Comparison of microbial fermentation data from dual-flow continuous culture system and omasal sampling technique: A meta-analytical approach.

Although the omasal sampling technique (OST) has been successfully used to estimate ruminal fermentation and nutrient flow, alternatives to invasive animal trials should be pursued and evaluated. The objective of this study was to evaluate carbohydrate and N metabolisms using a meta-analytical approach to compare 2 methods: dual-flow continuous culture system (DFCCS) and OST. To be included, studies needed to report diet chemical composition and report at least 1 of the dependent variables of interest. A total of 155 articles were included, in which 97 used the DFCCS and 58 used the OST. The independent variables used were dietary nonfiber carbohydrate concentration, neutral detergent fiber (NDF) degradability, true crude protein (CP) degradability, and efficiency of microbial protein synthesis (EMPS). In addition, 12 dependent variables were used. Statistical analyses were performed using the Mixed procedure of SAS (SAS Institute Inc., Cary, NC). A random coefficients model was used considering study as a random effect and including the possibility of covariance between the slope and the intercept. The effect of method (DFCCS or OST) was included and tested in the estimates of the intercept, linear, and quadratic effects of the independent variable. There was no method effect when NDF degradability was regressed with total volatile fatty acids concentration, true CP degradability, and EMPS. Molar proportions of acetate and propionate were quadratically associated with NDF degradability. When NDF degradability was regressed with acetate and propionate there was a method effect, differing only in the intercept (ß0) estimate. True organic matter digestibility, bacterial N/total N, efficiency of N utilization, total volatile fatty acid concentration, and molar proportion of butyrate linearly increased as dietary nonfiber carbohydrate concentration increased, and none of these variables were affected by method. Concentration of ammonia N had a linear and positive association with true CP degradability. This was the only variable that had a method effect when regressed with true CP degradability, differing only in the estimate of the intercept (ß0). As EMPS increased, efficiency of N utilization also increased, and it was affected by method. Overall, the majority of DFCCS responses were similar to OST. When a method effect was observed, it was mainly on the estimate of the intercept, demonstrating that the magnitude of these responses was different. However, the relationships between independent and dependent variables were similar across methods.

Microbial diversity in the rumen, reticulum, omasum, and abomasum of yak on a rapid fattening regime in an agro-pastoral transition zone.

The ruminant digestive system harbors a complex gut microbiome, which is poorly understood in the case of the four stomach compartments of yak. High-throughput sequencing and quantitative PCR were used to analyse microbial communities in the rumen, reticulum, omasum, and abomasum of six domesticated yak. The diversity of prokaryotes was higher in reticulum and omasum than in rumen and abomasum. Bacteroidetes predominated in the four stomach compartments, with abundance gradually decreasing in the trend rumen > reticulum > omasum > abomasum. Microorganism composition was different among the four compartments, all of which contained high levels of bacteria, methanogens, protozoa and anaerobic fungi. Some prokaryotic genera were associated with volatile fatty acids and pH. This study provides the first insights into the microorganism composition of four stomach compartments in yak, and may provide a foundation for future studies in this area.

Microbial community composition along the digestive tract in forage- and grain-fed bison.

BACKGROUND: Diversity and composition of microbial communities was compared across the 13 major sections of the digestive tract (esophagus, reticulum, rumen, omasum, abomasum, duodenum, jejunum, ileum, cecum, ascending colon, transverse colon, descending colon, and rectum) in two captive populations of American bison (Bison bison), one of which was finished on forage, the other on grain. RESULTS: Microbial diversity fell to its lowest levels in the small intestine, with Bacteroidetes reaching their lowest relative abundance in that region, while Firmicutes and Euryarchaeota attained their highest relative abundances there. Gammaproteobacteria were most abundant in the esophagus, small intestine, and colon. The forage-finished bison population exhibited higher overall levels of diversity, as well as a higher relative abundance of Bacteroidetes in most gut sections. The grain-finished bison population exhibited elevated levels of Firmicutes and Gammaproteobacteria. Within each population, different sections of the digestive tract exhibited divergent microbial community composition, although it was essentially the same among sections within a given region of the digestive tract. Shannon diversity was lowest in the midgut. For each section of the digestive tract, the two bison populations differed significantly in microbial community composition. CONCLUSIONS: Similarities among sections indicate that the esophagus, reticulum, rumen, omasum, and abomasum may all be considered to house the foregut microbiota; the duodenum, jejunum, and ileum may all be considered to house the small intestine or midgut microbiota; and the cecum, ascending colon, transverse colon, descending colon, and rectum may all be considered to house the hindgut microbiota. Acid from the stomach, bile from the gall bladder, digestive enzymes from the pancreas, and the relatively low retention time of the small intestine may have caused the midgut's low microbial diversity. Differences in microbial community composition between populations may have been most strongly influenced by differences in diet (forage or grain). The clinical condition of the animals used in the present study was not evaluated, so further research is needed to establish whether the microbial profiles of some bison in this study are indeed indicative of dysbiosis, a predisposing factor to ruminal acidosis and its sequelae.

