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Precision dietary interventions (e.g., altering proportions of dietary protein fractions) has significant implications for the efficiency of nutrient use in ruminants, as well as lowering their environmental footprint, specifically nitrogen (N) emissions. Soluble protein (SP) is defined as the protein fraction that is rapidly degraded in the rumen (e.g., non-protein N and true protein), and our previous study found that regulating SP levels could improve N efficiency in Hu sheep. Thus, the present study was conducted to explore in vitro how protein fractions with different SP levels modulate the rumen microbial community and its association with N metabolism. Four dietary treatments with different SP proportions and similar crude protein (CP) content (~14%) were formulated (% of CP): 20 (S20), 30 (S30), 40 (S40) and 50 (S50). Results showed that NH3-N content increased with increasing SP levels at 4, 12 and 24 h; TVFA, acetate, propionate and valerate were higher in S30 and S40 (p < 0.05) and had quadratic effects (p < 0.05). Moreover, dry matter digestibility (DMD) and N digestibility (ND) were all decreased with S20 and S50 (p < 0.05). The S30 and S40 treatments increased the abundance of Bacteroidetes and Prevotella (Prevotella_ruminicola) but decreased the abundance of Firmicutes and Proteobacteria (p < 0.05). Bacterial pathways related to amino acid and fatty acid metabolism also were enriched with S30 and S40. The abundance of Entodinium was increased with S30 and S40 and had a positive correlation with Prevotella, and these two genera also played an important role in N metabolism and VFA synthesis of this study. In conclusion, bacterial and protozoal communities were altered by the level of SP (% of CP), with higher SP levels (~50% of CP) increasing the microbial diversity but being detrimental to rumen N metabolism.

Research Areas: Science & Technology - Other Topics This study investigated the associations between abundance of rumen ciliate protozoa and the proportion of the main bioactive fatty acids related to rumen biohydrogenation, as 18:0, t10-18:1, t11-18:1, c9,t11-18:2, 18:3n-3 and 18:2 n-6, in rumen and meat of growing lambs, using data derived from 3 production experiments. A global correlation analysis and a linear regression analysis considering the effect of the experiment were performed. Ten of the 86 lambs involved in the experiments did not present ciliate cells in rumen liquor and the remaining lambs presented an average of 1.35 x 106ciliates / ml rumen liquor. From the nine genera of ciliates identified, Entodinium was the most abundant, averaging 1.17 x 106 cells / ml of rumen liquor. A large variation among lambs was observed for both rumen concentration and community structure of ciliates. Rumen t11-18:1 (P< 0.001) and meat deposition of t11-18:1 (P < 0.001) and of c9,t11-18:2 (P < 0.001) increased linearly with total ciliates, whereas the t10/t11 ratio in rumen (P= 0.002) and in meat (P= 0.036) decreased linearly. Entodiniomorphids seems to be strongly related with meat deposition of t11-18:1 and c9,t11-18:2 and with the reduction of the trans-10 shifted pathway. Completeness of RBH decreased linearly with Holotrichs (P = 0.029), Entodiniomorphids (P = 0.029), Isotricha (P = 0.011) and Epidinium (P = 0.027) abundances. Rumen 18:0 also decreased linearly with increasing counts of total ciliates (P = 0.015), Holotrichs (P = 0.020), Entodiniomorphids (P= 0.010) and Isotricha (P= 0.014). Rumen protozoa were positively linked with the deposition of healthy bioactive FA and simultaneously negatively associated with the occurrence of trans-10 shift. info:eu-repo/semantics/publishedVersion

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is considered a re-emerging environmental pollutant, and exposure to environmentally relevant concentrations has been shown to cause individual developmental toxicity in zebrafish and the water flea ( Daphnia magna ). However, multigenerational effects during exposure to TDCIPP and after subsequent recovery were unknown. In the present study, individuals of a model aquatic organism, the ciliated protozoan, T. thermophila were exposed to environmentally-relevant concentrations of TDCIPP (0, 300 or 3000 ng/L) for 60 days (e.g., theoretically 372 generations) followed by a 60-day period of recovery, during which T. thermophila were not exposed to TDCIPP. During exposure and after exposure, effects at the molecular, histological, individual and population levels were examined. Multigenerational exposure to 300 or 3000 ng TDCIPP/L for 60 days significantly decreased numbers of individuals, sizes of individuals, expressed as length and width of bodies, number of cilia, and depth and diameter of basal bodies of cilia, and up-regulated expressions of genes related to assembly and maintenance of cilia. Complete or partial recoveries of theoretical sizes of populations as well as sizes of individuals and expressions of genes were observed during the 60-day recovery period. Effects on number of cilia and depth and diameter of basal body of cilia were not reversible and could still be observed long after cease of TDCIPP exposure. Collectedly, and shown for the first time, multigenerational effects to T. thermophila were caused by exposure to environmentally relevant concentrations of TDCIPP. Also, there were multi-generational effects at the population level that were not caused by carry-over exposure to TDCIPP. The “permanent” alterations and their potential significance are discussed.

The methods used for culturing rumen protozoa were found to be unsatisfactory for growth of ciliate protozoa from the kangaroo forestomach. Based on published measurements of physical parameters in the marsupial forestomach, several modifications were incorporated into the procedure, i.e., an increase in % hydrogen in the gas phase, adjustment of initial pH of the medium to 6.9–7.0 range, feed only forage as a substrate and incubate at a lower temperature (33–36 °C). Only incubation at the lower temperature increased survival time of the kangaroo protozoa. Two species of Bitricha were still viable after 28 d in culture. Cultures had to be terminated at that time. One of the species differed considerably in size and shape from previously described species and based on 18S rRNA data, may represent a new species of Bitricha. The second species, present in low numbers was identified as Bitricha oblata. In a separate trial, Macropodinium yalanbense survived for 11 d, at which time these cultures also had to be terminated.

