Simple Method to Remove Completely Ciliate Protozoa of Adult Ruminants

Dioctyl sodium sulfosuccinate (Aerosol OT) has been used in a remarkably short period to obtain rumen ciliate-free cows. Two 30-g doses of Aerosol OT given on consecutive days appeared to effectively eliminate all types of rumen ciliate protozoa without harming the health of the host animal. Measurement of the rumen metabolic parameters of gas production, carbon dioxide to methane ratio, pH value, volatile fatty acids, ammonia, and in vitro cellulose digestion, along with total amylolytic streptococci counts of the rumen contents, showed that conditions in rumens of defaunated animals are normal 4 days after such treatment. It appears that such animals may be used in ruminal studies that require defaunated animals.     

Effect of Absence of Ciliate Protozoa From the Rumen on Microbial Activity and Growth of Lambs

A survey of the components of the rumen ciliate population in a series of adult sheep, raised in the Faculty of Agriculture, University of Alexandria, has shown that a mixture of Entodinium, Isotricha, Ophryoscolex, Diplodinium, and Polyplastron species was found in the rumen contents of Egyptian sheep; no Epidinium and a negligible number of Dasytricha ruminantium were also observed. The microbial population, reducing sugars, ammonia, volatile fatty acids (VFA) production, and growth rate of 14 lambs inoculated with whole rumen contents from a mature sheep were compared over a 6-month period with those of 13 lambs maintained under the same conditions, except that they were strictly isolated from other ruminants. Certain large oval organisms and large numbers of flagellates and Oscillospira were frequently observed in the rumen contents of the isolated lambs. The reducing sugars, ammonia, and VFA levels, measured before and at intervals after feeding, in the inoculated lambs showed a pronounced rise above the values found in the ciliate-free animals. The propionic acid-acetic acid ratio in the rumen contents of the faunated lambs was considerably higher than in the nonfaunated controls. The inoculated lambs grew faster than the isolated lambs. Differences in weight gain which ranged from 15 to 17% were statistically significant. The inoculated animals impressed the observers by their good appearance which was superior to that of the ciliate-free lambs. It was, therefore, concluded that the rumen ciliate protozoa are essential for the metabolism and growth of young lambs.     

Conversion of cereal residues into biogas in a rumen-derived process

A recently developed high-rate, two-phase process, which employs rumen microorganisms for efficient acidogenesis, was tested for anaerobic degradation of barley straw, rye straw, and maize stover. Under conditions similar to those of the rumen and loading rates varying between 9.8 and 26.0 g of organic matter/I/day in the first phase (acidogenic reactor), total fibre degradation efficiencies ranged between 42% and 57%, irrespective of the loading rate applied. Average specific production of volatile fatty acids and biogas/g volatile solid digested in the acidogenic reactor varied between 6.9 and 11.2 mmol and 0.10 and 0.25 l, respectively.The effect of varying solid retention times on the extent of degradation of barley straw was examined. Changing of retention times in the range of 60 to 156 h had no effect on degradation efficiency, but a decrease in efficiency was observed at retention times below 60 h.By connecting the acidogenic reactor in series to an Upflow Anaerobic Sludge Blanket (UASB) methanogenic reactor the volatile fatty acids were converted into biogas. Average methane contents of the gases produced in the acidogenic reactor and in the UASB reactor were 30±3% and 78±3%, respectively.     

Maintenance of the Rumen Microbial Population in Continuous Culture

The design and use of an apparatus for continuous in vitro cultivation of the mixed bovine rumen microbial population are described. Data relative to the concentrations and proportions of volatile fatty acids produced, methanogenesis, carbon dioxide production, and survival of bacteria and protozoa indicated that an essentially normal in vivo fermentation pattern was maintained in vitro for experimental periods of from 3 to 10 days. The continuous cultures were responsive to major changes in type of feed intake. A change from grain to hay resulted in increased acetate and decreased propionate production, whereas decreased acetate and increased propionate resulted when feed intake was changed from hay to grain. Methanogenesis, volatile fatty acid production, and the numbers of microorganisms in the cultures were proportional to the amount of feed materials added up to levels calculated to approximate the in vivo maintenance requirement of the adult bovine.     

