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PubMed2004

In vitro and in vivo inhibition of Helicobacter pylori by Lactobacillus casei strain Shirota.

We studied the potential inhibitory effect of Lactobacillus casei strain Shirota (from the fermented milk product Yakult [Yakult Ltd., Tokyo, Japan]) on Helicobacter pylori by using (i) in vitro inhibition assays with H. pylori SS1 (Sydney strain 1) and nine H. pylori clinical isolates and (ii) the in vivo H. pylori SS1 mouse model of infection over a period of 9 months. In vitro activity against H. pylori SS1 and all of the clinical isolates was observed in the presence of viable L. casei strain Shirota cells but not in the cell-free culture supernatant, although there was profound inhibition of urease activity. In vivo experiments were performed by oral administration of L. casei strain Shirota in the water supply over a period of 9 months to 6-week-old C57BL/6 mice previously infected with H. pylori SS1 (study group; n = 25). Appropriate control groups of H. pylori-infected but untreated animals (n = 25) and uninfected animals given L. casei strain Shirota (n = 25) also were included in the study. H. pylori colonization and development of gastritis were assessed at 1, 2, 3, 6, and 9 months postinfection. A significant reduction in the levels of H. pylori colonization was observed in the antrum and body mucosa in vivo in the lactobacillus-treated study group, as assessed by viable cultures, compared to the levels in the H. pylori-infected control group. This reduction was accompanied by a significant decline in the associated chronic and active gastric mucosal inflammation observed at each time point throughout the observation period. A trend toward a decrease in the anti-H. pylori immunoglobulin G response was measured in the serum of the animals treated with lactobacillus, although this decrease was not significant.

Animals↗

PubMed1989

Regulation of Glycolytic Flux and Ethanol Production in Saccharomyces cerevisiae: Effects of Intracellular Adenine Nucleotide Concentrations on the In Vitro Activities of Hexokinase, Phosphofructokinase, Phosphoglycerate Kinase, and Pyruvate Kinase.

The progressive decline in the glycolytic activity of Saccharomyces cerevisiae during batch fermentation is accompanied by changes in adenine nucleotide pools. The relative activities of four glycolytic enzymes were examined in vitro in the presence of nucleotide concentrations equivalent to intracellular pools. Phosphofructokinase and pyruvate kinase were not inhibited. Phosphoglycerate kinase was inhibited by AMP but was judged unlikely to be of physiological consequence owing to enzyme abundance. Both isoenzymes of hexokinase were strongly inhibited by AMP. The degree of hexokinase inhibition was sufficient to account for the observed decline in glycolytic activity during batch fermentation.

Journal Article↗

PubMed2001

Prebiotic digestion and fermentation.

Prebiotics, as currently conceived of, are all carbohydrates of relatively short chain length. To be effective they must reach the cecum. Present evidence concerning the 2 most studied prebiotics, fructooligosaccharides and inulin, is consistent with their resisting digestion by gastric acid and pancreatic enzymes in vivo. However, the wide variety of new candidate prebiotics becoming available for human use requires that a manageable set of in vitro tests be agreed on so that their nondigestibility and fermentability can be established without recourse to human studies in every case. In the large intestine, prebiotics, in addition to their selective effects on bifidobacteria and lactobacilli, influence many aspects of bowel function through fermentation. Short-chain fatty acids are a major product of prebiotic breakdown, but as yet, no characteristic pattern of fermentation acids has been identified. Through stimulation of bacterial growth and fermentation, prebiotics affect bowel habit and are mildly laxative. Perhaps more importantly, some are a potent source of hydrogen in the gut. Mild flatulence is frequently observed by subjects being fed prebiotics; in a significant number of subjects it is severe enough to be unacceptable and to discourage consumption. Prebiotics are like other carbohydrates that reach the cecum, such as nonstarch polysaccharides, sugar alcohols, and resistant starch, in being substrates for fermentation. They are, however, distinctive in their selective effect on the microflora and their propensity to produce flatulence.

Cathartics↗

PubMed1981

Characteristics of the rumen proteolysis of fraction I (18S) leaf protein from lucerne (Medicago sativa L).

