EVALUATION OF A DEHYDROGENASE ASSAY BASED ON TETRAZOLIUM REDUCTION FOR RAPID IN VITRO ESTIMATION OF FERMENTATION ACTIVITY IN RUMEN CONTENTS

1978 ◽  
Vol 58 (3) ◽  
pp. 501-511 ◽  
Author(s):  
G. A. JONES ◽  
B. A. HUMPHREY
Keyword(s):  
Dehydrogenase Activity ◽  
Rumen Fluid ◽  
Stomach Tube ◽  
Triphenyltetrazolium Chloride ◽  
Activity Maximum ◽  
Alfalfa Hay ◽  
The Mean ◽  
Ph Range ◽  
Second Peak

Rumen contents obtained from fistulated steers fed brome–alfalfa hay was used to evaluate a dehydrogenase assay based on the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) to triphenylformazan (TPF) for rapid in vitro estimation of fermentation activity. Maximum absorbance of TPF occurred at 485 nm over the pH range 3–10. TPF formed from TTC was stable to atmospheric O2. Absorbance decreased if TPF was exposed to light. In the presence of excess TTC, production of TPF in strained rumen fluid (SRF) at 39 °C was linear with elapsed incubation times up to at least 5 min. Dehydrogenase activity was abolished in the presence of air. It was significantly increased if the CO2 gas phase above SRF was replaced with N2 or H2, owing, at least in part, to an increase in the pH of the fermentation. The dehydrogenase activity of SRF was maximal at pH 7.8–8.0. Fractionation of SRF by differential centrifugation showed that about 50% of the dehydrogenase activity was associated with a protozoal fraction sedimenting at 188 × g, and 42% with a bacterial fraction sedimenting at 1,475 × g. Neither the cell-free supernatant obtained by centrifuging SRF at 21,500 × g, nor boiled SRF, nor the hay fed to the animals showed dehydrogenase activity. Whole rumen contents (WRC) showed a mean dehydrogenase activity that was 58% higher than that of SRF. The dehydrogenase activity of SRF was increased in the presence of substrates representing major plant carbohydrates, the greatest stimulatory effect being shown by ground brome–alfalfa hay. When rumen contents were sampled at the same time by stomach tube and through the fistula, the mean dehydrogenase activity of SRF prepared from the stomach tube samples was 137% higher than that of SRF prepared from the fistula samples. In an animal fed at 12-h intervals, the dehydrogenase activity of SRF rose within 2 h after feeding, declined for the next 6 h, and showed a second peak at 9 h. Activity then declined to the pre-feeding level. The dehydrogenase activity of WRC showed a similar pattern but the peaks of activity were less pronounced. It was concluded that dehydrogenase activity based on TTC reduction provides a simple and rapid means of estimating the fermentation activity of rumen contents which may reflect the availability of energy substrates to the microbiota.

scholarly journals 372 Effect of rumen fluid collection methodology on extent and variance of in vitro dry matter disappearance and interaction with substrate and incubation time

2019 ◽  
Vol 97 (Supplement_2) ◽  
pp. 155-156
Author(s):  
Emma L Rients ◽  
Sara M Tondini ◽  
Daniel W Shike ◽  
Joshua C McCann
Keyword(s):  
Incubation Time ◽  
Dry Matter ◽  
Rumen Fluid ◽  
Fluid Collection ◽  
Stomach Tube ◽  
Time Points ◽  
Substrate Interaction ◽  
Time Interaction ◽  
Tube Suction