Survival of the recombinant Bacteroides thetaiotaomicron strain BTX in in vitro rumen incubations

The survival of Bacteroides thetaiotaomicron strain BTX under rumen-simulating conditions was studied. Strain BTX is a recombinant variant of strain 5482 engineered for the production of high levels of xylanase, an enzyme important in the degradation of hemicellulose. Strain BTX was not inhibited by compounds present in rumen fluid and it grew well in media containing rumen fluid (up to 75%) or high concentrations of volatile fatty acids (total concentration, 100 mmol l⁻¹). The ability of strain BTX to compete with other micro-organisms under rumen-like conditions was studied in in vitro incubations of rumen contents. These experiments employed a consecutive batch culture (CBC) system consisting of alfalfa suspended in a rumen fluid buffer inoculated with blended rumen contents and maintained by transfers (10%, v/v) at 48 h intervals. CBC cultures contained a diversity of microbial morphotypes and accumulated fermentation products in rumen-like proportions. When added alone, the numbers of BTX cells were maintained for only a few hours, and then declined precipitously until undetectable after 48 h. If CBC cultures were also supplemented with chondroitin sulphate (a mucopolysaccharide used by Bact. thetaiotaomicron ), strain BTX grew and the pattern of its population generally followed that of the total population of ruminal bacteria in these cultures. When transferred into fresh CBC cultures containing chondroitin sulphate, BTX was again able to grow and increase in numbers, but to a diminished degree. Although BTX was able to survive and maintain itself in chondroitin sulphate supplemented cultures, this was at a very low level (10¹⁰ ml⁻¹). The potential for manipulation of rumen function by inoculation with recombinant bacteria is discussed.     

Ruminal methanogenesis as influenced by individual fatty acids supplemented to complete ruminant diets

The objective of the present study was to investigate the effects of seven different pure fatty acids on rumen fermentation using the rumen simulation technique (RUSITEC). The fatty acids were supplied to a complete ruminant diet at a proportion of 50 g x kg(-1) dietary dry matter and compared with an unsupplemented control. Methane release and methanogenic counts were suppressed by the fatty acids C12 : 0, C14 : 0 and C18 : 2 whereas C8 : 0, C10 : 0, C16 : 0 and C18 : 0 showed no corresponding effects. Apart from C12 : 0 and C18 : 2, C8 : 0 and C10 : 0 also adversely affected ciliate protozoa suggesting independence from the methane-suppressing effect of medium-chain fatty acids (MCFA). Although MCFA but not C18 : 2 reduced ruminal fibre degradation, the influence on other fermentation traits remained low. In conclusion, the supply of certain fatty acids to ruminant diets seems to have the potential to reduce methane release.     

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 protozoa in vitro, on their co-culture bacterial population, and volatile fatty acid concentration. Nisaplin did not affect the in vitro growth of Entodinium 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 the Epidinium ecaudatum forma caudatum et ecaudatum but not growth of E. caudatum. Nisaplin moderately supported the growth of E. 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 Nisaplin 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.     

Effect of glyphosate contaminated feed on rumen fermentation parameters and in sacco degradation of grass hay and corn grain

