Physicochemical and microbiological parameters of rumen fluid of lactating goats fed diets containing crude glycerin from waste frying oil used in biodiesel production.

Crude glycerin from the biodiesel industry is a alternative energy source used to replace part of the energy components of the diet, since the nutritional value of glycerol makes it suitable for use in ruminant diets. The objective was to analyze the effects including crude glycerin (CG) derived from the production of biodiesel from residual frying oil, in diets of lactating goats on the physicochemical and microbiological parameters of rumen fluid. Eight 2-year-old goats weighting approximately 42.06 ± 3.5 kg of body weight (BW) were kept in individual stalls, receiving diets containing 0, 7, 14 and 21% of CG (% DM). The experiment was carried out according to a double 4 × 4 Latin square and lasted for 80 days. Orthogonal contrasts were tested to determine linear and quadratic effects, as well as the effects of 0% CG in the diet in comparison to the other treatments. There was a linear decreasing effect on DM intake (P = 0.001) and neutral detergent fiber (P = 0.0001), as well as a quadratic effect (P = 0.0002) on ether extract (EE) intake with increasing level of CG in the diet. Including CG affected motility and density of the protozoa (P = 0.0001). Treatments had no effect on pH, N-NH3 concentration and percentage of protozoa in the rumen liquid. There was increasing linear effect on sedimentation and flotation time (P = 0.001). Therefore, including 14 and 21% CG in the diet as a source of glycerol decreased nutrient intake and altered the ruminal microbiota, but pH and N-NH3 were not affected. Thus, to avoid impairment to the physicochemical and microbiological parameters of the rumen liquid of goats, the inclusion of CG should not exceed 7% DM.

Performance and microbiota of the digestive tract of Nellore calves supplemented with fungi isolated from bovine rumen.

BACKGROUND AND AIM: In tropical semiarid regions, supplementation with fungi could contribute to rumen modulation, promoting greater production of fibrolytic enzymes and degradation of forage. The objective of this study was to analyze the effect of supplementation with fungi, isolated from the bovine rumen, on the performance and microbiota of the digestive tract of Nellore calves. MATERIALS AND METHODS: The experiment was conducted in randomized blocks evaluating eight Nellore calves that were daily supplemented with isolates of Aspergillus terreus and Trichoderma longibrachiatum, along with eight calves that were not supplemented. After 55 days, the animals were weighed, and samples of rumen fluid and feces were collected for analysis. The characteristics that showed normal distribution were subjected to analysis of variance and compared using Tukey's test. Whereas, the variables that did not show normal distribution were subjected to the Kruskal-Wallis test, and the frequencies of the bacterial and fungal genera were compared using the Chi-square test. RESULTS: Supplementation with fungi promoted the reduction in ruminal pH (p<0.05). However, the final live weight; average daily weight gain; total weight gain; rumen protozoa; and the count of Enterobacteriaceae, mycelial fungi, and yeasts of ruminal fluid and feces were not influenced by supplementation (p>0.05). Moreover, the protozoa Eodinium spp. was identified only in supplemented calves (p<0.05). CONCLUSION: Supplementation with the fungi presented the potential for use as possible additives because it did not alter the physiological parameters of the facultative anaerobic microbiota composition in the rumen and feces. In addition, it favored the presence of the ciliate genus Eodinium. However, further studies should be performed to better define suitable dosages for supplementation.

Effects of vegetable oil supplementation on rumen fermentation and microbial population in ruminant: a review.

Understanding the nature of ruminant nutrition and digestion is essential to improve feeding management and animal production. Among many approaches, manipulating ruminant nutrition and fermentation through feed supplementation is being practised and researched. Over the last decade, the utilization of vegetable oils in feed formulation and their effects on various aspects of ruminants have been reported by many researchers. It is important to understand the lipid metabolism in ruminants by microorganisms because it affects the quality of ruminant-derived products such as meat and milk. Majority of vegetable oil supplementation could reduce rumen protozoa population in ruminants due to the effects of medium-chain fatty acids (FAs). However, vegetable oil also contains unsaturated FAs that are known to have a negative effect on cellulolytic bacteria which could show inhibitory effects of the fibre digestion. In this paper, the physiology of nutrient digestion of ruminants is described. This paper also provides a current review of studies done on improvement and modification of rumen fermentation and microbial population through vegetable oil supplementation.

