Effects of rumen fluid pre-incubation on in vitro proteolytic activity of enzymatic extracts from rumen microorganisms

Mixed rumen microorganisms present in bovine rumen fluid were extracted with butanol-acetone to provide a dry powder which retained 75–80% of the proteolytic activity of strained rumen fluid (SRF). Sixty percent of the proteolytic activity of the powder was extracted with water and concentrated on an Amicon XM-300 filter to give a protease preparation which had about 30% of the activity present in the SRF. The protease preparation was used for the determination of the rates of feed protein degradation in vitro by incubating at pH 6.8 in 0.1 M potassium phosphate buffer and measuring the rate of amino acid (and ammonia) production by the ninhydrin method. The relative degradation rates of the true proteins from feedstuffs tested were: soybean meal > fish meal > linseed meal and blood meal > canola meal > corn gluten meal. Substituting Streptomyces griseus protease for the rumen protease gave results which were very different from those obtained with the rumen enzyme. The advantages of using the rumen protease over other in vitro methods and the practical applicability of the procedure are discussed. Key words: Rumen, protease, preparation, in vitro, feed protein degradability

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The evaluation of Acacia and other tree pods for goats: influence of rumen fluid source and polyethylene glycol addition on in vitro gas production

Proceedings of the British Society of Animal Science ◽

10.1017/s1752756200007882 ◽

2002 ◽

Vol 2002 ◽

pp. 132-132 ◽

Cited By ~ 1

Author(s):

V. Mlambo ◽

F. L. Mould ◽

T. Smith ◽

E. Owen ◽

I. Mueller-Harvey

Keyword(s):

Polyethylene Glycol ◽

Prolonged Exposure ◽

Rumen Fluid ◽

Gas Production ◽

Rumen Microorganisms ◽

Protein Supplements ◽

In Vitro Gas Production ◽

Adaptive Mechanisms ◽

Acacia Pods

After prolonged exposure to tanniniferous diets, it has been reported that some rumen microorganisms acquire defensive mechanisms against tannins (Brooker et al., 2000) or produce tannin-degrading enzymes. Such rumen microorganisms are said to be “tannin resistant” as their fermentation activity is less inhibited by the presence of tannins in the host’s diet. As acacia pods contain tannins their use as protein supplements for goats in the dry season may require that they be first detannified e.g. by using polyethylene glycol (PEG). However, goats with prior exposure to tanniniferous diets may have developed adaptive mechanisms to deal with tannins. This study, therefore, investigated the need for tannin inactivation in feeds given to ‘adapted’ animals by comparing the effect on the in vitro fermentation of tree pods incubated with and without PEG using rumen fluid from adapted and unadapted goats.

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TRYPTOPHAN UTILIZATION BY RUMEN MICROORGANISMS IN VITRO

Canadian Journal of Animal Science ◽

10.4141/cjas70-049 ◽

1970 ◽

Vol 50 (2) ◽

pp. 331-335 ◽

Cited By ~ 2

Author(s):

ELIZABETH CANDLISH ◽

T. J. DEVLIN ◽

L. J. LaCROIX

Keyword(s):

Benzene Ring ◽

Continuous Flow ◽

Rumen Fluid ◽

Rumen Microorganisms

A continuous flow, artificial rumen apparatus was used to investigate the utilization of DL-tryptophan–14C(U)–benzene ring labelled material by rumen microorganisms. Half of the labelled material left the rumen sac in the first six hours. After a 24-hour period, less than 4% of the label was associated with either the protozoal or the bacterial fraction of the rumen fluid.

