The use of Prevotella bryantii 3C5 for modulation of the ruminal environment in an ovine model

Abstract

In the Southern Hemisphere, ruminants are mostly raised in grazing systems where animals consume forage and are supplemented with low amounts of concentrates. Concentrates are usually given separately and are rapidly ingested. This practice leads to changing rumen environment conditions during the day, may alter the rumen microbial metabolism and could affect host performance. The native ruminal Prevotella bryantii strain 3C5 was administered every 48 h to wethers under experimental conditions simulating Southern-Hemisphere feeding to evaluate its potential as a rumen fermentation modulator. The inoculum potential was assessed on day 17. The ammonia nitrogen (NH₃-N), volatile fatty acids and ruminal pH were monitored on a 24-h basis 19 days after the beginning of the experiment, and the microbial community structure was assessed by pyrosequencing. The administration of P. bryantii modified the fermentation products and daily pH values compared to the control. The NH₃-N concentration in the rumen of treated animals was significantly higher than that of the untreated animals. Modification of the ruminal environment and fermentation pathways was achieved without altering the general structure of the microbial community or the potential methane production. P. bryantii 3C5 could be considered in potential probiotic formulations for ruminants in semi-intensive systems.

Keywords
Probiotics; Rumen; Fermentation; Prevotella bryantii; Ovine model

Introduction

Ruminants are raised in intensive and semi-intensive production systems that include the supplementation with protein-energy concentrates. These practices can generate a misbalance of rumen microorganisms’ metabolism, and therefore, production yields are usually affected. To overcome these limitations, feed additives such as antibiotics or ionophores have been extensively used.¹ Due to the emergence risks of resistant strains and antibiotic residues in animal products and byproducts, the use of antibiotics as feed additives has been progressively restricted. The use of antibiotics has been banned in the European Union since January 2006 (Directive 1831/2003/CEE, European Commission 2003). Therefore, alternatives to the use of antibiotics have been proposed, including the use of probiotics.^2,3

Probiotics, live microorganisms that confer benefits to the host,⁴ could be designed to modulate the rumen microbiota, improving feed utilization. This goal could be achieved by enhancing fiber and starch digestion, promoting volatile fatty acid (VFA) synthesis and diminishing or buffering lactate accumulation to avoid acidification of ruminal contents.^2,5 Some of these issues have been assessed in previous studies using different native and exogenous microorganisms with diverse results.^6–8

Prevotella is the most represented bacterial genus in the rumen,⁹ and in particular, Prevotella bryantii strains have been tested on high-producing dairy cows to evaluate the potential of the strains to prevent subacute acidosis.^10,11 Although P. bryantii was not effective at preventing ruminal acidosis, the authors observed that its administration could improve the ruminal environment.

In the Southern Hemisphere, ruminants are mostly raised in grazing systems. Animals usually consume forage all day long and are supplemented with low amounts of concentrates generally provided separately from forage, once or twice a day. This practice leads to daily variations of rumen environment conditions.^12,13 Therefore, in these conditions, the use of probiotics could help to stabilize the rumen environment and feed digestion.

The objective of this work was to evaluate the ability of a native P. bryantii strain to modulate the ruminal environment and fermentation and the bacterial microbiota structure in a model based on sheep consuming forage and supplemented twice a day with a high-starch concentrate.

Materials and methods

Bacterial cultures and inoculum preparation

P. bryantii 3C5, used in the administration trial, was previously isolated from the rumen of an only pasture-consuming cow at the Experimental Station at Libertad (San José, Uruguay), Faculty of Veterinary, University of Uruguay, and identified by sequencing of the 16S ribosomal RNA gene (JQ674698.1). This strain grew in a culture medium containing lactate as the sole carbon source and exhibited antimicrobial activity against Escherichia coli and Streptococcus bovis. Bacterial cultures were grown using a modified rumen-fluid-free broth according to Caldwell and Bryant¹⁴ (Table 1). For inoculum preparation, Pb3C5 was inoculated in CO₂-gassed culture bottles containing 50 mL of broth and incubated at 39 °C until a density of 1 × 10⁸ cells/mL was achieved.

Table 1
Medium composition.