The macronuclear genome of anaerobic ciliate Entodinium caudatum reveals its biological features adapted to the distinct rumen environment.

Entodinium caudatum is an anaerobic binucleated ciliate representing the most dominant protozoal species in the rumen. However, its biological features are largely unknown due to the inability to establish an axenic culture. In this study, we primally sequenced its macronucleus (MAC) genome to aid the understanding of its metabolism, physiology, ecology. We isolated the MAC of E. caudatum strain MZG-1 and sequenced the MAC genome using Illumina MiSeq, MinION, and PacBio RSII systems. De novo assembly of the MiSeq sequence reads followed with subsequent scaffolding with MinION and PacBio reads resulted in a draft MAC genome about 117 Mbp. A large number of carbohydrate-active enzymes were likely acquired through horizontal gene transfer. About 8.74% of the E. caudatum predicted proteome was predicted as proteases. The MAC genome of E. caudatum will help better understand its important roles in rumen carbohydrate metabolism, and interaction with other members of the rumen microbiome.

Extending Burk Dehority's Perspectives on the Role of Ciliate Protozoa in the Rumen.

Dr. Burk Dehority was an international expert on the classification and monoculture of ruminal ciliated protozoa. We have summarized many of the advancements in knowledge from his work but also in his scientific way of thinking about interactions of ruminal ciliates with the entire rumen microbial community and animal host. As a dedication to his legacy, an electronic library of high-resolution images and video footage catalogs numerous species and techniques involved in taxonomy, isolation, culture, and ecological assessment of ruminal ciliate species and communities. Considerable promise remains to adapt these landmark approaches to harness eukaryotic cell signaling technology with genomics and transcriptomics to assess cellular mechanisms regulating growth and responsiveness to ruminal environmental conditions. These technologies can be adapted to study how protozoa interact (both antagonism and mutualism) within the entire ruminal microbiota. Thus, advancements and limitations in approaches used are highlighted such that future research questions can be posed to study rumen protozoal contribution to ruminant nutrition and productivity.

Inhibition of Rumen Protozoa by Specific Inhibitors of Lysozyme and Peptidases in vitro.

Defaunation studies have shown that rumen protozoa are one of the main causes of low nitrogen utilization efficiency due to their bacterivory and subsequent intraruminal cycling of microbial protein in ruminants. In genomic and transcriptomic studies, we found that rumen protozoa expressed lysozymes and peptidases at high levels. We hypothesized that specific inhibition of lysozyme and peptidases could reduce the activity and growth of rumen protozoa, which can decrease their predation of microbes and proteolysis and subsequent ammoniagenesis by rumen microbiota. To test the above hypothesis, we evaluated three specific inhibitors: imidazole (IMI), a lysozyme inhibitor; phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor; and iodoacetamide (IOD), a cysteine protease inhibitor; both individually and in combinations, with sodium dodecyl sulfate (SDS) as a positive control. Rumen fluid was collected from two Jersey dairy cows fed either a concentrate-based dairy ration or only alfalfa hay. Each protozoa-enriched rumen fluid was incubated for 24 h with or without the aforementioned inhibitors and fed a mixture of ground wheat grain, alfalfa, and grass hays to support microbial growth. Live protozoa cells were morphologically identified and counted simultaneously at 3, 6, 12, and 24 h of incubation. Fermentation characteristics and prokaryotic composition were determined and compared at the end of the incubation. Except for IOD, all the inhibitors reduced all the nine protozoal genera identified, but to different extents, in a time-dependent manner. IOD was the least inhibitory to protozoa, but it lowered ammoniagenesis the most while not decreasing feed digestibility or concentration of volatile fatty acids (VFA). ANCOM analysis identified loss of Fibrobacter and overgrowth of Treponema, Streptococcus, and Succinivibrio in several inhibitor treatments. Functional prediction (from 16S rRNA gene amplicon sequences) using the CowPI database showed that the inhibitors decreased the relative abundance of the genes encoding amino acid metabolism, especially peptidases, and lysosome in the rumen microbiota. Overall, inhibition of protozoa resulted in alteration of prokaryotic microbiota and in vitro fermentation, and peptidases, especially cysteine-peptidase, may be targeted to improve nitrogen utilization in ruminants.

The transcriptome of the rumen ciliate Entodinium caudatum reveals some of its metabolic features.

