Comparisons of bacterial and archaeal communities in the rumen and a dual-flow continuous culture fermentation system using amplicon sequencing.

Dual-flow continuous culture (CC) fermenters are commonly used to study rumen fermentation in vitro. Research using culture-based and oligonucleotide techniques has shown that certain microbial populations within fermenters may be maintained at abundances similar to those observed in vivo. In this study, bacterial and archaeal communities in the rumen of dairy cattle and in a dual-flow CC fermentation system were compared using high-throughput amplicon sequencing targeting the V4 hypervariable region of 16S rRNA. We hypothesized that the in vitro system harbored a comparable bacterial and archaeal community to that observed in the rumen. Members of the Bacteroidetes and Firmicutes made up the 2 most abundant phyla in the rumen, inoculum, and fermenters and did not differ among sample types (P > 0.10). Similarly, Prevotellaceae, the most abundant family in all 3 sample types, did not differ based on source (P = 0.80). However, beta diversity analyses revealed that bacterial and archaeal communities differed between fermenters and rumen samples (P ≤ 0.001), but fermenter bacterial and archaeal communities stabilized by day 4 of each period. While the overall bacterial and archaeal community differs between natural rumens and those detected in in vitro fermenter systems, several prominent taxa were maintained at similar relative abundances suggesting that fermenters may provide a suitable environment in which to study shifts among the predominant members of the microbial community.

Differences in in vitro hydrolysis and fermentation among and within high-fiber ingredients using a modified three-step procedure in growing pigs.

In vitro DM disappearance (IVDMD) and gas production can be used to rapidly estimate apparent total tract digestibility of DM and GE in feed ingredients used in swine diets. However, the accuracy of the system in estimating ME among sources feed ingredients with high content of dietary fiber is not clear. Objectives of this study were 1) to measure IVDMD of feed ingredients with high insoluble fiber content and determine and compare in vitro gas production kinetics from fiber fermentation among wheat straw (WS; 16 sources; 69.0-83.4% NDF), soybean hulls (SBH; 16 sources; 60.9-67.7% NDF), and corn distiller's dried grains with solubles (DDGS; 16 sources; 28.8-44.0% NDF); and 2) to estimate ME contributions resulting from gas production of DDGS. Each 2-g sample was hydrolyzed for 2 h with pepsin and for a subsequent 4 h with pancreatin. Hydrolyzed residues were filtered, washed, dried, weighed, pooled within the same sample, and used for subsequent fermentation using swine fecal inocula. Volume of gas produced was recorded at 11 time points during 72 h of incubation. Parameters of gas production kinetics were calculated using a nonlinear monophasic model, and differences among ingredients were compared using a mixed model. The IVDMD from simulated gastric and small intestinal hydrolysis (IVDMDh) in DDGS (55.7%) was greater (P < 0.05) than that in SBH (19.7%), which was greater (P < 0.05) than that in WS (14.5%). In vitro DM digestibility from simulated large intestine fermentation (IVDMDf) of SBH (68.5%) was greater (P < 0.05) than that of DDGS (52.7%), which was greater than that of WS (41.8%). In vitro DM digestibility from simulated total tract digestion (IVDMDt) was greatest (P < 0.01) in DDGS (79.2%) followed by SBH (74.8%), and both were greater than that in WS (50.2%). The asymptotic gas production (mL/g substrate) was greater (P < 0.05) for SBH (293) than for DDGS (208) and WS (53). There were differences (P < 0.01) in IVDMDh among sources of WS, SBH, and DDGS, whereas IVDMDf and IVDMDt were different (P < 0.01) among sources of SBH but not among sources of DDGS or WS. There were no differences in asymptotic gas production among sources of WS, SBH, or DDGS. In conclusion, the modified 3-step procedure allowed for characterizing the variability of DM digestibility and asymptotic gas production resulting from residue fermentation among WS, SBH, and DDGS and among sources of each ingredient.

Effects of bismuth subsalicylate and dietary sulfur level on fermentation by ruminal microbes in continuous culture.

