Rumen bacterial community evaluated by 454 pyrosequencing and terminal restriction fragment length polymorphism analyses in dairy sheep fed marine algae

Castro-Carrera, T.; Toral, Pablo G.; Frutos, Pilar; McEwan, Neil R.; Hervás, Gonzalo; Abecia, Leticia; Pinloche, Eric; Girdwood, Susan Elizabeth; Belenguer, Álvaro

doi:10.3168/jds.2013-7243

Cited by 67 publications

(62 citation statements)

References 41 publications

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“…This have been observed by Castro-Carrera et al (2014) who reported that the effect of diet supplementation with marine lipids, evaluated by 454 pyrosequencing, was rather consistent in rumen content or fluid samples despite inherent differences between these fractions in their bacterial composition. However, caution should be taken when the composition of the diets do not differ to a great extent, as occurs in the present study.…”

Section: Quantification Of Microorganisms and Structure And Diversitysupporting

confidence: 60%

Use of stomach tubing as an alternative to rumen cannulation to study ruminal fermentation and microbiota in sheep and goats

Ramos-Morales

Arco-Pérez

Martín-García

et al. 2014

Animal Feed Science and Technology

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120

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Rumen cannulation is the reference method for collection of representative samples of rumen digesta. However, it is not always viable, which obliges to depend on less invasive techniques, such as stomach tubing. The aim of this work was to study if the differences in fermentation parameters and rumen microbial populations observed between species (sheep and goats), diets (forage and forage plus concentrate) and sampling times (pre-and post-feeding) are consistent when collecting the samples through stomach tube or rumen cannula, in an attempt to validate the use of the former as an alternative to the latter. Four sheep and four goats, fitted with ruminal cannula, were fed either forage (F diet; alfalfa hay) or forage plus concentrate (1:1; FC diet), in two 15-d periods. At the end of each period (d 14 and 15), samples of rumen digesta were taken by stomach tube and rumen cannula, before and 4 hours after morning feeding, for determination of ruminal fermentation parameters (pH, and lactate, ammonia and total VFA concentrations). The three main rumen microbial groups (bacteria, protozoa and methanogenic archaea) and two fibrolytic bacteria (Ruminococcus flavefaciens and Fibrobacter succinogenes) were quantified by real time PCR and, additionally, PCR-DGGE analysis of the bacterial community on the rumen digesta samples collected post-feeding was carried out. Overall, sampling through ruminal cannula and stomach tube gave similar results regarding fermentation parameters when comparing species, diets and sampling times. Despite samples for microbiology assays contained liquid plus solid fractions when collected through rumen cannula and mostly liquid when collected through stomach tube, both techniques showed certain consistency in the effects of treatments on the rumen microbiota (e.g., both revealed no differences between species in total bacteria, archaea and R. flavefaciens concentrations, and higher protozoa numbers in goats than 3 in sheep). However, there was also some discrepancy regarding microorganism concentrations, particularly concerning sampling times (e.g., differences between preand post-feeding samplings were only observed in rumen cannula samples for total bacteria and methanogenic archaea, and in stomach tube samples for R. flavefaciens concentrations). Therefore, this study supports that non-invasive stomach tubing is a feasible alternative to surgical rumen cannulation in sheep and goats to examine ruminal fermentation. Nonetheless, caution should be taken when using this technique to assess the structure and composition of the rumen microbial community.

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Section: Quantification Of Microorganisms and Structure And Diversitysupporting

confidence: 60%

Use of stomach tubing as an alternative to rumen cannulation to study ruminal fermentation and microbiota in sheep and goats

Ramos-Morales

Arco-Pérez

Martín-García

et al. 2014

Animal Feed Science and Technology

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120

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“…This protocol was further used to extract sorghum root and stem mDNA to perform high-throughput pyrosequencing using 16S rRNA marker analysis. Sorghum-associated bacterial communities were shown to be highly diverse, and root and stem communities were significantly different (Castro-Carrera et al, 2014;Lagos et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench)

Maropola

Ramond

Trindade

2015

Journal of Microbiological Methods

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Keywords:Metagenomic DNA extraction Endophytic bacteria Sorghum root and stem t-RFLP Pyrosequencing Culture-independent studies rely on the quantity and quality of the extracted environmental metagenomic DNA (mDNA). To fully access the plant tissue microbiome, the extracted plant mDNA should allow optimal PCR applications and the genetic content must be representative of the total microbial diversity. In this study, we evaluated the endophytic bacterial diversity retrieved using different mDNA extraction procedures. Metagenomic DNA from sorghum (Sorghum bicolor L. Moench) stem and root tissues were extracted using two classical DNA extraction protocols (CTAB-and SDS-based) and five commercial kits. The mDNA yields and quality as well as the reproducibility were compared. 16S rRNA gene terminal restriction fragment length polymorphism (t-RFLP) was used to assess the impact on endophytic bacterial community structures observed. Generally, the classical protocols obtained high mDNA yields from sorghum tissues; however, they were less reproducible than the commercial kits. Commercial kits retrieved higher quality mDNA, but with lower endophytic bacterial diversities compared to classical protocols. The SDS-based protocol enabled access to the highest sorghum endophytic diversities. Therefore, "SDS-extracted" sorghum root and stem microbiome diversities were analysed via 454 pyrosequencing, and this revealed that the two tissues harbour significantly different endophytic communities. Nevertheless, both communities are dominated by agriculturally important genera such as Microbacterium, Agrobacterium, Sphingobacterium, Herbaspirillum, Erwinia, Pseudomonas and Stenotrophomonas; which have previously been shown to play a role in plant growth promotion. This study shows that DNA extraction protocols introduce biases in culture-independent studies of environmental microbial communities by influencing the mDNA quality, which impacts the microbial diversity analyses and evaluation. Using the broad-spectrum SDSbased DNA extraction protocol allows the recovery of the most diverse endophytic communities associated with sorghum tissues and, as such, establishes a reliable basis for future study of endophytic communities.