Trichodinids, which are ciliate protists, are causative agents of an aquatic animal disease, trichodiniasis, especially among both captive and wild fish. This disease can adversely affect aquaculture and have economic impacts. The objectives of this study were to evaluate the prevalence and mean intensity of Trichodina unionis infection, describe qualitative and quantitative morphological characters, and perform a molecular phylogenetic analysis. The gastropod samples were randomly collected by hand-picking and a hand net. Trichodina unionis was collected by the crushing method under a stereomicroscope. Among all 4977 examined gastropods, 55 individuals of two gastropod species, Gyraulus siamensis and Physella acuta, were found to be infected by T. unionis, with overall prevalence and mean intensity of infection of 1.11% and 16.65, respectively. The characteristics of the denticles indicated T. unionis as having moderately wide blades and moderately curved blade margins, with distinctive bend angles near the distal end. The quantitative characters showed some variations, which could be due to food availability. Molecular phylogenetic analysis conducted with 18S rRNA provided a monophyletic tree of our specimens and previously identified T. unionis, confirming species identification. This study represents the first record of T. unionis in Thailand.

Medium-chain fatty acids (MCFA), for example, capric acid (C10:0), myristic (C14:0) and lauric (C12:0) acid, have been suggested to decrease rumen archaeal abundance and protozoal numbers. This study aimed to compare the effect of MCFA, either supplied through krabok (KO) or coconut (CO) oil, on rumen fermentation, protozoal counts and archaeal abundance, as well as their diversity and functional organization. KO contains similar amounts of C12:0 as CO (420 and 458 g/kg FA, respectively), but has a higher proportion of C14:0 (464 v. 205 g/kg FA, respectively). Treatments contained 35 g supplemental fat per kg DM: a control diet with tallow (T); a diet with supplemental CO; and a diet with supplemental KO. A 4th treatment consisted of a diet with similar amounts of MCFA (i.e. C10:0+C12:0+C14:0) from CO and KO. To ensure isolipidic diets, extra tallow was supplied in the latter treatment (KO+T). Eight fistulated bulls (two bulls per treatment), fed a total mixed ration predominantly based on cassava chips, rice straw, tomato pomace, rice bran and soybean meal (1.5% of BW), were used. Both KO and CO increased the rumen volatile fatty acids, in particular propionate and decreased acetate proportions. Protozoal numbers were reduced through the supplementation of an MCFA source (CO, KO and KO+T), with the strongest reduction by KO. Quantitative real-time polymerase chain reaction assays based on archaeal primers showed a decrease in abundance of Archaea when supplementing with KO and KO+T compared with T and CO. The denaturing gradient gel electrophoresis profiles of the rumen archaeal population did not result in a grouping of treatments. Richness indices were calculated from the number of DGGE bands, whereas community organization was assessed from the Pareto-Lorenz evenness curves on the basis of DGGE band intensities. KO supplementation (KO and KO+T treatments) increased richness and evenness within the archaeal community. Further research including methane measurements and productive animals should elucidate whether KO could be used as a dietary methane mitigation strategy.

The flow of ciliate protozoa from the reticulo-rumen is significantly less than expected given the total density of rumen protozoa present. To maintain their numbers in the reticulo-rumen, protozoa can be selectively retained through association with feed particles and the rumen wall. Few mathematical models have been designed to model rumen protozoa in both the free-living and attached phases, and the data used in the models were acquired using classical techniques. It has therefore become necessary to provide an updated model that more accurately represents these microorganisms and incorporates the recent literature on distribution, sequestration, and generation times. This paper represents a novel approach to synthesizing experimental data on rumen microorganisms in a quantitative and structured manner. The development of a linear programming model of rumen protozoa in an approximate steady state will be described and applied to data from healthy ruminants consuming commonly fed diets. In the model, protozoa associated with the liquid phase and protozoa attached to particulate matter or sequestered against the rumen wall are distinguished. Growth, passage, death, and transfer of protozoa between both pools are represented. The results from the model application using the contrasting diets of increased forage content versus increased starch content indicate that the majority of rumen protozoa, 63 to 90%, are found in the attached phase, either attached to feed particles or sequestered on the rumen wall. A slightly greater proportion of protozoa are found in the attached phase in animals fed a hay diet compared with a starch diet. This suggests that experimental protocols that only sample protozoa from the rumen fluid could be significantly underestimating the size of the protozoal population of the rumen. Further data are required on the distribution of ciliate protozoa in the rumen of healthy animals to improve model development, but the model described herein does indicate that the attached protozoal population is a significant component of the total rumen protozoal community. Key words: linear programming , protozoa , reticulorumen , sequestration