In ruminants, excess dietary sulfur can be associated with a reduction in DM intake, poor feedlot performance and sulfur-associated polioencephalomalacia. Bismuth subsalicylate (BSS) has been shown to decrease hydrogen sulfide in vitro. The objective of this experiment was to evaluate effects of BSS inclusion (0 or 0.5% of diet DM) and dietary sulfur (0.21 or 0.42% of diet DM) on microbial fermentation in continuous culture. Treatments were arranged in a 2 × 2 factorial design. Eight dual-flow continuous culture fermenters were used during 2 consecutive 10-d periods consisting of 7 d for stabilization followed by 3 d of sampling. A pelleted feedlot diet containing 39% dry rolled corn, 32% earlage, 21% wet distillers grains, 3.2% corn silage, 1.5% soybean meal, 0.6% urea and 2.7% mineral premix (DM basis) was provided as substrate for microbes at a rate of 75 g of DM × fermenter-1 × d-1. Effluents from sampling days were composited by fermenter within period, resulting in 4 replicates/treatment. Bismuth subsalicylate inclusion decreased (P < 0.01) true OM digestion, while no effects were observed for NDF and ADF digestion. Total VFA concentrations, molar proportions of acetic, propionic, and branched-chained VFA decreased (P < 0.01) with BSS addition. The ratio of acetic to propionic acid and the molar proportion of butyric acid increased (P < 0.01) with BSS addition. In regard to nitrogen metabolism, BSS increased NH3-N concentration, NH3-N and dietary-N flows (P < 0.01), and decreased non-NH3-N flow, microbial-N flow, CP degradation, and efficiency of microbial protein synthesis (P < 0.01). Inclusion of BSS increased mean, minimum, and maximum fermentation pH (P < 0.01). Amount of dietary sulfur and BSS inclusion influenced flows of amino acids and fatty acids from fermenters. Influences on fatty acid biohydrogenation and amino acid flows demonstrated an overall suppression of microbial fermentation. Results from this experiment indicate that BSS inclusion at 0.5% of diet DM has detrimental effects on in vitro rumen fermentation in continuous culture.

Effects of dietary roughage and sulfur in diets containing corn dried distillers grains with solubles on hydrogen sulfide production and fermentation by rumen microbes in vitro.

Dried distillers grains with solubles (DDGS) have been used in production animal diets; however, overuse of DDGS can cause toxic concentrations of ruminal hydrogen sulfide gas (HS), resulting in polioencephalomalacia, a deleterious brain disease. Because HS gas requires an acidic rumen environment and diet can influence ruminal pH, it has been postulated that dietary manipulation could help mitigate HS production. The objective of this study was to assess the effect of dietary roughage and sulfur concentrations on HS production and rumen fermentation. In Exp. 1, 7 dual-flow continuous culture fermenters were used in 4 consecutive 9-d periods consisting of 6 d of adaptation followed by 3 d of sampling. At the conclusion of each 9-d continuous culture period, adapted rumen fluid was used for inoculation of 24-h batch culture incubations for Exp. 2. For both experiments, 6 dietary treatments were formulated to consist of 0.3%, 0.4%, or 0.5% dietary sulfur (LS, MS, and HS, respectively) and 3% or 9% dietary roughage (LR and MR, respectively), using grass hay as the roughage source. A corn-based diet without DDGS was used as a control diet. Headspace gas was sampled to determine HS production and concentration. In Exp. 1, greater dietary roughage had no effect ( = 0.14) on HS production but did create a less acidic environment because of an increase ( < 0.01) in the in vitro pH. In Exp. 2, an increase in dietary sulfur caused an increase ( = 0.04) in ruminal HS production, but there was no direct effect ( = 0.25) of dietary roughage on HS production. Greater dietary roughage resulted in a less ( = 0.01) acidic final batch culture pH but a lower ( < 0.01) total VFA concentration. Further investigation is needed to determine a more effective way to mitigate ruminal HS production using dietary manipulation, which could include greater inclusion of dietary roughage or the use of different roughage sources.

Mitigation of in vitro hydrogen sulfide production using bismuth subsalicylate with and without monensin in beef feedlot diets.

The objective of this study was to determine if a sulfur binder, bismuth subsalicylate (BSS), alone or combined with monensin (MON) could decrease the production of HS by rumen microbes. In Exp. 1, two 24-h batch culture incubations were conducted using a substrate consisting of 50% corn, 40% distillers grains, 9.75% hay, and 0.25% mineral premix, on a DM basis. Five treatments including BSS concentrations of 0% (control), 0.5%, 1%, 2%, and 4% of DM were assigned in 5 replicates to 120-mL serum bottles containing rumen fluid, buffer, and 0.5 g of dietary substrate. Addition of 2% and 4% BSS decreased ( < 0.05) gas production, whereas all concentrations of BSS reduced ( < 0.05) HS production by 18%, 24%, 82%, and 99% for 0.5%, 1%, 2%, and 4% BSS, respectively. Final pH increased ( < 0.05) with 2% and 4% BSS treatments. At 4% of DM, BSS decreased ( < 0.05) total VFA concentration (m) and propionate (mol/100 mol) but increased acetate (mol/100 mol) and acetate to propionate ratio. Concentration of branched-chain VFA increased ( < 0.05) with the addition of 0.5% BSS, compared with the control. On the basis of these results, addition of BSS (1% of DM) and MON (5 mg/kg) were used to assess their effects on metabolism and HS release by rumen microbes in 8 dual flow continuous culture fermenters during two 10-d periods (Exp. 2). Treatments were arranged in a 2 × 2 factorial design. Substrate similar to that used in Exp. 1 was provided at 75 g DM/fermenter daily. Headspace HS concentration was reduced ( < 0.05) by 99% with BSS treatment but was not affected ( = 0.21) by MON. An overall increase ( < 0.05) in fermentation pH was found following addition of BSS. Addition of BSS increased ( < 0.05) digestion of NDF and ADF but decreased ( < 0.05) nonfiber carbohydrate digestion and total VFA concentration. Acetate and propionate (mol/100 mol) increased ( < 0.05) with BSS, whereas butyrate (mol/100 mol) and branched-chain VFA (m) decreased ( < 0.05). Addition of BSS increased ( < 0.05) NH-N concentration and NH-N outflow but decreased ( < 0.05) microbial N outflow. Results from this study showed no response to monensin addition, but BSS markedly reduced HS production and altered microbial fermentation during in vitro rumen fluid incubations.

