Substrates enriched by the fungus Cunninghamella echinulata: an in vitro study of nutrient composition, sheep rumen fermentation and lipid metabolism.

AIMS: Enrichment of wheat bran (WB), corn meal (CM) and barley flakes (BF) with the oleaginous fungus Cunninghamella echinulata (CE) might lead to effective use of these by-products in ruminant nutrition. We examined their effects on rumen fermentation and lipid metabolism. METHODS AND RESULTS: WB, CM and BF substrates without or with brewer's grains (WBG, CMG, BFG) and enriched with CE were incubated with meadow hay (MH, 500 : 500, w/w) in rumen fluid in vitro for 24 h. The dry matter of the CE-enriched substrates increased (by 2-4%); however, digestibility decreased (P < 0·01). Adverse effects of CE-enriched substrates on the rumen ciliate population were observed. Little effect on the rumen eubacterial population was detected by the 16S-polymerase chain reaction/denaturizing gradient gel electrophoresis method. The increase in γ-linolenic acid output in the MH + BFGCE diet (800 : 200, w/w) was accompanied by an increase in rumen biohydrogenation of polyunsaturated fatty acids. CONCLUSION: The diet substrates enriched with the fungus CE were less digestible than the untreated cereal substrates; no appreciable positive effect was observed on rumen fermentation patterns or the eubacterial and ciliate populations. SIGNIFICANCE AND IMPACT OF THE STUDY: The in vitro study showed that adding CE-enriched substrates to ruminant diets is not effective for improving rumen fermentation.

Fluorescence in situ hybridization probing of protozoal Entodinium spp. and their methanogenic colonizers in the rumen of cattle fed alfalfa hay or triticale straw.

AIMS: To develop and test a fluorescence in situ hybridization (FISH) based technique and to identify and quantify simultaneously those methanogenic populations colonizing Entodinium spp. in the rumen of cows fed different forages. METHODS AND RESULTS: New FISH probes targeting protozoal Entodinium spp. were designed and used together with FISH probes for methanogens in the cow rumen. The composition and relative abundance of methanogenic populations colonizing Entodinium simplex-, E. caudaum- and Entodinium furca-related populations were similar. Methanogens including Methanobrevibacter thaueri, Methanobrevibacter millerae and Methanobrevibacter smithii, and members of Methanomicrobium and Methanosphaera were generally the predominant colonizers of protozoa, regardless of the forage fed to cattle. Individual animals appeared to differ in which ruminal methanogenic populations colonized each of the individual Entodinium spp. CONCLUSIONS: Simultaneous FISH probing is shown here to be a reliable and effective approach to investigate the dynamics of symbiotic relationships between ruminal protozoa and methanogens at a single cell level. Phylogenetically closely related Entodinium spp. were colonized by similar methanogenic populations regardless of the forage fed. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the methanogenic archaeal populations that specifically colonize Entodinium spp. as identified using simultaneous FISH probing.

The ciliate, Troglodytella abrassarti, contributes to polysaccharide hydrolytic activities in the chimpanzee colon.

Entodiniomorphid ciliates are intestinal protists inhabiting the colons of African great apes. The participation of intestinal entodiniomorphid ciliates in ape hindgut digestion has been proposed, but little data have been available to support the hypothesis. We measured the specific activities of carboxymethyl cellulase, xylanase, inulinase, and α-amylase against different polysaccharides in the feces of captive chimpanzees and evaluated the participation of the entodiniomorphid ciliate, Troglodytella abrassarti, in these activities. T. abrassarti contributed to the total fecal hydrolytic activities of CM-cellulase by 16.2%, α-amylase by 5.95%, and xylanase by 0.66%. Inulinase activity in T. abrassarti samples was not measurable at reaction conditions used. The ciliates, T. abrassarti, actively participate in the chimpanzee hindgut fermentation of fiber and starch.

Effects of prefermented cereal-derived substrates (ground barley and rye bran) enriched with fungal γ-linolenic acid on rumen fermentation parameters and lipid metabolism in vitro.