Resume Une ration de base, constituee de 900 g d'aliments concentres et de 100 g de foin a ete supplementee avec 0,3 %, 0,8 % et 1,2 % de methionine hydroxyanalogue (M. H. A.) et distribuee a quatre moutons adultes, munis d'une fistule du rumen, et pesant en moyenne 60 kg. On a observe que l'addition de M. H. A. a raison de 0,8 % de la ration augmente considerablement le nombre total de ciliates du rumen, tandis que le taux de 1,2 % n'entraine plus de nouvelle augmentation. Chaque taux de supplementation a entraine une elevation considerable de la concentration totale en methionine du jus de rumen, ce qui permet de penser qu'une partie de la M. H. A. a ete transformee en methionine dans le rumen. Zusammenfassung Der Einflus einer Methioninhydroxyanalog-Erganzung auf die Protozoenkonzentration im Schafpansen 1 Eine Grundration aus 900 g Konzentrat und 100 g Heu wurde mit 0,3 %, 0,8 % und 1,2 % Methioninhydroxyanalog (M. H. A.) erganzt und an 4 fistulierte Schafe im Gewicht von 60 kg verfuttert. 2 Die Erganzung von 0,8 % M. H. A. fuhrte zu einer merklichen Anreicherung von Ciliaten im Pansen. Eine 1,2%oige M. H. A.-Erganzung erbrachte keinen weiteren Anstieg in der Protozoenkonzentration. 3 Die Erganzung von M. H. A. fuhrte auserdem zu einem Anstieg von Methionin in der Pansenflussigkeit. Daraus ist zu schliesen, das M. H. A. zu Methionin im Pansen umgewandelt werden kann.

A new approach to the enumeration of ciliate protozoa in activated sludge is described. A 25 microL sub-sample volume is optimal for routine analysis using a standard slide and 24 x 24 mm cover slip requiring between 20 and 40 min per sub-sample for full enumeration and identification of species. However, to achieve high probability (or=95%) of recovering all species large numbers of replicates are required (i.e. 23-47). To achieve high probabilities of recovery using less replicates it is necessary to neglect rare species with low densities (0.5%); based on the assumption that they do not play a significant role in plant performance. The precise number of replicates required for different probabilities of recovering species is determined by conducting an initial pilot survey analysing a minimum of 8 replicates and using a probability equation to determine the optimum replicate number for that particular plant. Six replicate 25 microL sub-samples provided excellent species recovery (90-95% excluding up to 3 rare species), while analysing just two or three replicates, as commonly used in previous wastewater studies, only gave probabilities of 25 and 50% respectively for the same recovery. Ciliate analysis should be completed within 8h of collection with significant changes in community structure occurring beyond this period.

The morphology and infraciliature of the soil hypotrich ciliate, Gonostomum albicarpathicum nov. spec., collected from Biele Karpaty (White Carpathian Mts.), Slovakia, were investigated using living observations and the protargol impregnation method. This new species is distinguished from its congeners by a combination of characters including arrangement of macronuclear nodules forming a pair of nodules each ahead of and behind mid-body; 6-10 frontoventral cirri and 3 frontoterminal cirri forming a short row; first frontoterminal cirrus separated from the second by a slightly greater distance than between second and third.

The ability of rumen ciliate protozoa to move in a unidirectional electrical field from the anode to the cathode was tested in large-volume electromigration equipment; the aim was to concentrate the microorganisms and clean them of impurities. During galvanotaxis in the freshly harvested rumen fluid and at a voltage of 10 V (I=0.8 mA), cells of Isotricha (Isotricha prostoma, Isotricha intestinalis) were the first to swim towards the cathode; 1 min later, they were followed by Dasytricha ruminantium. Entodiniomorphous ciliates (small Entodiniae as well as large species) displayed minimum movement towards the cathode. The yield of electromigration of Entodinium caudatum from the in vitro culture ranged within 2-6% when using 75-100 V (10 mA) and 60 V (5 mA) voltage, respectively. At 10 V (0.8 mA), E. caudatum did not move towards the cathode. It is shown that the behavior of Trichostomatids (Dasytricha and Isotricha) facilitates separation by means of the tested large-volume equipment. Concentration and cleaning of Entodiniomorphous ciliates using galvanotaxis in the tested equipment proved to be ineffective.

Species composition and concentration of rumen ciliate protozoa were investigated in the rumen contents of 14 domestic sheep and 1 goat living in Bishkek, Kyrgyzstan. This is the first report on rumen ciliates from ruminants living in Kyrgyzstan. In sheep 12 genera, 28 species and 12 morphotypes were detected, whereas in goat 8 genera, 12 species and 4 morphotypes were detected. The density of ciliates in sheep was (28.1 ± 20.0) × 10(4) cells mL(-1) and in goat was 37.0 × 10(4) cells mL(-1). Dasytricha ruminantium, Isotricha prostoma, Entodinium simulans and Ophryoscolex caudatus were major species (100%) in sheep, and for the first time, Diplodinium rangiferi was detected in a domestic goat.

Protozoa species were identified in rumen contents of four domestic sheep (Ovis aries) from South Africa. All animals were fed a forage diet which consisted of 50% lucerne and 50% teff hay. Ten new host records were identified, bringing the total number of species and forms observed in sheep in South Africa to 30. The occurrence and geographic distribution of ciliate protozoa in both domestic and wild sheep from around the world are summarised. It was found that 15 genera and 131 species occur in domestic sheep globally.

Changes in the microbial populations, their activities, and the ruminal fermentation were monitored for 50 d following the reintroduction of ciliate protozoa into four defaunated sheep. A protozoal population was reestablished successfully in each recipient, using a washed inoculum containing approximately 103 cells, although there were between-animal differences in the rates of recolonization and genus establishment. Entodinium spp. predominated in the initial stages of the refaunation period and had an apparent maximal generation time of 9–10 h. Bacterial and fungal numbers did not decline following the reintroduction of protozoa and a small transient increase in the numbers of amylolytic and xylanolytic bacteria and fungal zoospores occurred in the early stages of refaunation when the protozoal population was 5/g ruminal contents, but these subsequently declined as the protozoa established. Although the fibrolytic bacterial population was lowest in period 3 (> 105 protozoa/g), the in sacco ruminal digestion of Lolium perenne hay and polysaccharolytic enzyme activities in the solids-associated populations were either maintained or increased when protozoa were present confirming the important contribution of protozoa to fibre breakdown in the rumen. Significant changes in ruminal microbial activities occurred after protozoal reinoculation but before the rumen had refaunated completely. Arylamidase activities in the liquor-phase population and ruminal ammonia concentrations increased significantly within 48 h of transfaunation; the magnitude of the effects became more pronounced as the protozoal population developed. However, volatile fatty acid formation and ruminal pH were not affected after the reintroduction of protozoa.Key words: rumen, sheep, ciliate protozoa.