Effects of Temperature during Moist Heat Treatment on Ruminal Degradability and Intestinal Digestibility of Protein and Amino Acids in Hempseed Cake.

The objective of this study was to evaluate ruminal degradability and intestinal digestibility of crude protein (CP) and amino acids (AA) in hempseed cake (HC) that were moist heat treated at different temperatures. Samples of cold-pressed HC were autoclaved for 30 min at 110, 120 or 130°C, and a sample of untreated HC was used as the control. Ruminal degradability of CP was estimated, using the in situ Dacron bag technique; intestinal CP digestibility was estimated for the 16 h in situ residue using a three-step in vitro procedure. AA content was determined for the HC samples (heat treated and untreated) of the intact feed, the 16 h in situ residue and the residue after the three-step procedure. There was a linear increase in RUP (p = 0.001) and intestinal digestibility of RUP (p = 0.003) with increasing temperature during heat treatment. The 130°C treatment increased RUP from 259 to 629 g/kg CP, while intestinal digestibility increased from 176 to 730 g/kg RUP, compared to the control. Hence, the intestinal available dietary CP increased more than eight times. Increasing temperatures during heat treatment resulted in linear decreases in ruminal degradability of total AA (p = 0.006) and individual AA (p<0.05) and an increase in intestinal digestibility that could be explained both by a linear and a quadratic model for total AA and most individual AA (p<0.05). The 130°C treatment decreased ruminal degradability of total AA from 837 to 471 g/kg, while intestinal digestibility increased from 267 to 813 g/kg of rumen undegradable AA, compared with the control. There were differences between ruminal AA degradability and between intestinal AA digestibility within all individual HC treatments (p<0.001). It is concluded that moist heat treatment at 130°C did not overprotect the CP of HC and could be used to shift the site of CP and AA digestion from the rumen to the small intestine. This may increase the value of HC as a protein supplement for ruminants.

Effects of two halophytic plants (kochia and atriplex) on digestibility, fermentation and protein synthesis by ruminal microbes maintained in continuous culture.

Eight continuous culture fermenters were used in a completely randomized design to evaluate various nutritional values of Kochia (Kochia scoparia) compared with Atriplex (Atriplex dimorphostegia). Dried and pelleted samples (leaves and stems) provided substrate for metabolism by ruminal microbes maintained in a continuous culture fermentation system. Results indicated that there were no differences (p>0.05) in dry matter (DM) and crude protein (CP) digestibility between the two halophytic plants. Atriplex had higher (p<0.05) organic matter (OM) digestibility compared with Kochia. Neutral detergent fiber (aNDF) digestibility of Atriplex (411 g/kg) was higher (p<0.05) than that of Kochia (348 g/kg), however acid detergent fiber (ADF) digestibility was higher (p<0.05) in Kochia compared with Atriplex (406 vs. 234 g/kg). There were no differences (p>0.05) between the two halophytic plants in molar proportion of acetate and propionate, but the concentration of butyrate and valerate in Kochia were about two fold of Atriplex (p<0.05). When Kochia provided substrate to the microbes, protein synthesis was higher (p<0.05) compared with feeding Atriplex (5.96 vs. 4.85 g N/kg of OM truly digested). It was concluded that Kochia scoparia and Atriplex dimorphostegia had similar digestibility of DM and CP. It appears that these halophytic plants may not have enough digestible energy for high producing ruminants.

Intestinal digestibility of amino acids in rumen-undegraded protein estimated using a precision-fed cecectomized rooster bioassay: II. Distillers dried grains with solubles and fish meal.