BACKGROUND: Rumen ciliates play important roles in rumen function by digesting and fermenting feed and shaping the rumen microbiome. However, they remain poorly understood due to the lack of definitive direct evidence without influence by prokaryotes (including symbionts) in co-cultures or the rumen. In this study, we used RNA-Seq to characterize the transcriptome of Entodinium caudatum, the most predominant and representative rumen ciliate species. RESULTS: Of a large number of transcripts, > 12,000 were annotated to the curated genes in the NR, UniProt, and GO databases. Numerous CAZymes (including lysozyme and chitinase) and peptidases were represented in the transcriptome. This study revealed the ability of E. caudatum to depolymerize starch, hemicellulose, pectin, and the polysaccharides of the bacterial and fungal cell wall, and to degrade proteins. Many signaling pathways, including the ones that have been shown to function in E. caudatum, were represented by many transcripts. The transcriptome also revealed the expression of the genes involved in symbiosis, detoxification of reactive oxygen species, and the electron-transport chain. Overall, the transcriptomic evidence is consistent with some of the previous premises about E. caudatum. However, the identification of specific genes, such as those encoding lysozyme, peptidases, and other enzymes unique to rumen ciliates might be targeted to develop specific and effective inhibitors to improve nitrogen utilization efficiency by controlling the activity and growth of rumen ciliates. The transcriptomic data will also help the assembly and annotation in future genomic sequencing of E. caudatum. CONCLUSION: As the first transcriptome of a single species of rumen ciliates ever sequenced, it provides direct evidence for the substrate spectrum, fermentation pathways, ability to respond to various biotic and abiotic stimuli, and other physiological and ecological features of E. caudatum. The presence and expression of the genes involved in the lysis and degradation of microbial cells highlight the dependence of E. caudatum on engulfment of other rumen microbes for its survival and growth. These genes may be explored in future research to develop targeted control of Entodinium species in the rumen. The transcriptome can also facilitate future genomic studies of E. caudatum and other related rumen ciliates.

Influence of Farming Conditions on the Rumen of Red Deer (Cervus elaphus).

The red deer is an intermediate feeder, showing a marked degree of forage selectivity, with seasonal morphological adaptations due to changes in food quality and availability. In captivity, deer have a limited choice of habitat and food, and we hypothesize that this condition affects the rumen environment. Rumen samples were collected from 20 farmed and 11 wild red deer in autumn 2018 in Poland, and analyzed for chemical composition, food residues, microbial population, and rumen papillation. Farmed deer had the highest Campylobacter spp., and total anaerobic bacteria, but lower Clostridium spp. Moreover, they showed a decrease in Diplodininae protozoa, and the presence of holotrichs that were absent in the wild animals. The rumen digesta of farmed animals had lower dry matter and acid detergent fiber than the wild ones. The analysis of food residues underlined the poor variety of the diet for animals in the farm. This apparently affected the papillation of the rumen, with animals of the farm having the shortest papillae of the Atrium ruminis. Overall, results suggest that red deer kept in farms, with a diet based mainly on grass, tree leaves, and some concentrate supplements, undergo a small modification of the rumen compared to the wild conspecifics.

Specific inhibitors of lysozyme and peptidases inhibit the growth of the rumen protozoan Entodinium caudatum without decreasing feed digestion or fermentation in vitro.

AIMS: Experiments were designed to determine the effects of different chemical inhibitors of lysozyme and peptidases on rumen protozoa and the associated prokaryotes, and in vitro fermentation using Entodinium caudatum as a model protozoan species. METHODS AND RESULTS: Imidazole (a lysozyme inhibitor), phenylmethylsulphonyl fluoride (PMSF, a serine peptidase inhibitor) and iodoacetamide (IOD, a cysteine peptidase inhibitor) were evaluated in vitro both individually and in two- and three-way combinations using E. caudatum monocultures with respect to their ability to inhibit the protozoan and their effect on feed digestion, fermentation and the microbiota. All the three inhibitors, both individually and in combination, decreased E. caudatum counts (P < 0·001), and IOD and its combinations with the other inhibitors significantly (P < 0·01) decreased ammonia concentration, with the two- and three-way combinations showing additive effective. Feed digestion was not affected, but fermentation and microbial diversity were affected mostly by PMSF, IOD and their combinatorial treatments potentially due to the overgrowth of Streptococcus luteciae accompanying with the disappearance of host ciliates. CONCLUSIONS: Entodinium caudatum depends on lysozyme and peptidase for digestion and utilization of the engulfed microbes and specific inhibition of these enzymes can inhibition E. caudatum without adversely affecting feed digestion or fermentation even though they changed the microbiota composition in the cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The peptidase inhibitors may have the potential to be used in controlling rumen protozoa to improve ruminal nitrogen utilization efficiency.

