In vivo vs. in vitro Continuous Culture of Ruminal Microbial Populations

ABSTRACT In the intestinal tracts of animals, methanogenesis from CO2 and other C1 compounds strictly depends on the supply of electron donors by fermenting bacteria, but sources and sinks of reducing equivalents may be spatially separated. Microsensor measurements in the intestinal tract of the omnivorous cockroachBlaberus sp. showed that molecular hydrogen strongly accumulated in the midgut (H2 partial pressures of 3 to 26 kPa), whereas it was not detectable (<0.1 kPa) in the posterior hindgut. Moreover, living cockroaches emitted large quantities of CH4 [105 ± 49 nmol (g of cockroach)−1h−1] but only traces of H2. In vitro incubation of isolated gut compartments, however, revealed that the midguts produced considerable amounts of H2, whereas hindguts emitted only CH4 [106 ± 58 and 71 ± 50 nmol (g of cockroach)−1 h−1, respectively]. When ligated midgut and hindgut segments were incubated in the same vials, methane emission increased by 28% over that of isolated hindguts, whereas only traces of H2 accumulated in the headspace. Radial hydrogen profiles obtained under air enriched with H2 (20 kPa) identified the hindgut as an efficient sink for externally supplied H2. A cross-epithelial transfer of hydrogen from the midgut to the hindgut compartment was clearly evidenced by the steep H2 concentration gradients which developed when ligated fragments of midgut and hindgut were placed on top of each other—a configuration that simulates the situation in vivo. These findings emphasize that it is essential to analyze the compartmentalization of the gut and the spatial organization of its microbiota in order to understand the functional interactions among different microbial populations during digestion.

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78 INTERFERON-τ SECRETION OF CRYOPRESERVED BOVINE TROPHOBLASTIC VESICLES DERIVED FROM IN VIVO

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab78 ◽

2012 ◽

Vol 24 (1) ◽

pp. 151

Author(s):

Y. Hashiyada ◽

H. Takahashi ◽

K. Imai ◽

M. Geshi

Keyword(s):

Continuous Culture ◽

Culture Media ◽

Calf Serum ◽

Measure Concentration ◽

Fetal Bovine ◽

The Mean ◽

Student’S T ◽

Student’S T Test

The co-transfer of bovine trophoblastic vesicles (bTVs) prepared from in vivo recovered conceptuses is known to promote the successful implantation of embryos, which expected lower viability, through the effects of interferon-τ (IFN-τ) secreted by bTVs. We have reported that the pregnancy rate was improved for co-transferred embryos with frozen-thawed bTVs using the direct-transfer technique (Hashiyada et al. 2008, 41st SSR). However, the IFN-τ secretion level from cryopreserved bTVs is not well known. The objective of the present study was to measure concentration of IFN-τ released from frozen-thawed bTVs individually cultured in vitro. bTVs were prepared from elongating blastocysts 3 to 20 mm in length, following superstimulatory treatment and recovered on Day 16 post-AI, by dissection using a surgical blade. Each trophoblastic fragment, 1 to 1.5 mm in width, was cultured in a well of a 96-well plate using TCM-199 supplemented with 20% (v/v) fetal bovine serum and 0.1 mM β-mercaptoethanol. Formed vesicles after 24 or 48 h of culture were cryopreserved using D-PBS supplemented with 20% calf serum, 1.5 M ethylene glycol (EG) and 0.1 M sucrose or 1.8 M EG. After thawing, each bTVs was cultured for 2 days to compare IFN-τ secretion between the 2 cryoprotectants. Furthermore, transition of IFN-τ level was assessed in continuous culture until Day 10 (the day of thawing was defined as Day 0). The volume of culture medium was 100 μL well–1 day–1 until Day 2 and thereafter changed to 200 μL well–1 day–1 until termination. Exchange and collection of culture media were performed on Day 1, 2, 4, 6, 8 and 10. Collected culture media were stored at –30°C until use. IFN-τ was measured by RIA (Takahashi et al. 2005 Theriogenology 63, 1050–1060). Data were analysed by Student's t-test. Initial IFN-τ secretion from bTVs before cryopreservation did not differ between 24 and 48 h of culture period to form vesicles, 44.0 ± 2.9 (mean ± standard error of the mean, n = 64) and 52.8 ± 6.4 ng mL–1 (n = 27), respectively. IFN-τ secretion was no difference between the 1.5 M EG group and the 1.8 M EG group on Day 1 (41.2 ± 4.9 ng mL–1, n = 42 and 30.4 ± 2.2 ng mL–1, n = 31) and on Day 2 (38.0 ± 5.4 and 38.2 ± 4.5 ng mL–1), respectively. In the continuous culture group (n = 28), IFN-τ secretion tended to increase from Day 2 (25.2 ± 3.4 ng mL–1) to Day 4 (51.8 ± 12.3 ng mL–1) and 6 (55.4 ± 13.3 ng mL–1) (P < 0.05). However, this amount of IFN-τ on Day 6 significantly decreased on Day 8 (25.6 ± 2.7 ng mL–1; P < 0.05) and Day 10 (15.5 ± 2.2 ng mL–1; P < 0.01), gradually. These results indicate that cryopreserved bTVs could secrete IFN-τ at the same level as fresh bTVs on Day 4 to 6 after thawing and then these amounts of IFN-τ significantly decrease in vitro.

