activities (alanine aminotransferase, aspartate transaminase, alkaline phosphatase, creatine phosphokinase, γ-glutamyl transferase), and heterophils/lymphocytes ratio. In the meanwhile, there was an increase (P ≤ 0.05) in egg production, egg mass, and weight with the improvement of egg quality, serum sex hormones level and ileal lactic acid bacteria for G5 followed by G4 and G3. Moreover, G5 enhanced (P ≤ 0.05), the total antioxidant capacity of egg and serum glutathione, superoxide dismutase, catalase, glutathione peroxidase, protein and calcium levels. Therefore, dietary CrMCAR, CAR and CrM have analogous influence to control by improving the antioxidant and physiological parameters which resulted in better productive performance and egg characteristics of stressed quails. These antioxidants, especially in their equal combination, are beneficial to alleviate oxidative stress incidence and can be recommended for poultry feeding under various aspects of environmental stresses.
... Individual boli from each cow were processed for enumeration of total viable anaerobic bacteria and cellulolytic bacteria immediately after collection using the most probable number (MPN) method of Dehority et al. [15]. Samples (5 g) of bolus or forage (as a negative control) were placed in a bag, which was gassed extensively with carbon dioxide prior to anaerobic diluting solution [16] (45 mL) being added. The bag contents were then stomached for 3 min prior to serial dilution and inoculation into MPN broth for simultaneous total anaerobic bacteria and cellulolytic bacteria counts [15]. ...
Sampling of ruminant saliva has gained interest as a non-invasive proxy for exploring the structure of the rumen microbiome. However, the subsequent data analysis assumes that bacteria originating from the oral cavity are merely passengers in the rumen and play no active role. In this study, it was hypothesised that metabolically active oral bacteria present in the salivary microbiome play a role in the ruminal degradation of plant material. In vitro cultivation-based enumeration confirmed that the ruminant oral cavity harbours a significant number of anaerobic and cellulolytic bacteria that are metabolically active under ruminal conditions. Bacterial 16S rRNA gene profiling of in vitro enrichments also confirmed that oral-derived bacteria were capable of colonising plant material. Preliminary analysis of the colonising bacteria indicated that bacteria belonging to the genus Streptococcus were of particular interest. In conclusion, the findings of the current study clearly indicate that bolus-associated bacteria have the potential to play a metabolically active role in terms of ruminal colonisation and the degradation of plant material. This evidence confirms the merit of the hypothesis that the metabolically active oral bacteria present in the salivary microbiome may play a role in the ruminal degradation of plant material.
... Rumen contents were incubated with cellulose powder for 10 min at 37 °C, before harvesting cellulose and the adherent bacteria by centrifugation (250 g for 10 min). After washing the cellulose pellet five times with phosphate-buffered saline, the pellet was resuspended in anaerobic dilution solution [7]. Serial dilutions of the resuspended pellet were then used as inoculum for isolations using the anaerobic roll-tube method with rumen fluid-glucose-cellobiose-maltose-starch-agar (RGCMSA) medium, as previously described [8]. ...
Two strains of Gram-negative, anaerobic, rod-shaped bacteria, from an abundant but uncharacterized rumen bacterial group of the order 'Christensenellales', were phylogenetically and phenotypically characterized. These strains, designated R-7T and WTE2008T, shared 98.6-99.0 % sequence identity between their 16S rRNA gene sequences. R-7T and WTE2008T clustered together on a distinct branch from other Christensenellaceae strains and had <88.1 % sequence identity to the closest type-strain sequence from Luoshenia tenuis NSJ-44T. The genome sequences of R-7T and WTE2008T had 83.6 % average nucleotide identity to each other, and taxonomic assignment using the Genome Taxonomy Database indicates these are separate species within a novel family of the order 'Christensenellales'. Cells of R-7T and WTE2008T lacked any obvious appendages and their cell wall ultra-structures were characteristic of Gram-negative bacteria. The five most abundant cellular fatty acids of both strains were C16 : 0, C16 : 0 iso, C17 : 0 anteiso, C18 : 0 and C15 : 0 anteiso. The strains used a wide range of the 23 soluble carbon sources tested, and grew best on cellobiose, but not on sugar-alcohols. Xylan and pectin were fermented by both strains, but not cellulose. Acetate, hydrogen, ethanol and lactate were the major fermentation end products. R-7T produced considerably more hydrogen than WTE2008T, which produced more lactate. Based on these analyses, Aristaeellaceae fam. nov. and Aristaeella gen. nov., with type species Aristaeella hokkaidonensis sp. nov., are proposed. Strains R-7T (=DSM 112795T=JCM 34733T) and WTE2008T (=DSM 112788T=JCM 34734T) are the proposed type strains for Aristaeella hokkaidonensis sp. nov. and Aristaeella lactis sp. nov., respectively.
... From the same birds per replicate (n=2/replicate) which bled for carcass quality determination, they were subjected for microbial examination at the end of the experiment, the ileal contents were extracted separately from each replicate and collected gently by squeezing into the sterile tubes. The method of Bryant and Burkey (1953) was followed, briefly, before inoculation of microbial isolations onto Petri dishes with sterile agar, a serial dilution (10 -4 to 10 -6 ) of ileal collections in anaerobic diluents was performed. The diluents were prepared from 9 ml phosphate buffer solution in sterile test tubes. ...
This study was aimed to investigate the effects of L-carnitine (Car), creatine monohydrate (CrM) and their combination (CarCrM) as dietary antioxidants materials on productive performance, digestibility, ileal eubiosis, blood chemistry and redox system of stressed quails challenged by lead acetate (LA) in drinking water. In total, 600 quails were assigned into 5 treatments with 4 replications each and 30 chicks per replication from 1 until 42 days old. The treatments involved control (T1), stressed treatment (adding 2.5 ppm of LA in drinking water only; T2) and treatments of adding 500 mg/kg Car+LA (T3), 500 mg/kg CrM+LA (T4) and CarCrM (250 mg/kg Car plus 250 mg/kg CrM)+LA (T5). A completely randomized design was followed to analyze treatment influences on variables. In comparison to T2, the results showed that T5 and T4 had equivalent positive influence followed by T3 to increase (p≤0.05) body weight, feed intake, survivability, carcass yield and digestibility accompanied with increase lactic acid bacteria and reduced total coliform, E.coli in ileum. Also, increased levels (p≤0.05) of glutathione peroxidase, superoxide dismutase, catalase and glutathione in serum and ferric-reducing ability of plasma were obtained by T5 followed by T4. Moreover, T5 and T4 achieved low values (p≤0.05) of feed efficiency and serum lipid hydroperoxide, malondialdehyde, cortisol, aspartate transaminase, alanine aminotransferase, alkaline phosphatase,gamma-glutamyl transferase, creatine kinase and creatinine. The results confirm that dietary CarCrM or CrM mitigated stress and reinforced antioxidant pool which was reflected by supported productive and physiological aspects of birds. Dietary Car seemed less powerful effect than CarCrM and CrM but without negative influence compared with stressed treatment.
... Rats were weaned at 3-4 weeks of age and inoculated with 0.2 mL of infant fecal slurries from single donors by a single intragastric gavage [100-fold dilution of fecal sample in anaerobic mineral solution (27) that was composed of 7.5% v/v mineral solution I (0.3% w/v K 2 HPO 4 ), 7.5% v/v mineral solution II [0.3% w/v K 2 HPO 4 ; 0.6% w/v NaCl; 0.6% w/v (NH 4 ) 2 SO 4 ; 0.06% w/v MgSO 4 ; 0.06% w/v CaCl 2 ], 0.01% w/v resazurin, 0.4% w/v NaHCO 3 , 0.05% w/v cysteine hydrochloride, and distilled water; all chemicals were obtained from Sigma-Aldrich]. IMA rats were placed in cages housing two animals. ...
