Lyophilization of rumen fluid for use in culture media.

The supernatant from centrifugation at 1,000 x g of strained rumen fluid was lyophilized, and the residue and sublimate fractions were used to replace fresh rumen fluid in a complete roll tube medium for enumeration of total rumen bacteria. Most of the growth-supporting nutrients in fresh rumen fluid were found in the residue fraction. With one exception, no significant differences were found in total bacterial numbers either by roll tube or most-probable-number procedures when lyophilized rumen fluid residue was substituted for fresh rumen fluid. Lyophilized rumen fluid residue was stable for at least 5 months at room temperature. Rumen fluid supernatant from centrifugation at 1,000 x g had a mean density of 1.005 +/- 0.03 g/ml and contained 1.56% +/- 0.30% dry matter. On the basis of these values, 15.68 mg of lyophilized rumen fluid residue is equivalent to 1 ml of rumen fluid supernatant from centrifugation at 1,000 x g.     

Variation in colony counts of total viable anaerobic rumen bacteria as influenced by media and cultural methods.

Volume and type of medium, carbohydrate concentration, carbohydrate ratios, and inoculum level were investigated as possible factors influencing total colony counts of anaerobic rumen bacteria obtained in roll tubes (18 by 150 mm). Colony counts were lower when the rumen fluid was clarified by centrifugation before inclusion in the medium; however, decreasing the volume of 40% rumen fluid glucose-cellobiose-starch-agar medium (RGCSA medium with 0.025% each of glucose and cellobiose and 0.05% starch, 4 ml per tube) was compared to the clarified rumen fluid medium and non-rumen fluid medium (medium 10) of Caldwell and Bryant (1966), 9 ml of each per tube. Total counts of rumen contents from sheep consuming four different types of rations were higher with the 4 ml of RGCSA medium than with the other two media. Dilution of the basal medium as a result of inoculum volume, as much as 1.5 ml per 4 ml of medium, did not appear to affect total counts. Colony counts and the simplicity of medium preparation and inoculation would favor the present method for routine use in estimating numbers of total viable anaerobic rumen bacteria, especially when large numbers of samples are involved.     

Metabolism of toxic pyrrolizidine alkaloids from tansy ragwort (Senecio jacobaea) in ovine ruminal fluid under anaerobic conditions.

The ability of ovine ruminal fluid to metabolize pyrrolizidine alkaloid (PA) from Senecio jacobaea under anaerobic conditions was evaluated. Four fistulated sheep fed PA served as individual sources of ruminal fluid, which was incubated in a defined minimal salts medium under two different anaerobic conditions, denitrifying and methanogenic. Anaerobic cultures amended with ovine ruminal fluids (20%), PA (100 micrograms/ml), and a defined minimal salts medium were monitored for a period of several days. These cultures revealed that while PA was not depleted in sterile, autoclaved controls or under denitrifying conditions, it was metabolized during periods of active methanogenesis under methanogenic conditions. In addition, samples of ruminal fluid were separated by differential centrifugation under anaerobic conditions, and the resultant supernatants were tested for their ability to metabolize PA as compared with those of the respective uncentrifuged control fluids. Uncentrifuged controls exhibited a PA depletion rate of -4.04 +/- 0.17 micrograms of PA per ml per h. Supernatants 1 (centrifuged at 41 x g for 2 min), 2 (centrifuged at 166 x g for 5 min), and 3 (centrifuged at 1,500 x g for 10 min) exhibited significantly slower depletion rates, with slopes of data representing -1.64 +/- 0.16, -1.44 +/- 0.16, and -1.48 +/- 0.16 micrograms of PA metabolized per ml per h, respectively, demonstrating no statistically significant difference among the supernatant cultures. Microscopic evaluations revealed that protozoa were present in the control whole ruminal fluid and to a lesser extent in supernatant 1, while supernatants 2 and 3 contained only bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of toxic pyrrolizidine alkaloids from tansy ragwort (Senecio jacobaea) in ovine ruminal fluid under anaerobic conditions.