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Study of the effects of PR toxin, mycophenolic acid and roquefortine C on in vitro gas production parameters and their stability in the rumen environment

The Journal of Agricultural Science ◽

10.1017/s0021859614000343 ◽

2014 ◽

Vol 153 (1) ◽

pp. 163-176 ◽

Cited By ~ 12

Author(s):

A. GALLO ◽

G. GIUBERTI ◽

T. BERTUZZI ◽

M. MOSCHINI ◽

F. MASOERO

Keyword(s):

Mycophenolic Acid ◽

Rumen Fluid ◽

Rumen Fermentation ◽

Gas Production ◽

Farm Animals ◽

Marginal Effect ◽

Rumen Microorganisms ◽

Marginal Effects ◽

Roquefortine C

SUMMARYMoulds belonging to Penicillium section roqueforti are common contaminants of feedstuffs and produce several mycotoxins that can cause health hazards when ingested by farm animals. Among these, PR toxin (PR), mycophenolic acid (MY) and roquefortine C (RC) have been frequently detected in forages, particularly silages. The aims of the current trials were to study the effects of the presence of pure mycotoxins on in vitro rumen fermentation parameters and to assess their stability in the rumen environment. Two successive in vitro gas production experiments were carried out: a central composite design with four replications of central point (CCD) and a completely randomized design with a fully factorial arrangement of treatments (FFD). In CCD, the effects of PR, MY and RC concentrations in diluted rumen fluid (i.e. 0·01, 0·30, 1·01, 1·71 and 2·00 μg of each mycotoxin/ml) were tested. Gas volume produced after 48 h of incubation (Vf) decreased linearly as concentrations of RC and MY in diluted rumen fluid increased, with marginal effects similar for two mycotoxins, being respectively −14·6 and −13·4 ml/g organic matter (OM) for each 1·0 μg/ml of increment in mycotoxin concentration. Similarly, total volatile fatty acid (VFA) production decreased quadratically as concentrations of RC and MY increased, with marginal effects about two times higher for MY than RC, being −4·22 and −2·62 mmol/l for each 1·0 μg/ml of increment in mycotoxin concentration. With respect to maximum Vf (i.e. 410·6 ml/g OM) and VFA (98·06 mmol/l) values estimated by the model, decreases of 13·6 and 15·2% were obtained when incubating the highest RC and MY concentrations, respectively. The PR did not interfere with rumen fermentation pattern and it was not recovered after 48 h of incubation, whereas the stabilities of MY and RC in rumen fluid were similar and on average equal to about 50%. On the basis of CCD results, a second experiment (FFD) was carried out in which only effects of MY and RC concentrations (i.e. 0, 0·67, 1·33 and 2·00 μg of each mycotoxin/ml of diluted rumen fluid) were tested. Data from FFD showed Vf decreased linearly when concentrations of MY and RC increased, with marginal effect two-folds higher for MY than for RC (−11·1 ml/g OM and −6·7 ml/g OM, respectively). Similar marginal effects of MY and RC in decreasing VFA production were recorded: −2·38 and −2·86 mmol/l for each 1·0 μg/ml of increment in mycotoxin concentration, respectively. At the highest RC and MY tested concentrations, Vf and VFA decreased by 8·7 and 10·7%, respectively, over maximum estimated values. In FFD, the average amounts of MY and RC recovered in rumen fluid after 48 h of incubation were 79·0 and 40·6%, respectively. In conclusion, the MY and RC from standards interfered with rumen microorganisms at relatively low levels and were partially stable in the rumen environment after 48 h of incubation. These findings suggested that MY and RC could interfere with digestive processes and might represent a potential risk for ruminants fed diets containing feeds contaminated by mycotoxins produced by P. roqueforti.

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Comparison of fermentation characteristics and bacterial diversity in the rumen of sheep and in batch cultures of rumen microorganisms

The Journal of Agricultural Science ◽

10.1017/s0021859615000167 ◽

2015 ◽

Vol 153 (6) ◽

pp. 1097-1106 ◽

Cited By ~ 7

Author(s):

I. MATEOS ◽

M. J. RANILLA ◽

C. SARO ◽

M. D. CARRO

Keyword(s):