Establishing the relationship between gut microbiota and host health has become a main target of research in the last decade. Human gut microbiota-associated animal models represent one alternative to human research, allowing for intervention studies to investigate causality. Recent cohort and in vitro studies proposed an altered gut microbiota and lactate metabolism with excessive H2 production as the main causes of infant colic. To evaluate H2 production by infant gut microbiota and to test modulation of gut colonizer lactose- and lactate-utilizer non-H2-producer, Cutibacterium avidum P279, we established and validated a gnotobiotic model using young germ-free rats inoculated with fecal slurries from infants younger than 3 months. Here, we show that infant microbiota-associated (IMA) rats inoculated with fresh feces from healthy (n = 2) and colic infants (n = 2) and fed infant formula acquired and maintained similar quantitative and qualitative fecal microbiota composition compared to the individual donor’s profile. We observed that IMA rats excreted high levels of H2, which were linked to a high abundance of lactate-utilizer H2-producer Veillonella. Supplementation of C. avidum P279 to colic IMA rats reduced H2 levels compared to animals receiving a placebo. Taken together, we report high H2 production by infant gut microbiota, which might be a contributing factor for infant colic, and suggest the potential of C. avidum P279 in reducing the abdominal H2 production, bloating, and pain associated with excessive crying in colic infants.
... Microbial status: At the end of trial (42 days), eight birds from each dietary treatment were sacrificed by cervical dislocation; crop and jejunum scraping were collected in sterile vials for evaluation of total microbial load colonization. Microbial populations were determined by serial dilution (10 4 to 10 6 ) of crop and jejunum samples in anaerobic diluents before inoculation onto petri dishes of sterile agar as described by Bryant and Burkey [5]. Total bacterial count and Lactobacilli was grown on nutrient agar and rogosa agar respectively [8]. ...
A biological experiment of six weeks was undertaken with completely randomized design (CRD) on broiler chickens to investigate the effects of rice gluten meal (RGM) feeding on gut health, immunity and intestinal histomorphometry in broilers. A total of 192 day old chicks were taken and divided into six treatments with 32 chicks with four replicates per treatment. Six experimental diets as per ICAR (2013) were prepared by incorporating five different levels of RGM (0, 10, 15, 20, 25 and 30 %). Chemical analysis on as such basis indicated that RGM contained crude protein 49.94% and gross energy 4742 kcal/kg. In crop, total viable count (TVC) decreased significantly (P<0.01) at 30% RGM inclusion level as compared to control and other dietary treatments. Lactobacillus count were significantly (P<0.01) increased in 25 and 30% RGM levels. In jejunum, no significant (P>0.05) difference were observed in TVC and Lactobacillus count in control and other dietary treatments. Humoral immunity was significantly (P<0.05) better in 30% RGM group as compared to control but cell mediated immunity did not show any significant (P>0.05) difference between control and other dietary treatments. Villus height (VH) decreased significantly (P<0.01) in 20, 25 and 30% RGM levels but villus depth (VD) and VH/VD did not show any significant(P>0.05) difference between control and other dietary treatments. Thus, it is concluded that RGM can safely can be incorporated in broiler diet at the inclusion level of 15% without any adverse effect on humoral immunity, gut health and intestinal histomorphometry. INTRODUCTION Poultry industry is the fastest growing sector in Indian agriculture. Feed is the major constituent in the poultry production accounts for 65-75% of total recurring expenditure. Feed costs are primarily driven by the cost of protein sources. Substitution of expensive protein sources with lower cost ingredients would potentially reduce the cost of the feed. Soybean meal (SBM) is the major protein source used in poultry diet. Instability in its production, indiscriminate exports and higher demand has resulted in its shortage for the poultry industry leading to its higher price. Substitution of SBM at reasonable price will lead to economic broiler production [3, 4, 13]. India is second largest producers of rice in world after China, producing approximately 106.65 MT rice in 2013-14 [1]. Now days, certain newer rice by products are available in appreciable quantities and cheaper rate that can be utilized from rice processing industries such as rice gluten meal (RGM). Rice gluten meal is a by-product of wet-milling of rice obtained after starch extraction and syrup preparation. It is relatively a new feedstuff having brownish color and coarse powdery texture. Initial research finding showed that RGM can be included up to 10% level in broiler chicken without affecting feed efficiency and dressing percentage [20]. Metwally and Farahat [18] found that broiler fed RGM with different inclusion rates up to 12.5% had the same growth performance. Kumar et al. [15] found that RGM could replace ground nut cake (GNC) in the concentrate mixture of growing calves up to 75% level without any adverse effect on growth performance and nutrient utilization. Malik et al. [16] reported that replacement of ground nut cake (GNC) by RGM and maize gluten meal (MGM) at 75% level did not differ in dry matter intake, feed efficiency, average daily gain (ADG) in growing Sahiwal cattle [17].
... Eleven bacterial strains, including R5019 T , isolated from the rumen of two lactating Holstein cows maintained with total mixed ration at the Institute of Livestock and Grassland Science of NARO, were subjected to the genomic and physiological analyses. Briefly, strains were isolated and identified using the following procedures: rumen fluid of the cows was collected through the mouth using a rumen catheter (Sanshin Industrial), filtered through three layers of gauze, serially diluted (10-fold) with anaerobic dilution solution [7], and a roll tube with modified yeast extract-trypticase-rumen fluid (YTR) agar medium. Colonies that appeared after 48-72 h incubation at 37 °C were isolated. ...
The genus Prevotella plays an important role in polysaccharide degradation and fermentation in the rumen. To further understand the function of the phylogenetically diverse genus Prevotella , it is necessary to explore the individual characteristics at the species level. In this study, Gram-negative anaerobic bacterial strains isolated from the rumen of Holstein cows were identified. Strain R5019 T was classified within the genus Prevotella based on 16S rRNA gene sequence-based phylogenetic analysis. The values of 16S rRNA gene sequence similarity, average nucleotide identity and digital DNA–DNA hybridization between strain R5019 T and its phylogenetically nearest species Prevotella multisaccharivorax PPPA20 T were 89.8, 82.6, and 29.3 %, respectively. The genome size of R5019 T was estimated to be ca. 4.19 Mb with a genomic G+C content of 49.5 mol%. The major cellular fatty acids and menaquinones were C 15 : 0 anteiso and C 17 : 0 anteiso and MK-11 and MK-12, respectively. Succinate, lactate, malate, acetate and formate were produced as the fermentation end products using glucose. Based on phylogenetic, physiological, biochemical and genomic differences between 11 strains and other phylogenetically related Prevotella species, a novel species, Prevotella lacticifex sp. nov., is proposed within the genus Prevotella . The type strain is R5019 T (=JCM 34664 T =DSM 112675 T ).
... In 1953, a medium to cultivate anaerobe bacteria of the rumen was using rumen fluid, glucose, cellobiose, and agar (RGCA medium), which allowed the isolation of cellulolytic bacterial strains without a source of cellulose. 81 In all three cases, the method used for the culture and isolation of anaerobic bacteria was the roll tube method. This method comprises cooling tubes containing agar medium and the bacterial inoculum that are turned rapidly in cold water to give an even dispersion of substrate and inoculum in the agar. ...
The utilization of dietary cellulose by resident bacteria in the large intestine of mammals, both herbivores and omnivores (including humans), has been a subject of interest since the nineteenth century. Cellulolytic bacteria are key participants in this breakdown process of cellulose, which is otherwise indigestible by the host. They critically contribute to host nutrition and health through the production of short-chain fatty acids, in addition to maintaining the balance of intestinal microbiota. Despite this key role, cellulolytic bacteria have not been well studied. In this review, we first retrace the history of the discovery of cellulolytic bacteria in the large intestine. We then focus on the current knowledge of cellulolytic bacteria isolated from the large intestine of various animal species and humans and discuss the methods used for isolating these bacteria. Moreover, we summarize the enzymes and the mechanisms involved in cellulose degradation. Finally, we present the contribution of these bacteria to the host.