AIMS: To increase rumen output of γ-linolenic acid (GLA), we used two cereal-derived substrates, ground barley (GB) and rye bran (RB), enriched with fungal GLA as components of feed rations. We examined their effects on rumen fermentation patterns, lipid metabolism and the ciliated protozoan population in an artificial rumen. METHODS AND RESULTS: Four diets consisting of meadow hay (MH) plus unfermented (GB or RB) or prefermented (GB - TE or RB - TE) cereal-derived substrates were fermented in an artificial rumen with ovine rumen inoculum. The cereal-derived substrates were prefermented with the fungus Thamnidium elegans (TE) by fungal solid-state fermentation. The diets with TE increased the rumen input of dietary GLA (mg day(-1)) from 0 to 21 (GB - TE) or 26 (RB - TE). Both experimental diets increased the rumen output of GLA (P < 0.001). Adverse effects on the ciliate population were observed. Both diets also had an effect on the fatty acids profile. Fermentation patterns were also affected with MH + RB - TE. CONCLUSION: Cereal-derived substrates enriched with GLA effectively enhanced the output of GLA in artificial rumen. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability of the fungal strain T. elegans to grow and utilize various agro-industrial substrates might be useful in developing potential new animal diets enriched in GLA.

Bacterial-protozoal interactions in a microbial community of rumen ciliate Entodinium caudatum culture under mercury stress.

We examined the role of rumen ciliates, using Entodinium caudatum as a model organism, in the detoxification of soluble mercury(II) in vitro under conditions with enhanced or reduced diversity of a co-culture bacterial population as well as the effects of long-term mercury(II) stress on in vitro fermentation parameters and major mercury detoxification products. The E. caudatum growth depended on the capability of the co-culture bacterial population to develop resistance to mercury(II) chloride and on culture conditions. The production of fermentation gas was reduced (P < 0.01) in contrast to methane production. Proportions of volatile fatty acids were affected; however, the total concentration of volatile fatty acids was not influenced. No organic mercury species were detected after long-term application (>1 month) of mercury(II) chloride. The major mercury species was inorganic mercury(II) with substantial accumulation in the bacterial fraction (70%) and less in black sediment (21%) and ciliate fraction (9%) at the 25 micromol/L mercury(II) dose. The data indicate that free-living bacteria protect the ciliate cells by transforming mercury(II) into its insoluble forms.

Comparison of nisin and monensin effects on ciliate and selected bacterial populations in artificial rumen.

The effect of daily supplementation of nisin (2 mg/L), monensin (5.88 mg/L) and nisin and monensin (2 + 5.88 mg/L) on ovine ruminal ciliates and bacteria was investigated using the artificial rumen RUSITEC. Major groups in RUSITEC were Entodinium spp. and Dasytricha ruminantium. The supplementation of nisin significantly increased the population of both major ciliate groups. The supplementation of monensin significantly decreased the population of both groups. The combined effect of nisin and monensin was similar to the effect of monensin. Monensin had strong antiprotozoic effects in contrast to the stimulatory effects of nisin. D. ruminantium followed by Entodinium spp. appeared more resistant to tested compounds than other rumen ciliates. Tested additives did not significantly influence the presence and growth of amylolytic streptococci and enterococci but nisin showed a tendency to decreasing the concentration of Escherichia coli and lactobacilli.

Distinctive archaebacterial species associated with anaerobic rumen protozoan Entodinium caudatum.

The diversity of archaebacteria associated with anaerobic rumen protozoan Entodinium caudatum in long term in vitro culture was investigated by denaturing gradient gel electrophoresis (DGGE) analysis of hypervariable V3 region of archaebacterial 16S rRNA gene. PCR was accomplished directly from DNA extracted from a single protozoal cell and from total community genomic DNA and the obtained fingerprints were compared. The analysis indicated the presence of a solitary intensive band present in Entodinium caudatum single cell DNA, which had no counterparts in the profile from total DNA. The identity of archaebacterium represented by this band was determined by sequence analysis which showed that the sequence fell to the cluster of ciliate symbiotic methanogens identified recently by 16S gene library approach.

Highly efficient galvanotaxis apparatus for cleaning and concentrating rumen ciliates.

Galvanotaxis was shown to be an efficient method for cleaning and concentrating rumen ciliate protozoa whose harvesting (centrifugation of large volumes of in vitro cultures followed by repeated washing of the sediment to remove plant debris) is time consuming. We suggested the use of a new galvanotaxis apparatus (a small-capacity two-way glass stopcock) to improve cell yield in concentrating the rumen ciliate protozoa and cleaning them from impurities. Migration of the ciliates (Entodinium caudatum, Entodinium furca monolobum and Diploplastron affine) into the cathode compartment under different electric currents (0, 5, 10, and 15 mA) and intervals (5, 10, 20, and 30 min) was evaluated. The lethal current level was 20 mA. Cell yield was 9-81%, depending on ciliate species, migration time and current. The migration time significantly affected both E. caudatum and D. affine. The electric current-migration time interplay was shown to be significant in both E. caudatum and D. affine. The advantages and disadvantages of the tested apparatus were determined; the two-way glass stopcock was very convenient for both cleaning and concentrating rumen ciliate in vitro cultures by galvanotaxis.