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Rumen methanogens, and inhibition of methanogenesis

Australian Journal of Agricultural Research ◽

10.1071/ar99005 ◽

1999 ◽

Vol 50 (8) ◽

pp. 1293 ◽

Cited By ~ 21

Author(s):

S. K. Baker

Keyword(s):

Normal Component ◽

Distinct Group ◽

Microbial Populations ◽

Ch4 Production ◽

Enhanced Production ◽

Wide Range ◽

Gross Energy ◽

Strong Inverse Relationship

Methane-producing archaea (known as methanogens) are a distinct group of organisms which are a normal component of the rumen microbial ecosystem. Hydrogen and carbon dioxide are the principal substrates used by rumen methanogens to produce methane (CH4). Because hydrogen and formate are products of fermentation by other microorganisms in the rumen, inhibition of fermentation by other members of the rumen microbial population may in turn inhibit methanogenesis. As well, compounds that inhibit the activity of methanogens directly are likely to reduce or eliminate CH4 production. A strong inverse relationship between the molar proportion of propionate and CH4 production is predicted from knowledge of the interactions among microbial populations in the rumen, and compounds that promote greater production of propionate in the rumen may also have the effect of decreasing CH4 production. Although a wide range of ionophores, antibiotics, and other compounds have been evaluated in vivo and in vitro for their influence on rumen microbial populations and on propionate production, re-evaluation of some of these has been prompted by a search for compounds that both enhance propionate production and decrease production. Where there is a focus on CH4 production by livestock, interest necessarily is in the total amount of CH4 produced per day as a proportion of gross energy intake (%GEI) and its relationship with animal productivity. Because enhanced production of propionate in the rumen also can be associated with an increase in the flow of microbial protein from the rumen, evaluation or re-evaluation of compounds that may be effective in reducing methane production should also include evaluation of the effects on animal productivity, and appropriate approaches are discussed.

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THE FATE OF MYCOBACTERIUM TUBERCULOSIS IN MOUSE TISSUES AS DETERMINED BY THE MICROBIAL ENUMERATION TECHNIQUE

Journal of Experimental Medicine ◽

10.1084/jem.104.5.763 ◽

1956 ◽

Vol 104 (5) ◽

pp. 763-802 ◽

Cited By ~ 152

Author(s):

Robert M. McCune ◽

Ralph Tompsett ◽

Walsh McDermott

Keyword(s):