First insights into the microbial diversity in the omasum and reticulum of bovine using Illumina sequencing.

The digestive systems of mammals harbor a complex gut microbiome, comprising bacteria and other microorganisms that confer metabolic and immunological benefits to the host. Ruminants that digest plant-based foods have a four-compartment stomach consisting of the rumen, reticulum, omasum, and abomasum. The microorganisms in the stomach are essential for providing the host with critical nutrients. However, the majority of these microorganisms are unknown species. The microbiome of the stomach is diverse, and the majority of these organisms cannot be cultured. Next-generation sequencing (NGS) combined with bioinformatic analysis tools have allowed the dissection of the composition of the microbiome in samples collected from a specific environment. In this study, for the first time, the bacterial composition in two compartments, the reticulum and the omasum, of bovine were analyzed using a metagenomic approach and compared to the bacterial composition of the rumen. These data will assist in understanding the biology of ruminants and benefit the agricultural industry. The diversity and composition of the bacterial community in samples collected from the rumen, reticulum, and omasum of bovines in the Changchun Region of Northeast China were analyzed by sequencing the V3 region of the 16S rRNA gene using a barcoded Illumina paired-end sequencing technique, and the primary composition of the microbiome in the rumen, reticulum, and omasum of the bovines was determined. These microbiomes contained 17 phyla and 107 genera in all three samples. Five phyla, Bacteroidetes, Firmicutes, Proteobacteria, Spirochaetes, and Lentisphaerae, were the most abundant taxonomic groups. Additionally, the different stomach compartments harbored different compositions of the microorganisms.

Effect of replacing grass silage with red clover silage on nutrient digestion, nitrogen metabolism, and milk fat composition in lactating cows fed diets containing a 60:40 forage-to-concentrate ratio.

Diets based on red clover silage (RCS) typically increase the concentration of polyunsaturated fatty acids (PUFA) in ruminant meat and milk and lower the efficiency of N utilization compared with grass silages (GS). Four multiparous Finnish Ayrshire cows (108 d postpartum) fitted with rumen cannulas were used in a 4 × 4 Latin square design with 21-d periods to evaluate the effect of incremental replacement of GS with RCS on milk production, nutrient digestion, whole-body N metabolism, and milk fatty acid composition. Treatments comprised total mixed rations offered ad libitum, containing 600 g of forage/kg of diet dry matter (DM), with RCS replacing GS in ratios of 0:100, 33:67, 67:33, and 100:0 on a DM basis. Intake of DM and milk yield tended to be higher when RCS and GS were offered as a mixture than when fed alone. Forage species had no influence on the concentration or secretion of total milk fat, whereas replacing GS with RCS tended to decrease milk protein concentration and yield. Substitution of GS with RCS decreased linearly whole-tract apparent organic matter, fiber, and N digestion. Forage species had no effect on total nonammonia N at the omasum, whereas the flow of most AA at the omasum was higher for diets based on a mixture of forages. Replacing GS with RCS progressively lowered protein degradation in the rumen, increased linearly ruminal escape of dietary protein, and decreased linearly microbial protein synthesis. Incremental inclusion of RCS in the diet tended to lower whole-body N balance, increased linearly the proportion of dietary N excreted in feces and urine, and decreased linearly the utilization of dietary N for milk protein synthesis. Furthermore, replacing GS with RCS decreased linearly milk fat 4:0 to 8:0, 14:0, and 16:0 concentrations and increased linearly 18:2n-6 and 18:3n-3 concentrations, in the absence of changes in cis-9 18:1, cis-9, trans-11 18:2, or total trans fatty acid concentration. Inclusion of RCS in the diet progressively increased the apparent transfer of 18-carbon PUFA from the diet into milk, but had no effect on the amount of 18:2n-6 or 18:3n-3 at the omasum recovered in milk. In conclusion, forage species modified ruminal N metabolism, the flow of AA at the omasum, and whole-body N partitioning. A lower efficiency of N utilization for milk protein synthesis with RCS relative to GS was associated with decreased availability of AA for absorption, with some evidence of an imbalance in the supply of AA relative to requirements. Higher enrichment of PUFA in milk for diets based on RCS was related to an increased supply for absorption, with no indication that forage species substantially altered PUFA bioavailability.