Effect of insulin on in vitro fermentation activity of microrganism community of rumen ciliate Entodinium caudatum culture.

The influence of insulin (17.4 nmol l-1) on total gas and methane production, the concentration of total and individual fatty acids and dry matter degradability was investigated in the rumen ciliate culture of Entodinium caudatum. The experimental groups consisted of control group (without insulin) and two groups with insulin application--single shot and long-term application (over 30 days). Fermentation activity of each experimental group was observed on two subgroups: whole protozoan culture (protozoa plus bacteria) and bacterial fraction (bacteria without protozoa). Long-term application of insulin significantly increased methane production and DM degradability in the whole protozoan culture. Total VFA concentration was significantly increased by long-term as well as single-dose application of insulin (by 255% and 158%, respectively). The growth of the protozoa was not influenced by insulin treatments. It can be concluded that the fermentation activity of the community of the rumen ciliate Entodinium caudatum culture was marked stimulated by application of insulin.

The effects of organic selenium supplementation on the rumen ciliate population in sheep.

The effect of selenium supplementation on the rumen protozoan population of sheep was demonstrated. Both the total and generic counts of rumen ciliates in sheep fed a diet with basal Se content (70 microg/kg dry matter) were compared to those of animals given feed supplemented with inorganic (disodium selenite) or organic Se (selenized yeast) (310 microg/kg dry matter). The genera of Entodinium, Isotricha, Dasytricha, Ophryoscolex, Diploplastron and Polyplastron occurred in all sheep except for the control, in which Ophryoscolex was not observed. The population of Ophryoscolex caudatus f. tricoronatus was significantly higher in sheep supplemented with organic Se than in animals given inorganic Se (by 160 %). Supplementation of feed with selenized yeast induced significant growth in the Diploplastron population (by 63 %) while no change occurred in sheep given selenite. The populations of Dasytricha ruminantium and Polyplastron multivesiculatum were higher than control in both Se-supplemented groups. The ciliate population of Entodinium spp. was not influenced by Se supplements. Our results suggest a protective effect of Se feed supplementation on the development of some rumen ciliate species in young ruminants.

A re-appraisal of the diversity of the methanogens associated with the rumen ciliates.

The diversity of methanogenic archaea associated with different species of ciliated protozoa in the rumen was analysed. Partial fragments of archaeal SSU rRNA genes were amplified from DNA isolated from single cells from the rumen protozoal species Metadinium medium, Entodinium furca, Ophryoscolex caudatus and Diplodinium dentatum. Sequence analysis of these fragments indicated that although all of the new isolates clustered with sequences previously described for methanogens, there was a difference in the relative distribution of sequences detected here as compared to that of previous work. In addition, many of the novel sequences, although clearly of archaeal origin have relatively low identity to the sequences in database which are most closely related to them.

Two-step freezing procedure for cryopreservation of rumen ciliates, an effective tool for creation of a frozen rumen protozoa bank.

The present study aimed at the long-term storage of rumen protozoa as living cells in liquid nitrogen. The two-step or interrupted slow freezing procedure was used to cryopreserve six of the dominant species of rumen ciliates isolated from monofaunated animals, Dasytricha ruminantium, Entodinium caudatum, Epidinium ecaudatum caudatum, Eudiplodinium maggii, Isotricha prostoma, and Polyplastron multivesiculatum. We optimized the first step in the interrupted slow freezing procedure, from the extracellular ice nucleation temperature to the holding temperature, and studied the effects of the cooling rates on survival. In addition to the nature of the cryoprotectant (dimethyl sulfoxide), the equilibration temperature and equilibration time (25 degrees C and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S. Kisidayová, J. Microbiol. Methods 22:185-192, 1995), we found that a holding temperature of -30 degrees C, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2 degrees C/min and 2.5 degrees C/min, depending on the ciliate, and rumen juice as the freezing and thawing medium markedly improved the survival rate. Survival rates determined after 2 weeks in liquid nitrogen were 100% for Isotricha, 98% for Dasytricha, 85% for Epidinium, 79% for Polyplastron, 63% for Eudiplodinium, and 60% for Entodinium. They were not significantly modified after a period of 1 year in liquid nitrogen. Four of the five ciliate species cryopreserved for 8 months in liquid nitrogen successfully colonized the rumen when inoculated into defaunated animals. These results have made it possible to set up a bank of cryopreserved rumen protozoa.