1. The rate of proteolysis of fraction I (18S) leaf protein in the rumen of sheep of cattle was affected by diet ans the rate on fresh lucerne (medicago sativa L) was three to nine times the rate on a hay + concentrate diet. 2. Simultaneous rumen fermentations in vivo and in an artificial rumen showed that the rates of proteolysis of fraction I in vitro was approximately 30% of the rates in sheep. 3. Using 14 C uniformly-labelled fraction I protein at low concentrations, proteolysis exhibited 1st-order kinetics. Over a wide range of protein concentrations the velocity v. substrate concentration curve showed Michaelis-Menten characteristics typical of an enzyme-catalysed reaction. With rumen fluid from a hay + concentrate-fed sheep the maximum velocity was 2.6 mg protein nitrogen/1 per min and the Michaelis constant was 75 mg nitrogen/l. 4. Rapid absorption of 14C-labelled fraction I protein onto bacterial cells preceded proteolysis. 5. Sucrose-density-gradient analysis showed initial incorporation of 14C from protein into rumen bacteria followed by partial transfer to rumen protozoa. 6. No peptides were detected during proteolysis showing that the rate-limiting step occurred during the initial stages of proteolysis. Only small amounts of free amino acids were released except for leucine, isoleucine, valine and ornithine, which showed significantly increased levels. 7. Volatile fatty acids were the main 14C-labelled end products and were rapidly produced in descending concentrations: acetate greater than propionate greater than 3-methyl + 2-methyl butyrate greater than butyrate greater than isobutyrate greater than valerate.

Amino Acids↗

PubMed1999

Processing of quinoa (Chenopodium quinoa, Willd): effects on in vitro iron availability and phytate hydrolysis.

The effect of different processing techniques was studied on in vitro iron availability and phytate hydrolysis in high and low saponin content quinoa (Chenopodium quinoa, Willd) seeds. Water slurries of ungerminated and germinated quinoa flour were processed by cooking, soaking, and fermentation using Lactobacillus plantarum as starter. Iron solubility under physiological conditions (in vitro) was measured and used as an estimation of iron availability. Phytate (inositol hexaphosphate/IP6) and its degradation products were analysed by an HPLC method. The IP6 + IP5 content was reduced by cooking with 4 to 8%, germination with 35 to 39%, soaking with 61 to 76% and by fermentation with 82 to 98%. The highest reduction, about 98%, was obtained after fermentation of the germinated flour. Cooking had no effect on the amount of soluble iron. Iron solubility increased, however, two to four times after soaking and germination, three to five times after fermentation and five to eight times after fermentation of the germinated flour samples and was highly correlated to the reduction of IP6 + IP5 (P < 0.001). There was no difference between the quinoa varieties with regard to phytate reduction and iron solubility. The pH in fermented samples was reduced from 6.5 to about 3.5, due to lactic acid formation.

Chenopodiaceae↗

PubMedDate not supplied

Xylulokinase activity in various yeasts including Saccharomyces cerevisiae containing the cloned xylulokinase gene. Scientific note.

D-Xylose is a major constituent of hemicellulose, which makes up 20-30% of renewable biomass in nature. D-Xylose can be fermented by most yeasts, including Saccharomyces cerevisiae, by a two-stage process. In this process, xylose is first converted to xylulose in vitro by the enzyme xylose (glucose) isomerase, and the latter sugar is then fermented by yeast to ethanol. With the availability of an inexpensive source of xylose isomerase produced by recombinant E. coli, this process of fermenting xylose to ethanol can become quite effective. In this paper, we report that yeast xylose and xylulose fermentation can be further improved by cloning and overexpression of the xylulokinase gene. For instance, the level of xylulokinase activity in S. cerevisiae can be increased 230fold by cloning its xylulokinase gene on a high copy-number plasmid, coupled with fusion of the gene with an effective promoter. The resulting genetically-engineered yeast can ferment xylose and xylulose more than twice as fast as the parent yeast.

Cloning, Molecular↗

PubMed1984

Potential cariogenicity of Lycasin 80/55 before and after repeated transmissions of the dental plaque flora in rats.

Five successive experiments, with rats fed ad libitum on diets containing sucrose or Lycasin 80/55, were carried out. In experiment I, the rats were inoculated with Streptococcus mutans alone or with Strep. mutans in combination with Actinomyces viscosus. In three successive transmission experiments (II, III, IV), the rats were inoculated with plaque of the rats of the preceding experiment. The rats of experiment V were inoculated with the original strains or with plaque derived from experiment IV. After five successive transmissions of the plaque flora, no alterations were demonstrated in the numbers and percentages of Strep. mutans and A. viscosus or in the fermentation rate of Lycasin by the plaque flora in vitro. Lycasin 80/55 was virtually non-cariogenic compared with sucrose (p less than 0.001) irrespective of whether the rats were inoculated with Strep. mutans alone or in combination with A. viscosus, with the original strains or with plaque from the preceding experiments with rats on a Lycasin 80/55-containing diet.