Microbial Populations ◽

Complete Disappearance ◽

Antituberculous Drugs ◽

Mouse Tissues ◽

Two Factors ◽

Posttreatment Period ◽

Definition Of ◽

Microbial Enumeration

Populations of tubercle bacilli of human origin exposed in vivo to pyrazinamide and a companion drug, vanished from the tissues of the mouse in so far as could be determined by microscopy, culture, or guinea pig subinoculation. The vanishing did not represent a complete elimination of the tubercle bacilli from all the animals. 90 days after the completion of treatment, tubercle bacilli could be cultured from approximately one-third of the animals examined at that time. This complete disappearance of the tubercle bacilli thus meets the definition of a truly latent infection in that the infection is present but is hidden beyond the limits of diagnostic reach. All but one of the strains of tubercle bacilli which survived in the animals and were detectable in the posttreatment period, were susceptible to pyrazinamide when tested under appropriate conditions in vitro. Only two factors could be identified which were essential for the uniform occurrence of the disappearance of tubercle bacilli: the administration of the pyrazinamide in a high daily dosage for at least eight of a total of 12 weeks of antimicrobial therapy; and the concurrent or prior exposure of the microbial populations to isoniazid or in some cases to other antituberculous drugs. The observations suggest that the ability of pyrazinamide-containing chemotherapies to bring about the disappearance of a tubercle bacillus is closely related to the occurrence of some alteration in the bacillus, essential for maximal pyrazinamide action, in response to environmental influences, including other antituberculous drugs present in the environment.

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In Vitro Fermentation Properties of Selected Fructooligosaccharide-Containing Vegetables and In Vivo Colonic Microbial Populations Are Affected by the Diets of Healthy Human Infants

Journal of Nutrition ◽

10.1093/jn/132.8.2188 ◽

2002 ◽

Vol 132 (8) ◽

pp. 2188-2194 ◽

Cited By ~ 18

Author(s):

Elizabeth A. Flickinger ◽

Terry F. Hatch ◽

Rachel C. Wofford ◽

Christine M. Grieshop ◽

Sean M. Murray ◽

...

Keyword(s):

Microbial Populations ◽

Human Infants ◽

Healthy Human ◽

In Vitro Fermentation

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Some effects of potassium chlorate administration onin vitroandin vivorumen fermentation

The Journal of Agricultural Science ◽

10.1017/s0021859600055453 ◽

1978 ◽

Vol 90 (2) ◽

pp. 345-353 ◽

Cited By ~ 3

Author(s):

T. N. Barry ◽

F. J. Harte ◽

B. N. Perry ◽

D. G. Armstrong

Keyword(s):

Continuous Culture ◽

Propionic Acid ◽

Microbial Population ◽

Rumen Fluid ◽

Rumen Fermentation ◽

Potassium Chlorate ◽

Temporary Reduction ◽

Cellulose Digestion

SummaryHay was fed to anin vitrocontinuous culture of the rumen microbial population and to sheep kept in metabolism cages, and the effects of potassium chlorate addition on the rumen fermentation were studied. The compound was given for 8 daysin vitroand for either 3 or 8 daysin vivo.Potassium chlorate additionin vitro(13·7 mg/g hay D.M.) depressed the production of CH4and acetate, had little effect on propionate production and caused a small increase in the production ofn-butyrate andn-valerate. The treatment also depressed cellulose digestion and the concentration of DNA in fermentor liquor, but increased the CO2:CH4ratio in fermentor gas.When givenin vivofor 8 days at 6·7 mg/g hay D.M., potassium chlorate progressively depressed total VFA concentration in rumen fluid, had no effect on VFA molar proportions but caused a small increase in the CO2:CH4ratio in rumen gas. When administeredin vivofor 3 days at 14·4–15·3 mg/g hay D.M. the treatment increased the molar proportions of propionate and depressed those of acetate in rumen fluid without affecting total VFA concentration. There were considerable differences between animals in propionate response, and the maximum responses were generally obtained 2–5 days after dosing had ceased. Potassium chlorate addition also caused a temporary reduction in appetite with some sheep.It was concluded that potassium chlorate was toxic to the rumen microbial population when given for 8 days, but that large doses given over 3 days could be used to increase the ratio of propionic acid relative to acetic and butyric acids produced from the rumen fermentation.