The objectives of this experiment were to measure intestinal digestibility of AA in the rumen-undegraded protein fraction (RUP-AA) of distillers dried grains with solubles (DDGS) and fish meal (FM) samples and to determine whether these feeds contain a constant protein fraction that is undegradable in the rumen and indigestible in the small intestine, as assumed in the French Institut National de la Recherche Agronomique (Paris, France) and Scandinavian AAT-PBV (AAT = AA absorbed from small intestine; PBV = protein balance in the rumen) models. Five sources of DDGS and 5 sources of FM were obtained from Feed Analysis Consortium, Inc. (Champaign, IL). To obtain the rumen-undegradable protein fraction, samples were ruminally incubated in situ for 16 h in 4 lactating cows, and the collected rumen-undegraded residues (RUR) were pooled by sample. Subsamples of the intact feeds and RUR were crop-intubated to 4 cecectomized roosters, and total excreta were collected for 48 h. Intact feeds, RUR, and excreta were analyzed for AA. Basal endogenous AA loss estimates were obtained from fasted birds and were used to calculate standardized digestibility of RUP-AA and AA in the intact feeds. Indigestibility coefficients of the intact feeds were calculated as (100 - % standardized AA digestibility), and indigestibility of the RUR was calculated as [(100 - % ruminal degradation of AA) x (100 - % standardized RUP-AA digestibility)/100]. Results indicate that standardized digestibility of feed-AA differs from RUP-AA for DDGS samples but not for FM samples, and that standardized digestibility of individual AA differs within samples. For the DDGS samples, standardized feed-AA and RUP-AA digestibility values were most often lowest for His and Lys and highest for Met and Trp. For FM samples, standardized feed-AA and RUP-AA digestibility values were most often lowest for His and highest for Trp. Results also indicate that DDGS and most FM samples do not contain a constant protein fraction that is both undegradable in the rumen and indigestible in the small intestine. Indigestibility values of RUR were lower than in intact feeds, suggesting that the feed ingredients used in this experiment contain a protein fraction that is indigestible in the intestine but partly degradable in the rumen or digestible in the intestine after rumen incubation, or both.

Intestinal digestibility of amino acids in rumen undegradable protein estimated using a precision-fed cecectomized rooster bioassay: I. Soybean meal and SoyPlus.

The objectives of this experiment were to measure intestinal digestibility of AA in rumen undegradable protein (RUP-AA) in soybean meal (SBM) and expeller SBM (SoyPlus, West Central, Ralston, IA; SP) and to determine if these feeds contain a constant protein fraction that is undegradable in the rumen and indigestible in the small intestine, as assumed in the French Institut National de la Recherche Agronomique (Paris, France) and Scandinavian AAT-PBV (AAT = AA absorbed from small intestine; PBV = protein balance in the rumen) models. Three samples of SBM and 3 samples of SP were obtained from the Feed Analysis Consortium Inc. (Savoy, IL). To obtain the RUP fraction, samples were ruminally incubated in situ for 16 h in 4 lactating cows, and the collected rumen undegraded residues (RUR) were pooled by sample. Subsamples of the intact feeds and RUR were crop intubated to 4 cecectomized roosters, and total excreta were collected for 48 h. Intact feeds, RUR, and excreta were analyzed for AA. Basal endogenous AA loss estimates were obtained from fasted birds and were used to calculate standardized digestibility of AA in the intact feeds and RUP-AA. Indigestibility coefficients of the intact feeds were calculated as (100 - % standardized AA digestibility), and indigestibility of the RUR was calculated as [(100 - % ruminal degradation of AA) x [(100 - % standardized RUP-AA digestibility)]/100]. Results indicated that standardized digestibility of feed-AA was similar to standardized digestibility of RUP-AA for SBM and SP samples and that standardized digestibility of individual AA differed within samples. Standardized feed-AA and RUP-AA digestibility values were lowest for Lys and Cys and highest for Trp and Met. Results also indicated that SBM and SP did not contain a constant protein fraction that was both undegradable in the rumen and indigestible in the small intestine. Indigestibility values of RUR were lower than in intact feeds, suggesting that SBM and SP contain a protein fraction that is indigestible in the intestine but partly degradable in the rumen, digestible in the intestine after ruminal incubation, or both.

In vitro digestibility of individual amino acids in rumen-undegraded protein: the modified three-step procedure and the immobilized digestive enzyme assay.