Component	% (m/v)
Yeast extract	0.05
Trypticase	0.2
Hemin	0.0001
Glucose	0.05
Cellobiose	0.05
Soluble starch	0.05
Mineral solutiona (mL)	3.75
VFA mixtureb (mL)	0.31
Na2S·9H2O	0.025
Cysteine hydrochloride	0.025
Na2CO3	0.4

• ^a
  Mineral solution composition (g/L): NaCl, 5.4; KH₂PO₄, 2.7; CaCl₂·2H₂O, 0.159; MgCl₂·6H₂O, 0.12; MnCl₂·4H₂O, 0.06; CoCl₂·6H₂O, 0.06; (NH₄)₂SO₄, 5.4.
• ^b
  VFA mixture: acetic acid, 5.80 mM; propionic acid, 1.60 mM; butyric acid, 0.86 mM; n-valeric acid, 0.18 mM; and isovaleric acid, 0.18 mM.

Animals, feeding and preparation of samples

Animals were cared for and handled according to the procedures approved by the Honorary Commission of Animal Experimentation (CHEA) of the University of Uruguay and by the National Commission of Animal Experimentation (CNEA, Uruguay). Ten eleven-month-old Corriedale wethers (Ovis aries; 33.8 ± 4.3 kg) with a rumen cannula were individually housed in metabolic cages at the Experimental Station at Libertad (San José, Uruguay), Faculty of Veterinary, University of Uruguay. Animals had access to water and were fed ad libitum on alfalfa hay. They were supplemented with cracked corn grain (1% metabolic weight basis) in two supplements, at 10 AM and 4 PM. The average intake was 1347 g per day, and the forage/concentrate ratio was 4.4/1 on a dry matter basis. Animals were randomly assigned to the Control or Pb3C5 group. Every 48 h, wethers in the Pb3C5 group received an intra-ruminal dose of 1 × 10⁹ Pb3C5 cells in 100 mL of culture medium, while animals in the Control group received 100 mL of sterile medium. This experimental procedure was designed after Chiquette et al.¹⁵

Wethers were adapted to housing, diet and treatments for 16 days. On day 17, samples were taken for inoculum potential experiments. On days 19 and 20, ruminal fluid samples were taken to measure pH, ammonia nitrogen (NH₃-N) and VFA for a 24 h period. Additionally, on day 19, ruminal fluid was taken from each wether to evaluate the microbial community.

Inoculum potential assay and methane production

The effect of P. bryantii 3C5 on the fermentative potential of ruminal fluid was assessed using an in vitro fermentation assay based on gas production experiments.¹⁶ Experiments were performed in 125 mL fermenters containing a substrate, buffer solution,¹⁷ reductive solution and fresh ruminal fluid from each wether. The substrate (0.5 g per fermenter) was a mix of alfalfa hay (70%) and corn (30%), ground to pass a 1 mm sieve, with 90% dry matter, 93.4% organic matter, 34.8% neutral detergent fiber, 21.4% acid detergent fiber, and 14.6% crude protein. This substrate was allowed to hydrate with the buffer solution (38 mL) and the reductive solution (2 mL) for 18 h at 4 °C (solutions from Oeztuerk et al.¹⁷) inside the fermenters. Then, 10 mL of fresh ruminal fluid, obtained from both groups of animals, was added to every fermenter. The headspace was saturated with CO₂, and the fermenters were sealed. All incubations were performed in individual batches. To deduct the gas production of the rumen fluid of the donors, two were incubated without substrate addition. Incubation was performed at 39 °C, and the internal pressure was measured with a manual manometer D1005PS (Ashcroft^®, Stratford, USA) coupled to a 0.6 mm needle. Measurements were taken at 2, 4, 8, 10, 12, 18, 24, 48, 72 and 96 h after inoculation, and gas was vented after pressure readings. At times of 4, 8 and 24 h, headspace gas samples from the fermenters were obtained for measuring methane concentration by GC as described before.¹⁸

Ruminal environment measures

On day 19, ruminal fluid samples were taken every 2 h, covering the first 12 h of the day and then every 4 h, completing the whole 24 h period. The pH was measured immediately. For VFA determination, the samples were mixed 1:1 with 0.1 M perchloric acid. For NH₃-N analysis, rumen fluid samples were preserved with H₂SO₄, 50% (v/v) in a 100:1 relation. Samples were stored at −20 °C until they were analyzed.

Lactic acid and VFA (acetic, propionic and butyric acids) were quantified by HPLC separation.¹⁹ The chromatogram peaks were integrated at 210 nm.²⁰ The concentration of NH₃-N was determined using a selective electrode (Thermo Scientific Orion) according to manufacturer's instructions.