Bacterial Diversity ◽

Rumen Fluid ◽

Similarity Index ◽

Bacterial Strains ◽

Rumen Microorganisms ◽

Batch Cultures ◽

Alfalfa Hay ◽

Grass Hay ◽

Forage Type

SUMMARYThe objective of the current study was to assess how closely batch cultures (BC) of rumen microorganisms can mimic the dietary differences in fermentation characteristics found in the rumen, and to analyse changes in bacterial diversity over the in vitro incubation period. Four ruminally and duodenally cannulated sheep were fed four diets having forage : concentrate ratios (FCR) of 70 : 30 or 30 : 70, with either alfalfa hay or grass hay as forage. Rumen fluid from each sheep was used to inoculate BC containing the same diet fed to the donor sheep, and the main rumen fermentation parameters were determined after 24 h of incubation. There were differences between BC and sheep in the magnitude of most measured parameters, but BC detected differences among diets due to forage type similar to those found in sheep. In contrast, BC did not reproduce the dietary differences due to FCR found in sheep for pH, degradability of neutral detergent fibre and total volatile fatty acid (VFA) concentrations. There were differences between systems in the magnitude of most determined parameters and BC showed higher pH values and NH3–N concentrations, but lower fibre degradability and VFA and lactate concentrations compared with sheep. There were significant relationships between in vivo and in vitro values for molar proportions of acetate, propionate and butyrate, and the acetate : propionate ratio. The automated ribosomal intergenic spacer analysis (ARISA) of 16S ribosomal deoxyribonucleic acid showed that FCR had no effect on bacterial diversity either in the sheep rumen fluid used as inoculum (IN) or in BC samples. In contrast, bacterial diversity was greater with alfalfa hay diets than those with grass hay in the IN, but was unaffected by forage type in the BC. Similarity index between the bacterial communities in the inocula and those in the BC ranged from 67·2 to 74·7%, and was unaffected by diet characteristics. Bacterial diversity was lower in BC than in the inocula with 14 peaks out of a total of 181 detected in the ARISA electropherograms never appearing in BC samples, which suggests that incubation conditions in the BC may have caused a selection of some bacterial strains. However, each BC sample showed the highest similarity index with its corresponding rumen IN, which highlights the importance of using rumen fluid from donors fed a diet similar to that being incubated in BC when conducting in vitro experiments.

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Lovastatin-Enriched Rice Straw Enhances Biomass Quality and Suppresses Ruminal Methanogenesis

BioMed Research International ◽

10.1155/2013/397934 ◽

2013 ◽

Vol 2013 ◽

pp. 1-13 ◽

Cited By ~ 11

Author(s):

Mohammad Faseleh Jahromi ◽

Juan Boo Liang ◽

Rosfarizan Mohamad ◽

Yong Meng Goh ◽

Parisa Shokryazdan ◽

...

Keyword(s):

Rice Straw ◽

Rumen Fluid ◽

Methanogenic Archaea ◽

Primary Objective ◽

Gas Production ◽

Cellulolytic Bacteria ◽

Hmg Coa Reductase ◽

Rumen Microorganisms ◽

Coa Reductase

The primary objective of this study was to test the hypothesis that solid state fermentation (SSF) of agro-biomass (using rice straw as model); besides, breaking down its lignocellulose content to improve its nutritive values also produces lovastatin which could be used to suppress methanogenesis in the rumen ecosystem. Fermented rice straw (FRS) containing lovastatin after fermentation withAspergillus terreuswas used as substrate for growth study of rumen microorganisms usingin vitrogas production method. In the first experiment, the extract from the FRS (FRSE) which contained lovastatin was evaluated for its efficacy for reduction in methane (CH4) production, microbial population, and activity in the rumen fluid. FRSE reduced total gas and CH4productions (P<0.01). It also reduced (P<0.01) total methanogens population and increased the cellulolytic bacteria includingRuminococcus albus,Fibrobacter succinogenes(P<0.01), andRuminococcus flavefaciens(P<0.05). Similarly, FRS reduced total gas and CH4productions, methanogens population, but increasedin vitrodry mater digestibility compared to the non-fermented rice straw. Lovastatin in the FRSE and the FRS significantly increased the expression of HMG-CoA reductase gene that produces HMG-CoA reductase, a key enzyme for cell membrane production in methanogenic Archaea.