Three soybean meal, 3 SoyPlus (West Central Cooperative, Ralston, IA), 5 distillers dried grains with solubles, and 5 fish meal samples were used to evaluate the modified 3-step in vitro procedure (TSP) and the in vitro immobilized digestive enzyme assay (IDEA; Novus International Inc., St. Louis, MO) for estimating digestibility of AA in rumen-undegraded protein (RUP-AA). In a previous experiment, each sample was ruminally incubated in situ for 16 h, and in vivo digestibility of AA in the intact samples and in the rumen-undegraded residues (RUR) was obtained for all samples using the precision-fed cecectomized rooster assay. For the modified TSP, 5 g of RUR was weighed into polyester bags, which were then heat-sealed and placed into Daisy(II) incubator bottles. Samples were incubated in a pepsin/HCl solution followed by incubation in a pancreatin solution. After this incubation, residues remaining in the bags were analyzed for AA, and digestibility of RUP-AA was calculated based on disappearance from the bags. In vitro RUP-AA digestibility estimates obtained with this procedure were highly correlated to in vivo estimates. Corresponding intact feeds were also analyzed via the pepsin/pancreatin steps of the modified TSP. In vitro estimates of AA digestibility of the feeds were highly correlated to in vivo RUP-AA digestibility, which suggests that the feeds may not need to be ruminally incubated before determining RUP-AA digestibility in vitro. The RUR were also analyzed via the IDEA kits. The IDEA values of the RUR were good predictors of RUP-AA digestibility in soybean meal, SoyPlus, and distillers dried grains with solubles, but the IDEA values were not as good predictors of RUP-AA digestibility in fish meal. However, the IDEA values of intact feed samples were also determined and were highly correlated to in vivo RUP-AA digestibility for all feed types, suggesting that the IDEA value of intact feeds may be a better predictor of RUP-AA digestibility than the IDEA value of the RUR. In conclusion, the modified TSP and IDEA kits are good approaches for estimating RUP-AA digestibility in soybean meal products, distillers dried grains with solubles, and fish meal samples.

Evaluation of the fermentation dynamics of soluble crude protein from three protein sources in continuous culture fermenters.

Eight dual-flow continuous culture fermenters (1.03 +/- 0.05 L) were used to assess differences in microbial degradation of the soluble CP fraction of canola meal (CMSCP), soybean meal (SBMSCP), and fish meal (FMSCP) using a completely randomized design with two 9-d experimental periods and a solution of tryptone as a control treatment (control). All fermenters received the same basal diet (58% ground corn, 40% canary grass hay, 0.4% vitamin-mineral premix, 1% CaCO(3), 0.6% salt on a DM basis) in 8 equal portions daily. During sampling on the last 3 d of each period, 90-mL doses containing soluble CP were infused into the fermenters 30 min after the beginning of the first and last feedings of the day. The total amount of soluble CP supplied by the infusions of FMSCP, CMSCP, and SBMSCP was 3.2 g/d, representing 24% of the daily dietary CP intake. Infusion of FMSCP resulted in the greatest (P < 0.05) NH(3)-N concentration (4.6 +/- 0.40 mg/dL) compared with the other treatments (0.5 +/- 0.40 mg/dL). Microbial N flow (g/d) from the fermenters was also greatest (P < 0.05) with FMSCP (1.42 +/- 0.062) compared with the other soluble CP fractions (1.08 +/- 0.062). The efficiency of microbial protein synthesis tended to be lowest with the control diet, and the efficiency of N utilization was lowest with FMSCP treatment. These results indicate that N was limiting microbial growth in the control diet, and there was more rumen-available N with the FMSCP diet compared with the other dietary treatments. The extent of degradation of the soluble CP fraction from fish meal, soybean meal, and canola meal was determined to be 99, 30, and 37% of soluble CP, respectively. These results indicate that the soluble CP fraction is not 100% degraded in all feeds and that assuming a high degradation extent of the soluble CP fraction from soybean meal and canola meal may result in an underestimation of the supply of undegradable protein from these protein sources.

Nitrogen metabolism in the rumen.

Protein metabolism in the rumen is the result of metabolic activity of ruminal microorganisms. The structure of the protein is a key factor in determining its susceptibility to microbial proteases and, thus, its degradability. Ruminal protein degradation is affected by pH and the predominant species of microbial population. Ruminal proteolytic activity decreases as pH decreases with high-forage dairy cattle-type rations, but not in high-concentrate beef-type rations. Accumulation of amino acid (AA) N after feeding suggests that AA uptake by rumen microorganisms could be the limiting factor of protein degradation in the rumen. In addition, there are several AA, such as Phe, Leu, and Ile, that are synthesized by rumen microorganisms with greater difficulty than other AA. The most common assessment of efficiency of microbial protein synthesis (EMPS) is determination of grams of microbial N per unit of rumen available energy, typically expressed as true organic matter or carbohydrates fermented. However, EMPS is unable to estimate the efficiency at which bacteria capture available N in the rumen. An alternative and complementary measure of microbial protein synthesis is the efficiency of N use (ENU). In contrast to EMPS, ENU is a good measurement for describing efficiency of N capture by ruminal microbes. Using EMPS and ENU, it was concluded that optimum bacterial growth in the rumen occurs when EMPS is 29 g of bacterial N/kg of fermented organic matter, and ENU is 69%, implying that bacteria would require about 1.31 x rumen-available N per unit of bacterial N. Because the distribution of N within bacterial cells changes with rate of fermentation, AA N, rather than total bacterial N should be used to express microbial protein synthesis.