Structure of the ruminal bacterial microbiota

Total DNA was extracted from 10 g of contents from each animal using the method proposed by Zhou et al.²¹ to assess the ruminal bacterial community of wethers by massive sequencing. Extracted DNA was used to amplify and sequence the 16S rDNA V1-V2 region in a GS FLX Titanium XLR70 as in Allai et al.²² The 16S rDNA sequences generated by pyrosequencing were subsequently analyzed running the Quantitative Insights into Microbial Ecology (QIIME, version 1.8.0) per scripted modules and workflow scripts.²³ The sequences were trimmed and then filtered by length (≥150 bp), quality (≥25 score), and the content of either one or more ambiguous bases or a long homopolymer (>6). Operational taxonomic units (OTU) were generated by aligning the reads to the GreenGenes database²⁴ and clustered at 97% sequence identity using the PyNAST tool²³ and the UCLUST algorithm,²⁵ respectively. An analysis of similarity was performed with QIIME using ANOSIM and ADONIS analysis.

Statistics

Cumulated gas volume along time was compared among treatments (control and Pb3C5). For this purpose, the gas produced at a specific time was considered to be dependent on the preceding time. Consequently, this variable was analyzed as a repeated measure over the fermenter substrate, according to the model:

$Y_{ijk}=\mu +SI+T_j+{(S*T)}_{ij}+{\epsilon }_{ijk}$

where Y_ijk is the volume of gas produced, µ is the overall mean, SI is the effect of Pb3C5 treatment (I = Control, Pb3C5) in k replicates (2 fermenters per wether), T_j is the fixed effect of time (j = 2, 4, 6, 8, 10, 18, 26, 48, 72 and 96 h), (S*T)_ij is the interaction between the strain and the time and ɛ_ijk is the residual error.

All data sets of pH, NH₃-N and VFA in ruminal contents were tested before statistical analysis to ensure that all the assumptions of analysis of variance (additive model, independence of errors, data normality and homoscedasticity) were met. After, they were analyzed using the MIXED procedure as repeated measures, using the wether as the subject for the repeated measurement, according to the following model:

$Y_{ijk}=\mu +S_i+h_j+{(S*h)}_{ij}+{\epsilon }_{ik}$

where Y_ijk is the variable, µ is the general mean, S_i the fixed effect of the treatment, h_j is the fixed effect of the time of measurement, (S*h)_ij is the interaction between treatment and time and ɛ_ik is the residual error.

The data were analyzed using SAS software (version 8.2; SAS 185 Institute, Cary, NC, USA), and differences among means with p < 0.05 were considered statistically significant.

Family and genus abundance values were compared using the non-parametric Kruskal–Wallis ANOVA test.

Results

Inoculum activity and potential methane production

No significant differences were observed between the groups in in vitro fermentation gas production associated with the addition of ruminal fluid of treated and untreated animals (p _treatment = 0.0622). The methane concentrations in the fermenters were similar in both groups (p > 0.05, data not shown).

Influence of added bacteria on the ruminal parameters (VFA, pH, NH₃-N)

Total VFA levels (considering acetic, propionic and butyric acids) tended to be higher (p = 0.0622) in the ruminal fluid of the treated animals. Acetic and butyric acid concentrations were significantly higher in the Pb3C5-treated animals than in the control group (p = 0.0187 and p = 0.0199, respectively; Fig. 1). No differences were observed when propionic acid levels were compared between the two groups (p > 0.05). Lactic acid was not detected in any sample.

Fig. 1
Total VFA, acetic, propionic and butyric acid concentration. Means from 5 animals in Control (dash line) and Pb3C5 (full line) treatments for each time are presented. Time 0 corresponds to the first feed intake (8 AM). p for treatment is inserted in each graphic.

The ruminal pH of the treated animals was significantly lower than that of the controls during most of the day (p _treatment = 0.006), and the treatment*time interaction was not significant (p > 0.05; Fig. 2).

Fig. 2
Daily ruminal pH. The means of five animals at every time point is presented for the control group (dashed line) and the Pb3C5-treated group (solid line). Time 0 corresponds to the first feed intake, 8 AM; p for treatment is inserted in the graphic.

The N-NH₃ concentration in the rumen of the animals treated with Pb3C5 was also significantly higher than recorded values in the ruminal fluid of the control animals (p = 0.05); the treatment*time interaction was not significant (p > 0.05, Fig. 3).

Fig. 3
Evolution of ruminal ammonia concentration during the day. The means of five animals are presented for each group. Time 0 corresponds to the first feed intake, 8 AM; p for treatment is inserted in the graphic.