The present results are the last part of a series of experiments conducted in this laboratory on metabolism of major individual rumen ciliate protozoa species and their combinations in sheep. These species were inoculated into the rumen of fauna-free (FF) sheep to study digestibility of dietary fibre and duodenal flow of N. Sixteen rumen- and duodenum-cannulated FF sheep were divided into four equal groups and used in the present experiment. One group remained FF, while the other three were each inoculated intraruminally with the Holotrich protozoa sp. (HT), Entodinium sp. (EN) or total mixed fauna (TF). In comparison with the FF group, the HT did not affect (P.0·05) the duodenal flow of non-NH3-N and bacterial-N, while the flow was decreased (P,0·05) by both the EN and TF populations. The digestibility of fibre was increased (P,0·05) by both the EN and TF populations, but the results for the HT group were mostly similar to those for the FF sheep. The fibre digestibility in the stomach for TF was intermediate between that for EN and FF. It was concluded that except when fed high-carbohydrate diets, the presence in the rumen of the HT protozoa are of no significant value or major detriment to ruminant production. Holotrich protozoa: Entodinium: Rumen ciliate protozoa: Sheep It is well established that the presence of mixed protozoa species populations in the rumen have a negative effect on the utilisation of dietary N by ruminants (1) . In comparison with defaunated (all existing protozoa eliminated from the rumen) or fauna-free (FF; protozoa never existed in the rumen) ruminants a number of experiments have shown a decreased flow of non-NH3-N (NAN) components from the stomach to the intestinal tract when mixed protozoa populations were present in the rumen (2‐5) . However, the species of the mixed population that contribute negatively to N utilisation and to what extent they do was not known. Although a number of studies involved individual species of protozoa such as Entodinium (EN) and Isotricha (IS) (6‐9) , the effect of these species on the duodenal flow of NAN components has not been measured. Therefore, we conducted a series of experiments in this laboratory on N metabolism by individual or combinations of ciliate protozoa species (IS, Dasytricha (DS), Polyplastron (PP), Epidinium (EP), Eudiplodinium (EU), EN and total mixed fauna (TF)). These species were established in the rumen of naturally FF sheep which originated from a sheep flock free of rumen ciliate protozoa for several generations (10) . The present work concludes the

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.

The aim of this work was to determine whether reductive acetogenesis can provide an alternative to methanogenesis in the rumen. Gnotobiotic lambs were inoculated with a functional rumen microbiota lacking methanogens and reared to maturity on a fibrous diet. Lambs with a methanogen-free rumen grew well, and the feed intake and ruminal volatile fatty acid concentrations for lambs lacking ruminal methanogens were lower but not markedly dissimilar from those for conventional lambs reared on the same diet. A high population density (10 7 to 10 8 cells g −1 ) of ruminal acetogens slowly developed in methanogen-free lambs. Sulfate- and fumarate-reducing bacteria were present, but their population densities were highly variable. In methanogen-free lambs, the hydrogen capture from fermentation was low (28 to 46%) in comparison with that in lambs containing ruminal methanogens (>90%). Reductive acetogenesis was not a significant part of ruminal fermentation in conventional lambs but contributed 21 to 25% to the fermentation in methanogen-free meroxenic animals. Ruminal H 2 utilization was lower in lambs lacking ruminal methanogens, but when a methanogen-free lamb was inoculated with a methanogen, the ruminal H 2 utilization was similar to that in conventional lambs. H 2 utilization in lambs containing a normal ruminal microflora was age dependent and increased with the animal age. The animal age effect was less marked in lambs lacking ruminal methanogens. Addition of fumarate to rumen contents from methanogen-free lambs increased H 2 utilization. These findings provide the first evidence from animal studies that reductive acetogens can sustain a functional rumen and replace methanogens as a sink for H 2 in the rumen.

Most previously reported generation times for rumen ciliate protozoa are longer than would be required to prevent their being flushed out of the rumen. In an earlier study from this lab, using a sequential transfer procedure, generation times between 12 and 13 h were determined for both Epidinium caudatum and Entodinium caudatum. This would permit these species to be maintained in a rumen with a fluid volume turnover rate as rapid as twice a day. In this study, generation times were estimated for Entodinium exiguum (13.2 h), Eudiplodinium maggii (26.8 h), and Ophryoscolex purkynjei (29 h), by sequential transfer at both 12 and 24 h time periods. The generation time for E. exiguum is lower than reported for this and other Entodinium species as determined by logarithmic growth from a small inoculum, but similar to that obtained for Ent. caudatum using sequential transfer. Eudiplodinium maggii and O. purkynjei generation times are similar to previous estimates of 24- and 24-48 h, respectively. However, it was observed that after an adaptation period of 36 to 48 h (generally 3-4 transfers) cell concentrations decreased and generation times were markedly decreased, i.e. 12.2 h for Ent. exiguum, 15.0 h for E. maggii and 12.8 h for O. purkynjei. In a separate study, varying both the concentration of Epidinium and the quantity of substrate fed per cell had no effect on generation time.