The elementary events of Ca2+ release elicited by membrane depolarization in mammalian muscle.

Cytosolic [Ca(2+)] transients elicited by voltage clamp depolarization were examined by confocal line scanning of rat skeletal muscle fibres. Ca(2+) sparks were observed in the fibres' membrane-permeabilized ends, but not in responses to voltage in the membrane-intact area. Elementary events of the depolarization-evoked response could be separated either at low voltages (near -50 mV) or at -20 mV in partially inactivated cells. These were of lower amplitude, narrower and of much longer duration than sparks, similar to 'lone embers' observed in the permeabilized segments. Their average amplitude was 0.19 and spatial half-width 1.3 microm. Other parameters depended on voltage. At -50 mV average duration was 111 ms and latency 185 ms. At -20 mV duration was 203 ms and latency 24 ms. Ca(2+) release current, calculated on an average of events, was nearly steady at 0.5-0.6 pA. Accordingly, simulations of the fluorescence event elicited by a subresolution source of 0.5 pA open for 100 ms had morphology similar to the experimental average. Because 0.5 pA is approximately the current measured for single RyR channels in physiological conditions, the elementary fluorescence events in rat muscle probably reflect opening of a single RyR channel. A reconstruction of cell-averaged release flux at -20 mV based on the observed distribution of latencies and calculated elementary release had qualitatively correct but slower kinetics than the release flux in prior whole-cell measurements. The qualitative agreement indicates that global Ca(2+) release flux results from summation of these discrete events. The quantitative discrepancies suggest that the partial inactivation strategy may lead to events of greater duration than those occurring physiologically in fully polarized cells.

Ca2+ sparks and embers of mammalian muscle. Properties of the sources.

Ca2+ sparks of membrane-permeabilized rat muscle cells were analyzed to derive properties of their sources. Most events identified in longitudinal confocal line scans looked like sparks, but 23% (1,000 out of 4,300) were followed by long-lasting embers. Some were preceded by embers, and 48 were "lone embers." Average spatial width was approximately 2 microm in the rat and 1.5 microm in frog events in analogous solutions. Amplitudes were 33% smaller and rise times 50% greater in the rat. Differences were highly significant. The greater spatial width was not a consequence of greater open time of the rat source, and was greatest at the shortest rise times, suggesting a wider Ca2+ source. In the rat, but not the frog, spark width was greater in scans transversal to the fiber axis. These features suggested that rat spark sources were elongated transversally. Ca2+ release was calculated in averages of sparks with long embers. Release current during the averaged ember started at 3 or 7 pA (depending on assumptions), whereas in lone embers it was 0.7 or 1.3 pA, which suggests that embers that trail sparks start with five open channels. Analysis of a spark with leading ember yielded a current ratio ranging from 37 to 160 in spark and ember, as if 37-160 channels opened in the spark. In simulations, 25-60 pA of Ca2+ current exiting a point source was required to reproduce frog sparks. 130 pA, exiting a cylindric source of 3 microm, qualitatively reproduced rat sparks. In conclusion, sparks of rat muscle require a greater current than frog sparks, exiting a source elongated transversally to the fiber axis, constituted by 35-260 channels. Not infrequently, a few of those remain open and produce the trailing ember.

Persistence and functional impact of a microbial inoculant on native microbial community structure, nutrient digestion and fermentation characteristics in a rumen model.

Small sub-unit (SSU) rRNA-targeted oligonucleotide probes were used to monitor the persistence of a genetically engineered bacterium inoculated in model rumens. Eight dual flow continuous culture fermenters were operated with either standard artificial saliva buffer or buffer with chondroitin sulfate (0.5 g/l) added. After 168 h of operation, fermenters were inoculated with Bacteroides thetaiotaomicron BTX (BTX), at approximately 1% of total bacteria. B. thetaiotaomicron was quantified using a species-specific probe and shown to persist in fermenters 144 h after inoculation (relative abundance 0.48% and 1.42% of total SSU rRNA with standard and chondroitin sulfate buffers, respectively). No B. thetaiotaomicron SSU rRNA was detected in fermenter samples prior to inoculation with strain BTX. Relative abundances of Bacteria, Eucarya and Archaea were not affected by either inoculation or buffer type. Fiber digestion, in particular the hemicellulose fraction, increased after strain BTX addition. Chondroitin sulfate addition to the buffer increased bacterial nitrogen flow in fermenters, but did not alter fiber digestion. Neither inoculum nor buffer type altered total short chain fatty acid (VFA) concentrations but proportions of individual VFA differed. In model rumens, B. thetaiotaomicron BTX increased fiber digestion when added to mixed ruminal microbes, independent of chondroitin sulfate addition; but further study is needed to determine effects on other fiber-digesting bacteria.