The utilization and digestion of cellulose by the rumen ciliate Diploplastron affine.

Rumen protozoa are known to contribute to fibre digestion, but the fibrolytic enzymes of the majority of ciliate species have been poorly recognised to date. The aims of the study were, first, to determine the influence of crystalline cellulose on the survival and population density of the ciliate Diploplastron affine when cultured in vitro, and second to identify and characterise the protozoal enzymes catalysing the hydrolysis of cellulose. It was found that crystalline cellulose, when added to a culture medium, increased the number of protozoa maintained in vitro. We observed that the bacteria-free ciliates fermented microcrystalline cellulose and produced 43.3 nmol volatile fatty acids/protozoon/h. A cell extract prepared from the bacteria-free ciliates degraded crystalline cellulose in the rate of 11.5 nmol released glucose/mg protein/min, whereas the degradation rates of carboxymethyl-cellulose (CMC), avicel and cellobiose were 343, 6.8 and 145 nmol released glucose/mg protein/min respectively. Two distinct peaks in the activity of relevant enzymes were identified following ion exchange chromatography of the protozoal cell extract and the presence of two different CMC-ases were confirmed by zymographic studies. CMC was mainly degraded to mono- and disaccharides but that some other oligosaccharides were also present. Cellobiose was the only product of avicel digestion.

Rumen digestion kinetics, microbial yield, and omasal flows of nonmicrobial, bacterial, and protozoal amino acids in lactating dairy cattle fed fermentation by-products or urea as a soluble nitrogen source.

The objective of this study was to evaluate the effect of a fermentation by-product on rumen function, microbial yield, and composition and flows of nutrients from the rumen in high-producing lactating dairy cattle. Eight ruminally cannulated multiparous Holstein cows averaging (mean ± standard deviation) 60 ± 10 d in milk and 637 ± 38 kg of body weight were randomly assigned to 1 of 2 treatment sequences in a switchback design. Treatment diets contained (dry matter basis) 44% corn silage, 13% alfalfa silage, 12% ground corn, and 31% protein premix, containing either a control mix of urea and wheat middlings (CON) or a commercial fermentation by-product meal (Fermenten, Arm and Hammer Animal Nutrition, Princeton, NJ) at 3% diet inclusion rate (EXP). The trial consisted of three 28-d experimental periods, where each period consisted of 21 d of diet adaptation and 7 d of data and sample collection. A triple-marker technique and double-labeled 15N15N-urea were used to were used to measure protozoal, bacterial, and nonmicrobial omasal flow of AA. Rumen pool sizes and omasal flows were used to determine digestion parameters, including fractional rates of carbohydrate digestion, microbial growth, and yield of microbial biomass per gram of degraded substrate. Fermentation by-product inclusion in EXP diets increased microbial N and amino acid N content in microbes relative to microbes from CON cows fed the urea control. Microbial AA profile did not differ between diets. Daily omasal flows of AA were increased in EXP cows as a result of decreased degradation of feed protein. The inclusion of the fermentation by-product increased nonmicrobial AA flow in cows fed EXP versus CON. Average protozoal contribution to microbial N flow was 16.8%, yet protozoa accounted for 21% of the microbial AA flow, with a range of 8 to 46% for individual AA. Cows in this study maintained an average rumen pool size of 320 g of microbial N, and bacterial and protozoal pools were estimated at 4 different theoretical levels of selective protozoa retention. Fractional growth rate of all microbes was estimated to be 0.069 h-1, with a yield of 0.44 g of microbial biomass per gram of carbohydrate degraded. Results indicated that fermentation by-product can increase omasal flow of AA while maintaining adequate rumen N available for microbial growth and protein synthesis. Simulations from a developmental version of the Cornell Net Carbohydrate and Protein System indicated strong agreement between predicted and observed values, with some areas key for improvement in AA flow and bacterial versus protozoal N partitioning.