Influence of added bacteria on the structure of ruminal bacterial microbiota

The microbial community structure, assessed eight hours after the first feed offer on day 19, was similar in both groups of animals (p _ADONIS = 0.837; p _ANOSIM = 0.712), and when comparing the structure at different phylogenetic levels, no differences were found (p > 0.05). An average 2588.6 (±645.9) sequences per animal were analyzed and were designated to 1987 OTUs. The microbiota of every animal was dominated by Bacteroidetes, representing more than 65% of all sequences. Firmicutes was the second most represented phylum in both groups of animals, while 15.8% and 18.8% of sequences in the control and treated groups, respectively, could not be classified. Prevotellacea was the most represented family in the rumen of animals in both treatment and control groups, with 56% and 50% of all the sequences, respectively.

Discussion

The rumen bears a complex ecosystem and, the modulation of ruminal fermentation and the microbiota by probiotic bacteria is a considerable challenge. However, in this work, the ruminal environment was modulated by adding repeated low doses of the native P. bryantii 3C5 strain (Pb3C5). Administration of this strain effectively modified the VFA profile in the treated animals compared to the non-treated controls. The modulation of total VFA in the ruminal contents of treated animals could reflect a fermentative shift associated with the influence of the administered bacteria. This effect was evidenced by a significantly higher concentration of acetic and butyric acids in the Pb3C5-treated animals. It is important to note that butyric acid, which significantly increased in the treated animals, plays an important role in the rumen, being the most energetic VFA along with propionic acid.⁵ Butyric acid is also metabolized in the rumen mucosae and intestinal epithelium^7,26,27 as an energy source and exerts mitotic and trophic effects on the host gut epithelia.^28,29 These effects may enhance digestion and absorption efficiency, which may contribute to a better nutrient absorption.³⁰

Animals that belonged to the Pb3C5-treated group showed lower ruminal pH values than those in the control group. This observation could be associated with the increase in VFA concentration. Although ruminal pH values were lower than in the control group, these values were never dangerous or harmful to the animals. Ruminants can show subclinical acidosis signs when pH values are between 5.2 and 5.6 for a long time,³¹ and they show acute acidosis signs when the ruminal pH reaches values lower than 5.2,¹ but in our conditions, the lowest mean ruminal pH observed was 5.9.

Modifications of the rumen environment were also seen in the daily ruminal NH₃-N concentration, with a higher daily concentration in the Pb3C5-treated animals than in the control group. These results are similar to those obtained when the probiotic potential of Bacillus subtilis natto was assessed using in vitro and in vivo approaches.^6,32 NH₃-N is a microbial protein precursor in the rumen; it is necessary for good microbial growth and microbial protein synthesis and is considered the most important source of nitrogen for protein synthesis, especially for fibrolytic bacteria.³³ Generally, the NH₃-N level is high when protein feedstuffs or good quality young forage are given.³⁴ The modulation induced by Pb3C5 administration could be associated with a better digestibility of the feed and an influence on host nitrogen and protein metabolism.

These ruminal environment modifications were obtained without altering the potential methane production nor the potential based on in vitro gas production experiments.

The structure of the ruminal microbial communities in both groups of animals was similar after the administration of Pb3C5.

The microbiota of all animals was dominated by Bacteroidetes and Firmicutes phyla, and Prevotella was the most abundant genus, which is in concordance with several analyses of the ruminal microbiota performed by other authors.^9,35,36

This result could be observed as a positive trait since the diversity of the rumen microbiota was not affected by the treatment. Considering these results, it is not possible to associate the predominance or the absence of certain groups of microorganisms with the changes observed in the rumen environment induced by Pb3C5 administration. In similar studies performed with human beings, researchers observed significant changes at the community transcriptome level, but they did not find any difference in the community structure.³⁷

Conclusion

In this work, the modulation of ruminal fermentation could be achieved by the addition of repeated doses of a native bacterial strain. These doses were small when compared with the whole ruminal microbial community, but a differential fermentation pattern could be observed. Pb3C5 administration modified fermentation products, acetic and butyric acids, which could explain the differences of the daily pH values in the treated animals. Additionally, a differential pattern of NH₃-N concentration in the rumen was observed. The modification of ruminal fermentation and environment was achieved by providing repeated low doses of Pb3C5. Although in this approach, the causes of the ruminal fermentation modulation could not be associated with changes in the microbiota structure, the effect may be associated with changes in ruminal microbial metabolism Additional studies should be performed to shed light on the specific activity of Pb3C5 and its possible use in probiotic formulations for ruminants in semi-intensive systems.