Effects of carbohydrates from citrus pulp and hominy feed on microbial fermentation in continuous culture.

Eight dual-flow continuous-culture fermenters were used to evaluate the effect of neutral detergent-soluble carbohydrates (NDSC) on fermentation by ruminal microorganisms. Citrus pulp and hominy feed were added to a basal diet as sources of NDSC, with citrus pulp providing neutral detergent-soluble fiber (NDSF) in the form of pectic substances and with hominy feed in the form of starch. The basal diet contained 26.7% corn silage, 6.0% alfalfa hay and 3.8% cottonseed hulls on a DM basis. The dried citrus pulp diet contained on a DM basis 17.2% CP, 34.7% NDF, 33.7% NDSC, and 14.4% NDSF, whereas the hominy feed diet contained 17.9% CP, 33.2% NDF, 35.9% NDSC, and 8.8% NDSF. Organic matter, DM, and NDF and ADF digestion were not affected by source of carbohydrate. Ammonia N concentration was greater (P < 0.05) for the hominy feed diet (14.2 mg/100 mL) than for the dried citrus pulp diet (9.3 mg/100 mL). Total N, nonammonia N, microbial N, and dietary N flows were not affected by treatments; however, the efficiency of microbial protein synthesis was greater (P = 0.055) for the dried citrus pulp diet than for the hominy feed diet (30.6 vs 27.8 g of bacterial N/kg of OM truly digested). Results from this experiment indicate that NDSF from citrus pulp can provide similar sources of energy compared with starch from hominy feed to support ruminal microbial growth.

Ion concentration-dependence of rat cardiac unitary L-type calcium channel conductance.

Little is known about the native properties of unitary cardiac L-type calcium currents (i(Ca)) measured with physiological calcium (Ca) ion concentration, and their role in excitation-contraction (E-C) coupling. Our goal was to chart the concentration-dependence of unitary conductance (gamma) to physiological Ca concentration and compare it to barium ion (Ba) conductance in the absence of agonists. In isolated, K-depolarized rat myocytes, i(Ca) amplitudes were measured using cell-attached patches with 2 to 70 mM Ca or 2 to 105 mM Ba in the pipette. At 0 mV, 2 mM of Ca produced 0.12 pA, and 2 mM of Ba produced 0.19 pA unitary currents. Unitary conductance was described by a Langmuir isotherm relationship with a maximum gammaCa of 5.3 +/- 0.2 pS (n = 15), and gammaBa of 15 +/- 1 pS (n = 27). The concentration producing half-maximal gamma, Kd(gamma), was not different between Ca (1.7 +/- 0.3 mM) and Ba (1.9 +/- 0.4 mM). We found that quasi-physiological concentrations of Ca produced currents that were as easily resolvable as those obtained with the traditionally used higher concentrations. This study leads to future work on the molecular basis of E-C coupling with a physiological concentration of Ca ions permeating the Ca channel.

A preferred amplitude of calcium sparks in skeletal muscle.

In skeletal and cardiac muscle, calcium release from the sarcoplasmic reticulum, leading to contraction, often results in calcium sparks. Because sparks are recorded by confocal microscopy in line-scanning mode, their measured amplitude depends on their true amplitude and the position of the spark relative to the scanned line. We present a method to derive from measured amplitude histograms the actual distribution of spark amplitudes. The method worked well when tested on simulated distributions of experimental sparks. Applied to massive numbers of sparks imaged in frog skeletal muscle under voltage clamp in reference conditions, the method yielded either a decaying amplitude distribution (6 cells) or one with a central mode (5 cells). Caffeine at 0.5 or 1 mM reversibly enhanced this mode (5 cells) or induced its appearance (4 cells). The occurrence of a mode in the amplitude distribution was highly correlated with the presence of a mode in the distribution of spark rise times or in the joint distribution of rise times and spatial widths. If sparks were produced by individual Markovian release channels evolving reversibly, they should not have a preferred rise time or amplitude. Channel groups, instead, could cooperate allosterically or through their calcium sensitivity, and give rise to a stereotyped amplitude in their collective spark.