The ability of ovine ruminal fluid to metabolize pyrrolizidine alkaloid (PA) from Senecio jacobaea under anaerobic conditions was evaluated. Four fistulated sheep fed PA served as individual sources of ruminal fluid, which was incubated in a defined minimal salts medium under two different anaerobic conditions, denitrifying and methanogenic. Anaerobic cultures amended with ovine ruminal fluids (20%), PA (100 micrograms/ml), and a defined minimal salts medium were monitored for a period of several days. These cultures revealed that while PA was not depleted in sterile, autoclaved controls or under denitrifying conditions, it was metabolized during periods of active methanogenesis under methanogenic conditions. In addition, samples of ruminal fluid were separated by differential centrifugation under anaerobic conditions, and the resultant supernatants were tested for their ability to metabolize PA as compared with those of the respective uncentrifuged control fluids. Uncentrifuged controls exhibited a PA depletion rate of -4.04 +/- 0.17 micrograms of PA per ml per h. Supernatants 1 (centrifuged at 41 x g for 2 min), 2 (centrifuged at 166 x g for 5 min), and 3 (centrifuged at 1,500 x g for 10 min) exhibited significantly slower depletion rates, with slopes of data representing -1.64 +/- 0.16, -1.44 +/- 0.16, and -1.48 +/- 0.16 micrograms of PA metabolized per ml per h, respectively, demonstrating no statistically significant difference among the supernatant cultures. Microscopic evaluations revealed that protozoa were present in the control whole ruminal fluid and to a lesser extent in supernatant 1, while supernatants 2 and 3 contained only bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of centrifugation on in vitro survival of fresh diluted canine spermatozoa

Prostatic fluid is unsuitable for preserving dog semen at 4 degrees C and exerts harmful effects upon the spermatozoa during the freezing process. Centrifugation immediately after sperm collection is a common method to remove prostatic admixture. In the present study, dog semen, diluted to 25 x 10(6)/ml, was exposed for 5 min to four different centrifugation speeds (180 x g, 720 x g, 1620 x g and 2880 x g) to determine subsequent sperm losses in the supernatant and to assess sperm survival over time. Using 180 x g as centrifugation speed, 8.9% of the sperm cells was lost upon supematant removal. Using 720 x g, 1620 x g or 2880 x g, sperm losses were lower, 2.3, 0.4 and 0.006%, respectively. After centrifugation, the sperm pellet was rediluted in egg-yolk-Tris extender, cooled and stored for 3 days at 4 degrees C. Motility, progressive motility, membrane integrity and sperm morphology were assessed daily. Acrosomal status was assessed after 3 days of storage. The only functional parameter which was influenced by centrifugation speed was membrane integrity as evaluated by means of SYBR14-PI staining: significantly more dead and moribund sperm cells were found after centrifugation at 1620 x g and 2880 x g after 48 and 72 h of storage at 4 degrees C. When higher initial sperm concentrations (50 x 10(6), 75 x 10(6) or 100 x 10(6)/ml) were evaluated for sperm losses, less than 2.3% of the initial total sperm cells was lost at lower centrifugation speeds. We conclude that centrifuging dog sperm for 5 min at 720 x g is the best strategy to remove prostatic fluid because the loss of sperm cells is acceptable and the functional parameters of the spermatozoa are well preserved, even after 3 days of storage.     

The suitability of a faecal suspension of sheep as inocula for the estimation of utilizable crude protein of feeds by in vitro incubation