Abstract The objective was to evaluate the interaction of rumen fluid collection methodology, substrate, and incubation time on the extent and variance of in vitro dry matter disappearance (IVDMD). A 4×2×2 factorial design was used to test the interaction between rumen fluid collection methodology, substrates (hay and silage), and incubation time-points (24 and 48 h). Four methods of rumen fluid collection were evaluated: stomach tube (TUBE), suction strainer through rumen cannula (STRN), ruminal contents collected through cannula and squeezed through cheesecloth (SQZ), and ruminal contents blended for 1 minute and squeezed through cheesecloth (BSQZ). Four replications of each treatment were used and repeated in three periods. A methodology by substrate interaction (P < 0.01) was observed for pH; hay had a greater pH than silage and the magnitude of this difference was greatest for STRN. A tendency for a methodology by substrate interaction (P = 0.06) was observed for ammonia. For TUBE ammonia was greater (P < 0.01) with hay while substrate did not affect ammonia for BSQZ, SQZ and STRN. A methodology by time interaction was observed (P < 0.01) for ammonia; TUBE was least at 24 h, but TUBE and BSQZ were lesser (P ≤ 0.01) than STRN and SQZ at 48 h. A tendency for a methodology by substrate interaction (P = 0.08) was observed in IVDMD; silage had greater IVDMD than hay and the magnitude of this difference was greatest for TUBE. Collection methodology affected (P < 0.01) IVDMD. The greatest IVDMD was observed for STRN (64.7%), with BSQZ (59.1%) and SQZ (57.7%) being intermediate and TUBE (54.3%) being the least. A tendency for a methodology by substrate interaction (P = 0.11) was observed for variation in IVDMD; silage had greater variation than hay for TUBE. Rumen fluid collection methodology affects IVDMD and may depend on substrate.

scholarly journals Relationship between In Vitro Activities of Amphotericin B and Flucytosine and pH for Clinical Yeast and Mold Isolates

2005 ◽  
Vol 49 (8) ◽  
pp. 3341-3346 ◽  
Author(s):  
D. T. A. te Dorsthorst ◽  
P. E. Verweij ◽  
J. F. G. M. Meis ◽  
J. W. Mouton
Keyword(s):  
Amphotericin B ◽  
Phosphate Buffer ◽  
Vitro Activity ◽  
Nonlinear Relationship ◽  
In Vitro Activity ◽  
The Mean ◽  
Ph Range ◽  
Citrate Phosphate ◽  
Citrate Phosphate Buffer

ABSTRACT In this study, we investigated the pH dependency of the in vitro activities of amphotericin B (AMB) and flucytosine (5FC) against Candida spp., Cryptococcus neoformans, Aspergillus fumigatus, Rhizopus spp., and Scedosporium prolificans in RPMI 1640 buffered with citrate buffer (pH 4.0, 5.0, 5.4, and 6.0), citrate-phosphate buffer (pH 5.4, 6.0, 6.4, and 7.0), and 3-[N-morpholino]propanesulfonic acid (MOPS) (pH 6.4, 7.0, 7.4, and 7.9). For 5FC, no significant differences were found between MICs obtained with the different buffers, while for AMB, significant differences were found. The MICs obtained with citrate-phosphate buffer were approximately 1 twofold-dilution step higher than the MICs obtained with MOPS. We demonstrated that the in vitro activities of AMB and 5FC against yeast and mold isolates were pH dependent. The in vitro activity of AMB decreased when the pH was lowered, while the in vitro activity of 5FC increased. The effect of the pH on the in vitro activities was dependent not only on the antifungal agent tested but also on the microorganism. For AMB, there was a nonlinear relationship (median r 2, 0.864) for Candida spp., C. neoformans, A. fumigatus, and Rhizopus spp. over the pH range tested. The mean MICs ranged from 0.5 to 2.52 μg/ml at pH 7.0 and from 20.16 to 32 μg/ml at pH 5.0. For S. prolificans, there was no relationship. For 5FC, there was a linear relationship for Candida spp. (median r 2, 0.767) and a nonlinear relationship for C. neoformans and A. fumigatus (median r 2, 0.882) over the pH range tested. The mean MIC values ranged from 0.125 to 1,024 μg/ml at pH 7.0 and from 0.02 to 4 μg/ml at pH 5.0. For Rhizopus spp. and S. prolificans, the relationship could not be determined, since the MIC was >1,024 μg/ml over a pH range of 4.0 to 7.9.