The composition of ciliates obtained from the forestomachs of eleven dromedary (one-humped) camels in Egypt was examined. As a result, eight genera containing 24 species with 11 forms were identified. Of them, one species was concluded to be new, then described as Dasytricha kabanii n. sp. This new species was clearly distinguished from D. ruminantium, the other species of the genus, by its lack of somatic cilia on the posterior one-fifth of the body surface. Entodinium nanellum and Epidinium ecaudatum f. caudatum were found in all camels examined. Although the percentage composition of respective species varied with the individual camel, the rate of Entodinium spp. was high in general. Total ciliate density in forestomach fluid was 1.9 x 10(5)/ml on average. Ciliate composition in Egyptian camels was similar to that in Bactrian camels, Camelus bactrianus, in China reported previously. However, more Entodinium species were detected from Egyptian camels than from Bactrian camels.

The composition of rumen ciliate protozoa was surveyed in 15 Tokara native goats, Capra hircus domesticus, inhibiting Kagoshima, Japan. As a result, of 11 species with 3 forms under 5 genera detected, Entodinium simplex, E. nanellum and E. rectangulatum were found in all the animals examined. The large ophryoscolecid ciliates detected were Polyplastron multivesiculatum and Metadinium affine alone, which have been regarded as the typical members of Type A composition proposed by Eadie. Since Tokara native goats have been bred isolated in a small islet for a long time, native goats that had been kept in south Japan might possess Type A fauna. Average ciliate density was 43.9 x 10(4) per 1 ml of rumen fluid. The number of species per host was 8.3 on average. Diversity index was 1.412 on average.

The composition of ciliate protozoa in the rumen of 30 Japanese beef black cattle (Bos taurus taurus) fed in Yoron Island, Kagoshima, was compared with that of 125 Holstein-Friesian cattle (Bos taurus taurus) fed in Hokkaido. Thirteen genera including 44 species with 32 formae detected from Japanese beef black cattle and 15 genera including 48 species with 25 formae from Holstein-Friesian cattle were identified. Thirty-six species with 19 formae were common to both hosts. A new species was recognized from Japanese beef black cattle and named Entodinium yunnense n. sp. This new species was classified into such four formae as yunnense n.f., spinonucleatum n.f., acutonucleatum n.f. and spinolobum n.f. A new forma of Entodinium bifidum having only one caudal spine was also recognized from Japanese beef black cattle and named monospinosum n.f. The average ciliate density in Japanese beef black cattle, 18.7 x 10(4)/ml, was lower than 40.3 x 10(4)/ml in Holstein-Friesian cattle. The average number of ciliate species per head in Japanese beef black cattle, 14.4 was also lower than 17.8 in Holstein-Friesian cattle. The average value of diversity index was 1.789 in Japanese beef black cattle and 1.718 in Holstein-Friesian cattle. Both incidence and composition ratio of Entodinium rectangulatum and E. ovinum were significantly higher in Japanese beef black cattle than in Holstein-Friesian cattle. On the other hand, Charonina ventriculi, E. caudatum, E. rostratum, E. parvum, E. bursa, Metadinium affine and Ostracodinium mammosum showed a reverse relation.

Ciliate composition in the forestomach was examined in four Bactrian camels, Camelus bactrianus, inhabiting Mongol and Steppes, then 7 genera containing 14 species with 5 formae were identified. One of these formae was a new forma, and described as Entodinium okoppensis f. cameli n.f. Six species containing one new forma were new records in the forestomach of camels. The percentage composition of genera varied with the individuals of host and the collection sites in the stomach but Entodinium was generally predominant. Total ciliate density per milliliter of stomach fluid was 2.1 x 10(5) on average.

1. 1. The rate of passage of chromic oxide (Cr) was similar in the pony and rhinoceros. 2. 2. Higher apparent amounts digested were found using 4N-HCl-insoluble ash (AIA) than those determined by Cr. but overall digestibilities were similar for the two species. 3. 3. An abrupt increase in the starch content of the horse diets increased the number of faecal ciliate protozoa. Only when the overall feed intake was increased in horses receiving a high dietary proportion of starch were the numbers depressed. 4. 4. When the rhinoceros received 109 kJ apparent DE per kg bodyweight daily (716 kJ/W0.75 dailyl it maintained normal condition.

During anaerobic fermentation of the filtrate juice of swine feces, mastigophoran and sarcodine protozoa were observed, while no ciliate protozoa appeared. Monocercomonas spp. and Amphimonas spp. of mastigophoran protozoa as well as small amoebae of sarcodine protozoa were predominant.

SYNOPSIS. Diplodinium flabellum, previously described on the basis of its occurrence in rumen contents from 3 hosts of Bos indicus, has been observed in domestic sheep. Numbers of this species were quite high, constituting up to 18.8% of the total fauna in one instance. Diurnal changes in numbers for sheep fed once daily were similar to the cycles reported for other Ophryoscolecids. No obvious relationship was found between the occurrence and numbers of D. flabellum and other genera of rumen ciliates. Two new morphologically distinct forms of this species were observed, one without any spines on the posterior left side and one with a single spine. This is in contrast to the original species description in which 2 spines occur on the posterior left side. Intermediate forms were observed which connected the 3 morphological types, indicating their conspecific relationship. It is proposed that the species description for Diplodinium flabellum be emended to include a total of 3 subspecies: Diplodinium flabellum Kofoid and MacLennan, 1932 flabellum subsp. n.; Diplodinium flabellum aspinatum subsp. n.; and Diplodinium flabellum monospinatum subsp. n. Considerable variation in the shape and length of the caudal fan was observed in specimens of the subspecies aspinatum. In three of the four sheep studied, the subspecies aspinatum predominated, while the species monospinatum was highest in the fourth animal. D. flabellum flabellum occurred in very low numbers, ranging from 0 to 2.5% of the total species number.