... De igual forma que con el método roll-tube, se inoculan 0,5 mL en el medio, que contiene agar líquido, se inocula por triplicado, y los tubos se incuban a 39 °C con una inclinación de 30°. Finalizado el tiempo de incubación, se cuentan las colonias por réplica, y este valor es promediado entre el número de réplicas para obtener la unidad formadora de colonias (ufc/mL) (Bryant & Burkey, 1953;Hungate, 1957). ...
Esta publicación hace parte de las acciones de promoción y divulgación del Banco de Germoplasma de Microorganismos en su interés de fortalecer capacidades técnicas, científicas y metodológicas en conservación, caracterización y uso de las colecciones a su cargo, además de promover condiciones para la interconectividad e interoperabilidad de estas. El conocimiento plasmado en este libro es producto de una trayectoria de años de investigación por parte de cada uno de los curadores, que han dejado su legado y sus enseñanzas en una acción de relevo generacional que esperamos que sea de gran ayuda tanto para los próximos curadores como para quienes buscan, en su hacer diario, aumentar y conservar la diversidad microbiana. Entregamos este libro como parte de una estrategia de gestión de conocimiento para garantizar y mantener un uso sostenible de la agrobiodiversidad y contribuir de forma eficiente al crecimiento económico del país.
... Total fourty two numbers of bacterial isolates were isolated using Hungate's anaerobic roll tube technique (Hungate, 1969) using carboxymethylcellulose as the substrate in the culture medium. The culture medium for isolation of anaerobic fibrolytic bacteria was prepared as described by Bryant and Burkey (1953). Pure cultures were obtained by repeated roll tube preparation on agar medium, picking off the single bacterial colony, and subsequent sub-culturing into the broth medium. ...
Dietary interventions aiming at increasing the number and activities of beneficial gut microbes could enhance digestive functions as well as the long-term welfare of dairy animals. In the present experiment, the effect of supplementation of a novel bacterial culture Ruminococcus flavefaciens FD-1 isolated from the rumen liquor of Murrah buffaloes was studied for its effect on rumen microbial populations enumerated using the most probable number technique. Three permanently fistulated buffaloes maintained on a high fibre diet were used as the source of rumen fluid for bacterial isolation that was subsequently tested in vivo. Twelve healthy mid-to-late lactating buffaloes in their second to third parity were divided into two similar groups of six each with a mean body weight of 601.5 kg. One group (LBC) was dosed with 300 mL of live bacterial culture, whereas an equal volume of autoclaved bacterial culture was dosed in another group (ABC) for a period of one month. The treatment-wise effect of supplementation of R. flavefaciens FD-1 culture was evident in an increase (P<0.05) in total rumen fungal population by 2.7 times in LBC than ABC in the post-dosing period. However, the period-wise comparison revealed that the population of bacteria augmented (P<0.05) by 2.16 and 3.17 times in both the groups ABC and LBC, respectively; whereas the magnitude of increase in fungal population was 5.04 and 10.3 times, respectively for both the groups. Post-dosing effects were evident for only group LBC in both bacteria and fungi, respectively increasing (P<0.05) the population by 2.16 and 8.28 times than pre-dosing period. These preliminary results may foster scope for developing species-specific (autochthonous) bacterial probiotic/direct-fed microbial based on R. flavefaciens FD-1 for large ruminants maintained on fibrous diets under tropical production systems.
... Freshly voided fecal samples from each cat were immediately placed into sterile sampling bags (Whirlpac, Fisher Scientific, Pittsburgh, PA) that were sealed after expressing excess air. Each sample then was diluted 1:10 (wt/vol) in previously warmed (39°C) anaerobic diluting solution (Bryant and Burkey, 1953) by blending it for 15 s in a Waring blender under a stream of CO 2 . Blended, diluted feces were filtered through 4 layers of cheesecloth and sealed in 100-mL serum bottles under CO 2 . ...
Nine young adult male cats were used in a 3 × 3 factorial design to determine the effects of microbial adaptation to fiber type on changes in pH, total and hydrogen gas, short‐chain fatty acid (SCFA), and branched‐chain fatty acid (BCFA) production. Cats were adapted to a diet containing 4% cellulose, fructooligosaccharides (FOS), or pectin for 30 d prior to fecal sampling. Each cat was used as a single donor, and fecal inoculum was subjected to each of the aforementioned fiber substrates. Fructooligosaccharides produced the greatest changes in pH, total gas, hydrogen gas, acetate, propionate, butyrate, total SCFA, valerate, and total BCFA production ( P <0.001) regardless of adaptation to diet. Adaptation to the FOS or pectin diets increased production of total ( P =0.043) and hydrogen ( P =0.008) gas. Greater changes in pH ( P <0.001), acetate ( P =0.001), and total SCFA ( P =0.003) were observed for FOS and pectin substrates with adaptation to these respective substrates. Adaptation to the cellulose diet increased production of propionate ( P =0.045) when exposed to the FOS or pectin substrates. Adaptation to the pectin diet increased production of valerate and total BCFA ( P =0.039 and 0.022, respectively) when exposed to the cellulose or pectin substrates. Overall, adaptation to either FOS or pectin appear to increase SCFA and gas production.
... Enumeration of bacteria from fecal samples was performed using conventional anaerobic culture techniques in roll tubes [29]. To maintain strict anaerobic conditions, fresh fecal samples were diluted 10-fold in a mineral solution under a continuous flow of CO 2 [30]. Samples were then inoculated on non-selective medium and on selective media containing starch and lactic acid, for enumeration of total anaerobic bacteria, amylolytic and lactic acid-utilizing bacteria, respectively [31], after 48 h at 38 • C. The cellulolytic bacteria were cultured anaerobically in roll tubes containing a complex liquid medium with one filter paper strip as cellulose source for 14 days at 38 • C [32]. ...
In horses, abrupt changes from high-fiber (HF) to high-starch (HS) diets can affect the cecal and colonic microbiota. This study investigated modifications and recovery of fecal microbiota after two consecutive abrupt dietary changes. Twelve horses fed HF for 2 weeks were changed to HS for 5 days then returned to HF for 7 weeks. Six received lactic acid bacteria supplementation. Bacterial population diversity, structure, and activity, especially fibrolysis, were assessed to obtain an overview of alteration in hindgut microbiota. Two days after the abrupt change from HF to HS, the findings in feces were consistent with those previously reported in the cecum and colon, with a decrease in fibrolytic activity and an increase in amylolytic activity. Fecal parameters stabilized at their basal level 3–4 weeks after the return to HF. A bloom of cellulolytic bacteria and lower pH were observed after 1.5 weeks, suggesting a higher level of fiber degradation. In supplemented horses the relative abundance of potentially fibrolytic genera was enhanced 2 days after HS and 2 days to 2–3 weeks after the return to HF. Fecal analysis could be a promising technique for monitoring hindgut microbial variations accompanying dietary changes.
... Enumeration from fecal samples was performed using conventional anaerobic culture techniques. To maintain strict anaerobic conditions, fresh fecal samples were diluted in a mineral solution under continuous flow of CO 2 (Bryant and Burkey, 1953). Total anaerobic, starch-utilizing, and lactic acidutilizing bacteria were inoculated on non-selective medium and on selective media containing starch and lactic acid, respectively (Grimm et al., 2020), in roll tubes (Hungate and Macy, 1973), and incubated for 48 h at 38 • C. Cellulolytic bacteria were cultured anaerobically in roll tubes containing a complex liquid medium with one filter paper strip as cellulose source for 14 days at 38 • C (Halliwell and Bryant, 1963). ...