Actinomyces↗

PubMed2004

Prevention of biofilm formation by dairy products and N-acetylcysteine on voice prostheses in an artificial throat.

OBJECTIVE: To evaluate the preventive effect of buttermilk, Yakult Light fermented milk drink and N-acetylcysteine on biofilm formation on voice prostheses in vitro. MATERIAL AND METHODS: Groningen button and Provox 2 voice prostheses were inoculated with a mixture of bacteria and yeasts isolated from previously explanted Groningen button voice prostheses. After 5 h, separate throats were flushed with buttermilk, Yakult Light fermented milk drink, N-acetylcysteine or phosphate-buffered saline, which served as a control. After 7 days, the microflora on each voice prosthesis was determined. RESULTS: On Groningen button voice prostheses, buttermilk, Yakult Light fermented milk drink and N-acetylcysteine all reduced the amount of both bacteria and yeasts. On Provox 2 voice prostheses, buttermilk, Yakult Light fermented milk drink and N-acetylcysteine reduced the amount of bacteria but, conversely, increased the amount of yeasts. CONCLUSION: These in vitro experiments demonstrate that biofilm formation on voice prostheses is reduced in an artificial throat by the use of buttermilk, Yakult Light fermented milk drink and N-acetylcysteine. However, the structural differences between the type of voice prostheses may influence the ultimate effects.

Acetylcysteine↗

PubMed2006

Transcarboxylase mRNA: a marker which evidences P. freudenreichii survival and metabolic activity during its transit in the human gut.

Dairy propionibacteria have recently been considered as probiotics which may beneficially modulate the intestinal ecosystem. However, appropriate vectors (food matrices containing the probiotic) which preserve their viability and offer good tolerance towards digestive stresses need to be developed. In addition, the development of efficient non-invasive methods which specifically monitor Propionibacterium freudenreichii concentration and activity within the human gut is required. To address this latter need, an enzyme involved in propionic fermentation, transcarboxylase, was evaluated in this study as molecular marker in P. freudenreichii. In vitro, the three transcarboxylase subunits were shown to be encoded by an operon and their expression regulated. It occurred during propionic fermentation, ceased in starved cells and was not affected by digestive stresses. The 5S subunit gene of transcarboxylase allowed specific detection of P. freudenreichii by real time PCR in the complex human faecal microbiota. A dairy vector harbouring P. freudenreichii was developed and afforded elevated probiotic faecal concentrations in humans. In vivo, this PCR method allowed rapid quantification of faecal P. freudenreichii in agreement with the cultural method (cfu counting). Moreover, real time Reverse Transcription (RT) -PCR evidenced transcription of the 5S subunit gene during transit through the human digestive tract. This work constitutes a methodological advance for survival and activity evaluation in human trials of the probiotics belonging to the P. freudenreichii species. It strongly suggests that this bacterium not only survives but remains metabolically active in the human gut.

Base Sequence↗

PubMed2005

Literature data may underestimate the actual antioxidant capacity of cereals.

Several recent articles have reported a significant antioxidant capacity of cereal products, determined in methanolic and ethanolic extracts. The aim of this work was to conduct an assessment of the antioxidant capacity of cereals using both chemical and in vitro digestive enzymatic extraction of antioxidants. Ferric reducing power (FRAP) and free radical scavenging capacity (DPPH) methods were used to determine the antioxidant capacity in wheat flour, bread, raw and boiled rice, wheat bran, and oat bran. The most efficient antioxidant extraction was achieved by using successively acidic methanol/water (50:50 v/v, pH 2) and acetone/water (70:30 v/v). The antioxidant capacity in these extracts ranged from 1.1 to 4.4 micromol Trolox/g dw. A significant amount of hydrolyzable phenolics with a high antioxidant capacity (from 5 to 108 micromol Trolox/g dw) was found in the residues of this aqueous-organic extraction. The antioxidant capacities of these nonextractable polyphenols are usually ignored in the literature, although they may have an antioxidant role in the gastrointestinal tract, especially after colonic fermentation, and may be fermentated to active metabolites. On the other hand, in vitro digestive enzymatic extracts obtained by enzymatic treatments that mimic conditions in the gastrointestinal tract showed that the amount of antioxidants released by the cereal matrix into the human intestine may be higher than the one that can be expected from measurements in the usual aqueous-organic extracts.