EFFECT OF BARLEY SUPPLEMENT TO THE RATION OF DONOR ANIMALS USED IN IN VITRO DIGESTIBILITY DETERMINATION

1970 ◽  
Vol 50 (2) ◽  
pp. 265-267 ◽  
Author(s):  
F. W. CALDER
Keyword(s):  
Hordeum Vulgare ◽  
Dry Matter ◽  
Rumen Fluid ◽  
Barley Grain ◽  
In Vitro Digestibility ◽  
Hordeum Vulgare L ◽  
Alfalfa Hay ◽  
Medicago Sativa L ◽  
Ad Libitum

Rumen fluid from two sheep was used to determine in vitro dry matter digestibilities of 25 samples each from alfalfa hay (Medicago sativa L.) and barley grain (Hordeum vulgare L.). The sheep were fed either a good quality hay ad libitum and 1 kg rolled barley per day, or only the hay ad libitum, in a switchback design. The in vitro dry matter digestibilities of both alfalfa hay and barley were lower (P < 0.01) when the donor animal was fed hay and barley than when fed only hay.

scholarly journals PSI-4 Effects of increasing metabolizable protein on starch digestion in lightweight beef cattle

2020 ◽  
Vol 98 (Supplement_3) ◽  
pp. 226-226
Author(s):  
Cierra Crowell ◽  
Sara Tondini ◽  
Miles Redden ◽  
Daniel W Shike ◽  
Joshua C McCann
Keyword(s):  
Fixed Effects ◽  
Rumen Fluid ◽  
Random Effect ◽  
Beef Steers ◽  
Starch Digestion ◽  
Stomach Tube ◽  
Metabolizable Protein ◽  
D 12 ◽  
Dietary Treatments

Abstract The objective was to evaluate the effect of increasing MP on starch digestion in lightweight beef steers. Greater provision of metabolizable protein (MP) may increase RUP and aid in pancreatic amylase production for optimum digestion of starch in the small intestine. Twelve crossbred steers were early weaned and used in a crossover design with two 18-day periods and 4 dietary treatments. Lightweight steers (BW = 153 kg) were randomly assigned to a treatment sequence for the 2 periods. Diets provided MP at: 0.59 (MP1), 0.69 (MP2), 0.85 (MP3), and 0.91 kg/d (MP4) based on observed DMI. Additional MP was provided through greater inclusion of blood meal. Feed and total feces were collected on d 12-18 to determine total tract starch digestion. Rumen fluid was sampled prior to feeding on d 17 via stomach tube to assess in vitro starch disappearance over 8 h for each steer. Data were analyzed using the Mixed procedure of SAS with fixed effects of treatment and period, and a random effect of steer. Starch intake was not affected (P = 0.18) by treatment with MP1 and MP4 having similar starch intake values (3.3 kg/d and 3.1 kg/d, respectively). In vitro starch disappearance and final in vitro pH were not affected (P ≥ 0.18) by increasing MP. Similarly, fecal starch output was not different (P = 0.65) among treatments with MP1 treated steers having 0.21 kg/d of starch output and MP4 steers having 0.18 kg/d of starch output. Low fecal starch output values resulted in high total tract starch digestion ranging from 90-96% and were not affected (P = 0.52) by treatment. Overall, increasing dietary MP did not affect ruminal in vitro or total tract starch digestion.

EFFECTS OF TAMOXIFEN ON THE BINDING AND METABOLISM OF TESTOSTERONE BY HUMAN PROSTATIC TISSUE AND PLASMA IN VITRO

1979 ◽  
Vol 83 (3) ◽  
pp. 369-378 ◽  
Author(s):  
F. K. HABIB ◽  
G. RAFATI ◽  
M. R. G. ROBINSON ◽  
S. R. STITCH
Keyword(s):  
Dehydrogenase Activity ◽  
Reductase Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Inhibitory Effect ◽  
Prostatic Tissue ◽  
Sex Hormone Binding Globulin ◽  
Benign Tissue ◽  
The Mean ◽  
Malignant Prostate