Rumen methanogen and protozoal communities of Tibetan sheep and Gansu Alpine Finewool sheep grazing on the Qinghai-Tibetan Plateau, China.

BACKGROUND: Tibetan sheep (TS) and Gansu Alpine Finewool sheep (GS) are both important plateau sheep raised and fed on the harsh Qinghai-Tibetan Plateau, China. Rumen methanogen and protozoal communities of plateau sheep are affected by their hosts and living environments, and play important roles in ruminant nutrition and greenhouse gas production. However, the characteristics, differences, and associations of these communities remain largely uncharacterized. RESULTS: The rumen methanogen and protozoal communities of plateau sheep were investigated by 16S/18S rRNA gene clone libraries. The predominant methanogen order in both sheep species was Methanobacteriales followed by Methanomassiliicoccales, which is consistent with those seen in global ruminants. However, the most dominant species was Methanobrevibacter millerae rather than Methanobrevibacter gottschalkii seen in most ruminants. Compared with GS and other ruminants, TS have more exclusive operational taxonomic units and a lower proportion (64.5%) of Methanobrevibacter. The protozoa were divided into Entodiniomorphida and Vestibuliferida, including nine genera and 15 species. The proportion of holotrich protozoa was much lower (1.1%) in TS than ordinary sheep. The most predominant genus was Entodinium (70.0%) in TS and Enoploplastron (48.8%) in GS, while the most common species was Entodinium furca monolobum (43.9%) and Enoploplastron triloricatum (45.0%) in TS and GS, respectively; Entodinium longinucleatum (22.8%) was only observed in TS. LIBSHUFF analysis indicated that the methanogen communities of TS were significantly different from those of GS, but no significant differences were found in protozoal communities. CONCLUSION: Plateau sheep have coevolved with unique rumen methanogen and protozoal communities to adapt to harsh plateau environments. Moreover, the host appears to have a greater influence on rumen methanogen communities than on rumen protozoal communities. The observed associations of methanogens and protozoa, together with the findings of previous studies on methane emissions from ruminant livestock, revealed that the lower proportion of Methanobrevibacter and holotrich protozoa may be responsible for the lower methane emission of TS. These findings facilitate our understanding of the rumen microbial ecosystem in plateau sheep, and could help the development of new strategies to manipulate rumen microbes to improve productivity and reduce the emission of greenhouse gases.

Aerobic cultivation of anaerobic rumen protozoa, Entodinium caudatum and Epidinium caudatum.

Rumen protozoa, primarily ciliates, are one of the important groups of strictly anaerobic microbes living in the rumen. Despite their ubiquitous occurrence in the rumen and significant contribution to host animals, it is still poorly understood why they live only in the rumen and similar environment. Because rumen protozoa require strict anaerobic conditions to sustain their viability and grow, only a few laboratories equipped with protozoology expertise and anaerobic facilities can grow rumen protozoa in laboratory. Also for the same reason, only a few species have been grown and maintained as laboratory cultures for research. Prompted by a recent study, we hypothesized that anaerobic rumen protozoa could also be cultivated aerobically if antioxidants were included in the media. Indeed, our experiments showed that the cultures of both Entodinium caudatum and Epidinium caudatum, two major rumen protozoal species, could be cultured successfully in aerobic media supplemented with ascorbic acid, glutathione and α-ketoglutarate as antioxidants. Anaerobic fermentation was maintained through the fermentation characteristics and microbial populations were altered to some extent under aerobic conditions. The antioxidants also enhanced the revival of cryopreserved stock cultures of both rumen protozoal species. The results of this study may facilitate and promote future research in which rumen protozoa need to be cultured in laboratory.

Using video microscopy to improve quantitative estimates of protozoal motility and cell volume.