The objective of the experiments was to study the suitability of using a faecal suspension of sheep for the estimation of the utilizable crude protein (uCP) of feeds for sheep by an in vitro incubation. Twenty-four single feeds and eight feed mixtures were used as incubation substrates. In Experiment 1, the gas production after the in vitro incubation with rumen fluid or with a faecal suspension of a sheep were compared using the Hohenheim gas test. It was found that there were significant linear regression between the 24, 48 and 72 h gas production with rumen fluid and those with faecal suspensions of 35, 50, 100 and 150 g wet faeces of sheep (which were 18.6, 23.5, 52.0 and 70.5 g faeces DM, respectively) per litre McDougall's buffer (P < 0.0001). The highest regression coefficient (r2) was calculated between the gas production after inoculation with a suspension of 100 g wet faeces per litre McDougall's buffer (x, ml x 200 mg (-1) feed DM) for 48 h and the gas production after inoculation with rumen fluid (y, ml x 200 mg (-1) feed DM) for 24 h: y = 0.82 (+/- 0.07)x + 9.87 (+/-3.83), r2 = 0.82, n = 32, P < 0.0001. Based on these results, in Experiment 2 the estimation of utilizable crude protein (uCP) of feeds was compared by using the in vitro incubation technique of Zhao and Lebzien (2000), where feeds were inoculated either with rumen fluid or with a faecal suspension (100 g wet faeces of sheep, i.e. 52 g faeces DM per litre McDougall's buffer). The results indicated that there were no significant differences of the estimated uCP after inoculation with rumen fluid or the faecal suspension (P > 0.05). A significant regression was found between the uCP after incubation for 48 h with 100 g wet faeces (x, g x kg (-1) DM) and the uCP after incubation for 24 h with rumen fluid (y, g x kg(-1) DM): y = 0.95 (+/-0.10)x - 4.90 (+/-26.70), r2 = 0.75, n = 32, Although this regression was significant, the coefficient r2 was not high. Therefore, further research is needed before sheep faeces could replace rumen fluid as an inocula for the estimation of uCP by the in vitro incubation technique.     

Rapid isolation of vesicular and micellar carriers of biliary lipids by ultracentrifugation

A simple, rapid, and new method has been developed to isolate and to quantitate the vesicular carrier of biliary lipids by isopycnic ultracentrifugation. The method combines the use of Metrizamide, as an inert centrifugation media to change the density of bile for isopycnic separation of vesicles, and a vertical rotor, to decrease both the time of centrifugation and the pressure of the hydrostatic column in the ultracentrifuge tube. Vesicles harvested from bile-Metrizamide density gradients were identified by negative staining electron microscopy. The buoyant densitites of biliary vesicles varied between 1.010 and 1.030 g/ml. The diameter of vesicles in fractions with d less than 1.020 g/ml was 82 +/- 10 nm and in fraction with d approximately 1.030 g/ml was 57 +/- 8 nm. Gel filtration chromatography with Ultrogel AcA 34 was used to validate the quantitive isolation of vesicles by the ultracentrifugal method. In experiments with bile-Metrizamide continuous preformed density gradients, greater than 93% of vesicular cholesterol was found in fractions with d less than 1.030 g/ml after 285 min of centrifugation at 50,000 rpm in a VTi vertical rotor (Beckman Instruments, Inc.). When 16% Metrizamide was dissolved in bile and centrifuged for 120 min, greater than 96% of total vesicular cholesterol was found in the top 0.4 ml of the 5-ml centrifuge tube, as assessed by gel filtration chromatography. This fraction contained less than 8% of cholesterol carried in micelles, as assessed by gel filtration chromatography. The variation coefficient of this short ultracentrifugal method to isolate biliary vesicles was 4.6%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Some Morphological Types of Bacteriophages in Bovine Rumen Contents

Six morphological types of bacteriophage were found in bovine rumen contents. Minimal total phage count was 5 x 10(7) per ml of rumen fluid.     

Isolation and identification of fecal bacteria from adult swine.

An examination of the fecal microflora of adult swine was made with regard to the efficiency of several roll tube media in enumeration and recovery of anaerobes, the effects of medium constituents on recovery, and the isolation and identification of the predominant kinds of bacteria. Total number of organisms by microscopic bacterial counts varied among fecal samples from 4.48 X 10(10) to 7.40 X 10(10) bacteria/g (wet weight). Comparison of different nonselective roll tube media indicated that about 30% of the fecal bacteria could be recovered with a rumen fluid (40%, vol/vol) medium (M98-5). Recoveries of 21 and 15%, respectively, were obtained with M10 and rumen fluid-glucose-cellobiose agar (RGCA) media. Rumen fluid, Trypticase, sugars, and CO2 gas phase were important components required for maximum recovery with this medium. Similar high recoveries of anaerobes were also obtained with M98-5 containing swine cecal extract of place in rumen fluid or M10 plus swine cecal extract. Significantly lower recoveries were observed with RCGA, media supplemented with swine fecal extracts, reinforced clostridial medium, brain heart infusion agar, and prereduced blood agar. Ninety percent of the bacteria isolated from roll tube media were gram positive and consisted of facultatively anaerobic streptococci, Eubacterium sp., Clostridium sp., and Propionibacterium acnes. The remainder of the flora (8%) included several other species of anaerobes and Escherichia coli. Rumen fluid (or volatile fatty acids), Trypticase, and yeast extract additions to basal media stimulated the growth of anaerobic strains. Variation in the relative proportions of the predominant fecal microflora was observed. This work indicates that satisfactory enumeration, isolation and cultivation of the predominant microflora in swine feces can be obtained when strict anaerobic culture methods and a rumen fluid medium are used.     