The rumen contents of 70 lactating Holstein cows fed five different rations were examined for ciliate protozoa. Species of eight genera of the family Ophryoscolecidae and two genera of the family Isotrichidae were observed, with Entodinium, Epidinium, Polyplastron, Ophryoscolex , and Isotricha predominating. Isotricha appeared frequently in the rumens of cows fed a ration containing cane molasses, whereas Polyplastron, Epidinium , and Ophryoscolex were frequently present in cows fed a ration containing hemicellulose extract. There were no differences in the frequency of appearance of most ciliate protozoa in the rumens of cows fed different quantities of calcium and phosphorus. Entodinium caudatum appeared frequently in the rumens of cows fed rations containing large quantities of grain starch. Polyplastron multivesiculatum or Ophryoscolex caudatus or both were not seen in cows containing Epidinium caudatum or Eudiplodinium maggii . However, O. caudatus was seen together with Epidinium tricaudatum. P. multivesiculatum was not seen together with Metadinium medium .

SUMMARY: The limitations of methods previously used for counting rumen ciliates, and the value of developing a method of known accuracy, are discussed. Two counting-chambers, one based on the Sedgwick-Rafter cell, the other on the MacMaster eelworm-counting cell, were constructed and tested. A MacMaster-type cell was adopted. The preparations of suitable suspensions for counting organisms from washed suspensions and from rumen contents are described. The counting technique adopted was found to be reliable; its accuracy depends upon the number of organisms counted. In one sheep on a constant diet, day-to-day differences in rumen ciliate population were far greater than the differences found throughout the rumen at any one time, or the variations due to technique.

SUMMARY: The times taken by Ophryoscolex tricoronatus and species of the genus Epidinium to establish in the rumens of young animals were found to differ from those of other rumen ciliate species. The antagonistic relationships between certain rumen ciliates were examined in vivo and concurrent experiments were made in small vessels termed vivars which were suspended within the rumen of a sheep. A Millipore membrane separated the rumen contents from the contents of the vivars. Within these vessels it was possible to reproduce population changes first observed in vivo and to determine with certainty that in a population change where Polyplastron multivesiculatum became irreversibly dominant this had been caused by predation by Polyplastron. This organism will eliminate Epidinium, Eudiplodinium maggii, Eremo-plastron and Ostracodinium from a population. The cause, factors affecting it and the ultimate effect on the population are discussed. It was not possible to cause a reversal of the population within a vivar suspended in sheep, but since the prey of Polyplastron still existed and since the reverse change was seen to occur in cattle, this problem still remains unsolved. There was also some evidence of antagonism between Epidinium and Ophryoscolex.

1. The dynamics of large ciliate (holotrich) protozoa (Isotricha and Dasytricha spp.) in the rumen of cattle given cut, fresh ryegrass (Lolium multiflorium Lam) were studied by means of a single intrarumen injection of14C-labelled protozoa prepared in vitro by adding [Me14C]choline to rurnen fluid containing protozoa and incubating at 39° for 2 h.2. An indication of the lysis rate of protozoa in the rumen was obtained from the radioactivity apparently lost through the methane pool.3. The turnover time of the holotrich protozoa indicates that these protozoa were extensively retained in the rumen and that only a small proportion of those produced in the rumen flowed out in the digesta. This was supported by the estimation of the rate of lysis which was approximately 85% of the turnover rate in the rurnen.4. The apparent production rate of the larger protozoa indicates that they contribute only about 9% of the predicted net microbial protein synthesis in the rumen.

In a first experiment 4 rams consumed over a period of 70 days 0; 82; 208 and 345 mg of monensin daily, respectively. All doses of monensin caused a marked decrease in total numbers of rumen protozoa in samples taken after this period. More than 90% of total protozoal numbers belonged to the genus Entodinium. The sensitivity of the genus Entodinium to monensin was found to be lower than that of genera Dasytricha and Isotricha. In the second experiment 24 lambs were fed hay and concentrates in the ratio 60:40% (groups 1 and 2) and 40:60% (groups 3 and 4). Groups 2 and 4 received 40 mg monensin per animal daily during 16 weeks. The decrease in protozoal numbers due to monensin in samples taken after this period was significant (P less than 0.001) in lambs fed the concentrate diet (groups 3 and 4) and also in lambs from groups 1 and 2 fed the roughage diet (P less than 0.05). The statistical evaluation of the inhibition of total protozoal numbers by monensin (%) in lambs fed both diets has shown that the antiprotozoal effect of monensin was significantly more intensive with the concentrate diet (P less than 0.025) than with the roughage diet.

Scanning electron, fluorescent and light microscopy of rumen ciliate protozoaof sheep revealed that spherical bacteria were attached to the surface of ciliates of variousspecies. These bacteria were cocci forming units, pairs or chains of varying length. Mostof them were identified as Streptococcus bovis and Ruminococcus albus by means of lm-munofluorescent miaoscopy. No rods were observed. Of the ciliates, Eudiplodiniummaggii had the largest number of bacteria attached to the settled dimensions of thesurface, and Epidinium ecaudatum the smallest number. In Hudiplodinium maggii andDiplopZastron affine, the attachement of bacteria was observed especially around the cyto-stome. It was considered that about 250 bacteria on the average might be attached to thesurface of one ciliate. The cause and the significance of the attachment of bacteria werediscussed.

