S. Kišidayová

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.

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 Dougalls 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 asin 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.

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 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 µg/kg dry matter) were compared to those of animals given feed supplemented with inorganic (disodium selenite) or organic Se (selenized yeast) (310 µg/kg dry matter). The genera ofEntodinium, Isotricha, Dasytricha, Ophryoscolex, Diploplastron andPolyplastron occurred in all sheep except for the control, in whichOphryoscolex was not observed. The population ofOphryoscolex 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 theDiploplastron population (by 63 %) while no change occurred in sheep given selenite. The populations ofDasytricha ruminantium andPolyplastron multivesiculatum were higher than control in both Se-supplemented groups. The ciliate population ofEntodinium 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.

Two-Step Freezing Procedure for Cryopreservation of Rumen Ciliates, an Effective Tool for Creation of a Frozen Rumen Protozoa Bank

ABSTRACT 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°C and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S. Kišidayová, J. Microbiol. Methods 22:185-192, 1995), we found that a holding temperature of −30°C, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2°C/min and 2.5°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.

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.

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

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.

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.

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 protozoain vitro, on their co-culture bacterial population, and volatile fatty acid concentration. Nisaplin did not affect thein vitro growth ofEntodinium 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 theEpidinium ecaudatum formacaudatum etecaudatum but not growth ofE. caudatum. Nisaplin moderately supported the growth ofE. 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 Nispalin 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.