Influence of Rumen Fluid Source upon Establishment and Cultivation in vitro of the Rumen Protozoon Entodinium

SYNOPSIS. Favorable conditions for establishing Entodinium cultures in vitro were provided by rendering the inoculum almost free of other genera of rumen ciliates, inoculating at a final concentration of 30,000 entodinia per ml, providing 0.2 mg rice starch per ml, but principally by including an appropriate rumen fluid in the media. Rumen fluids collected from animals with high concentrations of entodinia in vivo, in general, were superior in supporting entodinia in vitro than were those collected from animals having low concentrations of entodinia in vivo. Rumen fluids from lambs were improved by clarification (centrifuging at 25,000 × g for 20 min), but clarification destroyed the ability of rumen fluids from pregnant ewes to support entodinia in vitro. One or more factors occurring in the supernatant portion of pooled, clarified lamb rumen fluid stimulated entodinia in vitro.     

Lysis of Viable Rumen Bacteria in Bovine Rumen Fluid

Streptococcus bovis and Butyrivibrio sp. were labeled with thymidine-methyl-(3)H, washed, and resuspended in rumen fluid or rumen fluid fractions obtained from Holstein and Jersey cows fed alfalfa hay once daily. Factors affecting the lytic activity found in untreated rumen fluid were examined. Day to day variation and differences before and after feeding were observed for the same cow. There were also differences between cows on the same day. For a given rumen fluid, the rate of release of label was roughly proportional to the number of labeled cells present over a 100-fold range in concentration. Removal of protozoa largely abolished the lytic action of fresh rumen fluid for S. bovis, but some soluble lytic activity remained. Mixed rumen protozoa added to media containing labeled S. bovis caused label to appear in solution. In a sample of rumen fluid containing 4.3 x 10(4) protozoa/ml 5.2% of the S. bovis population were destroyed by protozoa per hr. The mean rate of destruction for 12 runs on whole rumen fluid was 8.7% per hr with a standard deviation of 6.05. Parallel experiments with Butyrivibrio indicated that soluble lytic factors were more important for this organism. They could be destroyed by autoclaving and were generated when viable rumen bacteria were resuspended in autoclaved rumen fluid. The lysis of S. bovis and Butyrivibrio, at equal cell densities, by mixed rumen protozoa was compared in 30% rumen fluid media, and Butyrivibrio appeared to be more readily lysed than S. bovis.     

Testicular FSH receptor numbers and affinity in bulls of various ages

Bulls (N = 42) ranging in age from 1 day to 5.5 years were used to determine whether a change in the concentration of FSH receptors in the bovine testis occurred as bulls matured. 125I-labelled human FSH was used as the ligand to evaluate binding to bovine testicular membranes. Membrane fractions were collected by centrifugation of testicular homogenates at 120 g and recentrifugation of the 120 g supernatant at 1250 g. Relative binding activity of membrane sedimented at 1250 g was determined after incubation of membranes with 125I-labelled FSH for 16--18h at 25 degrees C, followed by centrifugation (1250 g) to separate bound from free hormone. Specifically bound FSH when expressed as fmol/mg protein was negatively correlated with age (r = -0.73). The association constant (Ka) determined by Scatchard analysis was the same for bulls at all ages with a mean (+/- s.e.m.) Ka = 1.5 +/- 0.3 x 10(9) M-1. Concentration of FSH receptors on a per mg protein basis declined rapidly from birth to 2.5 years of age and remained low up to 5.5 years of age. On a whole testis basis the total number of receptors increased as the bulls matured. After 2.5 years of age total testicular binding did not change.     