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“…Hence, the effect of tannin extracts on the milk FA profile observed in the present experiment could be due to the modulation of rumen BH because of changes in the microbial ecosystem. Nevertheless, some in vivo studies on rumen bacterial diversity in cows and ewes suggest that B. fibrisolvens and B. proteoclasticus do not play a dominant role in rumen lipid metabolism and that other as-yet-uncultured bacteria phylogenetically classified as Prevotella, Lachnospinaceae incertae sedis, and unclassified Bacteroidales, Clostridiales, and Ruminococcaceae might be more relevant (Boeckaert et al, 2008;Belenguer et al, 2010;Huws et al, 2011;Castro-Carrera et al, 2014). The literature also provides evidence that alterations in rumen outflow of 18-carbon BH intermediates and 18:0 induced by diets based on different types of forages or supplemented with fish oil are not accompanied by significant changes in B. proteoclasticus group (Kim et al, 2008;Huws et al, 2010;Halmemies-Beauchet-Filleau et al, 2013).…”

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confidence: 99%

Milk fatty acid composition, rumen microbial population, and animal performances in response to diets rich in linoleic acid supplemented with chestnut or quebracho tannins in dairy ewes

Buccioni

Pauselli

Viti

et al. 2015

Journal of Dairy Science

141

130

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The aim of the study was to evaluate milk fatty acid (FA) profile, animal performance, and rumen microbial population in response to diets containing soybean oil supplemented or not with chestnut and quebracho tannins in dairy ewes. Eighteen Comisana ewes at 122 ± 6 d in milking were allotted into 3 experimental groups. Diets were characterized by chopped grass hay administered ad libitum and by 800 g/head and day of 3 experimental concentrates containing 84.5 g of soybean oil/kg of dry matter (DM) and 52.8 g/kg of DM of bentonite (control diet), chestnut tannin extract (CHT diet), or quebracho tannin extract (QUE diet). The trial lasted 4 wk. Milk yield was recorded daily, and milk composition and blood parameters were analyzed weekly. At the end of the experiment, samples of rumen fluid were collected to analyze pH, volatile fatty acid profile, and the relative proportions of Butyrivibrio fibrisolvens and Butyrivibrio proteoclasticus in the rumen microbial population. Hepatic functionality, milk yield, and gross composition were not affected by tannin extracts, whereas milk FA composition was characterized by significant changes in the concentration of linoleic acid (CHT +2.77% and QUE +9.23%), vaccenic acid (CHT +7.07% and QUE +13.88%), rumenic acid (CHT −1.88% and QUE +24.24%), stearic acid (CHT + 8.71% and QUE −11.45%), and saturated fatty acids (CHT −0.47% and QUE −3.38%). These differences were probably due to the ability of condensed versus hydrolyzable tannins to interfere with rumen microbial metabolism, as indirectly confirmed by changes in the relative proportions of B. fibrisolvens and B. proteoclasticus populations and by changes in the molar proportions of volatile fatty acids. The effect of the CHT diet on the milk FA profile and microbial species considered in this trial was intermediate between that of QUE and the control diet, suggesting a differential effect of condensed and hydrolyzable tannins on rumen microbes. Compared with control animals, the presence of B. fibrisolvens increased about 3 times in ewes fed CHT and about 5 times in animals fed QUE. In contrast, the abundance of B. proteoclasticus decreased about 5-and 15-fold in rumen liquor of ewes fed CHT and QUE diets, respectively. The use of soybean oil and a practical dose of QUE or CHT extract in the diet of dairy ewes can be an efficient strategy to improve the nutritional quality of milk.

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Rumen bacterial community evaluated by 454 pyrosequencing and terminal restriction fragment length polymorphism analyses in dairy sheep fed marine algae

Cited by 67 publications

References 41 publications

Use of stomach tubing as an alternative to rumen cannulation to study ruminal fermentation and microbiota in sheep and goats

Use of stomach tubing as an alternative to rumen cannulation to study ruminal fermentation and microbiota in sheep and goats

Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench)

Milk fatty acid composition, rumen microbial population, and animal performances in response to diets rich in linoleic acid supplemented with chestnut or quebracho tannins in dairy ewes

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