Four rumen fistulated wethers were used to investigate the effect of glyphosate contaminated feed on rumen fermentation. The rations were based on corn silage, urea and a vitamin-mineral premix, either in the absence or presence of 0.77?g glyphosate per kg DM. Furthermore, rations were fed either with or without aromatic amino acid supplementation. During four periods of 28 days, sheep received each of the four dietary treatments according to a Latin square. After 14 days of adaptation rumen fermentation parameters (pH, ammonia, volatile fatty acids) were measured on day 15 over a five-hour period after the morning feeding. The remaining 13 days served for in sacco degradation studies with grass hay and corn grain. Ammonia (NH₃) and pH of rumen fluid were within the normal range for all dietary treatments (NH₃: 9.1 – 32.3?mmol·l^???1, pH: 6.2 – 6.7). Neither rumen fermentation parameters nor in sacco DM and NDF degradation of incubated feedstuffs were significantly affected by glyphosate, with or without aromatic amino acid supplementation. Kinetic profiles of the in sacco dry matter and NDF degradation of grass hay were almost identical for the dietary treatments.     

Effect of glyphosate contaminated feed on rumen fermentation parameters and in sacco degradation of grass hay and corn grain

Abstract Four rumen fistulated wethers were used to investigate the effect of glyphosate contaminated feed on rumen fermentation. The rations were based on corn silage, urea and a vitamin-mineral premix, either in the absence or presence of 0.77 g glyphosate per kg DM. Furthermore, rations were fed either with or without aromatic amino acid supplementation. During four periods of 28 days, sheep received each of the four dietary treatments according to a Latin square. After 14 days of adaptation rumen fermentation parameters (pH, ammonia, volatile fatty acids) were measured on day 15 over a five-hour period after the morning feeding. The remaining 13 days served for in sacco degradation studies with grass hay and corn grain. Ammonia (NH3) and pH of rumen fluid were within the normal range for all dietary treatments (NH3: 9.1-32.3 mmol x l(- l), pH: 6.2-6.7). Neither rumen fermentation parameters nor in sacco DM and NDF degradation of incubated feedstuffs were significantly affected by glyphosate, with or without aromatic amino acid supplementation. Kinetic profiles of the in sacco dry matter and NDF degradation of grass hay were almost identical for the dietary treatments.     

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.     

Long-term cultivation of certain rumen protozoa in a continuous fermentation system supplemented with sponge materials

A be , M. & K urihara , Y. 1984. Long-term cultivation of certain rumen protozoa in a continuous fermentation system supplemented with sponge materials. Journal of Applied Bacteriology 56 , 201–213.
Inert polyurethane-sponge cubes were added to an 'artificial rumen' fermenter. With crushed barley as protozoal substrate this system maintained a population of Ento-dinia in both substrate bags and sponge cubes for 64 d in numbers (1 times 10⁶/ml) comparable with those found in vivo . This culture was more tolerant of variations in mechanical agitation and size and shape of substrate bags than a similar culture without the sponge cubes. The extent of protozoal sequestration, 2 h after feeding, on to the sponge cubes as well as on to acetone-treated hay was evaluated. The bags of acetone-treated hay and barley, and those of sponge cubes and barley could sequestrate the protozoa to the same extent as normal hay and barley. Based on the results of the short-term experiments and those of the long-term experiment with sponge cubes, a polyurethane-sponge wall was added to the inside of the fermenter. This system maintained a varied protozoal population including large protozoa, holotrichs and Entodinia with a normal fermentation. The molar percentages of butyrate in the total volatile fatty acid (VFA) in each part of the fermenter with a sponge wall were higher than those in a fermenter without a sponge wall, suggesting that there was high protozoal activity related to the sponge wall. These results show that the introduction of the sponge wall to the fermenter produced an enlargement of the area for protozoal sequestration and a suitable micro-habitat for the maintenance of Entodinia, large protozoa and holotrichs.     