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Influence of acetohydroxamic acid on some activities in vitro of the rumen microbiota

Canadian Journal of Microbiology ◽

10.1139/m68-065 ◽

1968 ◽

Vol 14 (4) ◽

pp. 409-416 ◽

Cited By ~ 12

Author(s):

G. A. Jones

Keyword(s):

Volatile Fatty Acids ◽

Urease Activity ◽

Rumen Fluid ◽

Selective Medium ◽

Colony Development ◽

Acetohydroxamic Acid ◽

Rumen Microorganisms ◽

Rumen Microbiota

When acetohydroxamic acid was incubated with washed suspensions of bovine rumen microorganisms the urease activity of the suspensions was depressed; activity could not be restored by the addition of divalent cations which, in the absence of acetohydroxamic acid, stimulated the urease activity of the cells. Acetohydroxamic acid was slowly degraded by the rumen microbiota. When the compound was incorporated into a non-selective medium for the enumeration of rumen bacteria it completely prevented visible colony development by some components of the inoculum and retarded the rate of multiplication of others. Acetohydroxamic acid inhibited the production of volatile fatty acids from added cellulose in strained rumen fluid and modified the molar proportions of acetate, propionate, and butyrate produced from the substrate; whereas in the absence of acetohydroxamic acid propionate production was favored at the expense of acetate; in its presence the acetate:propionate ratio remained constant. The effect of acetohydroxamic acid upon rumen microbial activities in vitro was therefore not limited to inhibition of rumen urease. It was impossible, however, to infer from the results obtained whether the potential value of the compound as a urease inhibitor in vivo would be diminished for this reason; this is because the influence of acetohydroxamic acid on the rumen microbiota in vivo is probably subject to modification by factors, such as the composition of the animal's diet, which were not investigated.

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In vitro Inoculation of Fresh or Frozen Rumen Fluid Distinguishes Contrasting Microbial Communities and Fermentation Induced by Increasing Forage to Concentrate Ratio

Frontiers in Nutrition ◽

10.3389/fnut.2021.772645 ◽

2022 ◽

Vol 8 ◽

Author(s):

Zhi Yuan Ma ◽

Ju Wang Zhou ◽

Si Yu Yi ◽

Min Wang ◽

Zhi Liang Tan

Keyword(s):

Bacterial Diversity ◽

Volatile Fatty Acids ◽

Rumen Fluid ◽

Starch Content ◽

Block Design ◽

Rumen Fermentation ◽

Rumen Microorganisms ◽

Fermentative Bacteria ◽

In Vitro Inoculation

In vitro rumen batch culture is a technology to simulate rumen fermentation by inoculating microorganisms from rumen fluids. Although inocula (INO) are commonly derived from fresh rumen fluids, frozen rumen fluids are also employed for the advantages of storing, transporting, and preserving rumen microorganisms. The effects of frozen INO on microbial fermentation and community may be interfered with by substrate type, which has not been reported. This study was designed to test whether rumen fluid treatments (i.e., fresh and frozen) could interact with incubated substrates. A complete block design with fractional arrangement treatment was used to investigate the effects of INO (fresh or frozen rumen fluids) and concentrate-to-forage ratios (C/F, 1:4 or 1:1) on rumen fermentation and microbial community. The effects of increasing C/F were typical, including increased dry matter (DM) degradation and total volatile fatty acids (VFA) concentration (P < 0.001), and decreased acetate to propionate ratio (P = 0.01) and bacterial diversity of richness and evenness (P ≤ 0.005) with especially higher fermentative bacteria such as genus Rikenellaceae_RC, F082, Prevotella, Bacteroidales_BS11, Muribaculaceaege, and Christensenellaceae_R-7 (P ≤ 0.04). Although frozen INO decreased (P < 0.001) DM degradation and altered rumen fermentation with lower (P ≤ 0.01) acetate to propionate ratio and molar proportion of butyrate than fresh INO, typical effects of C/F were independent of INO, as indicated by insignificant INO × C/F interaction on substrate degradation, VFA profile and bacterial community (P ≥ 0.20). In summary, the effect of C/F on fermentation and bacterial diversity is not interfered with by INO type, and frozen INO can be used to distinguish the effect of starch content.