The equine hindgut ecosystem is specialized in dietary fibers’ fermentation to provide horses’ energy and contribute to its health. Nevertheless, antibiotics are known to disrupt the hindgut microbiota, affecting the fibrolytic activity of bacteria and the intestinal immune balance, leading to diseases. This in vivo study used a general and comprehensive approach for characterizing the hindgut ecosystem of 9 healthy horses over 28 days in response to a 5-day challenge with oral trimethoprim-sulfadiazine (TMS), with a special emphasis on microbial fibrolytic activity and the host immune response. Horses were supplemented with two doses of Lactobacillus acidophilus , Ligilactobacillus salivarius (formerly L. salivarius ), and Bifidobacterium lactis blend or a placebo in a 3 × 3 Latin square design. Changes in fecal microbiota were investigated using 16S rRNA sequencing. Clostridioides difficile was quantified in feces using quantitative polymerase chain reaction. Anaerobic microbiological culture was used to enumerate functional bacterial groups (cellulolytic, amylolytic, and lactic acid-utilizing). The environmental dimensions were assessed by measuring the concentrations of volatile fatty acids (VFAs) and lactic acid using biochemical methods, and changes in pH and dry matter weight. Systemic and local inflammation was evaluated by determination of cytokine and immunoglobulin (Ig)A concentrations in the serum and secretory IgA (SIgA) concentrations in the feces using immuno-enzymatic methods. Oral TMS treatment strongly altered the whole hindgut ecosystem by 2 days after the first administration. Bacterial diversity decreased in proportion to the relative abundance of fibrolytic genera, which coincided with the decrease in the concentration of cellulolytic bacteria. At the same time, the composition of microbiota members was reorganized in terms of relative abundances, probably to support the alteration in fibrolysis. C. difficile DNA was not found in these horses, but the relative abundances of several potential pathobiont genera increased. 2 days after the first TMS administration, fecal concentrations of VFAs and SIgA increased in parallel with fecal water content, suggesting an alteration of the integrity of the hindgut mucosa. Recovery in bacterial composition, functions, and immune biomarkers took 2–9 days after the end of TMS administration. Supplementation with this bacterial blend did not limit bacterial alteration but might have interesting mucosal immunomodulatory effects.
... La fermentation des glucides conduit à la production de (Figure 4). Les IsoAGV et l'ammoniac sont essentiels à la croissance de certaines espèces bactériennes (Cotta & Hespell, 1986) (Bryant & Burkey, 1953;Bryant, 1959). Cependant, ces techniques, dites traditionnelles, ne permettent pas l'identification de bactéries non cultivables, donnant ainsi une fausse image de la diversité réelle. ...
... Crop and jejunum scraping were collected in sterile vials for evaluation of total microbial load colonization. Microbial populations were determined by serial dilution (10 4 to 10 6 ) of crop and jejunum samples in anaerobic diluents before inoculation onto Petri dishes of sterile agar as described by Bryant and Burkey (1953). Total bacterial count and Lactobacilli were grown on nutrient agar and Rogosa SL agar respectively (Deman et al., 1960). ...
... It is also apparent from Table 3 that the influence of the periods IV and V extended to periods VI and VII not only in numbers of bacteria, but also in types. Latham et al. found higher numbers of Butyrivibrio in cows fed 00 hay than when fed on cereal plus bay rations, and Butyrivibrio has been found to be the most common cellulolytic bacterium in the rumen at times (Gouws & Kistner 1965), although the more actively ceUulolytic ruminococci sometimes appear to be present in greater numbers (Bryant & Burkey 1953, Kistner et al. 1962 . ...
The composition of the rumen microflora and the volatile fatty acids were examined in cattle free-grazing on grass or stall-fed on hay, grass pellets, oats or dried beet pulp with molasses. Total and viable counts of anaerobic bacteria were highest on the grass feeding, but viable counts as a percentage of total counts were highest when oats or beet pulp with molasses were fed. Counts of cellulolytic bacteria were lowest on these latter 2 diets, and highest on grass or grass pellet diets. Studies of the anaerobic flora showed that the composition in animals fed on grass pellets resembled more that found in animals free-grazing on grass than in those fed on hay. Counts of aerotolerant bacteria were only a small percentage of the total count, but were highest on the hay diet. On this latter diet and on grass-feeding the streptococci (identified as Streptococcus bovis) were predominant, but contrary to expectation, streptococci were found only in small numbers on the oats diet, where coryneform rods were the major type present. Although a period of 4–6 weeks was allowed for the animals to adapt to the feeds, the 2 periods of feeding on oats and dried beet pulp with molasses markedly affected the composition of the rumen flora in the subsequent periods of feeding grass pellets and hay. Ruinen volatile fatty acid analysis showed a propionogenic effect of oats and the highest percentage of butyric acid when beet pulp with molasses was fed. The expected propionogenic effect of grass pellets was not observed.
... Since there are no digestive juices secreted into the rumen by the host, it was concluded that the agent responsible for this hydrolysis was of microbial origin. Considering protein as one of the nutrients undergoing hydrolysis, it can be stated that only a few proteolytic bacteria have been isolated from the rumen and their proteolytic activities have not been studied extensively (Appleby, 1955;Bryant and Burkey, 1953;Huhtanen and Gall, 1953). There are many more bacterial proteinases than can be found in plant and animal tissues. ...
... The medium was then tubed under O,-free CO, for use. The procedures were essentially those previously described (4)(5)(6). In most experiments, the same medium was used except for the use of glucose, cellobiose, or cellulose as energy sources as indicated. ...
Succinate is formed as an intermediate but not as a normal end product of the bovine rumen fermentation. However, numerous rumen bacteria are present, e.g., Bacteroides succinogenes, which produce succinate as a major product of carbohydrate fermentation. Selenomonas ruminantium, another rumen species, produces propionate via the succinate or randomizing pathway. These two organisms were co-cultured to determine if S. ruminantium could decarboxylate succinate produced by B. succinogenes. When energy sources used competitively by both species, i.e. glucose or cellobiose, were employed, no succinate was found in combined cultures, although a significant amount was expected from the numbers of Bacteroides present. The propionate production per S. ruminantium was significantly greater in combined than in single S. ruminantium cultures, which indicated that S. ruminantium was decarboxylating the succinate produced by B. succinogenes. S. ruminantium, which does not use cellulose, grew on cellulose when co-cultured with B. succinogenes. Succinate, but not propionate, was produced from cellulose by B. succinogenes alone. Propionate, but no succinate, accumulated when the combined cultures were grown on cellulose. These interspecies interactions are models for the rumen ecosystem interactions involved in the production of succinate by one species and its decarboxylation to propionate by a second species.
The use of anthelminthic drugs has several drawbacks, including the selection of resistant parasite strains. Alternative avenues to mitigate the negative effects of helminth infection involve dietary interventions that might affect resistance and/or tolerance by improving host immunity, modulating the microbiota, or exerting direct anthelmintic effects. The aim of this study was to assess the impact of diet on strongyle infection in horses, specifically through immune-mediated, microbiota-mediated, or direct anthelmintic effects. Horses that were naturally infected with strongyles were fed either a high-fiber or high-starch diet, supplemented with either polyphenol-rich pellets (dehydrated sainfoin) or control pellets (sunflower and hay). When horses were fed a high-starch diet, they excreted more strongyle eggs. Adding sainfoin in the high-starch diet reduced egg excretion. Additionally, sainfoin decreased larval motility whatever the diet. Moreover, the high-starch diet led to a lower fecal bacterial diversity, structural differences in fecal microbiota, lower fecal pH, lower blood acetate, and lower hematocrit compared to the high-fiber diet. Circulating levels of Th1 and Th2 cytokines, lipopolysaccharides, procalcitonin, and white blood cells proportions did not differ between diets. Overall, this study highlights the role of dietary manipulations as an alternative strategy to mitigate the effect of helminth infection and suggests that, in addition to the direct effects, changes in the intestinal ecosystem are the possible underlying mechanism.
Background
Methanomassiliicoccales are a recently identified order of methanogens that are diverse across global environments particularly the gastrointestinal tracts of animals; however, their metabolic capacities are defined via a limited number of cultured strains.