Effect of nisin on two cultures of rumen ciliates.

The effect of nisin (in the form of Nisaplin) was determined using two species of rumen ciliate protozoa in vitro, on their co-culture bacterial population, and volatile fatty acid concentration. Nisaplin did not affect the in vitro growth of Entodinium caudatum at concentrations of 50-400 mg/L during short-term treatment (5 d). Long-term application (30 d) of Nisaplin (100 mg/L) significantly decreased growth of the Epidinium ecaudatum forma caudatum et ecaudatum but not growth of E. caudatum. Nisaplin moderately supported the growth of E. caudatum after omission of wheat gluten (source of amino acids for protozoan growth). An inhibition of Gram-positive facultative anaerobic bacterial population in the protozoan cultures (lactobacilli, enterococci, staphylococci and amylolytic streptococci) was observed during long-term Nisaplin treatment. The concentration of volatile fatty acids significantly increased during the long-term Nisaplin treatment of both cultures. The propionate concentration in the mixture of volatile fatty acids was nearly twice higher on the account of the decreased concentration (from 74 to 63%) of acetate.

Effect of plant oils and organic acids on rumen fermentation in vitro.

We determined the effect of plant oils (rapeseed, sunflower, linseed) and organic acids (aspartic and malic) on the fermentation of diet consisting of hay, barley and sugar beet molasses. Rumen fluid was collected from two sheep (Slovak Merino) fed with the same diet twice daily. Mixed rumen microorganisms were incubated in fermentation fluid, which contained rumen fluid and Mc Dougall's buffer. All supplemented diets significantly increased pH, molar proportion of propionate, and numerically decreased methane production. Lactate production was also decreased significantly (except with malate). Incorporation of plant oils into aspartate- and malate-treated incubations negated the decrease of butyrate, lactate and the increase of pH and ammonia with malate treatment, as well as in vitro dry matter digestibility and pH with aspartate treatment. The effect of combined additives on methane production and molar proportion of propionate was lower compared with additives supplemented separately. Combination of additives had no additive effect on rumen fermentation. All additives decreased total protozoan counts in rumen fluid.

Effect of antibiotics, 2-bromoethanesulfonic acid and pyromellitic diimide on methanogenesis in rumen ciliate cultures in vitro.

The effects of penicillin G, streptomycin, chloramphenicol, 2-bromoethanesulfonic acid and pyromellitic diimide on total gas, methane, volatile fatty acid production and food degradability after 24 h of incubation in vitro were investigated in the cultures of two rumen ciliates. The inocula of both rumen ciliates Entodinium caudatum and Epidinium ecaudatum were used at a volume of 34 ml into the 50 ml glass syringes together with the feed and compounds tested. Despite penicillin G--streptomycin treatment methane production in both cultures was significantly decreased by the inhibitors for Epidinium ecaudatum. Methane production of the bacterial fraction of both protozoan species was significantly lower than in the whole cultures. No epifluorescence of methanogens on (or in) the cells of Entodinium caudatum was observed in contrast to Epidinium with which strong epifluorescence of methanogens on the cell surface was detected. Microscopic observation could indicate that the methane production by Entodinium caudatum was probably caused by their intracellular methanogenic activity, while methane production by Epidinium ecaudatum could be related to both the methanogenic bacterial fraction from their external surface and probably also to intracellular activity. Decreased feed degradability and differences in the fermentation end products accompanied the inhibition of methanogenesis in both in vitro cultures. Entodinium caudatum appeared to be more sensitive than Epidinium ecaudatum to the compounds tested.

Methanogenesis in rumen ciliate cultures of Entodinium caudatum and Epidinium ecaudatum after long-term cultivation in a chemically defined medium.