Distinct platelet-activating factor binding sites in synaptic endings and in intracellular membranes of rat cerebral cortex

The binding of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC or PAF, platelet-activating factor) to synaptic plasma membranes, microsomal membranes, and other rat cerebral cortex subcellular fractions was studied. Using several PAF-binding antagonists, three distinct sites were identified. Two of them were in intracellular membranes (microsomes) and one in synaptic plasma membranes. Microsomal membranes were prepared after obtaining a 43,500 x g pellet from the postmitochondrial supernatant and subsequent centrifugation at 105,000 x g of the resulting supernatant. Most plasma membrane markers were retained in the 43,500 x g pellet (Sun, G.Y., Huang, H.-M., Kelleher, J.A., Stubbs, E.B., Sun, A. Y. (1988) Neurochem. Int. 12, 69-77). Microsomes were purified by density-gradient centrifugation and marker enzymes showed relatively very low contamination by plasma membrane markers. Myelin and mitochondria were devoid of specific PAF binding. A site displaying the highest PAF-binding affinity reported to date in all cells and membranes (KD = 22.5 +/- 1.7 pM and Bmax 8.75 = fmol/mg protein), was found in the microsomal fraction. There was a second binding site in microsomal fractions (KD = 25.0 +/- 0.8 nM and Bmax = 0.96 pmol/mg protein. Ca2+ decreases PAF affinity for the microsomal binding sites. The third binding site displays relatively low specific PAF binding and is present in synaptosomal plasma membranes. Moreover, displacement curves by a wide variety of PAF antagonists indicated different affinities for each of the binding sites described here. These results indicate that PAF-binding sites are heterogeneous in rat cerebral cortex, and they imply that the microsomal membrane sites may be involved, at least in part, in intracellular events such as gene expression.     

Urease activity of adherent bacteria and rumen fluid bacteria

In experiments on six sheep fed on a low nitrogen diet (3.7 g N/day), urease (EC 3.5.1.5) activity (nkat X mg-1 bacterial dry weight) 3 h after feeding was found to be highest in the bacteria adhering to the rumen wall (13.25 +/- 2.10), lower in the rumen fluid bacteria (8.96 +/- 1.35) and lowest in the bacteria adhering to feed particles in the rumen (5.69 +/- 2.13). The urease activity of bacteria adhering to the rumen wall and of the rumen fluid bacteria of six sheep fed on a high nitrogen diet (21 g N/day) was significantly lower than in sheep with a low N intake and in both cases was roughly the same (3.81 +/- 1.37 and 3.76 +/- 1.02 respectively); it was lowest in bacteria adhering to feed particles in the rumen (1.92 +/- 0.90). It is concluded from the results that the urease activity of rumen fluid bacteria and of bacteria adhering to the rumen wall and to feed particles in the rumen is different and that it falls significantly in the presence of a high nitrogen intake. From the relatively high ureolytic activity of bacteria adhering to the rumen wall in the presence of a low nitrogen intake it is assumed that this is one of the partial mechanisms of the hydrolysis of blood urea entering the rumen across the rumen wall and of its reutilization in the rumen-liver nitrogen cycle in ruminants.     

Why does the establishment of the starch preferring<Emphasis Type="Italic">Entodinium caudatum</Emphasis> in the rumen decrease the numbers of the fibrolytic ciliate<Emphasis Type="Italic">Eudiplodinium maggii</Emphasis>?

The effect of the establishment of Entodinium caudatum on the population of Eudiplodinium maggii was examined in the rumen of three sheep fed a hay/ground barley diet. The cell concentration of E. maggii were 15.9-38.5 and 11.7-12.4 x 10(3) cells per g of the rumen contents in the absence and presence of E. caudatum, respectively. Microscopic analysis showed that starch was the only material engulfed by eudiplodinia irrespective of the time after feeding and the presence or absence of E. caudatum. Up to 82-93% of individuals contained starch grains when E. maggii was the only ciliate species in the rumen; the proportion was 70-77% after entodinia had been established. The largest quantity of starch engulfed by E. maggii ciliates was 12.4-19.0 and 6.7-7.6 mg per 100 mg protozoal dry mass in the absence and presence of entodinia, respectively. No visible engulfment of hay was observed in vivo in spite of the fact that hay particles up to 42 microns in length were dominating in rumen fluid. Ingestion of fresh particles of hay separated from the rumen digesta was found when they were added in the proportion of 1 g per 40 mL suspension of ciliates. No preferential intake of starch was observed when E. maggii ciliates were incubated in vitro with a mixture of hay and barley starch. It is suggested that competition for starch between the two ciliate species was responsible for the drop in the numbers of E. maggii. This could result from a too low concentration of small particles of hay in the rumen fluid.     