Results
Here, we profile and analyze 243 Methanomassiliicoccales genomes assembled from cultured representatives and uncultured metagenomes recovered from various biomes, including the gastrointestinal tracts of different animal species. Our analyses reveal the presence of numerous undefined genera and genetic variability in metabolic capabilities within Methanomassiliicoccales lineages, which is essential for adaptation to their ecological niches. In particular, gastrointestinal tract Methanomassiliicoccales demonstrate the presence of co-diversified members with their hosts over evolutionary timescales and likely originated in the natural environment. We highlight the presence of diverse clades of vitamin transporter BtuC proteins that distinguish Methanomassiliicoccales from other archaeal orders and likely provide a competitive advantage in efficiently handling B12. Furthermore, genome-centric metatranscriptomic analysis of ruminants with varying methane yields reveal elevated expression of select Methanomassiliicoccales genera in low methane animals and suggest that B12 exchanges could enable them to occupy ecological niches that possibly alter the direction of H2 utilization.
Conclusions
We provide a comprehensive and updated account of divergent Methanomassiliicoccales lineages, drawing from numerous uncultured genomes obtained from various habitats. We also highlight their unique metabolic capabilities involving B12, which could serve as promising targets for mitigating ruminant methane emissions by altering H2 flow.
Production of methane by methanogenic archaea, or methanogens, in the rumen of ruminants is a thermodynamic necessity for microbial conversion of feed to volatile fatty acids, which are essential nutrients for the animals. On the other hand, methane is a greenhouse gas and its production causes energy loss for the animal. Accordingly, there are ongoing efforts toward developing effective strategies for mitigating methane emissions from ruminant livestock that require a detailed understanding of the diversity and ecophysiology of rumen methanogens. Rumen methanogens evolved from free-living autotrophic ancestors through genome streamlining involving gene loss and acquisition. The process yielded an oligotrophic lifestyle, and metabolically efficient and ecologically adapted descendants. This specialization poses serious challenges to the efforts of obtaining axenic cultures of rumen methanogens, and consequently, the information on their physiological properties remains in most part inferred from those of their non-rumen representatives. This review presents the current knowledge of rumen methanogens and their metabolic contributions to enteric methane production. It also identifies the respective critical gaps that need to be filled for aiding the efforts to mitigate methane emission from livestock operations and at the same time increasing the productivity in this critical agriculture sector.
Fibrobacter succinogenes is a cellulolytic predominant bacterium that plays an essential role in the degradation of plant fibers in the rumen ecosystem. It converts cellulose polymers into intracellular glycogen and the fermentation metabolites succinate, acetate, and formate. We developed dynamic models of F. succinogenes S85 metabolism on glucose, cellobiose, and cellulose on the basis of a network reconstruction done with the Automatic Reconstruction of metabolic models (AuReMe) workspace. The reconstruction was based on genome annotation, 5 templates-based orthology methods, gap-filling and manual curation. The metabolic network of F. succinogenes S85 comprises 1565 reactions with 77% linked to 1317 genes, 1586 unique metabolites and 931 pathways. The network was reduced using the NetRed algorithm and analyzed for computation of Elementary Flux Modes (EFMs). A yield analysis was further performed to select a minimal set of macroscopic reactions for each substrate. The accuracy of the models was acceptable in simulating F. succinogenes carbohydrate metabolism with an average coefficient of variation of the Root mean squared error of 19%. Resulting models are useful resources for investigating the metabolic capabilities of F. succinogenes S85, including the dynamics of metabolite production. Such an approach is a key step towards the integration of omics microbial information into predictive models of the rumen metabolism.
IMPORTANCE
F. succinogenes S85 is a cellulose-degrading and succinate-producing bacterium. Such functions are central for the rumen ecosystem and are of special interest for several industrial applications. This work illustrates how information of the genome of F. succinogenes can be translated to develop predictive dynamic models of rumen fermentation processes. We expect this approach can be applied to other rumen microbes for producing a model on rumen microbiome that can be used for studying microbial manipulation strategies aimed at enhancing feed utilization and mitigating enteric emissions.
Condensed fermented corn extractives (liquid steep liquor) was assessed as an alternative to organic acids in broiler chicken. Day-old Ross 308 chicken (n = 180) were allocated into six treatments (basal diet with no supplement, basal diet supplemented with 1 and 2 cc/kg liquid corn steep liquor (CSL) or 1 and 2 cc/kg formic acid along with basal diet supplemented with probiotic (1 × 10⁸ CFU/g, Lactobacillus acidophilus, Lactobacillus casei) in a completely randomised design with five replicate pens per treatment and six chicks in each pen. Broilers were assessed for performance measures and intestinal and serum characteristics. The results indicated that inclusion of CSL increased (p < .05) feed intake (FI) on days 1–21. Relative weight and length of different organs showed great differences among the treatments (p < .01). The pH content of different parts of the gastrointestinal tract reduced by inclusion of 1 cc/kg of CSL and formic acid (p < .05). Formic acid inclusion reduced villi length in comparison to the control group (p < .05). The lowest villi length to crypt depth ratio was determined in chicks fed diets supplemented with 1 cc/kg of CSL. Ileal and caecal microbial population did not alter by CSL or formic acid supplementation. CSL supplementation reduced the concentration of blood triglyceride, total and LDL cholesterol compared to the control group (p < .05). Results obtained from current trial revealed that CSL is an organic substance that have an acidic pH and its inclusion (1 cc/kg of diet) improved body weight on starter period and reduced digesta pH in gastrointestinal tract comparable to liquid formic acid, however, supplementation of CSL on higher dose (2 cc/kg od diet) did not change digesta pH considerably.
• HIGHLIGHTS
• Condensed fermented corn extractives (liquid Steep Liquor) was produced from Wet-milling of corn.
• Inclusion of 1 cc/kg of condensed fermented corn extractives improved growth performance of broiler chicken on starter period.
• Inclusion of 1 cc/kg of condensed fermented corn extractives reduced digesta pH.
In order to decrease the time and cost of experiments as well as the use of animals in nutrition research, in vitro methodologies have become more commonplace in the field of ruminant nutrition. Therefore, the objectives of this review are i) describe the development of different in vitro methodologies, ii) discuss the application, utilization, and advantages of in vitro methodologies, iii) discuss shortcomings of in vitro methodologies, and iv) describe potential developments that may be able to improve in vitro methods. Having been used for decades, some in vitro methodologies such as pure, batch, and continuous cultures have been very well documented and utilized to investigate a wide array of different aspects of nutrition, including the effects of different dietary compositions, individual fermentation end products, and impacts on the microbiome of the rumen. However, both batch and pure cultures can result in a build-up of end products that may inhibit fermentation, as they culture ruminal contents or defined strains of bacteria, respectfully. Continuous culture; however, allows for the removal of end products but, similar to pure and batch cultures, is applicable only to ruminal fermentation and cannot provide information regarding intestinal digestion and bioavailability. This information for in vitro can only be provided using an assay designed for total tract digestibility, which is the three-step procedure (TSP). The TSP may be improved by coupling it with cell culture in order to investigate absorption of nutrients in both the ruminal and intestinal phases of the methodology; however, the TSP needs further development in order to investigate all nutrients and the methodologies available for cell culture are still relatively new to ruminant nutrition. Therefore, while in vitro methodologies provide useful data into the field of ruminant nutrition without the continuous use of animals, there is still much work to be done in order to improve the methodologies to further apply them.