The objective of this study was to apply digital imaging to improve quantification of rumen protozoal biomass and distinguish treatment differences in cell motility and volume among ruminal protozoa. Observations of protozoa in rumen fluid treated with essential oils (CinnaGar, CIN; Provimi North America, Brookville, OH) or an ionophore (monensin, MON) indicated possible cell shrinkage. We hypothesized that MON would decrease protozoal motility and interact with CIN on cell volume. In addition, we hypothesized that analysis of still frames from video of swimming protozoa would improve volume prediction accuracy. Flocculated rumen fluid was incubated in batch culture dosed with N-free feed only (control), MON, CIN, or a combination of MON+CIN. Samples were taken at 0, 3, or 6 h post-treatment and wet-mounted on a microscope fitted with a high-definition camera. At 3 h post-inoculation, there was a treatment interaction for average speed such that CIN attenuated the effect of MON, with treatment means of 243, 138, 211, and 183 µm/s for control, MON, CIN, and MON+CIN, respectively. At 6 h post-inoculation, MON decreased average speed by 79.2 µm/s compared with the main effect mean without MON. We measured both minimum and maximum diameters (depth and width, respectively) perpendicular to the longitudinal axis of swimming protozoa, yielding a 3-dimensional estimate of protozoal volume. The ellipsoid formula (4/3)πabc, where a = 1/2 length, b = 1/2 width, and c = 1/2 depth, was compared with previously published volume estimations using genera-specific coefficients (genera-specific coefficient × length × width2). Residuals (genera-specific coefficients - ellipsoid) were plotted against predicted (ellipsoid) and centered to the mean (Xi-x¯) to evaluate both mean and slope biases. For Entodinium spp., Y = 0.248 (±0.037) (Xi - 7.98 × 104) + 1.97 × 104 (±1.48 × 103); n = 100; r2 [coefficient of determination (squared correlation coefficient)] = 0.31, with significant slope and mean biases. For family Isotrichidae, Y = -0.124 (±0.068) (Xi - 2.54 × 106) - 1.21 × 104 (±4.86 × 104); n = 32; r2 = 0.10, where slope tended to be different from zero but with no mean bias. For Epidinium spp., Y = 0.375 (±0.056) (Xi - 2.45 × 105) + 6.65 × 104 (±0.28 × 104); n = 64; r2 = 0.43, with both mean and slope biases. The present regression analyses demonstrate that the genera-specific coefficient-based method more likely overestimates volume for Entodinium and Epidinium than for the teardrop-shaped Isotrichidae. Based on simulations derived from previous literature reporting treatments that depress protozoal populations or among-animal changes in protozoal population structures, our proposed ellipsoid method offers potential to advance the prediction of treatment effects on protozoal volume and to shift focus from the number of cells present to the diversity, function, and biomass of protozoa under various treatment conditions.

Description of Diploplastron dehorityi sp. nov. (Entodiniomorphida, Ophryoscolecidae), a new rumen ciliate from Brazilian sheep (Ovis aries).

To date the genus Diploplastron comprised only one species of ophryoscolecid ciliate, Diploplastron affine, which is characterized by having two retractable ciliary zones in the anterior end of the body, two slender and juxtaposed skeletal plates on the right side, a rod shape macronucleus, and two contractile vacuoles. During study on the characterization of rumen ciliate community composition in Brazilian domestic sheep, we observed ciliates with atypical morphology but with diagnostic features of genus Diploplastron. This study describes Diploplastron dehorityi, a new species of ophryoscolecid ciliate, that differs from D. affine, primarily, in the morphology of skeletal plates, morphology of nuclear apparatus and body shape. In addition to the similarities between the new species and congener species, D. dehorityi has some morphological similarities to species of genus Eremoplastron.

Effect of Entodinium caudatum on starch intake and glycogen formation by Eudiplodinium maggii in the rumen and reticulum.

This study aimed to quantify the engulfed starch and reserve α-glucans (glycogen) in the cells of the ciliates Eudiplodinium maggii, as well the α-glucans in defaunated and selectively faunated sheep. The content of starch inside the cell of ciliates varied from 21 to 183mg/g protozoal DM relative to the rumen fauna composition whereas, the glycogen fluctuated between 17 and 126mg/g dry matter (DM) of this ciliate species. Establishment of the population Entodinium caudatum in the rumen of sheep already faunated with E. maggii caused a drop in both types of quantified carbohydrates. The content of α-glucans in the rumen of defaunated sheep varied from 4.4 to 19.9mg/g DM and increased to 7.4-29.9 or 11.8-33.9mg/g DM of rumen contents in the presence of only E. maggii or E. maggii and E. caudatum, respectively. The lowest content of the carbohydrates was always found just before feeding and the highest at 4h thereafter. The α-glucans in the reticulum varied 7.5-40.1, 14.3-76.8 or 21.9-106.1mg/g DM of reticulum content for defaunated, monofaunated or bifaunated sheep, respectively. The results indicated that both ciliate species engulf starch granules and convert the digestion products to the glycogen, diminishing the pool of starch available for amylolytic bacteria.