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Glucose-Based Oligosaccharides Exhibit Different In Vitro Fermentation Patterns and Affect In Vivo Apparent Nutrient Digestibility and Microbial Populations in Dogs

Journal of Nutrition ◽

10.1093/jn/130.5.1267 ◽

2000 ◽

Vol 130 (5) ◽

pp. 1267-1273 ◽

Cited By ~ 70

Author(s):

Elizabeth A. Flickinger ◽

Bryan W. Wolf ◽

Keith A. Garleb ◽

JoMay Chow ◽

Gregory J. Leyer ◽

...

Keyword(s):

Nutrient Digestibility ◽

Microbial Populations ◽

In Vitro Fermentation

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Dilution as a Solution: Targeting Microbial Populations with a Simplified Dilution Strategy

mSphere ◽

10.1128/msphere.00701-20 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

Disha Bhattacharjee ◽

Anna M. Seekatz

Keyword(s):

Health Care ◽

Gut Microbiota ◽

Rumen fermentation studies on two contrasting diets. 2. Comparison of the performance of anin vitrocontinuous-culture fermentation within vivofermentation

The Journal of Agricultural Science ◽

10.1017/s0021859600027374 ◽

1977 ◽

Vol 89 (1) ◽

pp. 197-208 ◽

Cited By ~ 6

Author(s):

T. N. Barry ◽

A. Thompson ◽

D. G. Armstrong

Keyword(s):

Steady State ◽

Continuous Culture ◽

Osmotic Pressure ◽

Dna Concentration ◽

Initial Inoculum ◽

Low Levels ◽

Reduced Rate ◽

Time Required

SummaryAn impermeable continuous-culture artificial rumen was constructed and rumen microbial populations were culturedin vitrofor periods of up to 23 days. Diets of hay and 20:80 hay:cooked flaked maize (concentrate diet) were fed to thein vitrocultures and also to sheep, and the performance of thein vitrofermentation compared with that of thein vivofermentation.With the hay diet, changing the initial inoculum from strained rumen liquor to unmodified rumen contents (containing solid ingesta) reduced the time required to attain a steady-state fermentationin vitrofrom 13 to 4 days. VFA molar proportions and the CO2: CH4ratio indicated that the type of fermentation taking placein vitrowas remarkably close toin vivofermentation. However, rates of cellulose digestion, CH4production and number of bacteria/unit volume (as judged by DNA concentration) were much lessin vitrothanin vivo, showing that the rate of fermentation was reduced in the artificial rumen. Total VFA concentration and the osmotic pressure of fermentor liquor were slightly higher than the maximum values foundin vivo, and it was concluded that these were responsible for the reduced rate of fermentation.Regardless of the duration of culture, a satisfactory steady-state fermentation was never attained when the concentrate diet was administered. DNA concentration in fermentor liquor declined to very low levels after 4 days; CH4production rapidly declined and was replaced by H2production after 5 days. VFA molar proportions bore little resemblance to those foundin vivo. Both the osmotic pressure of fermentor contents and total VFA concentration rapidly increased with time, and by day 3 were approximately 2·5 times the values foundin vivo. It was concluded that high VFA concentration, and the associated increase in osmotic pressure, were the main factors responsible for the unrealisticin vitrofermentation, and that this problem was much greater than encountered with the hay diet due to starch being digested at a faster rate than cellulose and hemicellulose.Methods of regulating VFA concentration in continuous culturein vitrorumen systems are discussed and some improvements suggested.

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Induction and Differentiation of Dental Epithelium in Vivo and in Vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100053425 ◽

1982 ◽

Vol 40 ◽

pp. 142-145

Author(s):

E. J. Kollar

Keyword(s):

Connective Tissue ◽

Oral Mucosa ◽

Basal Lamina ◽

Functional Derivatives ◽

Specific Source ◽

Dental Epithelium ◽

Connective Tissue Stroma

The differentiation and maintenance of many specialized epithelial structures are dependent on the underlying connective tissue stroma and on an intact basal lamina. These requirements are especially stringent in the development and maintenance of the skin and oral mucosa. The keratinization patterns of thin or thick cornified layers as well as the appearance of specialized functional derivatives such as hair and teeth can be correlated with the specific source of stroma which supports these differentiated expressions.

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