The in-vitro metabolism of testosterone in benign and malignant prostatic tissue was examined and distinct quantitative differences between the two types of specimens were observed. The major metabolite of testosterone in the hyperplastic prostate was 5α-dihydrotestosterone and a high 3α(β)-hydroxysteroid dehydrogenase activity was also detected. In the malignant tissue, 5α-reductase activity was considerably reduced and there was little or no androstanediol formed; the 17β-dehydrogenase activity was, however, higher than in the benign tissue. The decrease in 5α-reductase was always followed by a compensatory change in the 3α(β)-hydroxysteroid dehydrogenase of the malignant prostate. The present study revealed that the ratio of the mean activities of 5α-reductase to 3α(β)-hydroxysteroid dehydrogenase in the two types of specimen always remained a constant. Although the antioestrogen, tamoxifen, induced an inhibitory effect on the activities of 5α-reductase and 17β-hydroxysteroid dehydrogenase in the gland, the present investigation also suggested that tamoxifen stimulated the activity of 3α(3β)-hydroxysteroid dehydrogenase. In blood, the action of tamoxifen appeared to be confined to the displacement of androgens from the binding sites on the sex hormone binding globulin.

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

2015 ◽  
Vol 153 (6) ◽  
pp. 1097-1106 ◽  
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.

scholarly journals Dynamics of protozoa in the rumen of cattle

1988 ◽  
Vol 59 (3) ◽  
pp. 429-436 ◽  
Author(s):  
D. Ffoulkes ◽  
R. A. Leng
Keyword(s):  
Production Rate ◽  
Rumen Fluid ◽  
Specific Radioactivity ◽  
Live Weight ◽  
Basal Diet ◽  
Order Kinetic ◽  
First Order ◽  
The Mean ◽  
Lower Digestive Tract

1. The dynamics of protozoa were studied in two groups of rumen-fistulated cattle fed on a basal diet of molassesad 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 measuring14C loss via the methane pool.3. After a single injection of labelled protozoa, the decline in the specific radioactivity (μCi/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.

scholarly journals Nilai VFA dan NH3 Rumput Alam Padang Penggembalaan Kecamatan Haharu Kabupaten Sumba Timur

2021 ◽  
Vol 5 (1) ◽  
pp. 8-12
Author(s):  
Marselinus Hambakodu ◽  
Elvis Pati Ranja ◽  
Made Adi Sudarma
Keyword(s):  
Fatty Acids ◽  
Volatile Fatty Acids ◽  
Steam Distillation ◽  
Rumen Fluid ◽  
Descriptive Analysis ◽  
Survey Method ◽  
Bothriochloa Ischaemum ◽  
Natural Grass ◽  
The Mean

This study aims to determine the value of volatile fatty acids (VFA) and ammonia (NH3) of natural grass in grazing fields in vitro. The study used a direct survey method in the field. Natural grass was tested in vitro using Bali cattle rumen fluid. The data obtained were analyzed using descriptive analysis based on the mean and standard deviation. Natural grass consisted of Heteropogon insignis, Bracharia decumbens, Bothriochloa ischaemum. The measurement of the value of VFA uses steam distillation, while the method of measuring the value of NH3 uses Conway micro diffusion. The results showed that Bracharia decumbens grass had a total VFA value of 74.48 mM and an NH3 value of 8.50 mM which was higher than Heteropogon insignis grass (total VFA 65.79 mM and NH3 6.67 mM), and Bothriochloa ischaemum grass (total VFA 60.64 mM and NH3 5.34 mM). The conclusion of this study was based on the value of VFA and NH3, Bracharia decumbens grass was a natural grass that may be cultivated for ruminants.