Lipid Metabolism of Rumen Ciliates and Bacteria: II. Uptake of Fatty Acids and Lipid Analysis of Isotrichia intestinalis and Rumen Bacteria with Further Information on Entodinium simplex

The total lipid and free fatty acid contents of Isotricha intestinalis, Entodinium simplex, and the rumen bacterial flora of the respective protozoa were determined. Warburg manometric data showed that the sodium salts of tributyrin, oleic, and acetic acids stimulated gas production in I. intestinalis, whereas tributyrin was stimulatory with E. simplex and less active with oleic and acetic acids. Rumen bacteria provided fatty acids produced lower manometric gaseous increases when compared with the protozoa. Volatile fatty acids were produced by I. intestinalis and rumen bacteria with tributyrin, but not with tripalmitin. Sodium oleate gave little volatile fatty acid response with I. intestinalis or rumen bacteria. Washed suspensions of I. intestinalis and rumen bacteria concentrated C¹⁴-labeled oleic, palmitic, stearic, and linoleic acids within the cells during short incubation periods. Autoradiographs demonstrated the conversion of C¹⁴-labeled oleic, palmitic, stearic, linoleic, and acetic acids in the rumen protozoa and bacterial cells.     

Effect of pH on Population and Fermentation in a Continuously Cultured Rumen Ecosystem

The effect of pH on rumen fermentation and microbial population was studied in a continuously cultured rumen ecosystem. A marked decrease in the production of volatile fatty acids and methane from alfalfa hay occurred when the cultures were maintained at pH values below 6.0. The decrease in acetate and methane production was greater than that of propionate production. The culture maintained at pH 6.7 contained the types of bacteria often found in high concentration in the rumen, whereas the culture maintained at pH 5.0 had a high percentage of bacteria which could not be identified with the major rumen bacteria found in rumens of animals fed alfalfa hay. Replacement of the bicarbonate-phosphate buffer used to maintain fermentor pH at 6.7 with phosphate alone did not greatly alter the fermentation products produced from a hay-concentrate substrate.     

Development of a two-phase combination fermenter for the conversion of cellulose to methane

Cellulose degradation to methane under continuous fermentation conditions was compared using fully mixed, fully mixed with solids return, sludge-blanket, and fixed-film fermenters. In fully mixed fermenters, a decrease in hydraulic retention time (HRT) of two weeks or less caused the wash out of anaerobes capable of converting volatile fatty acids to methane, while at increased feeding rates over 1 g/L day the rapid growth of cellulolytic anaerobes upset the balance between acid formation and its conversion to CH(4). Circulation of cellulose and difficulty in settling of cellulose with attached bacteria imposed problems in the use of other types of fermenters. On the basis of information obtained from this study, a fermenter which combined a fully mixed phase for cellulose degradation and a fixed-film phase with pre-immobilized bacteria for converting fatty acids to CH(4) in one vessel, was devised. Using this fermenter, a mixed culture converted cellulose to CH(4) at 4.8 g/L day at a HRT of six days as compared to 0.7 g/L day at a HRT of 28 days in the fully mixed fermenter.     

Contribution of rumen protozoa to fibre degradation and cellulase activity in vitro

Abstract The contribution of ciliates to rumen fermentation was estimated by determination of overall fibre degradation and cellulase activities (determined as carboxymethylcellulase activity) in faunated and defaunated 'artificial rumen' cultures. Experiments performed at loading rates of 22.5 and 35 g per liter per day of a grass-grain substrate revealed that fibre degradation was significantly lower in the absence of ciliates only at the high loading rate. This effect of defaunation was smaller at dilution rates below 1.7 fermenter volume turnovers per day. Bacterial numbers were higher in all experiments after removal of ciliates. Fractionation studies demonstrated that ciliates accounted for 19–28% of the total cellulase activity in faunated cultures fed on filter paper cellulose.     