Isolation, Enumeration, and Maintenance of Rumen Anaerobic Fungi in Roll Tubes

Anaerobic phycomycetous fungi have been isolated from rumen fluid by using the Hungate roll tube technique. Cultures on solid substrates in roll tubes can be stored for long periods, and fungal zoospores can be enumerated in roll tubes.     

Ruminal microbial digestion in free-living, in captive lichen-fed, and in starved reindeer (Rangifer tarandus tarandus) in winter.

In free-living (FL) reindeer eating a natural mixed winter diet dominated by lichens, captive (CF) reindeer fed pure lichens ad libitum, and CF reindeer subsequently starved for 1 day (CS1 reindeer) or 4 days (CS4 reindeer), the dominant rumen anaerobic bacteria were characterized, their population densities were estimated, and ruminal pH and volatile fatty acid concentrations were determined. In the FL reindeer, the total median viable anaerobic bacterial population ranged from 18 x 10(8) to 35 x 10(8) cells per ml of rumen fluid (n = 4), compared with 26 x 10(8) to 34 x 10(8) and 0.09 x 10(8) to 0.1 x 10(8) cells per ml of rumen fluid in CF reindeer (n = 2) and CS4 reindeer (n = 2), respectively. The median bacterial population adhering to the rumen solids ranged from 260 x 10(8) to 450 x 10(8), 21 x 10(8) to 38 x 10(8), and 0.5 x 10(8) cells per g (wet weight) of rumen solids in FL, CF, and CS4 reindeer, respectively. Although there were variations in the rumen bacterial composition among the FL reindeer (n = 4), strains of Bacteroides, Fibrobacter, Streptococcus, and Clostridium dominated in the rumen fluid. Streptococcus spp. and Clostridium spp. were the dominant bacteria in the CF reindeer (n = 2), while in the CS4 reindeer (n = 2) the dominant bacteria were Fusobacterium spp., members of the family Enterobacteriaceae, and Eubacterium spp. Transmission electron micrographs of lichen particles from the rumen of one FL reindeer, one CF reindeer, and one CS4 reindeer show bacteria resembling Bacteroides spp. adhering to the lichen particles, evidently digesting the lichen hyphae from the inside.(ABSTRACT TRUNCATED AT 250 WORDS)     

Splenic denervation worsens lipopolysaccharide-induced hypotension, hemoconcentration, and hypovolemia

During lipopolysaccharide (LPS)-induced endotoxemia, increased intrasplenic fluid efflux contributes to a reduction in plasma volume. We hypothesized that splenic sympathetic nerve activity (SSNA), which increases during endotoxemia, limits intrasplenic fluid efflux. We reasoned that splenic denervation would exaggerate LPS-induced intrasplenic fluid efflux and worsen the hypotension, hemoconcentration, and hypovolemia. A nonlethal dose of LPS (150 microg x kg(-1) x h(-1) for 18 h) was infused into conscious male rats bearing transit time flow probes on the splenic artery and vein. Fluid efflux was estimated from the difference in splenic arterial inflow and venous outflow (A-V). LPS significantly increased the (A-V) flow differential (fluid efflux) in intact rats (saline -0.01 +/- 0.02 ml/min, n = 8 vs. LPS +0.21 +/- 0.06 ml/min, n = 8); this was exaggerated in splenic denervated rats (saline -0.03 +/- 0.01 ml/min, n = 7 vs. LPS +0.41 +/- 0.08 ml/min, n = 8). Splenic denervation also exacerbated the LPS-induced hypotension, hemoconcentration, and hypovolemia (peak fall in mean arterial pressure: denervated 19 +/- 3 mmHg, n = 10 vs. intact 12 +/- 1 mmHg, n = 8; peak rise in hematocrit: denervated 6.7 +/- 0.3%, n = 8 vs. intact 5.0 +/- 0.3%, n = 8; decrease in plasma volume at 90-min post-LPS infusion: denervated 1.08 +/- 0.15 ml/100 g body wt, n = 7 vs. intact 0.54 +/- 0.08 ml/100 g body wt, n = 8). The exaggerated LPS-induced hypovolemia associated with splenic denervation was mirrored in the rise in plasma renin activity (90 min post-LPS: denervated 11.5 +/- 0.8 ng x ml(-1) x h(-1), n = 9 vs. intact 6.6 +/- 0.7 ng x ml(-1) x h(-1), n = 8). These results are consistent with our proposal that SSNA normally limits LPS-induced intrasplenic fluid efflux.     