Equines and ruminants have evolved as grazing herbivores with specialized gastrointestinal tracts capable of utilizing a wide range of fibrous feeds. In China, agricultural by-products, including corn straw, wheat straw, peanut vine, wheat husk, rice husk, and grass hay, have been extensively included in both equine and ruminant diets. These plant materials, which are composed predominantly of cellulose, hemicellulose, noncellulosic polysaccharides, and lignin, are largely undegradable by equines and ruminants themselves. Their breakdown is accomplished by communities of resident microorganisms that live in symbiotic or mutualistic associations with the host. Information relating to microbial composition in the hindgut and rumen has become increasingly available. Rumen fermentation is unique in that plant cell wall breakdown relies on the cooperation between microorganisms that produce fibrolytic enzymes and that ruminant animals provide an anaerobic fermentation chamber. Similar to the rumen, the equine hindgut is also an immensely enlarged fermentative chamber that includes an extremely abundant and highly complex community of microorganisms. However, few studies have characterized the microbial functions and their utilization process of lignocellulosic feeds within the equine hindgut. The process of understanding and describing plant cell wall degradation mechanisms in the equine hindgut ecosystem is important for providing information for proper feeding practices to be implemented. In the present study, we gather existing information on the rumen and equine ecosystem and provide scientific insights for understanding the process of plant cell wall breakdown within the hindgut.
Nitroethane is a potent methane-inhibitor for ruminants but little is known regarding simultaneous effects of repeated administration on pre- and post-gastric methane-producing activity and potential absorption and systemic accumulation of nitroethane in ruminants. Intraruminal administration of 120 mg nitroethane/kg body weight per day to Holstein cows (n = 2) over a 4-day period transiently reduced (P < 0.05) methane-producing activity of rumen fluid as much as 3.6-fold while concomitantly increasing (P < 0.05) methane-producing activity of feces by as much as 8.8-fold when compared to pre-treatment measurements. These observations suggest a bacteriostatic effect of nitroethane on ruminal methanogen populations resulting in increased passage of viable methanogens to the lower bovine gut. Ruminal VFA concentrations were also transiently affected by nitroethane administration (P < 0.05) reflecting adaptive changes in the rumen microbial populations. Mean (± SD) nitroethane concentrations in plasma of feedlot steers (n = 6/treatment) administered 80 or 160 mg nitroethane/kg body weight per day over a 7-day period were 0.12 ± 0.1 and 0.41 ± 0.1 μmol/mL 8 h after the initial administration indicating rapid absorption of nitroethane, with concentrations peaking 1 day after initiation of the 80 or 160 mg nitroethane/kg body weight per day treatments (0.38 ± 0.1 and 1.14 ± 0.1 μmol/mL, respectively). Plasma nitroethane concentrations declined thereafter to 0.25 ± 0.1 and 0.78 ± 0.3 and to 0.18 ± 0.1 and 0.44 ± 0.3 μmol/mL on days 2 and 7 for the 80 or 160 mg nitroethane/kg body weight per day treatment groups, respectively, indicating decreased absorption due to increased ruminal nitroethane degradation or to more rapid excretion of the compound.
Spoiled silages can harbor pathogenic and antimicrobial-resistant microbes. The potential of some antimicrobial additives to inhibit certain pathogenic and antimicrobial-resistant bacteria in air-exposed silage was measured using pure and mixed bacterial cultures. With pure cultures, laurate and monolaurin (5 mg·mL⁻¹) caused decreases (P < 0.05) of 4 to >7 log10 colony-forming units (CFU)·mL⁻¹ in Listeria monocytogenes and Enterococcus faecalis compared to controls. Ten-fold higher amounts of these inhibitors were needed to equivalently decrease staphylococci. 2-Nitropropanol (1 mg·mL⁻¹) decreased (P < 0.05) E. faecalis and L. monocytogenes 2.9–3.8 and 2.4–7.2 log10 CFU·mL⁻¹ after 6 and 24 h incubations, respectively. In air-exposed whole-plant corn silage the inhibitors caused decreases, although not necessarily significant, of 0.7–2.2 log10 CFU·mL⁻¹ in L. monocytogenes, staphylococci and culturable aerobes after 24 h incubation, with modest yet significant (P < 0.05) inhibition (<0.1–0.3 log10 CFU·mL⁻¹) of yeasts and molds. Tests for carry-over effects against ruminal microbes revealed laurate, monolaurin, and 2-nitropropanol inhibited methanogenesis by >50% (P < 0.05) after 24 h incubation and inhibited L. monocytogenes and enterococci. The antimicrobial activities exhibited by these compounds may yield opportunities to optimize their use to rescue spoiled silages.
The growth and morphology of rumen methanogenic archaea (15 strains of 10 species in 5 genera, including 7 strains newly isolated in the present study) and bacteria (14 species in 12 genera) were investigated using unsupplemented in vitro pure cultures and cultures supplemented with cashew nut shell liquid (CNSL) and its phenolic compound components, anti-methanogenic agents for ruminant animals. Growth of most of the methanogens tested was inhibited by CNSL and alkylphenols at different concentrations ranging from 1.56 to 12.5 μg/ml. Of the alkylphenols tested, anacardic acid exhibited the most potent growth inhibition. Three gram-negative bacterial species involved in propionate production were resistant to CNSL and alkylphenols (>50 μg/ml). All the methanogens and bacteria that were sensitive to CNSL and alkylphenols exhibited altered morphology; disruption of the cell surface was notable, possibly due to surfactant activity of the tested materials. Cells division was inhibited in some organisms, with cell elongation and unclear septum formation observed. These results indicate that CNSL and alkylphenols, particularly anacardic acid, inhibit both rumen bacteria and methanogens in a selective manner, which could help mitigate rumen methane generation.
Galactooligosaccharides (GOS), gum arabic (GA), HP inulin (HP), 2′‐fucosyllactose (2FL), 6′‐sialyllactose (6SL), and lacto‐N‐neotetraose (LNnT) were incubated (37°C) in semi‐defined medium with fecal inocula from healthy breast‐fed (n=4) or formula‐fed (n=4) infants, and samples collected over 12 h. LNnT, 2FL, and GOS led to rapid drops in pH (P<0.01; 3, 6, and 12 h), with intermediate changes for HP and 6SL, and no change for GA. GOS led to higher acetate production than 2FL at 3 h (P=0.02), and both 2FL and LNnT at 6 h (P<0.05). At 12 h, GOS led to greater production of acetate than the other substrates (GOS>LNnT=2FL>6SL>HP=GA; P<0.05). Other substrates produced more propionate than GA, but only detected at 12 h (6SL>LNnT=GOS=2FL=HP>GA; P<0.05). Butyrate increased to the same extent (P<0.01) for all substrates except for GA. More lactate accumulated for GOS, 2FL, and LNnT compared to HP, 6SL, and GA (P<0.01). GOS led to greater (P<0.01) lactate production compared to HP, 6SL, and GA at 3 h with this trend continuing through 12 h. This study suggests that the human milk oligosaccharides LNnT, 2FL, and 6SL are highly fermentable by infants. This research was supported by Abbott Nutrition.