Isolation and characterization of Shiga toxin-producing Escherichia coli (STEC) in retail edible beef by-products.

The prevalence of Shiga toxin-producing Escherichia coli (STEC) was investigated in 350 edible beef intestinal samples, including omasum (n=110), abomasum (n=120), and large intestines (n=120), collected from traditional beef markets in Seoul, Korea. A total of 23 STEC strains were isolated from 15 samples (four strains from three omasa, 10 from five abomasa, and nine from seven large intestines). The O serotypes and toxin gene types of all STEC isolates were identified, and antimicrobial resistance was assessed using the disk diffusion method. The isolation rates of STEC from edible beef intestines were 2.8% in omasum, 4.2% in abomasums, and 5.9% in large intestines. All STEC isolates harbored either stx1, or both stx1 and stx2 genes simultaneously. Among the 23 isolates, 13 strains were identified as 11 different O serogroups, and 10 strains were untypable. However, enterohemorrhagic Esherichia coli O157, O26, and O111 strains were not isolated. The highest resistance rate observed was against tetracycline (39%), followed by streptomycin (35%) and ampicillin (22%). Of the 23 isolates, 12 isolates (52%) were resistant to at least one antibiotic, nine (39%) isolates were resistant to two or more antibiotics, and one isolate from an abmasum carried resistance against nine antibiotics, including beta-lactam/beta-lactamase inhibitor in combination and cephalosporins. This study shows that edible beef by-products, which are often consumed as raw food in many countries, including Korea, can be potential vehicles for transmission of antimicrobial-resistant pathogenic E. coli to humans.

Technical note: The persistence of microbial-specific DNA sequences through gastric digestion in lambs and their potential use as microbial markers.

Two groups of 5 lambs were euthanized at the weaning (T45) and fattening stages (T90) to evaluate the use of microbial ribosomal DNA (rDNA) sequences as potential microbial markers in relation to purine bases (PB) as a conventional marker. Both microbial markers originated similar microbial N concentrations (mg/g of DM), although T45 showed decreased values compared with the T90 group when either PB or rDNA were considered (P = 0.02). The survival of microbial rDNA was determined in 3 digestive sites (omasum, abomasum, and duodenum), but no substantial differences were observed, indicating that rDNA maintains the molecular stability along the sampling sites analyzed. Contrarily PB concentration increased successively along the digestive tract (P < 0.05), likely as a consequence of the endogenous PB secretion. Undegraded milk PB may also explain the overestimation of the microbial N concentration (2.8 times greater) using PB than rDNA sequences. Abomasum was the sampling site where the best agreement between PB and rDNA estimations was observed. Protozoal N concentration was irrelevant in T45 animals, although substantial in T90 lambs (18% of microbial N). In conclusion, bacterial 16S and protozoal 18S rDNA sequences may persist through the gastric digestive tract and their utilization as a highly specific microbial marker should not be neglected.

Short communication: Detection of yeast DNA in omasal digesta of dairy cows consuming dried distillers grains and solubles.