Effect of dietary sodium bicarbonate supplementation on fermentation characteristics and ciliate protozoal population in rumen of lambs

Effects of sodium bicarbonate (NaHCO₃) inclusion on rumen fermentation characteristics and ciliate protozoal population in high concentrate-fed lambs were studied. Twenty-four weaner (90 days old) Malpura lambs divided into four equal groups (G1, G2, G3 and G4) were fed basal (25:75 roughage and concentrate) diet (G1) or basal diet supplemented with 0.75% (G2), 1.50% (G3) and 2.25% (G4) sodium bicarbonate for 90 days. Daily dry matter (DM) intake and digestibility of organic matter (OM), crude protein (CP), gross energy and plane of nutrition were similar in all the groups while cellulose digestibility was higher in G4 than in G2 and G1. Ruminal pH increased (P<0.05) with increasing levels of dietary sodium bicarbonate. Concentrations of total volatile fatty acids (TVFA), total nitrogen and trichloroacetic acid-precipitable nitrogen (TCA-ppt.-N) were higher while ammonia nitrogen was lower in the rumen fluid of G4 and G3 than in G2 and G1. The number of total protozoa, Isotricha, Dasytricha, large and small spirotrichs were higher (P<0.01) in the rumen of G4 and G3 than in G2 and G1. Total live weight gain and average daily gain were also higher in lambs supplemented with sodium bicarbonate. It is concluded that sodium bicarbonate inclusion at the rate of 1.50% of total ration increased cellulose digestibility, ciliate protozoal number, ruminal pH and total nitrogen concentration resulting in improved growth of lambs maintained on high concentrate diet.     

A simulation model “CTR Dairy” to predict the supply of nutrients in dairy cows managed under discontinuous feeding patterns

A simulation rumen model has been developed to function under non-steady state conditions in order to allow prediction of nutrient availability in dairy cows managed under discontinuous feeding systems. The model simulates availability of glycogenic, aminogenic and lipogenic nutrients to lactating dairy cows fed discontinuously. The model structure considers input of up to three different feeds fed independently at any time during the day. Feeds are described by their nitrogen (N), carbohydrate and fatty acid fractions. The N containing feed fractions include ruminally undegraded crude protein (CP), ruminally insoluble but potentially degradable CP, ruminally soluble CP and ammonia N. The feed carbohydrate fractions include ruminally undegradable neutral detergent fibre (NDF), ruminally degradable NDF, ruminally insoluble starch, ruminally soluble starch and sugars. The fatty acids in the feeds are divided between long chain fatty acids and volatile fatty acids (VFA). Additionally four pools were defined representing absorption of amino acids, glucose, long chain fatty acids and volatile fatty acids. The rumen microbial population is represented as a single pool. Besides a flexible structure, new features to the extant model include adoption of the concept of chewing efficiency (or chewing effectiveness) during eating, variable fractional ruminal absorption rates of VFA and variable fractional ruminal degradation rates of NDF as a function of rumen liquid pH, as well as a variable rumen volume which directly affects rumen concentrations of metabolites. The model continuously (i.e., by minute) predicts release of soluble components from the feeds in the rumen, concentration and absorption of fermentation end products in the rumen, rumen pools of nutrients and microbial biomass dynamics, as well as passage of microbial biomass and non-fermented nutrients from the rumen, in response to various feeding strategies. Model evaluation covered a wide range of feeding strategies that included pasture and housed feeding systems. Overall, the mean square prediction error (MSPE) as a percentage of the observed mean was relatively low (<10%) with a high amount of the total variation explained by random variation (>65%). Deviation from unity varied between 23% (rumen dry matter content) and 25% (NDF), indicating some consistent over and/or under prediction. A more detailed evaluation was done based on studies available that reported diurnal behaviour of key model outputs such as rumen pools, rumen pH, and rumen VFA. The predictions broadly simulated the observed values quantitatively, relative to general diurnal patterns, and relative to differences between treatments in the predicted diurnal patterns. Results show that the model provides a tool to assess potential outcomes of changing feeding strategies which may be particularly valuable in assessing selection of feeds, amounts and times of the day to offer the feeds. The continuous nature of the simulated output also allows determination of the time(s) of the day that ruminal (and/or post-ruminal) delivery of nutrients may limit ruminal output of nutrients (and/or availability of nutrients) to support milk nutrient synthesis.     