Effects of fiber source with various particle size and carbohydrase enzyme on growth criteria, carcass and blood characteristics, digestive enzyme activity, cecal microbial population and ileal morphology of chukar partridge were evaluated for a total period of 90 days. 300 day-old partridge were randomly allotted to a 5 × 2 factorial arrangement with 4 different fiber inclusion with different mean particle size (MPS) (none; rice hulls (RH) (711 ± 1.8 μm); wheat straw (WS) Coarse (3211 ± 1.2 μm); wheat straw Fine (755 ± 1.9 μm); and wheat bran (WB) (767 ± 1.8 μm)) along with a low-fiber control diets and two levels of carbohydrase enzyme (0 or 50 mg/kg). There were 10 treatments and 5 pens with 6 partridge chicks per each treatment. The results indicated that fiber inclusion regardless of fiber source significantly increased body weight (BW) of birds during the whole experimental period (P < 0.01). Among the fiber sources, the highest BW attained when fine WS and WB was incorporated into the low fiber diet (P < 0.01). The effect of enzyme on body weight was significant on days 60 and 90. Moreover, there were interactive effects (P< 0.01) between fiber sources and enzyme on BW during the trial. Feed intake significantly (P< 0.001) increased at starter (30 days) phase of trial by inclusion of fiber to control diet, but no significant differences were noted after on days 60 and 90. Enzyme addition had no effect on feed intake and also no interactive effect was observed between fiber source and enzyme for feed intake. Feed conversion ratio (FCR) was reduced by inclusion of fiber into control low fiber diet especially at late stage of rearing period (P< 0.05). Plasma uric acid concentration was reduced by inclusion of fiber on day 28 (P< 0.05). Inclusion of fine WS increased plasma triglyceride and very low-density lipoprotein cholesterol (VLDL-C) concentration on day 90 (P< 0.05). There were interactive effects (P< 0.05) between fiber sources and enzyme on total cholesterol and very low-density lipoprotein cholesterol (LDL-C) cholesterol on day 90. Jejunum and ileum relative weight were reduced by inclusion of fiber to control diet and the effect was more pronounced for coarse wheat straw (P< 0.05). Duodenum and ileum weight were increased by addition of enzyme to diets (P< 0.05). Fiber inclusion regardless of fiber source and particle size significantly (P< 0.01) reduced duodenum, jejunum and ileum relative length. Addition of coarse WS and RH into the low fiber diet significantly increased alkaline protease, trypsin and α-amylase activity (P< 0.01). There were interactive effects (P< 0.05) between fiber sources and enzyme on digestive enzyme activity (P< 0.01). Enzyme addition reduced trypsin activity (P< 0.05). Ceca E. coli population was increased when fiber sources were incorporated into the low fiber diet (P< 0.05). Villi length, crypt depth and villi to crypt ratio were reduced (P< 0.01) considerably regardless of fiber source compared to the low fiber diet. Inclusion of coarse WS increased and RH reduced (P< 0.01) submucosa, lamina propria and epithelial cells layer thickness compared to the low fiber diet. The results confirmed that partridge growth performance was improved by inclusion of fiber. Results obtained from current trial suggest that the fiber is a very important part of partridge diets which could improve the performance with advancing age especially in long-lived partridge.
A study was made of the cecal microflora isolated from broilers (5-week-old) reared under typical commercial husbandry conditions. Three hundred and twenty-five bacterial strains (randomly isolated from colonies representing 49 to 81% of the microscopic count) were isolated from cecal digesta of six animals on a rumen fluid roll tube medium (M98-5). Seventy-seven percent of these strains consisted of strict anaerobes: gram-negative, pleomorphic cocci (5.2%), Peptostreptococcus (1.5%), gram-positive rods (36.1% as Propionibacterium acnes and Eubacterium sp.), gram-negative rods (18.6% as Bacteroides clostridiiformis, B. hypermegas and B. fragilis) and sporeforming rods (15.7% as Clostridium sp.). Two types of facultatively anaerobic bacteria (gram-positive cocci and Escherichia coli) were also isolated and constituted 17.5% of the remaining flora. The distribution of the bacterial groups isolated from six cecal samples varied considerably. Data on the growth requirements of anaerobic strains indicated that many could be cultured in a simple medium consisting of an energy source, minerals, reducing agent, Trypticase, and yeast extract (or a vitamin mixture in place of yeast extract). The growth of some of these bacteria was also enhanced by CO2 and rumen fluid. These preliminary data suggest that some of the more numerous anaerobes isolated from the chicken cecum may not require complex nutrients for growth and, in fact, may be nutritionally similar to rumen anaerobes.
When three sheep were abruptly changed from a ration of 100% orchardgrass hay to 60% cracked corn-40% orchardgrass hay, fed at equal dry-matter intakes, significant increases in concentration were observed in the rumen microbial population. Bacterial numbers (colony counts) per gram of rumen contents did not appear to have stabilized within 21 days after the ration change; however, protozoan numbers per milliliter plateaued after 5 days. The concentration of cellulose-digesting bacteria varied considerably between animals and decreased in all animals with the change. Changes were observed in total and molar percentages of volatile fatty acids, which were typical for the two types of rations. Although the concentration of protozoa increased after the ration change, only minor differences were observed in their percent generic distribution. A significant decrease in rumen volume was measured in two of the three sheep with the change in ration; however, fluid turnover rates were not significantly affected. Rates of rumen dry-matter turnover were slower with the concentrate ration, although rumen dry-matter digestion was increased. Calculation of total bacterial numbers based on total rumen volume completely negated the effect of ration change in one animal, whereas total numbers in the other two animals were still significantly different between rations and very similar between animals. Adjustment of total protozoa numbers did not alter the trends seen previously with concentration values.
The fatty acid compositions of 21 pure cultures of rumen bacteria, representing 12 genera and 14 species, were compared as methyl esters. Each organism possessed a consistent and reproducible fatty acid profile. Overlapping similarities and differences in composition did not allow differentiation between families or genera. Although species differentiation was possible, fatty acid composition appeared to be only an aid in the identification of bacteria.
Changing the diet of five lactating cows and one nonlactating cow from high to low roughage induced milk fat depression in the lactating cows and altered the composition of the rumen microflora. While the numbers of lactic and propionic acid-producing bacteria increased, the numbers of Butyrivibrio spp. decreased. The numbers of lipolytic bacteria and the in vitro lipolytic activity of the rumen fluid were also decreased, as was the extent of hydrogenation of linoleic and linolenic acids combined in soybean oil incubated in vitro with rumen fluid. It is suggested that among the bacterial population in the rumen the vibrios, which were adversely affected by the low-roughage diets, may contribute significantly to both lipolysis and hydrogenation in the rumen.
Mixtures of ruminal bacteria degraded benzo(b)thien-4-yl methylcarbamate (Mobam) to 4-hydroxybenzothiophene, CO2, and polar product(s). The metabolite, 4-hydroxybenzothiophene, was identified (after acetylation) by comparative infrared and mass spectrometry with an authentic sample. Carbon dioxide and polar product(s) were produced by degradation of the methylcarbamate moiety. Ten previously characterized strains of ruminal bacteria with diverse physiological capabilities did not degrade Mobam. However, three tributyrin-hydrolyzing strains were isolated that did degrade Mobam. Mobam inhibited growth of two of ten strains isolated on Mobam-free glycerol-tributyrin enrichment medium. One of these strains was also sensitive to 2-carbomethoxy-propene-2yl dimethyl phosphate (Phosdrin). Mobam prevented some ruminal bacteria from producing zones of hydrolysis in tributyrin emulsion media and inhibited some ruminal bacteria from degrading 1-naphthyl acetate and fluorescein-3′,6′-diacetate.
Samples of rumen liquor were taken by stomach tube, at approximately monthly intervals for a period of 19 months, from three mature Merino wethers grazing annual pastures in a Mediterranean-type climate in Western Australia. Counts were made, on the rumen liquor samples, of the total numbers of 'free' microorganisms, of protozoa, and of the organism Oscillospira guilliermondi. Qualitative variations in the organisms present were examined by means of wet iodine preparations and the Gram stain. Marked seasonal fluctuations in the numbers of total organisms were found. Two definite levels were evident, a high level associated with the green grazing in winter, with a peak in the spring, and a low level associated with the dry grazing. The lowest mean count obtained was 32 million per cu. mm. in April 1949, and the highest was 88 million per cu. mm. in November 1948. The numbers of protozoa and of Oscillospira were found to follow a similar seasonal cycle, the former showing an extremely high spring peak. Except for minor additions during the period of green grazing, all morphological types were found to be constantly present, but the balance of the many constituent organisms changed at different times of the year. The relation of these various changes in the numbers and balance of the ruminal microorganisms to changes in the chemical composition of the grazing, particularly the protein content, is briefly discussed and the need for further investigation of the influence of dietary changes on these organisms is stressed.