Purine analysis is widely used to estimate microbial crude protein (MCP) flow, and the method assumes that all purines contained in feed are degraded in the rumen and that purines detected are of microbial origin. The objectives of our experiment were (1) to determine if DNA from yeast (Saccharomyces cerevisiae) contained in dried distillers grains and solubles (DDGS) escapes degradation in the rumen and (2) to estimate the proportion of yeast DNA compared with total bacterial DNA in omasal samples. Two ruminally fistulated Holstein dairy cows averaging 649 kg (SD = 42.0) and 126 d in milk (SD = 28.9) were fed in a crossover design during 2 periods of 21 d each. Treatments were (1) control, a total mixed ration (TMR) not containing DDGS and (2) a DDGS-based diet, a TMR in which DDGS were included at 30% of diet dry matter (DM). On d 20 and 21 at 0400 and 1600 h, omasal digesta samples were collected via a ruminal cannula, and DNA was extracted from each sample in duplicate. The DNA samples were subjected to a real-time PCR assay to detect the presence of DNA from yeast. Forward and reverse primers and a probe were designed to target a DNA segment contained on the second chromosome of Saccharomyces cerevisiae. Real-time PCR amplification curves indicated the presence of yeast DNA in samples from both treatments. Specifically, the estimate of relative abundance of yeast DNA from digesta samples collected from animals consuming the diet containing DDGS was 9.46 ± 0.67/g of DM and was significantly higher than that from animals consuming no DDGS, which was observed to be 0.091 ± 0.67/g of DM. Omasal samples were also analyzed for total bacterial DNA. Primers and a probe were designed from DNA encoding part of the 16S rRNA. When the DDGS-based diet was fed, the relative abundance of total bacterial DNA tended to increase from 610 to 626±3.82/g of DM. Results suggest that yeast DNA is detected in the omasum and this is increased when cows consume DDGS but it does not represent a significant proportion of total microbial DNA in the omasal digesta samples.

Quantifying ruminal nitrogen metabolism using the omasal sampling technique in cattle--a meta-analysis.

Mixed model analysis of data from 32 studies (122 diets) was used to evaluate the precision and accuracy of the omasal sampling technique for quantifying ruminal-N metabolism and to assess the relationships between nonammonia-N flow at the omasal canal and milk protein yield. Data were derived from experiments in cattle fed North American diets (n=36) based on alfalfa silage, corn silage, and corn grain and Northern European diets (n=86) composed of grass silage and barley-based concentrates. In all studies, digesta flow was quantified using a triple-marker approach. Linear regressions were used to predict microbial-N flow to the omasum from intake of dry matter (DM), organic matter (OM), or total digestible nutrients. Efficiency of microbial-N synthesis increased with DM intake and there were trends for increased efficiency with elevated dietary concentrations of crude protein (CP) and rumen-degraded protein (RDP) but these effects were small. Regression of omasal rumen-undegraded protein (RUP) flow on CP intake indicated that an average 32% of dietary CP escaped and 68% was degraded in the rumen. The slope from regression of observed omasal flows of RUP on flows predicted by the National Research Council (2001) model indicated that NRC predicted greater RUP supply. Measured microbial-N flow was, on average, 26% greater than that predicted by the NRC model. Zero ruminal N-balance (omasal CP flow=CP intake) was obtained at dietary CP and RDP concentrations of 147 and 106 g/kg of DM, corresponding to ruminal ammonia-N and milk urea N concentrations of 7.1 and 8.3mg/100mL, respectively. Milk protein yield was positively related to the efficiency of microbial-N synthesis and measured RUP concentration. Improved efficiency of microbial-N synthesis and reduced ruminal CP degradability were positively associated with efficiency of capture of dietary N as milk N. In conclusion, the results of this study indicate that the omasal sampling technique yields valuable estimates of RDP, RUP, and ruminal microbial protein supply in cattle.

Technical note: A new high-performance liquid chromatography purine assay for quantifying microbial flow.

An HPLC method was developed to quantify the purines adenine and guanine and their metabolites xanthine and hypoxanthine in hydrolysates of isolated bacteria and omasal digesta and to assess the effect of using either purines only or purines plus metabolites as microbial markers for estimating microbial flow from the rumen. Individual purines and their metabolites were completely resolved on a C18 column using gradient elution with 2 mobile phases. Intraassay coefficient of variation ranged from 0.6 to 3.1%. Hydrolytic recovery of the 4 purine bases from their corresponding nucleosides averaged 101% (control), 103% (when added to bacterial isolates), and 104% (when added to omasal digesta). Mean concentrations of adenine, guanine, xanthine, and hypoxanthine were, respectively, 53, 58, 2.8, and 3.5 micromol/g of dry matter in omasal bacteria and 10, 12, 7.5, and 7.5 micromol/g of dry matter in omasal digesta, indicating that xanthine plus hypoxanthine represented 5% of total purines in bacterial hydrolysates but 41% of total purines in digesta hydrolysates. A significant negative relationship (R(2) = 0.53) between the sum of adenine and guanine and the sum of xanthine and hypoxanthine in digesta samples (but not bacterial isolates) indicated that 89% of the adenine and guanine originally present in ruminal microbes were recovered as xanthine and hypoxanthine. These results suggested that, when total purines are used as the microbial marker, both purines and their metabolites should be quantified and used to compute microbial non-ammonia N and organic matter flows.