Der Einfluß der Verfütterung von Silage aus frischem bzw. angewelktem Gras oder Heu auf die Futteraufnahme,Verdaulichkeit, Milchleistung und Blutzusammensetzung der Milchkühe

A survey is given of research results on ruminant lipid digestion obtained at the authors’ laboratory. Results are presented in terms of lipid changes occurring in the rumen and in terms of effects on nature, extent and site of digestion.

The rumen can be adapted to an extremely high capacity for triglyceride lipolysis, preferentially releasing polyunsaturated fatty acids that are then further hydrogenated with accumulation of oleic acid isomers in vitro only. Evidence was obtained for both microbial incorporation and synthesis of polyunsaturated acids. In vitro lipolysis is inhibited by pH values below 6.3 and by ionophores. Free fatty acids inhibit methanogenesis with associated increases in propionate production and decreases in acetate and butyrate productions; the latter being related to their defaunating effect. Both in the faunated and defaunated rumen, free fatty acids decrease fibre digestion, which is shifted to the hindgut, at least in sheep. Defaunation increases rumen microbial growth efficiency and may result in a higher duodenal flow of both feed and microbial protein, provided these increases are not overcome by a decreased apparent rumen OM digestibility. Considerable between animal variability exists for these effects, associated with variable effects on rumen particle and liquid volumes and outflow rates.     

Dynamic role of single-celled fungi in ruminal microbial ecology and activities

In ruminants, high fermentation capacity is necessary to develop more efficient ruminant production systems. Greater level of production depends on the ability of the microbial ecosystem to convert organic matter into precursors of milk and meat. This has led to increased interest by animal nutritionists, biochemists and microbiologists in evaluating different strategies to manipulate the rumen biota to improve animal performance, production efficiency and animal health. One of such strategies is the use of natural feed additives such as single-celled fungi yeast. The main objectives of using yeasts as natural additives in ruminant diets include; (i) to prevent rumen microflora disorders, (ii) to improve and sustain higher production of milk and meat, (iii) to reduce rumen acidosis and bloat which adversely affect animal health and performance, (iv) to decrease the risk of ruminant-associated human pathogens and (v) to reduce the excretion of nitrogenous-based compounds, carbon dioxide and methane. Yeast, a natural feed additive, has the potential to enhance feed degradation by increasing the concentration of volatile fatty acids during fermentation processes. In addition, microbial growth in the rumen is enhanced in the presence of yeast leading to the delivery of a greater amount of microbial protein to the duodenum and high nitrogen retention. Single-celled fungi yeast has demonstrated its ability to increase fibre digestibility and lower faecal output of organic matter due to improved digestion of organic matter, which subsequently improves animal productivity. Yeast also has the ability to alter the fermentation process in the rumen in a way that reduces methane formation. Furthermore, yeast inclusion in ruminant diets has been reported to decrease toxins absorption such as mycotoxins and promote epithelial cell integrity. This review article provides information on the impact of single-celled fungi yeast as a feed supplement on ruminal microbiota and its function to improve the health and productive longevity of ruminants.     

The ability of the rumen protozoan <Emphasis Type="Italic">Eudiplodinium maggii</Emphasis> to utilize chitin

The ability was determined of the rumen ciliate Eudiplodinium maggii to utilize chitin from fungal cell wall. Cultivation experiments shoved that the population concentration (number of ciliates in vitro) was positively correlated with chitin doses. Cell extract prepared from the bacteria-free ciliates degraded colloidal chitin releasing 2.0 μmol reducing sugar per mg protein per h. End products of this reaction were chitotriose and N-acetylglucosamine. Incubation of the bacteria-free ciliates with chitin resulted in an increase in the concentration of acetic, propionic and butyric acids in the incubation medium. The production rate of total volatile fatty acids (VFA) by ciliates incubated with and without chitin was 45.0 and 30.5 pmol VFA per protozoan, respectively, the molar proportion of particular acids remaining unchanged.