The effect of rumen ciliates on chitinolytic activity, chitin content and the number of fungal zoospores in the rumen fluid of sheep.

The objective of this study was to investigate the effect of selected protozoa on the degradation and concentration of chitin and the numbers of fungal zoospores in the rumen fluid of sheep. Three adult ewes were fed a hay-concentrate diet, defaunated, then monofaunated with Entodinium caudatum or Diploplastron affine alone and refaunated with natural rumen fauna. The average density of the protozoa population varied from 6.1 · 10(4) (D. affine) to 42.2 · 10(4) cells/ml rumen fluid (natural rumen fauna). The inoculation of protozoa in the rumen of defaunated sheep increased the total activity of chitinolytic enzymes from 2.9 to 3.6 µmol N-acetylglucosamine/g dry matter (DM) of rumen fluid per min, the chitin concentration from 6.3 to 7.2 mg/g DM of rumen fluid and the number of fungal zoospores from 8.1 to 10.9 · 10(5) cells/ml rumen fluid. All examined indices showed diurnal variations. Ciliate population density was highest immediately prior to feeding and lowest at 4 h thereafter. The opposite effects were observed for the numbers of fungal zoospores, the chitin concentration and chitinolytic activity. Furthermore, it was found that chitin from zoospores may account for up to 95% of total microbial chitin in the rumen fluid of sheep. In summary, the examined ciliate species showed the ability of chitin degradation as well as a positive influence on the development of the ruminal fungal population.

Microorganisms in the rumen and reticulum of buffalo (Bubalus bubalis) fed two different feeding systems.

BACKGROUND: The community of microorganisms in the rumen and reticulum is influenced by feeding as well as the species and geographical distribution of ruminant animals. Bacteria, methanogenic archaea and ciliate protozoa existing in the rumen and reticulum were evaluated by real-time polymerase chain reaction and light microscopy in buffalo in two feeding systems, grazing and feedlot. RESULTS: No significant differences were observed in the total concentrations of bacteria/mL and archaea between rumen and reticulum, and between pasture and feedlots, or interactions between variables. However, the largest density of bacteria and smallest density of archaea was observed in the rumen of grazing animals. The total ciliates protozoa community was higher in grazing buffalo than those in the feedlot on a concentrated diet. There were significant interactions between location in the gastrointestinal tract (rumen vs reticulum) and types of diets (grazing vs feedlot) in the composition of ciliates. CONCLUSIONS: Our data showed differences in the microbial community of the rumen and reticulum between grazing and feedlot feeding systems demonstrating relevant changes in the microorganism:host relationship existing on rumen-reticulum ecosystem.

Rumen microbial communities influence metabolic phenotypes in lambs.

The rumen microbiota is an essential part of ruminants shaping their nutrition and health. Despite its importance, it is not fully understood how various groups of rumen microbes affect host-microbe relationships and functions. The aim of the study was to simultaneously explore the rumen microbiota and the metabolic phenotype of lambs for identifying host-microbe associations and potential biomarkers of digestive functions. Twin lambs, separated in two groups after birth were exposed to practices (isolation and gavage with rumen fluid with protozoa or protozoa-depleted) that differentially restricted the acquisition of microbes. Rumen microbiota, fermentation parameters, digestibility and growth were monitored for up to 31 weeks of age. Microbiota assembled in isolation from other ruminants lacked protozoa and had low bacterial and archaeal diversity whereas digestibility was not affected. Exposure to adult sheep microbiota increased bacterial and archaeal diversity independently of protozoa presence. For archaea, Methanomassiliicoccales displaced Methanosphaera. Notwithstanding, protozoa induced differences in functional traits such as digestibility and significantly shaped bacterial community structure, notably Ruminococcaceae and Lachnospiraceae lower up to 6 folds, Prevotellaceae lower by ~40%, and Clostridiaceae and Veillonellaceae higher up to 10 folds compared to microbiota without protozoa. An orthogonal partial least squares-discriminant analysis of urinary metabolome matched differences in microbiota structure. Discriminant metabolites were mainly involved in amino acids and protein metabolic pathways while a negative interaction was observed between methylotrophic methanogens Methanomassiliicoccales and trimethylamine N-oxide. These results stress the influence of gut microbes on animal phenotype and show the potential of metabolomics for monitoring rumen microbial functions.