THE kinds and numbers of bacteria in the rurnen of cattle and sheep .l. on practical farm rations is a question of considerable interest. Some workers, such as Baker (i943), have studied the kinds of bacteria micro~ scopically, while others have cultured these bacteria. Hungate (I947) has succeeded in isolating cellulose~digesting bacteria from high dilutions of rumen contents. Van der Wath et al. (1948) have grown several tureen bac- teria, one being an iodophilic coccus isolated from sheep and (Van der Wath, I948) has also made direct counts of tureen microorganisms finding an average of I-~ biliion per ml. rumen contents. KShler (194o) studied numbers of rurnen bacteria by several technics. By direct count he found about I3 billion per gram, but by indirect means he calculated that there should be over Ioo billion per gram. However, KShler (I94o) was able by aerobic cub turing to grow only ~5 million organisms per gram. Both Van der Wath (I948) and KShler (i94o) stated in their publications that their direct counts were too low, and KShler (I94o) also recognized that he was not cul- turing all of the important bacteria. Gall et al. (i947), using a direct slide count, found bacteria in numbers ranging from 5o-Ioo billion per gram of fresh rumen contents, and by anaerobic cultural methods have frequently been able to culture the bacteria in the dilution indicated by the slide count. This survey was undertaken in an effort to obtain information concerning the kinds and numbers of bacteria present in the tureen of cattle and sheep Under practical farm conditions, both winter and summer. Procedure
About 5,000 isolations of bacteria from the rumens of approximately 350 cattle and sheep from several herds in three states have been studied.
The data from these studies have been used as the basis for setting up these five criteria from judging true rumen bacteria: (a) Anaerobiosis; (b) presence in numbers of 1 million or more per gram of fresh rumen contents; (c) isolation of a similar type bacterium at least ten times from at least two animals; (d) isolation from animals in at least two geographical locations; and (e) production by the organism of end-products found in the rumen from substrates found in the rumen.
A description of some physiological characteristics of five rumen bacteria is included.
The importance of microorganisms in ruminant digestion generally is accepted, but little is known about the physiological activities of the pre- dominating flora. This fact is emphasized in Hastings' recent review (3). Some studies have been carried out to determine the role played by rumen bacteria in urea utilization (7, 9, 13, 15), and in cellulose digestion (5, 8, 10, 14). Studies were initiated in 1945 to develop techniques for the culturing of the predominating bacteria in order that their physiological activities could be investigated. The successful procedures include : (a) a direct slide counting technique using an indirect stain, to serve as a guide for the suc- cess or failure of the cultural work; (b) a method for obtaining a sample suitable for culture from a fistulated animal; (c) an anaerobic procedure for growing the bacteria in the numbers indicated by the direct count; (d) methods for studying urea utilization; and (e) methods for cellulose break- down. EXPERIMENTAL RESULTS Technique for slide counts. The direct counting technique found most successful involved the use of nigrosine, which colored the background but left the bacteria unstained. Rumen contents were diluted with 1 per cent glucose solution to 1 : 1,000 or 1 : 10,000, depending upon the expected bac- terial content of the sample, and shaken for 5 minutes at a speed of 240 oscillations per minute. The vigorous shaking was very essential in order to free the bacteria from the food particles and break up clumps of bacteria. After shaking, 0.01 ml. of the sample was transferred by a standard loop to a thoroughly cleaned slide upon which an area of 2 x 2 cm. had been marked off. A similar loopful of one-half saturated methyl alcohol solution of nigrosine was mixed thoroughly with the sample, and spread evenly over the 4 cmY area. The slide was placed on a very hot electric plate so that drying occurred within 2-3 seconds. The slide now was ready to count, the bacteria appearing white against a black background. Counts were made from several fields on three to five different slides,
THE elucidation of the factors concerned in the digestive assimilation by cattle of starch, cellulose, and also of protein substitutes, necessitates an accurate knowledge of the functional activities of the rumen micro-organisms. On this subject very conflicting opinions have been advanced1,2,3 based in many cases upon the results of inoculation of rumen-contents into artificial media. The value of these results, moreover, must always remain uncertain4,2 until the characteristics of the normal rumen microflora and microfauna have been independently determined by direct microscopical observation. Appropriate methods have been applied by me to the investigation of the gastro-intestinal contents of various herbivora4,5,6,7,8.
Two strains of Ruminococcus pavefadens, an important cellulosedecomposing bacterium, were isolated, one from the rumen of a sheep, the other from the rumen of a cow. Pure cultures were obtained by using the dilution method in agar media containing a strip of filter-paper. These strictly anaerobic, Grampositive streptococci attack cellulose and cellobiose, but not starch, maltose, lactose or xylose. Only one strain could use glucose. Colonies on cellulose media were characterized by the formation of a yellow pigment ; in cellobiose media the colonies were white. Growth on cellulose was favoured by addition of CZostridium sporogenes or a certain amount of sterilized medium in which CZ. sporogenes had previously grown. Estimations of the end-products of fermentation of cellulose and cellobiose showed that at least 25 yo of the carbon could be recovered as succinic acid, c. 23 yo as acetic acid and c. 10 yo as formic acid; ethanol was absent and gas formation very limited. A description of the genus and the species is given. Our knowledge of the bacteria that effect the breakdown of cellulose in the rumen of ruminants is still very meagre. Hungate (1947) was the first to obtain pure cultures of several types of these bacteria, in the form of asporogenous rods or cocci. Using a similar technique the present author (1948) isolated from a bovine rumen a cellulose-decomposing streptococcus, to which the name Ruminococcus JEavefuciens was given. This is a small Gram-positive, obligate anaerobic streptococcus which forms yellow colonies on filter-paper. This organism must be considered one of the predominant species of cellulosedecomposing bacteria in the rumen. As growth in pure culture was very poor, little information was then obtained about them and an analysis of the products of their metabolism was impossible. In combined culture with CZ. sporogenes, however, cellulose was rapidly attacked by the streptococci, acetic and succinic acids being the main end-products. The present work was carried out with two other strains of Rum. Jlavefmiens, and more information obtained about the species. During this investigation a further paper was published by Hungate (1950), in which he described more closely four bacterial types that play an important role in the decomposition of cellulose in bovine rumen. Two of these are cocci; it is not excluded that one of them is identical with Rum. Juvefmiens. MATERIAL AND METHODS
Special Methods for Rumen Bacterial Studies in the Field
- L S Gall
- W Bui~rougtis
- P Gei~lairgh
- B H And Edging~on
GALL, L. S., BUI~ROUGtIS, W., GEI~LAIrGH, P., AND EDGING~ON, B. H. Special Methods for Rumen Bacterial Studies in the Field. J. Animal Sci., 8: 433-440. 1949.
Microbiological and Physiological Changes Associated with Acute Indigestion in Sheep The Seasonal Variation in the Ruminal Microorganisms of Grazing Sheep On R~lmi~wcocc~ls flavefaciens, a Cellulose-Decomposing Bacterium from the Rumen of Sheep and Cattle
- R E Hungate
- ~ W Douc~he~ty
- M P B~yant
- ~ J And Cello13 ) Moir
HUNGATE, R. E., DOUC~HE~TY, 1~. W., B~YANT, M. P., AND CELLO, ~:~. Microbiological and Physiological Changes Associated with Acute Indigestion in Sheep. Cornell Vet-erinarian, 42: 423-449. 1952. (13) MOIR, ]~. J. The Seasonal Variation in the Ruminal Microorganisms of Grazing Sheep. Australian J. Agr. Research, 2: 322-330. 1951. (14) POUNDEN, W. D. Demonstration of Collection of Rumen Samples for Examination and Transfusion. Am. Vet. Med. Assoc. Proc. 49-50. 1952. (15) SIJPESTEIJN, A. K. On R~lmi~wcocc~ls flavefaciens, a Cellulose-Decomposing Bacterium from the Rumen of Sheep and Cattle. J. Gen. Microbiol., 5: 869-879. 1951. (16) VAN DER WATH, J. G. Studies on the Alimentary Tract of the Merino Sheep in South Africa. XII. A Technique for the Counting of Ruminal Bacteria. Onderstepoort J. Vet. Sci. and Animal Ind., 23: 385-387. 1948.
Special Methods for Rumen Bacterial Studies in the Field
- Gall
Demonstration of Collection of Rumen Samples for Examination and Transfusion
- Pounden