Rumen Ciliate Fauna of Alaskan Moose (Alces americana), Musk-Ox (Ovibos moschatus) and Dall Mountain Sheep (Ovis dalli)*

Total numbers, generic distribution and percentage species distribution were determined for the ciliate protozoa in rumen contents obtained from Alaskan moose (Alces americana), musk-ox (Ovibos moschatus) and Dall mountain sheep (Ovis dalli). The musk-ox has a fauna somewhat similar to that previously observed in reindeer and caribou. In contrast, only protozoa in the genus Entodinium were observed in moose, while Dall mountain sheep have a fauna unique among Alaskan ruminants studied to date. Other than Entodinium exiguum which was common to all animals, only 2 additional species of Entodinium, observed in the moose and musk-ox, occurred in more than one animal species. Four new species of protozoa are described, Entodinium dalli sp.n., Entodinium constrictum sp.n. and Polyplastron alaskum sp.n. from the Dall mountain sheep and Entodinium alces sp.n. from moose.     

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.     

Some rumen ciliates have endosymbiotic methanogens

Abstract Most of the small ciliate protozoa, including Dasytricha ruminantium and Entodinium spp. living in the rumen of sheep, were found to have intracellular bacteria. These bacteria were not present in digestive vacuoles. They showed characteristic coenzyme F₄₂₀ autofluorescence and they were detected with a rhodamine-labelled Archaea-specific oligonucleotide probe. The measured volume percent of autofluorescing bacteria (1%) was close to the total volume of intracellular bacteria estimated from TEM stereology. Thus it is likely that all of the bacteria living in the cytoplasm of these ciliates were endosymbiotic methanogens, using H₂ evolved by the host ciliate to form methane. Intracellular methanogens appear to be much more numerous than those attached to the external cell surface of ciliates.     

The 3' Untranslated Region of Messages in the Rumen Protozoan Entodinium caudatum

The 3' untranslated regions of a number of cDNAs from the rumen protozoal species Entodinium caudatum were studied with a view to characterising their preference for stop codons, general length, nucleotide composition and polyadenylation signals. Unlike a number of ciliates, Entodinium caudatum uses UAA as a stop codon, rather than as a codon for glutamine. In addition, the 3' untranslated region of the message is generally less than 100 nucleotides in length, extremely A+T rich, and does not appear to utilise any of the conventional polyadenylation signals described in other organisms.     

Activité Cellulolytique des Ciliés Entodiniomorphes

RESUME. Dans nos travaux antérieurs, nous avons observé dans les cultures in vitro que la disparition de la cellulose (languettes de papier filtre) est 2 fois plus grande lorsque les 2 micropopulations, Entodinium et bactéries, sont présentes que lorsque les bactéries sont seules ou en présence de cellules mortes de ciliés. Les filtrats des broyats cellulaires des ciliés entodiniomorphes de la panse des ruminants et du caecum des équidés possèdent une activité cellulolytique indépendante des bactéries endocellulaires. Il s'agit essentiellement d'une polyglucosidase (Cx) qui dégrade les dérivés de la cellulose native. On observe également une légère activité cellulolytique C₁ sur la cellulose native.
SYNOPSIS. We have observed previously that twice as much cellulose disappears from cultures containing both Entodinium and bacteria than from those which contain bacteria alone or with dead ciliates. Filtrates of cell-free homogenates of the entodiniomorphid ciliates from sheep rumen and horse cecum have a hydrolytic effect upon cellulose, hydrolytic activity being independent of the intracellular bacteria. The predominant enzyme involved in the reaction is a polyglucosidase (Cx) which acts upon derivatives of native cellulose. Low level activity of enzyme C₁, which attacks native cellulose, also is found in the filtrate.     