Summary: Azocasein-degrading proteinase activity was detected in all rumen ciliate protozoa that were examined from four entodiniomorphid and two holotrich genera. All of the activities were optimal in the range pH 4.0-5.0 and were inhibited by cysteine proteinase inhibitors, notably leupeptin. The inhibition profiles and extent of inhibition observed with the different groups of inhibitors were organism-specific. Gelatin-SDS-polyacrylamide gel electrophoresis of protozoal lysates revealed multiple forms of the proteinases in the species examined. The number of enzymes detected, their molecular masses, the level of activity and inhibitor susceptibility was genus-dependent. The proteinase profiles of the two holotrich species differed and inter-species differences were also apparent among species of the genus Entodinium. The characteristics and molecular size distribution of rumen bacterial proteinases were different to the protozoal proteinases. Low levels of proteinase activity, of apparently bacterial origin, were detected by gelatin-SDS-PAGE analysis of cell-free rumen liquor.

The concentration of 2-aminoethylphosphonic acid has been measured in seven genera of rumen ciliate protozoa. Expressed as milligrams per gram of total nitrogen, 2-aminoethylphosphonic acid concentrations ranged from 17.2 in Ophryoscolex spp. to 72.4 in Eremoplastron spp.

1. Rams, fauna-free from birth and initially of 48–65 kg live weight, were allocated to two groups of ten each and given a diet containing 14 μg copper/g dry matter; five additional rams were killed and their livers were analysed for Cu.2. One group (faunated) was inoculated with a mixed population of ciliate protozoa, and contained between 60x105and 195 x 105protozoa/ml rumen fluid throughout the 184 d experiment. The other group remained fauna-free. Following blood sampling, three rams in each group were killed on day 63, two on day 125 and four on day 184. One sheep in each group died during the experiment.3. Faunated rams showed higher weight gains and feed consumption than fauna-free rams.4. Plasma Cu concentration (μg/ml) increased from an initial 0.82to a final 1.00 in faunated and 1.36 in fauna-free rams. Liver Cu concentration (μg/g dry matter) increased from an initial 745 to a final 962 and 1684 in faunated and in fauna-free sheep respectively, representing a 4.3-fold greater increase in the fauna-free than in the faunated group. The absorption and retention of Cu was 38–50% higher in the fauna-free than in the faunated rams.5. It was suggested that rumen ciliate protozoa increased rumen production of sulphide (through increased breakdown of soluble proteins) which complexed part of the Cu, making it unavailable for absorption and utilization. Therefore, ciliate protozoa could determine susceptibility to chronic Cu toxicity in sheep.

The establishment of ciliate protozoa in the rumen was studied in conventional lambs reared under different conditions of management. The role of the microflora in the kinetics of this establishment was also investigated in conventionalized lambs. In lambs reared under farm conditions ciliate protozoa appeared in the following order: Entodinium (15-20 days), Polyplastron, Eudiplodinium, and Epidinium (20–25 days), and Isotricha (50 days). Entodinium was the most abundant (105–106 ciliates mL−1). During the 3rd month, ciliates disappeared spontaneously in about 60% of the lambs during a period that varied from 1 to 4 weeks. In lambs fed only cow's milk Entodinium spp. and Polyplastron multivesiculatum became established at low levels. The results obtained with the conventionalized lambs demonstrate that the establishment of the ciliates in the rumen requires that the bacterial flora be well established beforehand.

SYNOPSIS. Ciliate protozoa from the rumen of the tsessebe, Damaliscus lunatus lunatus, all belong to the family Ophryoscolescidae and no holotrichs were found in any of the 16 animals. Eighteen species were identified of which Entodinium fyferi sp. n., Enoploplastron garstangi sp. n. and Epidinium lunatus sp. n. are new and have been described for the first time. A short redescription of Eremoplastron gigantium Kofoid & MacLennan is given.

The composition of rumen ciliate protozoa from the Japanese serow was investigated. The average number of total ciliates per milliliter of rumen contents was 1.4×105. Eleven species, one of trichostomatid and ten of entodiniomorphid ciliates, were recognized. Of them, ten species had already been described in the domestic ruminants in Japan, but one was detected specifically from the Japanese serow. This species was described as the new form, Epidinium ecaudatum forma capricornisi forma nov. This form had bifurcated and trifurcated caudal spines. Species or formae with trifurcated spines had been described not in the genus Epidinium but in the genus Ophryoscolex. The fact may be one of the characters showing the phylogenetical relationship between these two geneta.

A NUMBER of workers have compared the general performance of domestic ruminants with and without ciliate protozoa. In no case was any striking difference observed. Hungate1 has suggested that bacteria may take over the activities of the normal ciliate protozoan population, and also Bryant and Small2 observed a distinct drop in the count of cellulolytic bacteria in two unfaunated calves after they had been inoculated with whole rumen contents containing protozoa. Biochemical work also suggests that the bacteria and ciliate protozoa digest similar substrates.

Thirty-eight female water buffalo calves were divided into four groups. In Group 1 (control) each calf received 340kg of milk in 120 days. In Groups 2, 3, and 4 each calf was fed on 103kg of milk in 45 days, plus a high quality ration which was gradually replaced by a low quality ration after 45 days, 75 days, and 120 days, respectively. Half of the calves in each group were inoculated with the rumen contents obtained from a fistulated adult buffalo. The establishment of rumen ciliate protozoa, microbial activity, and growth of inoculated calves were compared with uninoculated animals in the four groups. No active protozoa were established in half of the uninoculated calves before four months of age; Entodinium appeared in the other half at about two months of age. On the other hand, a thriving mixed population of protozoa developed within six days of inoculation. There was a tendency for higher VFA and ammonia-N concentrations in the rumen of inoculated calves. There was no difference between inoculated and uninoculated calves in blood urea and blood reducing sugars. The inoculated calves showed faster growth rates than the uninoculated calves. The effect was particularly obvious in groups where the good quality ration was replaced with a low quality one after 45 and 75 days. It was less pronounced for calves given the high quality ration for 120 days.

