Quantitative and differential analysis of ciliate protozoa in rumen content samples filtered before and after fixation

Rossi, Mariana Fonseca; Martinele, Isabel; D’Agosto, Marta

doi:10.1590/s1516-35982013001100010

Cited by 4 publications

(3 citation statements)

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“…Contrary to archaea and bacteria, ciliate protozoa vary across ruminants of different species as well as individuals of the same species [19]. A study in cattle indicated that Entodinium, Diplodinium, Eremoplastron, Ostracodinium, Eodinium, Epidinium, Isotricha, and Dasytricha were among the most abundant genera (> 1%) of a total of 13 identified in the ruminal fluid by microscopic identification and counting [38]. Conversely, in sheep and goats, 12 and 8 genera were identified, being Dasytricha, Entodinium, Eudiplodinum, Diplodinium, Isotricha, and Metadinium the more abundant (> 1%) in both species, and Enoploplastron, Ophryoscolex, and Polyplastron only in sheep [39].…”

Section: Protozoamentioning

confidence: 99%

Alternative pathways for hydrogen sink originated from the ruminal fermentation of carbohydrates: Which microorganisms are involved in lowering methane emission?

2022

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Agriculture is responsible for a great share of the anthropogenic sources of greenhouse gases that, by warming the earth, threaten its biodiversity. Among greenhouse gas emissions, enteric CH4 from livestock is an important target to slow down climate changes. The CH4 is originated from rumen fermentation and its concentration is affected by several factors, including genetics and nutrition. Ruminants have an extraordinary symbiosis with microorganisms (bacteria, fungi, and protozoa) that ferment otherwise indigestible carbohydrates, from which they obtain energy to grow and continue actively producing, among other products, volatile fatty acids, CO2 and H2. Detrimental ruminal accumulation of H2 is avoided by methanogenesis carried out by Archaea methanogens. Importantly, methanogenesis is not the only H2 sink pathway. In fact, other bacteria can reduce substrates using metabolic hydrogen formed during carbohydrate fermentation, namely propionate production and reductive acetogenesis, thus lowering the CH4 produced. Although the complexity of rumen poses challenges to mitigate CH4 production, the emergence of sequencing techniques that allow the study of microbial communities, gene expression, and metabolome are largely contributing to unravel pathways and key players in the rumen. Indeed, it is now recognized that in vivo emissions of CH4 are correlated to microbial communities, and particularly with the abundance of methanogens, several bacterial groups, and their genes. The goal of CH4 mitigation is to work in favor of the natural processes, without compromising rumen function, animal health, and productivity. Notwithstanding, the major challenge continues to be the feasibility and affordability of the proposed solutions.

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Section: Protozoamentioning

confidence: 99%

Alternative pathways for hydrogen sink originated from the ruminal fermentation of carbohydrates: Which microorganisms are involved in lowering methane emission?

2022

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“…A procedure developed for isolation of cultivatable mixed ruminal protozoa for quantitative PCR and competi-tion studies (Sylvester et al, 2004(Sylvester et al, , 2005Denton et al, 2015) can be used to isolate protozoa for more accurate nutrient analysis. In addition, several of the methods employ the use of formalin in the isolation and storage of microbial and specifically protozoal samples (Martin et al, 1994;Rossi et al, 2013); this might be problematic, as formaldehyde is known to react with AA (Barry, 1976).…”

Section: Introductionmentioning

confidence: 99%

Ruminal bacteria and protozoa composition, digestibility, and amino acid profile determined by multiple hydrolysis times

Fessenden

Hackmann

Ross

et al. 2017

Journal of Dairy Science

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Microbial samples from 4 independent experiments in lactating dairy cattle were obtained and analyzed for nutrient composition, AA digestibility, and AA profile after multiple hydrolysis times ranging from 2 to 168 h. Similar bacterial and protozoal isolation techniques were used for all isolations. Omasal bacteria and protozoa samples were analyzed for AA digestibility using a new in vitro technique. Multiple time point hydrolysis and least squares nonlinear regression were used to determine the AA content of omasal bacteria and protozoa, and equivalency comparisons were made against single time point hydrolysis. Formalin was used in 1 experiment, which negatively affected AA digestibility and likely limited the complete release of AA during acid hydrolysis. The mean AA digestibility was 87.8 and 81.6% for non-formalin-treated bacteria and protozoa, respectively. Preservation of microbe samples in formalin likely decreased recovery of several individual AA. Results from the multiple time point hydrolysis indicated that Ile, Val, and Met hydrolyzed at a slower rate compared with other essential AA. Singe time point hydrolysis was found to be nonequivalent to multiple time point hydrolysis when considering biologically important changes in estimated microbial AA profiles. Several AA, including Met, Ile, and Val, were underpredicted using AA determination after a single 24-h hydrolysis. Models for predicting postruminal supply of AA might need to consider potential bias present in postruminal AA flow literature when AA determinations are performed after single time point hydrolysis and when using formalin as a preservative for microbial samples.