The effects of hay and silage on growth and rumen function in young Holstein calves

1991 ◽  
Vol 71 (1) ◽  
pp. 145-153
Author(s):  
Roy S. Bush
Keyword(s):  
Plant Cell Wall ◽  
Rumen Fluid ◽  
Gas Production ◽  
Wall Material ◽  
Animal Performance ◽  
Grass Silage ◽  
Alfalfa Hay ◽  
In Vitro Gas Production ◽  
Rumen Development

Newborn calves were fed one of four different roughages (timothy hay, alfalfa hay, grass silage or two parts grass silage mixed with one part alfalfa hay) and a starter ration to a maximum of 2000 g d−1 beginning at 10 d of age along with whole milk to weaning at 28 d of age to measure the effect of forage type and method of conservation on voluntary intake and animal performance up to 112 d of age. Rumen fluid was sampled to determine whether the type of forage had a measureable effect on some gross parameters associated with rumen development. Forage dry matter became a larger proportion of total feed consumed as the animals became older. Overall alfalfa hay and silage:alfalfa mixture were consumed more than timothy hay (P < 0.01). Calves fed silage:alfalfa grew more rapidly than those fed timothy (P < 0.01). These differences were attributable to differences in forage protein and plant cell wall material. The differences in rumen fluid volatile fatty acid composition, pH, or in vitro gas production were found for treatments in only two sampling periods. In the 3rd period, gas production was more rapid from alfalfa hay than from silage (P < 0.05) and in the 4th period, pH was greater for alfalfa hay than timothy hay (P < 0.05). Rumen pH increased in the 3rd and 4th periods (P < 0.01). The acetate:propionate ratio increased over time when the propionate values decreased from the 2nd to the 3rd period (P < 0.01) and acetate values increased from the 3rd to the 4th period (P < 0.01). Silage was equal to hay in terms of consumption, animal performance and those rumen fluid parameters examined and can be fed to young calves without any anticipated problems. Key words: Silage, hay, rumen, development, methane, calves

scholarly journals 53 Comparison of digestibility of forages in Jersey steers supplemented with a yeast fermentation product on a high and low quality forage diet

2019 ◽  
Vol 97 (Supplement_1) ◽  
pp. 84-85
Author(s):  
Anissa R Garcia ◽  
Jamie Boyd ◽  
Rebecca Splan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rumen Fluid ◽  
Statistical Significance ◽  
Yeast Fermentation ◽  
Analysis Model ◽  
Orchard Grass ◽  
Alfalfa Hay ◽  
Fermentation Product ◽  
Yeast Product

Abstract The objective of this study was to determine the effect on digestibility of forages using a daisy jar system with inoculum from steers supplemented with the yeast fermentation product Saccharomyces cerevisiae. Forages tested included ryegrass, wheat, orchard grass, alfalfa, and sorghum sudan hays. Samples were ground through a 1mm screen, placed in Ankom F57 bags, and run in triplicate to increase statistical significance. Two ruminally cannulated jersey steers were used in a switchback design. Animals were housed in a dry lot pen. The study consisted of 2 three-week periods, 2-week standardization followed by a 1-week test period. Diets offered for period 1 were steer A = 100% alfalfa hay w/o yeast product and steer B = 100% wheat hay w/o yeast product. Diets offered for period 2 were steer A =100% alfalfa hay +15g/h/d yeast product and steer B =100% wheat hay +15g/h/d yeast product. During the test week, rumen fluid contents were collected from each donor via rumen cannula approximately 4 h after feeding and taken to the lab to be composited. The samples were incubated for 0 h, 24 h, 48 h, 72 h, and 96 h. At the end of the incubation period bags were collected and cold shocked to stop microbial digestion. Samples were dried at 55oC and stored for later analysis. Model included trt (+/-yeast), forage, hr, and interactions forage*hr and forage*trt. No significant differences were observed (P = 0.38) on DM digestibility or NDF digestibility (P = 0.76) with the supplementation of yeast fermentation product. Forage*Trt was significant (P = 0.03 DMdig, P = 0.02 NDFdig). Implications of this study are the inclusion of a Saccharomyces cerevisiae fermentation product at a rate of 15g/h/d may not impact overall forage NDF or DM digestibility but may impact types of forage differently in an in vitro system using composited inoculum from donors on a forage diet.

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