Establishment of ciliate protozoa in the rumens of cow and buffalo calves kept under early and late weaning systems was studied. The results showed that the rumens of the buffalo calves harbored active populations of ciliate protozoa at earlier ages than those of cow calves, probably indicating earlier rumen development in buffalo calves. Early weaning promoted faster establishment of ciliate protozoa than late weaning, especially in cow calves. The effect was not so clear in buffalo calves. Entodinium spp. were first to appear in the rumen. Eudiplodinium was established as soon as Isotricha in the early-weaned cow calves, while large oligotrichs appeared at an earlier date in buffaloes. Cross-inoculations among buffaloes, cows, and sheep showed that Ophryoscolex and Polyplastron from sheep failed to survive in adult or young cows and buffaloes, thus demonstrating host specificity. Epidinium was not observed in the rumen contents of sheep, cows, or buffaloes maintained under Egyptian conditions.

1. A method is described for separating and enumerating the ciliate Protozoa which occur in the large intestine of the horse.2. The concentration of ciliates varied in the different regions of the large intestine, being low in the caecum and highest in the left dorsal colon.3. Two distinct populations of ciliates occurred in the large intestine of the horse, one from the caecum to the pelvic flexure and the other from the pelvic flexure to the rectum.

DURING a recent investigation at the University Botany School, Cambridge, of the association of ciliate protozoa and bacteria in a chalk stream, Styl nichia mytilus in common with other ciliate protozoa exhibited a series of ‘pulses’, or maximum waves when it spread down the stream, and minimum phases when it could be found only in the upper reaches. Forms in binary fission or conjugation were sometimes noted at the end of the maximum phase, whereas the minimum phase was generally marked by the appearance of small forms. The pulse of Stylonichia could not be related to any constant climatic or physical factor (rainfall, pH, temperature, etc.) or to total bacterial numbers at 22° C. or any particular group of bacteria. However, the maximum phase often occurred about a week to ten days after one of the lunar periods. From May to December 1947, the maximum phases occurred on twelve out of thirteen occasions a week to ten days after a new moon or a full moon: the greatest maximum peak was attained nine days after a new moon in July and four days after a full moon in December. By contrast with the dry weather and clear nights of 1947, the year 1948 was wet and dull. Stylonichia was only recorded between May and September, yet on five occasions it reached its maximum phase on an average eight days after a new or a full moon.

A new temporary stain for the demonstration of nuclear and cytoplasmic structures in Paramecium and other protozoan ciliates during both vegetative reproduction and meiotic reorganization consists of a mixture of 10.5 parts of acetocarmine, 4.5 parts of 45% acetic acid, 2 parts of 1 N HCl and 1 part of 1% solution of fast green FCF in 95% alcohol. This stain replaces the acetocarmine and acidified methyl green nuclear stains commonly employed and has the following advantages: (1) it affords simultaneous differential stainability of nucleus and cytoplasm (brown-red and green to grey-green, respectively); (2) it provides differential stainability of newly developing macronuclei (homogeneous pale green), fragments of the old macronucleus (brown-red), and food vacuoles (granular, bright blue-green); and (3) it results in a delicate and more transparent stain which affords greater clarity of internal structure. Proportions may be shifted slightly to achieve the optimum results for any particular organism. Conc...

THE extensive biochemical investigations recently carried out with pure cultures of the free-living, aerobic ciliate Tetrahymena geleii (see Kidder1 for summary) raise the question whether similar studies are possible with the obligatory anaerobic ciliates of the sheep's rumen. So far we have not been able to induce these Protozoa consistently to multiply in vitro, although some success has attended our efforts at keeping them alive for long periods in the presence of rumen bacteria2. One of us, however, observed3 that the holotrich ciliates in sheep's rumen liquor, if allowed to attack glucose, rapidly became so dense through polysaccharide storage that they formed a separate white layer at the bottom of the vessel.

Hogg and Elliott1 noted that indole and skatole were both highly toxic towards the aerobic free-living ciliate Tetrahymena pyriformis. A concentration of 0.01 per cent of indole, for example, killed all organisms in a vigorous culture in two hours, but these workers made no mention of any characteristic change in appearance of the cells. We have now shown that these substances are highly toxic to the anaerobic ciliates of the sheep's rumen, particularly the holotrichs (Isotricha and Dasytricha) and also Ophryoscolex. These ciliates had been separated from strained rumen contents, washed and maintained alive and active for several hours in a phosphate buffer (without antibiotic) at pH 7.2 as described by Heald, Sugden and Oxford2. On a molecular basis skatole was much more toxic than indole to the rumen ciliates; but if the comparison was made at concentrations near saturation in the buffer employed, indole being the more soluble compound, the respective effects upon the final appearance of the cells were almost equally spectacular. Briefly, not only did all ciliary motion cease in two hours at 37°, but also a phenomenon resembling a drastic mechanical disintegration took place. The outer pellicles of the holotrichs, and also of Ophryoscolex, were torn asunder with liberation of the internal contents of the cells, so that after gently shaking the tube, instead of the rapid re-formation of a layer of ciliates at the bottom below a clear supernatant, there was a dense uniform turbidity throughout due to the release of storage polysaccharide granules and other inclusions from the disrupted cells.

IN the last decade the study of the ciliate protozoa of the rumen has emphasized the investigation of individual species, rather than the ciliate population as a whole.