Antioxidants↗

PubMed2004

In vitro and in vivo anti-allergic activity of soy sauce.

Soy sauce (Shoyu) is a traditional fermented seasoning of Japan and available throughout the world. Polysaccharides were obtained from dialysate of Shoyu, and these Shoyu polysaccharides (SPS) were examined for anti-allergic activity in vitro and in vivo. The SPS originated from partially-degraded polysaccharides of soybeans by mold enzymatic hydrolyses, and Shoyu contained about 1% (w/v) SPS. First, the inhibitory effects of SPS on hyaluronidase, which is known to be related to inflammation and allergic responses, were as potent as those of an anti-allergic medicine, disodium cromoglycate. Second, SPS significantly inhibited the release of histamine from rat basophilic leukemia (RBL-2H3) cells, which had been induced by the antigen. Third, orally administered SPS had a significant suppressive effect on passive cutaneous anaphylaxis induced in the ears of mice. These results suggest that SPS may have anti-allergic activities, and soy sauce is a potentially promising seasoning for the treatment of allergic diseases through food.

Animals↗

PubMed1992

Development of buffer systems for pH control and evaluation of pH effects on fiber digestion in vitro.

An in vitro buffering system capable of pH control between pH 5.8 and 6.8 was developed to examine the effect of media pH on disappearance of NDF at various times of fermentation and to assess initially the effect of media pH on kinetics of NDF digestion. The pH conditions selected for evaluation of these buffer systems were 5.8, 6.2, and 6.8. Use of McIlvaine's solution with sodium bicarbonate was not successful because of rapid drifting of pH downward during fermentation. To evaluate the effectiveness of citric or phosphoric acids as components of phosphate-bicarbonate buffer systems, alfalfa silage and a mixture of alfalfa silage and corn grain (1:1 mixture, dry basis) were fermented for 0, 12, 24, 48, and 72 h. The pH of each flask was measured at 0, 4, 12, 24, 48, and 72 h postinoculation, and pH was readjusted with bicarbonate solution when necessary. Drifting of media pH downward was more noticeable when phosphoric acid was used to adjust the buffer pH than with citric acid. Citric acid had no adverse effects on NDF digestion compared with phosphoric acid when used to adjust a phosphate-bicarbonate buffer system. Alfalfa hay, bromegrass hay, and corn silage were incubated for 0, 12, 24, 48, 72, or 96 h at pH 5.8 or 6.8 using the phosphate-bicarbonate buffer system adjusted with citric acid. Estimation of kinetics of NDF digestion indicated that a decrease in media pH from 6.8 to 5.8 resulted in a marked reduction in NDF digestion; the largest apparent difference was extended digestion lag time.

Animal Feed↗

PubMed2006

Effect of selected fungi on the reduction of gossypol levels and nutritional value during solid substrate fermentation of cottonseed meal.

The objective of this work was to investigate the effect of six individual strains of fungi on the reduction of gossypol levels and nutritional value during solid substrate fermentation of cottonseed meal (CSM). Six groups of disinfected CSM substrate were incubated for 48 h after inoculation with either of the fungi C. capsuligena ZD-1, C. tropicalis ZD-3, S. cerevisae ZD-5, A. terricola ZD-6, A. oryzae ZD-7, or A. niger ZD-8. One not inoculated group (substrate) was used as a control. Levels of initial and final free gossypol (FG), crude protein (CP), amino acids (AA) and in vitro digestibility were assayed. The experiment was done in triplicate. The experimental results indicated that microbial fermentation could greatly decrease (P<0.05) FG levels in CSM. The detoxification efficiency differed between the species of microorganisms applied. From the perspective of reducing CSM potential toxicity, C. tropicalis ZD-3 was most successful followed by S. cerevisae ZD-5 and A. niger ZD8. They could reduce FG levels of CSM to 29.8, 63.07 and 81.50 mg/kg based on DM (dry matter), respectively, and their detoxification rate were 94.57%, 88.51% and 85.16%, respectively. If crude protein, amino acids content and their in vitro digestibility were also taken into account, A. niger ZD-8 may be the best choice. The CP content of CSM substrate fermented by C. tropicalis ZD-3 and A. niger ZD-8 were improved by 10.76% and 22.24%; the TAA (total amino acids) contents were increased by 7.06% and 11.46%, and the EAA (essential amino acids) were raised by 7.77% and 12.64%, respectively. Especially, the levels of methionine, lysine and threonine were improved greatly (P<0.05). The in vitro CP digestibility of CSM fermented by C. tropicalis ZD-3 and A. niger ZD-8 was improved by 13.42% and 18.22%, the TAA were increased by 17.75% and 22.88%, and the EAA by 16.61% and 21.01%, respectively. In addition, the in vitro digestibility of methionine, lysine and threonine was also improved greatly (P<0.05).