A cryopreservation procedure for the rumen protozoon Entodinium caudatum: estimation of its viability by fluorescence microscopy

AIMS: To study the viability of a culture of the rumen protozoon Entodinium caudatum after a cryopreservation procedure by a fluorescence microscopy staining method. METHODS AND RESULTS: Fluorescence method is based on the different colour of cells depending on their membrane integrity. When the temperature effect was studied either by fluorescence or motility, the techniques were correlated (r = 0.727) and their slopes and intercepts were not different (P > 0.05). However, motility showed a higher variation coefficient (0.40 vs 0.12). There were no differences between cooling rates at cryopreservation (1 and 4 degrees C min-1) at 38, 15 or 5 degrees C, nor after thawing. CONCLUSIONS: Fluorescence staining is more accurate than motility for assessing protozoal viability. Viability after thawing was 0.50, and the number of viable cells per 250 microl straw was 320 and 420 for 1 and 4 degrees C min-1. SIGNIFICANCE AND IMPACT OF THE STUDY: This cryopreservation procedure seems to ensure culture recovery for E. caudatum.     

An NAD(+)-dependent glutamate dehydrogenase cloned from the ruminal ciliate protozoan, Entodinium caudatum

An NAD(+)-dependent glutamate dehydrogenase (GDH; EC 1.4.1.24) was cloned from the ruminal ciliate protozoan, Entodinium caudatum. The gene had high sequence similarity to GDH genes from the Bacteroides (class)--a class of bacteria which is highly represented in the rumen. When expressed in Escherichia coli the enzyme had a high affinity for ammonia and alpha-ketoglutarate (apparent K(m) of 2.33 and 0.71 mM, respectively) and a low affinity for glutamate (apparent K(m) of 98 mM). GDH activity and GDH mRNA concentration were increased by incubating washed E. caudatum cells with ammonia and antibiotics. These results suggest that the GDH is an anabolic enzyme catalysing the assimilation of ammonia by E. caudatum in the rumen and that the gene was probably acquired by lateral gene transfer from a ruminal bacterium.     

Methane production and substrate degradation by rumen microbial communities containing single protozoal species in vitro

AIMS: To assess the effect of protozoal species on rumen fermentation characteristics in vitro. METHODS AND RESULTS: Entodinium caudatum, Isotricha intestinalis, Metadinium medium, and Eudiplodinium maggii from monofaunated wethers and mixed protozoa from conventional wethers were obtained by centrifugation, re-suspended at their normal densities in rumen fluid supernatants from defaunated or conventional wethers and incubated in vitro. The presence of protozoa increased the concentration of ammonia and altered the volatile fatty acids balance with more acetate and butyrate produced at the expense of propionate. Differences among species were observed, notably in the production of methane, which increased with E. caudatum as compared to other ciliates and to defaunated and mixed protozoa treatments (P < 0.05). The increased methanogenesis was not correlated to protozoal biomass indicating that the metabolism of this protozoan and/or its influence on the microbial ecosystem was responsible for this effect. CONCLUSIONS: Entodinium caudatum stimulated the production of methane, a negative effect that was reinforced by a concomitant increase in protein degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison of individual species of protozoa highlighted the particular influence of E. caudatum on rumen fermentation. Its elimination (targeted defaunation) from the rumen could reduce methane production without affecting feed degradation.     

Light microscopical observation of infraciliary bands of Eodinium posterovesiculatum in comparison with Entodinium bursa and Diplodinium dentatum

Morphological features of the four morphotypes of the rumen ciliate, Eodinium posterovesiculatum, are described from pyridinated silver carbonate-impregnated specimens. Infraciliary bands are compared with those of Entodinium bursa and Diplodinium dentatum. In Entodinium bursa, the adoral polybrachykinety is "C" shaped and the vestibular polybrachykinety extends from the dorsal extremity of the adoral polybrachykinety as in other Entodinium species. In Diplodinium dentatum, the adoral polybrachykinety encircles most of the circumference of the vestibular opening and the vestibular polybrachykinety extends from the inner side of the adoral polybrachykinety as in other Diplodinium species. Infraciliary bands in Eodinium posterovesiculatum encircle the whole circumference of the vestibular opening and are, therefore, distinct from Entodinium, Diplodinium and other ciliates in the family Ophryoscolecidae. In Eodinium posterovesiculatum, the adoral polybrachykinety and the anterior part of the vestibular polybrachykinety encircle the vestibular opening. The wide and long vestibular polybrachykinety extends along the right wall of the tubular vestibulum and is bordered by a kinety. Kinetids in the central part of the vestibular polybrachykinety are randomly disposed. The genus Eodinium is valid because of this characteristic polybrachykinety arrangement in Eodinium posterovesiculatum.