The methanogenic activity in the presence of Entodinium caudatum and Epidinium ecaudatum was well preserved after long-term cultivation. Microscopic observation revealed that methane production in the presence of E. caudatum was probably caused by their intracellular methanogenic activity, while methane production in the presence of E. ecaudatum f caudatum et ecaudatum could be attributed to both the methanogenic bacterial fraction of their external surface and their intracellular activity. Methane production per protozoan cell of E. caudatum and E. ecaudatum was 2.1 nmol per cell per d and 6.0 nmol per cell per d, respectively. E. caudatum was responsible for almost the entire methane production in the culture. The activity of free methanogens constituted approximately 50% of the total methane production in the E. ecaudatum culture. Decrease of digestibility of substrates and differences in the fermentation end products accompanied the inhibition of methanogenesis in both cultures by penicillin G, streptomycin, chloramphenicol, 2-bromoethanesulfonate, and pyromellitic diimide. E. caudatum appeared to be more sensitive than E. ecaudatum to the compounds tested. Hydrogen recoveries based on both volatile fatty acids and methane production suggested that the methanogenic population appeared not to be fully able to consume hydrogen produced in the protozoan cultures. The culture conditions tested were found to be suitable for experiments on the relationship between rumen ciliates and rumen bacteria.

The cryopreservation of some large ciliate entodiniomorphid protozoa taken from the rumen.

Large entodiniomorphid ciliates taken from the rumen cannot be reliably kept in vivo, and cryopreservation was investigated as an alternative approach to storage. Three species of large entodiniomorphid ciliated (Polyplastron multivesiculatum, Epidinium ecaudatum forma caudatum and Ophryoscolex caudatus forma tricoronatus) were cryopreserved by one-step freezing, with mean recoveries of 43, 51 and 80%, respectively. For Ophryoscolex caudatus forma tricoronatus two-step freezing was investigated. The mean recovery was 30%, and of tested parameters, the equilibration time and temperature of the cryoprotective additive of dimethyl sulphoxide and the length of holding phase at -40 degrees C were found to be critical for the success of the method.

[The effect of cadmium on the protozoan population and rumen fermentation of feed in an artificial rumen]. / Vplyv kadmia na protozoárnu populáciu a bachorovú fermentáciu krmnej dávky v umelom bachore.

In our experiment, the effects of 5, 10 and 20 mg cadmium per kg dry matter (DM) on protozoan population and rumen fermentation of feed ration consisting of 11.7 g DM of hay and 2.8 g DM of barley (80: 20%) were followed in artificial rumen (Rusitec). The results of the experiment showed that the addition of 5 and 10 mg cadmium per kg DM less significantly (P < 0.05) decreased the digestibility of DM, organic matter and neutral detergent fibre and significantly (P < 0.01) decreased the digestibility of cellulose of feed ration. It is interesting that the addition of 20 mg cadmium per kg DM did not influence these parameters. Although the total gas production showed a tendency of decrease due to the influence of the addition of 5, 10 and 20 mg cadmium/kg DM, methane and CO2 production was not altered. However, cadmium significantly influenced the production of individual volatile fatty acids-VFA's (mmol/day). The addition of 5, 10 and 20 mg cadmium per kg DM significantly increased acetic acid production (by 10-20%) and decreased propionic acid production (by 18-30%). Therefore, the acetate: propionate ratio was significantly increased (from 1.57 to 2.45-2.60) by the addition of cadmium. The production of n-valeric and iso-valeric acids was also significantly decreased in comparison with the control. The decrease of individual VFA's was more significant at the higher amount of cadmium added into the fermentation system. The proportions of individual VFA's in total VFA production expressed in molar % showed a similar character. The changes in the production of individual VFA's due to the influence of the addition of 5, 10 and 20 mg cadmium per kg DM caused a decrease in energetic efficiency of VFA's (P < 0.001) in comparison with the control. The other parameters of rumen fermentation - utilization of glucose, adenosine triphosphate (ATP) production, fermented hexose, fermented amino acids and fermented organic matter (OMF) were not influenced by the cadmium addition. Although the total microbial matter production in effluent and undigested feed (residues) was significantly (P < 0.001) decreased by the addition of 5, 10 and 20 mg cadmium per kg DM, the efficiency of microbial synthesis expressed as YATP and g microbial N/kg OMF was not changed and achieved the values 11.78-12.68 (YATP), 25.56-28.35 (g microbial N/kg OMF), respectively. The total protozoan population was significantly decreased by the cadmium addition mainly as a result of the decrease of infusoria of Entodinium spp.(ABSTRACT TRUNCATED AT 400 WORDS)