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.

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.

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.

In vitro fermentation of cellulosis amorphous and meadow hay in experimentally Ascaris suum-infected lambs.

The objective of this experiment was to determine the effect of rumen inocula from experimentally Ascaris suum (AS)-infected lambs on cellulose amorphous (CA) and meadow hay (MH) used as substrates during 72h incubation in vitro. The rumen inocula were obtained post-mortem from eight lambs that had been experimentally infected with 1000 AS eggs per lamb daily for 3 weeks. Samples of rumen inocula were obtained from the lambs weekly throughout the experiment for 8 weeks. Two lambs were kept as uninfected control animals. The substrates were incubated together with buffered rumen fluid in sealed fermentation bottles. In vitro dry matter degradability (IVDMD), total gas, methane and total and individual fatty acid (VFA) for both incubated substrates were measured and compared by the pressure transducer technique. Comparison of the values for the controls, total gas, methane and VFA revealed significant differences (P<0.05, P<0.01, P<0.001, respectively) for both substrates. Pronounced differences (P<0.001) were also found between CA and MH especially for IVDMD, total gas and total VFA production. A decrease (P<0.001) of IVDMD, total gas, methane and total VFA was observed from week 6 to week 8. Restriction of fermentation was evident from week 7 to week 8. The results suggest that the functional damage arising from pathological lesions within the parasite-infected organs considerably affected the fermentation parameters of the incubated substrates.

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 comparison of in vitro fermentation kinetics estimated by three different methods.

Three different methods for the estimation of in vitro fermentation kinetics are compared. The glass syringe, flow gasometer and pressure transducer methods were used for measurement of gas production. The rumen fluid from fistulated Merino sheep mixed with McDougall's buffer (1:1) was used as an inoculum and added at an amount of 35 ml into the fermentation vessels containing 0.25 g of meadow hay. The total gas produced was recorded after 12, 24, 36, 48, 60, 72 and 96 h of incubation. Hay dry matter degradability was the same with all three methods and achieved 56.5 to 58.2%. Total volatile fatty acids were significantly lower with the pressure transducer method than with the syringe and flow gasometer method. Lower values of mol% of butyrate and valerate obtained with the flow gasometer and pressure transducer methods in comparison with the syringe were also observed. Total gas production estimated by the flow gasometer method was lower than that stated by the two other methods. With regard to precision of the used methods syringe method was the best followed by the pressure transducer and flow gasometer method. It can be concluded that in spite of some limitations the pressure transducer method used in this experiment can be regarded as suitable for total gas estimation in in vitro rumen fermentation experiments.