Nitrogen metabolism of early lactation cows fed diets with two different levels of protein and different amino acid profiles.

Four multiparous Holstein cows (569+/-122 kg) surgically prepared with indwelling catheters in the mesenteric, portal, and hepatic veins and carotid artery were allocated in a 4 x 4 Latin square to determine the effects of dietary crude protein (CP) level and amino acid (AA) profile on N metabolism during early lactation (from 25 to 65 d in milk). Cows received their diets in two equal meals and were milked twice daily. The dietary treatments were: 18% CP with a high (18H) or a low (18L) quality AA profile, and 15% CP with a high (15H) or a low (15L) quality AA profile. The four diets were similar in net energy for lactation (1.75 NEL Mcal/kg) and contained the same amount of RUP (34% of CP). The quality of the AA profile pertained only to the essential AA (EAA), and was assessed by comparison with the EAA profile of casein and considered the potential contribution of EAA from ruminal bacteria. The 18H and 15H diets were supplemented with 50 and 25 g/d of ruminally protected Met, respectively. After 10 d on treatment, a blood flow marker (p-amino-hippurate) was infused into a mesenteric vein, and arterial, portal, hepatic, and mammary blood samples were obtained at 3, 6, and 12 h after feeding. Dry matter intake was similar across treatments (23.4+/-0.5 kg/d). Amino acid oxidation, and consequent urea production, in the liver were numerically greater with the 18% CP rations, and, as a result, arterial urea concentrations were greatest (P < 0.01) with these rations. The amount of total AA extracted by the mammary gland tended to be greater with the H than with the L diets (21.4 vs. 18.2 mmol/ h, respectively). Milk yield tended to be greater (P = 0.16) with the 18H and 15H diets (47.7 and 46.3 kg/d, respectively) compared with the 18L and 15L diets (45.9 and 44.6 kg/d, respectively). Also, milk CP and casein contents were greatest (P = 0.09) with the H diets compared with the L diets. Milk and plasma urea N were greatest (P < 0.01) with the 18% CP diets. The efficiency of N utilization for milk protein synthesis was greatest (P < 0.09) with the 15% CP diets. It is concluded that milk protein production during early lactation is less susceptible to variations in dietary CP contents than variations in the AA profile of the dietary protein.

Voltage-independent changes in L-type Ca(2+) current uncoupled from SR Ca(2+) release in cardiac myocytes.

To determine the effect of voltage-independent alterations of L-type Ca(2+) current (I(Ca)) on the sarcoplasmic reticular (SR) Ca(2+) release in cardiac myocytes, we measured I(Ca) and cytosolic Ca(2+) transients (Ca(i)(2+); intracellular Ca(2+) concentration) in voltage-clamped rat ventricular myocytes during 1) an abrupt increase of extracellular [Ca(2+)] (Ca(o)(2+)) or 2) application of 1 microM FPL-64176, a Ca(2+) channel agonist, to selectively alter I(Ca) in the absence of changes in SR Ca(2+) loading. On the first depolarization in higher Ca(o)(2+), peak I(Ca) was increased by 46 +/- 6% (P < 0.001), but the increases in the maximal rate of rise of Ca(i)(2+) (dCa(i)(2+)/dt(max), where t is time; an index of SR Ca(2+) release flux) and the Ca(i)(2+) transient amplitude were not significant. Rapid exposure to FPL-64176 greatly slowed inactivation of I(Ca), increasing its time integral by 117 +/- 8% (P < 0.001) without significantly increasing peak I(Ca), dCa(i)(2+)/dt(max), or amplitude of the corresponding Ca(i)(2+) transient. Prolongation of exposure to higher Ca(o)(2+) or FPL-64176 did not further increase peak I(Ca) but greatly increased dCa(i)(2+)/dt(max), Ca(i)(2+) transient amplitude, and the gain of Ca(2+) release (dCa(i)(2+)/dt(max)/I(Ca)), evidently due to augmentation of the SR Ca(2+) loading. Also, the time to peak dCa(i)(2+)/dt(max) was significantly increased in the continuous presence of higher Ca(o)(2+) (by 37 +/- 5%, P < 0.001) or FPL-64176 (by 63 +/- 5%, P < 0.002). Our experiments provide the first evidence of a marked disparity between an increased peak I(Ca) and the corresponding SR Ca(2+) release. We attribute this to saturation of the SR Ca(2+) release flux as predicted by local control theory. Prolongation of the SR Ca(2+) release flux, caused by combined actions of a larger I(Ca) and maximally augmented SR Ca(2+) loading, might reflect additional Ca(2+) release from corbular SR.