Effects of different protein supplements on omasal nutrient flow and microbial protein synthesis in lactating dairy cows.

Eight ruminally cannulated Holstein cows that were part of a larger lactation trial were used in 2 replicated 4 x 4 Latin squares to quantify effects of supplementing protein as urea, solvent soybean meal (SSBM), cottonseed meal (CSM), or canola meal (CM) on omasal nutrient flows and microbial protein synthesis. All diets contained (% of dry matter) 21% alfalfa silage and 35% corn silage plus 1) 2% urea plus 41% high-moisture shelled corn (HMSC), 2) 12% SSBM plus 31% HMSC, 3) 14% CSM plus 29% HMSC, or 4) 16% CM plus 27% HMSC. Crude protein was equal across diets, averaging 16.6%. The CSM diet supplied the least rumen-degraded protein and the most rumen-undegraded protein. Microbial nonammonia N flow was similar among the true protein supplements but was 14% lower in cows fed urea. In vivo ruminal passage rate, degradation rate, and estimated escape for the 3 true proteins were, respectively, 0.044/h, 0.105/h, and 29% for SSBM; 0.051/h, 0.050/h, and 51% for CSM; and 0.039/h, 0.081/h, and 34% for CM. This indicated that CSM protein was less degraded because of both a faster passage rate and slower degradation rate. Omasal flow of individual AA, branched-chain AA, essential AA, nonessential AA, and total AA all were lower in cows fed urea compared with one of the true protein supplements. Among the 3 diets supplemented with true protein, omasal flow of Arg was greatest on CSM, and omasal flow of His was greatest on CSM, intermediate on CM, and lowest on SSBM. Lower flows of AA and microbial nonammonia N explained lower yields of milk yield and milk components observed on the urea diet in the companion lactation trial. These results clearly showed that supplementation with true protein was necessary to obtain sufficient microbial protein and rumen-undegraded protein to meet the metabolizable AA requirements of high-producing dairy cows.

Omasal flow of soluble proteins, peptides, and free amino acids in dairy cows fed diets supplemented with proteins of varying ruminal degradabilities.

Three ruminally and duodenally cannulated cows were assigned to an incomplete 4 x 4 Latin square with four 14-d periods and were fed diets supplemented with urea, solvent soybean meal, xylose-treated soybean meal (XSBM), or corn gluten meal to study the effects of crude protein source on omasal canal flows of soluble AA. Soluble AA in omasal digesta were fractionated by ultrafiltration into soluble proteins greater than 10 kDa (10K), oligopeptides between 3 and 10 kDa (3-10K), peptides smaller than 3 kDa (small peptides), and free AA (FAA). Omasal flow of total soluble AA ranged from 254 to 377 g/d and accounted for 9.2 to 15.9% of total AA flow. Averaged across diets, flows of AA in 10K, 3-10K, small peptides, and FAA were 29, 217, 50, and 5 g/d, respectively, and accounted for 10.3, 71.0, 17.5, and 1.6% of the total soluble AA flow. Cows with diets supplemented with solvent soybean meal had higher flows of Met, Val, and total AA associated with small peptides than those whose diets were supplemented with XSBM, whereas supplementation with corn gluten meal resulted in higher total small peptide-AA flows than did XSBM. Averaged across diets, 27, 75, and 93% of soluble AA in 10K, 3-10K, and peptides plus FAA flowing out of the rumen were of dietary origin. On average, 10% of the total AA flow from the rumen was soluble AA from dietary origin, indicating a substantial escape of dietary soluble N from ruminal degradation. Omasal concentrations and flows of soluble small peptides isolated by ultrafiltration were substantially smaller than most published ruminal small peptide concentrations and outflows measured in acid-deproteinized supernatants of digesta.

A comparison of sampling sites, digesta and microbial markers, and microbial references for assessing the postruminal supply of nutrients in dairy cows.