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Changes with time in the short chain fatty acid profile during in vitro incubations of feeds with rumen fluid and their effect on the prediction of ATP production

Proceedings of the British Society of Animal Science ◽

10.1017/s1752756200000259 ◽

2000 ◽

Vol 2000 ◽

pp. 24-24

Author(s):

C. Rymer ◽

D.I. Givens ◽

B.R Cottrill

Keyword(s):

Fatty Acid ◽

Short Chain Fatty Acid ◽

Rumen Fluid ◽

Gas Production ◽

Chain Fatty Acid ◽

Short Chain ◽

Rumen Microorganisms ◽

Atp Production ◽

Atp Supply

The in vitro gas production technique is a means of measuring the dynamics of fermentation. It is related to short chain fatty acid (SCFA) production, and so could be used to estimate ATP supply for rumen microorganisms. However, different fermentation patterns produce different amounts of gas. No fermentation gas is associated with the production of propionate, and so an increase in the proportion of propionic: (acetic+butyric) (P:AB) would be associated with a decrease in the volume of gas produced. If the molar proportions of SCFA changed during a fermentation, then this would complicate the interpretation of the gas production profile (GPP). If the GPP, combined with a measure of SCFA concentrations at the end of the incubation, was used to estimate ATP yield during the incubation, then changes in P:AB during the incubation may affect these estimates. The objectives of this experiment were therefore to determine whether P:AB did change during an in vitro incubation, and whether any such change affected the accuracy of the prediction of ATP yield with time.

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MICROBIAL DEGRADATION OF VITAMIN A IN RUMEN FLUID FROM STEERS FED CONCENTRATE, HAY OR STRAW DIETS

Canadian Journal of Animal Science ◽

10.4141/cjas90-026 ◽

1990 ◽

Vol 70 (1) ◽

pp. 227-233 ◽

Cited By ~ 17

Author(s):

L. M. RODE ◽

T. A. McALLISTER ◽

K. -J. CHENG

Keyword(s):

Vitamin A ◽

High Performance ◽

Rumen Fluid ◽

Biologically Active ◽

Retinyl Acetate ◽

Rumen Microorganisms ◽

Propionate Degradation ◽

Ruminal Degradation ◽

High Forage

The effect of diet on degradation of vitamin A by rumen microorganisms in vitro was investigated. Retinyl acetate was incubated in vitro with rumen fluid from cattle fed high-concentrate (C), hay (H) or straw (S) diets. Residual retinyl acetate and retinol were measured by high-performance liquid chromatography (HPLC), 24 h after retinyl acetate addition to rumen fluid. Retinyl acetate was degraded faster and more extensively in C rumen fluid than in H or S rumen fluid. Retinol levels increased in H and S rumen fluid throughout the incubation period but decreased in C rumen fluid (P < 0.05). Estimated effective ruminal degradation of biologically active vitamin A was 67, 16 and 19% for cattle fed C, H and S diets, respectively. Thirty-three strains of rumen bacteria were surveyed for their ability to degrade retinyl propionate. Of the nine strains that were positive for retinyl propionate degradation, seven were strains associated with starch digestion. The results imply that cattle fed high-concentrate diets will have considerably greater vitamin A requirements than those fed high-forage diets, if oral supplementation of vitamin A is used. Because of the extensive ruminal destruction of vitamin A, intramuscular injection would be a more efficient mode of supplementation for cattle fed high-concentrate diets. Key words: Retinol, retinyl acetate, vitamin A, concentrate, ruminai degradation, cattle, HPLC

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