Amino Acids↗

PubMed1993

Engineering Fab' fragments for efficient F(ab)2 formation in Escherichia coli and for improved in vivo stability.

We previously developed an efficient route to humanized F(ab')2 fragments by high level secretion of the Fab' arms from Escherichia coli followed by directed chemical coupling in vitro. Here the number and type of interchain linkages in F(ab')2 molecules has been modified to simplify their production and improve their serum stability. All F(ab')2 variants had comparable binding affinity for the p185HER2 Ag and antiproliferative activity against p185HER2-overexpressing tumor cells. This was anticipated since the modifications are distant from the Ag-binding loops. Replacement of a single disulfide bridge between Fab' arms with a more stable thioether bridge increased the serum permanence time in normal mice by threefold to 2.1 h. Removal of the disulfide bond between L and H chains in the thioether-bridged F(ab')2 did not affect the pharmacokinetics, suggesting that the L chain remains associated with the H chain. An additional Fab' variant containing three repeats of the motif, CysProPro, was constructed with the aim of promoting efficient formation of F(ab')2 in E. coli. This Fab' (CPP)3 variant was recovered predominantly (up to 70%) as F(ab')2 directly from fermentation cell pastes, thus circumventing the need for in vitro coupling. The F(ab')2 (CPP)3 variant has a similar serum pharmacokinetics to the thioether-bridged molecules. The improvements described here for deriving F(ab')2 fragments from E. coli should enhance the clinical potential of these molecules.

Amino Acid Sequence↗

PubMed1995

Additives containing bacteria and enzymes for alfalfa silage.

First-cutting alfalfa was wilted, harvested from alternate rows, left untreated or treated with additives containing lactic acid bacteria and enzymes (cellulase, amylase, and pectinase), and ensiled in bag silos. Inoculation increased lactic acid bacteria from 5 x 10(4) to 1 x 10(6) cfu/g of forage. Because treatments were bagged consecutively, the DM of treated silages was higher than that of untreated silage. However, after 4 d of ensiling, the pH of treated silage, about 4.3, was lower than that of untreated silage, 4.7, and remained lower throughout the ensiling period. After 177 d of ensiling, total lactate was about 25% higher, and ammonia N was about 40% lower, in treated silage. In addition, NDF and ADF contents were lower in treated than in untreated silage. Between 51 and 177 d of storage, glucose content increased in treated silage, but not in untreated silage, suggesting that some plant cell-wall hydrolysis occurred during prolonged storage. In vitro digestion of NDF did not differ among treatments during early incubation, but the extent of digestion after 36 and 48 h was lower in treated than in untreated silage. The microbial and enzyme silage additives used in this study improved fermentation characteristics and reduced fiber content of silage but decreased the in vitro digestibility of fiber.

Amylases↗

PubMed1999

Effect of three strains of Pleurotus tuber-regium (Fr.) Sing. on chemical composition and rumen fermentation of wheat straw.