Hemodynamics of anesthetized ventilated mouse models: aspects of anesthetics,fluid support,and strain

This study evaluates the effects of anesthesia and fluid support on hemodynamic parameters of the mechanically ventilated mouse of four different strains. All experiments were performed at a similar surgical level of anesthesia, as indicated by the probing of the pedal withdrawal reflex. Three anesthetic regimens [fentanyl-fluanisone-midazolam (FFM), ketamine-medetomidine-atropine (KMA), and isoflurane (ISO)], four commonly used mouse strains (Swiss, CD-1, BalbC, and C57Bl6), and three different fluid support strategies (no fluid, 0.2 ml x h(-1) x 10 g(-1) of 6% polystarch solution, and 0.5 ml x h(-1) x 10 g(-1) saline) were studied. Mean arterial pressure (MAP) or heart rate (HR) was similar among the four strains of mice except a trend toward lower HR for the BalbC mice. In terms of MAP, KMA is the preferred anesthetic for the Swiss and CD-1 mice, whereas KMA or ISO are recommended for BalbC or C57Bl6 mice. In terms of HR, ISO is the preferred anesthetic for the Swiss, CD-1, and C57Bl6 strains. No differences in HR for the three anesthetics were observed for the BalbC strain. Compared with administration of no fluid, both saline and polystarch administration similarly increased MAP by 7 +/- 2, 10 +/- 2, and 11 +/- 2 mmHg at t = 1, 2, and 3 h, respectively, whereas fluid administration was without effect on HR. Saline supplementation resulted in an increased dry-to-wet ratio of the heart and both fluid regimens decreased total hemoglobin in the blood from 12.6 +/- 0.5 to 10.4 +/- 0.5 g/100 ml. Saline administration was associated with blood acidosis (pH 7.20 +/- 0.03) compared with the Haes (pH 7.29 +/- 0.02) or no-fluid group (pH 7.34 +/- 0.03), whereas PCO(2) was approximately 30 mmHg for all groups. We conclude that at similar surgical levels of anesthesia, the preferable type of anesthesia (ISO or KMA, but never FFM) depends on the strain used and whether MAP or HR is the focus of study. Additional fluid support is beneficial in terms of raising arterial blood pressure, although this is at the cost of changes in organ water content and increased anemia.     

Enumeration and selective isolation of rumen spirochetes.

Enumeration by means of light microscopy showed that from 0.4 x 10(8) to 2.0 x 10(8) spirochetes were present per ml of bovine rumen fluid. Viable cell counts yielded slightly lower values, ranging from 0.1 x 10(8) to 1.2 x 10(8) spirochetes per ml of rumen fluid. The antibiotic rifampin, which served as a selective agent for rumen spirochetes, was added to agar media used in the estimation of viable spirochete numbers in rumen fluid. Morphologically diverse spirochetes were isolated from rumen fluid by means of a procedure involving the use of rifampin as a selective agent in agar media. The strains isolated represented seven morphological types of spirochetes differing in cell size, cell coiling pattern, and number of periplasmic fibrils per cell. Electron microscopy showed that the number of periplasmic fibrils present in the different morphological types of rumen spirochetes ranged from 2 to more than 20 per cell. The results of this study indicate that the bovine rumen is a highly favorable environment for a number of morphologically diverse spirochetes.