Z. Váradyová

Effects of high- and low-fiber diets on fecal fermentation and fecal microbial populations of captive chimpanzees.

We examined fiber fermentation capacity of captive chimpanzee fecal microflora from animals (n=2) eating low‐fiber diets (LFDs; 14% neutral detergent fiber (NDF) and 5% of cellulose) and high‐fiber diets (HFDs; 26% NDF and 15% of cellulose), using barley grain, meadow hay, wheat straw, and amorphous cellulose as substrates for in vitro gas production of feces. We also examined the effects of LFD or HFD on populations of eubacteria and archaea in chimpanzee feces. Fecal inoculum fermentation from the LFD animals resulted in a higher in vitro dry matter digestibility (IVDMD) and gas production than from the HFD animals. However, there was an interaction between different inocula and substrates on IVDMD, gas and methane production, and hydrogen recovery (P<0.001). On the other hand, HFD inoculum increased the production of total short‐chain fatty acids (SCFAs), acetate, and propionate with all tested substrates. The effect of the interaction between the inoculum and substrate on total SCFAs was not observed. Changes in fermentation activities were associated with changes in bacterial populations. DGGE of bacterial DNA revealed shift in population of both archaeal and eubacterial communities. However, a much more complex eubacterial population structure represented by many bands was observed compared with the less variable archaeal population in both diets. Some archaeal bands were related to the uncultured archaea from gastrointestinal tracts of homeothermic animals. Genomic DNA in the dominant eubacterial band in the HFD inoculum was confirmed to be closely related to DNA from Eubacterium biforme. Interestingly, the predominant band in the LFD inoculum represented DNA of probably new or yet‐to‐be‐sequenced species belonging to mycoplasms. Collectively, our results indicated that fecal microbial populations of the captive chimpanzees are not capable of extensive fiber fermentation; however, there was a positive effect of fiber content on SCFA production. Am. J. Primatol. 71:548–557, 2009.

A Survey of Entodiniomorphid Ciliates in Chimpanzees and Bonobos

Intestinal entodiniomorphid ciliates are commonly diagnosed in the feces of wild apes of the genera Pan and Gorilla. Although some authors previously considered entodiniomorphid ciliates as possible pathogens, a symbiotic function within the intestinal ecosystem and their participation in fiber fermentation has been proposed. Previous studies have suggested that these ciliates gradually disappear under captive conditions. We studied entodiniomorphid ciliates in 23 captive groups of chimpanzees, three groups of captive bonobos and six populations of wild chimpanzees. Fecal samples were examined using Sheathers flotation and Merthiolate-Iodine-Formaldehyde Concentration (MIFC) methods. We quantified the number of ciliates per gram of feces. The MIFC method was more sensitive for ciliate detection than the flotation method. Ciliates of genus Troglodytella were detected in 13 groups of captive chimpanzees, two groups of bonobos and in all wild chimpanzee populations studied. The absence of entodiniomorphids in some captive groups might be because of the extensive administration of chemotherapeutics in the past or a side-effect of the causative or prophylactic administration of antiparasitic or antibiotic drugs. The infection intensities of ciliates in captive chimpanzees were higher than in wild ones. We suppose that the over-supply of starch, typical in captive primate diets, might induce an increase in the number of ciliates. In vitro studies on metabolism and biochemical activities of entodiniomorphids are needed to clarify their role in ape digestion.

The symbiotic intestinal ciliates and the evolution of their hosts

The evolution of sophisticated differentiations of the gastro-intestinal tract enabled herbivorous mammals to digest dietary cellulose and hemicellulose with the aid of a complex anaerobic microbiota. Distinctive symbiotic ciliates, which are unique to this habitat, are the largest representatives of this microbial community. Analyses of a total of 484 different 18S rRNA genes show that extremely complex, but related ciliate communities can occur in the rumen of cattle, sheep, goats and red deer (301 sequences). The communities in the hindgut of equids (Equus caballus, Equus quagga), and elephants (Elephas maximus, Loxodonta africanus; 162 sequences), which are clearly distinct from the ruminant ciliate biota, exhibit a much higher diversity than anticipated on the basis of their morphology. All these ciliates from the gastro-intestinal tract constitute a monophyletic group, which consists of two major taxa, i.e. Vestibuliferida and Entodiniomorphida. The ciliates from the evolutionarily older hindgut fermenters exhibit a clustering that is specific for higher taxa of their hosts, as extant species of horse and zebra on the one hand, and Africa and Indian elephant on the other hand, share related ciliates. The evolutionary younger ruminants altogether share the various entodiniomorphs and the vestibuliferids from ruminants.

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 ofEntodinium caudatum andEpidinium ecaudatum was well preserved after long-term cultivation. Microscopic observation revealed that methane production in the presence ofE. caudatum was probably caused by their intracellular methanogenic activity, while methane production in the presence ofE. ecaudatum f.caudatum etecaudatum could be atributed to both the methanogenic bacterial fraction of their external surface and their intracellular activity. Methane production per protozoan cell ofE. caudatum andE. 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 thee. 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 diimideE. caudatum appeared to be more sensitive thanE. 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 ciliate and rumen bacteria.

In vitro study of the rumen and hindgut fermentation of fibrous materials (meadow hay, beech sawdust, wheat straw) in sheep

Abstract The influence of both rumen and hindgut inocula of sheep on fermentation of fibrous materials in vitro has been investigated. Different fibrous materials (meadow hay, beech sawdust, wheat straw) and cellulose were used as substrates. The study was carried out to compare: (1) fermentation of substrates with rumen and hindgut inocula, (2) fermentation of meadow hay (reference substrate) and other substrates, (3) fermentation of the two types of cellulose (amorphous and crystalline), and (4) fermentation of treated fibrous materials (treated beech sawdust by defibration and impregnation and fungal treated wheat straw) and untreated fibrous materials. Hindgut fermentation of fibrous materials was associated with decreased dry matter and neutral detergent fibre degradabilities, and also methane and total gas production. The calculated hydrogen recoveries with hindgut inoculum showed a tendency to lower values as compared to the rumen inoculum. Significant differences were found between meadow hay and other fibrous materials, between both celluloses and between treated and untreated fibrous materials. The positive correlation between hydrogen recoveries and methane production of untreated wheat straw with a hindgut inoculum suggested the presence of reductive acetogenesis with the hindgut inoculum. It can be concluded that reductive acetogenesis with a hindgut inoculum instead of methanogenesis may increase the energetic yield from VFA per substrate, and to some extent also the energetic yield for the host animal.

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.

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 McDougalls 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.

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.

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.