This study was conducted to investigate changes in in vitro dry matter digestibility (IVDMD) and cell wall constituent degradation in wheat straw treated with 3 strains of the fungus Pleurotus tuber-regium (PT). The incubation of wheat straw for 30 days at 28 degrees C improved IVDMD from 30.3% (UWS-untreated wheat straw) to 47.1% for strain PT1, to 48.5% for PT4, and was unchanged IVDMD-29.9% -for PT5. The growth of fungi was accompanied by the dry matter loss of wheat straw: 31.5% for PT1, 20.9% for PT4, and 4.8% for PT5. Fungal treatment was characterized by increased crude protein and ash contents (%) in all fungi-treated straws and reduced hemicellulose and lignin content. It is evident that enzymes of all 3 PT strains preferentially degraded hemicellulose and lignin over cellulose. Wheat straw treated with PT1 (TWS-PT1), PT4 (TWS-PT4), and PT5 (TWS-PT5) and barley (80% : 20%) were used as the experimental diets at the fermentation in the artificial rumen. UWS with barley (80% : 20%) served as the control diet. The fermentation of experimental diets was accompanied with increased IVDMD and a very low degree of hemicellulose degradation. Total gas and methane productions were similar in all diets. Moreover, total volatile fatty acid (VFA) production (mmol day(-1)), mol % of acetate, propionate, butyrate, isobutyrate, and isovalerate were not influenced during the fermentation of experimental diets. From the stoichiometric relations, production, utilization, and recovery of metabolic hydrogen and organic matter fermented were unchanged. Only the recovery of metabolic hydrogen in TWS-PT5 was significantly increased in comparison to control diet. Total microbial production showed the tendency of lower values in experimental diets, and it was accompanied with a significant decrease of ammonia nitrogen (mg L(-1)). Finally the results showed that the strains of Pleurotus tuber-regium can improve the quality of wheat straw, but the loss of dry matter (DM) (mainly hemicellulose) limits the effective utilization of fungi-treated straw in ruminant digestion.

Journal Article↗

PubMed1994

Effect of fiber source on short-chain fatty acid production and on the growth and toxin production by Clostridium difficile.

BACKGROUND: Fermentable fiber promotes the growth of resident gut microbes, which modify the environment of the gastrointestinal tract and thus prevent colonization by Clostridium difficile. METHODS: An in vitro system with pigs as fecal inoculum donors was used to estimate fiber fermentability and changes in intestinal microbiota. RESULTS: Acetate and propionate production (mumol/mg substrate fermented/day) was greatest for gum arabic (1013.4 and 704.1, respectively); butyrate production was greatest for xylo-oligosaccharide (345.6). Growth of total anaerobes and clostridia was greatest for gum arabic (21.2 and 16.2 x 10(8) counts/ml, respectively) and xylo-oligosaccharides (21.0 and 19.6 x 10(8) respectively); growth of acidogenic bacteria was greatest with fructo-oligosaccharide (6.7 x 10(8) counts/ml). No culturable counts of C. difficile were obtained, nor was toxin A detected. CONCLUSIONS: Fermentable fibers support the growth of indigenous intestinal bacteria, particularly acidogenic bacteria, and yield large amounts of short-chain fatty acids with decreased gut pH. These factors contribute to the prevention of growth and toxin elaboration by C. difficile.

Animals↗

PubMed1988

Dynamics of protozoa in the rumen of cattle.

1. The dynamics of protozoa were studied in two groups of rumen-fistulated cattle fed on a basal diet of molasses ad lib., with oaten chaff given at 6 or 18 g/kg live weight. This diet resulted in different mixtures of protozoal species in the populations in the rumen. 2. The rumen protozoa were studied by intrarumen injections of protozoa labelled in vitro with [14CH3]choline. An indication of protozoal death and fermentation of protozoal cell residues was obtained by measuring 14C loss via the methane pool. 3. After a single injection of labelled protozoa, the decline in the specific radioactivity (microCi/g nitrogen) of the protozoal pool in the rumen indicated that first-order kinetic processes applied. Conversely the specific radioactivity of protozoa, incubated in rumen fluid, remained constant indicating no growth in vitro, presumably owing to a rapid exhaustion of essential nutrients. 4. The protozoal populations in the rumen of cattle fed on the diet with the low level of oaten chaff were mainly small ciliates; but on the higher level of chaff in the diet, the large ciliates were a higher proportion of the total protozoal population present. 5. The mean pool size of protozoa in the rumen was significantly larger and the protozoal half-life tended to be longer for cattle fed on the higher level of chaff in the diet. The apparent production rate of protozoa in cattle fed on each diet was not significantly different and there were no differences in the production rate of methane. The percentage losses of label from protozoa in the rumen via the methane pool were not significantly different on the two diets and indicated that 74% of the protozoa that were apparently irreversibly lost from the rumen could be accounted for by death and lysis in the rumen and therefore only 26% of protozoa apparently entered the lower digestive tract.

Ammonia↗