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“…are dominant. According to previous reports, Entodinium can constitute up to 90% of the total ciliate protozoa 38 . However the abundance of Entodinium in our study was quite low (28.22%), which might be due to the fact that all previous studies were based on construction of clone libraries, which might have overrepresented the predominant genera and neglected rare organisms.…”

Section: Discussionmentioning

confidence: 96%

Analysis of Community Structure and Species Richness of Protozoa-Enriched Rumen Metagenome from Indian Surti by Shotgun Sequencing

et al. 2016

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The present study applies metagenomics to characterize the diversity and relative abundance of protozoa residing in the rumen of Indian Surti buffalo (Bubalus bubalis). To increase protozoa representation in the metagenome, protozoa enrichment was performed by density sedimentation and confirmed by quantitative real time PCR. The highly enriched metagenome sample was subjected to shotgun sequencing by Ion Torrent PGM which resulted in 10,303,375 reads totalling 1.6 gigabases. The taxonomic profile obtained by comparison with SILVA SSU database showed predominance of the class Litostomatea (99.78%) followed by Coccidia (0.10%) and Aconoidasida (0.06%). At the genus level Isotricha (48.06%) followed by Polyplastron (9.90%), Dasytricha (9.87%) and Eudiplodinium (7.47%) were predominant. The taxonomic assignment based on protein coding regions showed discrepancy with the SSU-based assignments, possibly due to the absence of most eukaryotic genomes in public databases. According to the SEED subsystems annotation database, genes for protein metabolism were the most abundant followed by genes for RNA metabolism, regulation and cell signalling. The present study offers a preliminary snapshot of diversity, functional potential and relative abundance of protozoa within the Indian Surti buffalo rumen and also expands our knowledge of these unicellular eukaryotes present in the rumen ecosystem.Keywords: Buffalo rumen, metagenome, protozoa enrichment, quantitative real time PCR, shotgun sequencing.RUMEN, one of the most diverse ecosystems in nature, harbours a complex consortium of interdependent anaerobic prokaryotes, protozoa, fungi, bacteriophages and methanogens. These act synergistically to convert lignocellulosic feeds into volatile fatty acids serving as an energy supplement for host ruminants 1 . Owing to the complexity of rumen ecosystem and the intricate interactions among various microbial groups within the rumen, it is difficult to decipher a precise role for each microorganism. Despite the fact that rumen microbiomes have been studied extensively, a majority of them have focussed on domain bacteria, because of their numerical abundance and metabolic diversity 2 , thus leaving rumen fungi, archaea, bacteriophages and protozoa underexplored.Rumen protozoa, besides contributing nutrients 3 to the host animal, help in digesting carbohydrate and protein containing feedstuff by secretion of various saccharolytic and proteolytic enzymes 4,5 in microbial protein turnover 6 , modulation of bacterial populations 7-9 and association with methanogens 8,10 . Numerous species of ciliates occupy the rumen, where they constitute about half of the total microbial biomass 11 . Although bacterial species play a vital role in digestion, protozoa are reported to digest 25-50% of the total fibre 12 . The polymorphic nature and complexity in cultivation of protozoa, has hampered the assessment of their taxonomy within the rumen ecosystem.Traditionally, microscopy has been the preferred method for identifying and enumerating protozoal ...

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Quantitative and differential analysis of ciliate protozoa in rumen content samples filtered before and after fixation

Cited by 4 publications

References 9 publications

Alternative pathways for hydrogen sink originated from the ruminal fermentation of carbohydrates: Which microorganisms are involved in lowering methane emission?

Alternative pathways for hydrogen sink originated from the ruminal fermentation of carbohydrates: Which microorganisms are involved in lowering methane emission?

Ruminal bacteria and protozoa composition, digestibility, and amino acid profile determined by multiple hydrolysis times

Analysis of Community Structure and Species Richness of Protozoa-Enriched Rumen Metagenome from Indian Surti by Shotgun Sequencing

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