• Funding
  This study was supported by Agencia Nacional de Investigación (ANII, Uruguay) [grants numbers: PR_FMV_2009_1_2799; MOV_CA_2013_1_10852; ANII POS_2011_3374].

Acknowledgements

The authors thank Luis Vázquez, Maximiliano Pastorini, Paola Delgado, Paola San Pedro, Diego Fernández, Cristina Manzzi, Bruno Cremella, Virginia Ramón, Ernesto Rosas and Elena de Torres for their support with the animal experiments and Luis Gustavo Corbellini for helping in statistical analysis

References

• ¹
  Mutsvangwa T, Walton JP, Plaizier JC, et al. Effects of a monensin controlled-release capsule or premix on attenuation of subacute ruminal acidosis in dairy cows. J Dairy Sci 2002;85:3454-3461.
• ²
  Chaucheyras-Durand F, Durand H. Probiotics in animal nutrition and health. Benef Microbes 2010;1:3-9.
• ³
  Allen HK, Levine UY, Looft T, Bandrick M, Casey TA. Treatment, promotion, commotion: antibiotic alternatives in food-producing animals. Trends Microbiol 2013;21:114-119.
• ⁴
  Joint FAO/WHO Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food; 2002:1–11.
• ⁵
  Calsamiglia S, Castillejos L, Busquet M. Alternatives to antimicrobial growth promoters in cattle. In: Smith P, Wiseman J, eds. Recent Advances in Animal Nutrition Nottingham: Nottingham University Press; 2006:129–167.
• ⁶
  Sun P, Wang JQ, Deng LF. Effects of Bacillus subtilis natto on milk production, rumen fermentation and ruminal microbiome of dairy cows. Animal 2013;7:216-222.
• ⁷
  Uyeno Y, Shigemori S, Shimosato T. Effect of probiotics/prebiotics on cattle health and productivity. Microbes Environ 2015;30:126-132.
• ⁸
  Malafaia PAM, Vieira RAM, Silva DO, Filho SDCV. In vitro degradation of coast-cross (Cynodon dactylon) by rumen microorganisms associated with Saccharomyces cerevisiae or Humicola sp. Rev Microbiol 1997;28:261-267.
• ⁹
  Henderson G, Cox F, Ganesh S, et al. Rumen microbial community composition varies with diet and host, but a core microbiome is found across a wide geographical range. Sci Rep 2015;5:14567.
• ¹⁰
  Chiquette J, Allison MJ, Rasmussen MA. Prevotella bryantii 25A used as a probiotic in early-lactation dairy cows: effect on ruminal fermentation characteristics, milk production, and milk composition. J Dairy Sci 2008;91:3536-3543.
• ¹¹
  Chiquette J, Allison MJ, Rasmussen M. Use of Prevotella bryantii 25A and a commercial probiotic during subacute acidosis challenge in midlactation dairy cows. J Dairy Sci 2012;95:5985-5995.
• ¹²
  Cajarville C, Aguerre M, Repetto JL. N concentration and forage degradation kinetics of cows grazing temperate pastures and supplemented with different sources of grain. Anim Res 2006;55:511-520.
• ¹³
  Tebot I, Cajarville C, Repetto JL, Cirio A. Supplementation with non-fibrous carbohydrates reduced fiber digestibility and did not improve microbial protein synthesis in sheep fed fresh forage of two nutritive values. Animal 2012;6:617-623.
• ¹⁴
  Caldwell DR, Bryant MP. Medium without rumen fluid for nonselective enumeration and isolation of rumen bacteria. Appl Microbiol 1966;14:794-801.
• ¹⁵
  Chiquette J, Talbot G, Markwell F, Nili N, Forster RJ. Repeated ruminal dosing of Ruminococcus flavefaciens NJ along with a probiotic mixture in forage or concentrate-fed dairy cows: effect on ruminal fermentation, cellulolytic populations and in sacco digestibility. Can J Anim Sci 2007;87:237-249.
• ¹⁶
  Mauricio RM, Mould FL, Dhanoa MS, Owen E, Channa KS, Theodorou MK. A semi-automated in vitro gas production technique for ruminant feedstuff evaluation. Anim Feed Sci Technol 1999;79:321-330.
• ¹⁷
  Oeztuerk H, Schroeder B, Beyerbach M, Breves G. Influence of living and autoclaved yeasts of Saccharomyces boulardii on in vitro ruminal microbial metabolism. J Dairy Sci 2005;88:2594-2600.
• ¹⁸
  Fraga M, Fernández S, Cajarville C, Martínez M, Abin-Carriquiry JA, Zunino P. In vitro modulation of rumen microbiota and fermentation by native microorganisms isolated from the rumen of a fed-exclusively-on-pasture bovine. Ann Microbiol 2015;65:2355-2362.
• ¹⁹
  Pérez-Ruchel A, Repetto JL, Cajarville C. Supplementing high-quality fresh forage to growing lambs fed a total mixed ration diet led to higher intake without altering nutrient utilization. Animal 2017;11:2175-2183.
• ²⁰
  Fraga M, Perelmuter K, Valencia MJ, et al. Evaluation of native potential probiotic bacteria using an in vitro ruminal fermentation system. Ann Microbiol 2014;64:1149-1156.
• ²¹
  Zhou J, Bruns MA, Tiedje JM. DNA recovery from soils of diverse composition. Appl Environ Microbiol 1996;62:316-322.
• ²²
  Allali I, Arnold JW, Roach J, et al. A comparison of sequencing platforms and bioinformatics pipelines for compositional analysis of the gut microbiome. BMC Microbiol 2017;17:194.
• ²³
  Caporaso JG, Kuczynski J, Stombaugh J, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods 2010;7:335-336.
• ²⁴
  DeSantis TZ, Hugenholtz P, Larsen N, et al. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 2006;72:5069-5072.
• ²⁵
  Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 2010;26:2460-2461.
• ²⁶
  Baldwin RL, Jesse BW. Propionate modulation of ruminal ketogenesis. J Anim Sci. 1996;74:1694-1700.
• ²⁷
  Baldwin VIRL, McLeod KR. Effects of diet forage: concentrate ratio and metabolizable energy intake on isolated rumen epithelial cell metabolism in vitro. J Anim Sci 2000;78:771-783.
• ²⁸
  Mentschel J, Leiser R, Mülling C, Pfarrer C, Claus R. Butyric acid stimulates rumen mucosa development in the calf mainly by a reduction of apoptosis. Arch Tierernaehr 2001;55:85-102.
• ²⁹
  Pryde SE, Duncan SH, Hold GL, Stewart CS, Flint HJ. The microbiology of butyrate formation in the human colon. FEMS Microbiol Lett 2002;217:133-139.
• ³⁰
  Neish AS. Microbes in gastrointestinal health and disease. Gastroenterology 2009;136:65-80.
• ³¹
  Duffield T, Plaizier JC, Fairfield A, et al. Comparison of techniques for measurement of rumen pH in lactating dairy cows. J Dairy Sci 2004;87:59-66.
• ³²
  Sun P, Li J, Bu D, Nan X, Du H. Effects of Bacillus subtilis natto and different components in culture on rumen fermentation and rumen functional bacteria in vitro. Curr Microbiol 2016;72:589-595.
• ³³
  Dixon RM, Egan AR. Strategies for optimizing use of fibrous crop residues as animal feeds. In: Dixon RM, ed. Ruminant Feeding Systems Utilizing Fibrous Agricultural Residues Canberra: International Development Program of Australian Universities and Colleges; 1988:11–26.
• ³⁴
  Erdman RA, Proctor GH, Vandersall JH. Effect of rumen ammonia concentration on in situ rate and extent of digestion of feedstuffs. J Dairy Sci 1986;69:2312-2320.
• ³⁵
  Castro-Carrera T, Toral PG, Frutos P, et al. Rumen bacterial community evaluated by 454 pyrosequencing and terminal restriction fragment length polymorphism analyses in dairy sheep fed marine algae. J Dairy Sci 2014;97:1661-1669.
• ³⁶
  Jami E, White BA, Mizrahi I. Potential role of the bovine rumen microbiome in modulating milk composition and feed efficiency. PLoS One 2014;9:e85423.
• ³⁷
  McNulty NP, Yatsunenko T, Hsiao A, et al. The impact of a consortium of fermented milk strains on the gut microbiome of gnotobiotic mice and monozygotic twins. Sci Transl Med 2011;3:106ra106.

Edited by

Associate Editor: Miliane Souza

Publication Dates

• Publication in this collection
  Nov 2018

History

• Received
  24 Apr 2018
• Accepted
  17 July 2018
• Published
  25 Aug 2018