This study evaluated the impact of some methodological factors on the flows of nutrients at the omasal canal and duodenum of dairy cows fed corn-based diets. Three ruminally and duodenally cannulated cows were assigned to an incomplete 4 x 4 Latin square with four 14-d periods and fed diets formulated to contain different amounts and ruminal degradabilities of crude protein. Samples from the omasal canal and duodenum were obtained and processed according to methodologies routinely used in our laboratories and elsewhere. Methodological factors that were evaluated included microbial references and markers, digesta markers, and sampling sites (techniques). Considerable variation was found for the compositions of microbial references and their impact on the intestinal supply of microbial nonammonia nitrogen. Likewise, it appears that variation in measuring the ruminal outflow of nitrogen fractions of microbial and dietary origin could be reduced by using 15N rather than purines as microbial markers. Sampling from the omasum and duodenum resulted in differences for ruminal outflow and site of digestion as well as digestibility of some nutrients, particularly nitrogen fractions and starch. A sizable portion of this variation was associated with deviations from the assumed ideal behavior of digesta markers and collection of samples that were unrepresentative of true digesta. Collectively, outcomes from this study indicate that more research will be required to determine the accuracy of nutrient flows and the agreement between measurements at the omasal canal and duodenum when dairy cows are fed a variety of diets under different feeding systems. Therefore, caution is recommended when extrapolating or interpreting the underlying biology of published results as well as the results of their application (e.g., model parameters and predictions).

Effects of feeding formate-treated alfalfa silage or red clover silage on omasal nutrient flow and microbial protein synthesis in lactating dairy cows.

Eight ruminally cannulated Holstein cows that were part of a larger lactation trial were blocked by days in milk and randomly assigned to replicated 4 x 4 Latin squares to quantify effects of nonprotein N (NPN) content of alfalfa silage (AS) and red clover silage (RCS) on omasal nutrient flows. Diets, fed as total mixed rations, contained 50% dry matter from control AS (CAS), ammonium tetraformate-treated AS (TAS), late maturity RCS (RCS1), or early maturity RCS (RCS2). Silages differed in NPN and acid detergent insoluble N (% of total N): 50 and 4% (CAS); 45 and 3% (TAS); 27 and 8% (RCS1); 29 and 4% (RCS2). The CAS, TAS, and RCS2 diets had 36% high-moisture shelled corn and 3% soybean meal, and the RCS1 diet had 31% high-moisture shelled corn and 9% soybean meal. All diets contained 10% corn silage, 27% neutral detergent fiber, and 17 to 18% crude protein. Compared with RCS, feeding AS increased the supply of rumen-degraded protein and omasal flows of nonammonia N and microbial protein, which may explain the improved milk yield observed in the companion lactation trial. However, omasal flow of rumen-undegraded protein was 34% greater on RCS. Except for Arg, omasal flows of individual AA, branched-chain AA, nonessential AA, essential AA, and total AA did not differ between cows fed AS vs. RCS. Within AS diets, no differences in omasal AA flows were observed. However, omasal flows of Asp, Ser, Glu, Cys, Val, Ile, Tyr, Lys, total nonessential AA, and total AA all were higher in cows fed RCS1 vs. cows fed RCS2. In this trial, there was no advantage to reducing NPN content of hay-crop silage.

Assessment of ruminal bacterial populations and protozoal generation time in cows fed different methionine sources.

Methionine supplemented as 2-hydroxy-4-(methylthio)-butanoic acid (HMB) has been suggested to alter bacterial or protozoal populations in the rumen. Our objective was to determine if source of Met would change microbial populations in the rumen and to compare those results to samples from the omasum. The ruminal and omasal samples were collected from cows fed control (no Met), dl-Met, HMB, or the isopropyl ester of HMB (HMBi; estimated 50% rumen protection) in a replicated 4 x 4 Latin square design. In one square, changes in protozoal populations were determined using microscopic counts and denaturing gradient gel electrophoresis (DGGE), whereas changes in bacterial populations were determined using DGGE and ribosomal intergenic spacer length polymorphism (RIS-LP). Neither the protozoal counts nor the DGGE banding patterns derived from protozoa were different among the dietary treatments or for ruminal vs. omasal samples. As revealed by both DGGE and RIS-LP, bacterial populations clustered by treatments in ruminal and especially in omasal samples. Using cows from both Latin squares, the flow of protozoal cells from the rumen was quantified by multiplying protozoal cell count in omasal fluid by the omasal fluid flow (using CoEDTA as a liquid flow marker) or was estimated by rumen pool size of cells multiplied by either the ruminal dilution rate of CoEDTA (after termination of CoEDTA dosing) or the passage rate of Yb-marked particles. Compared with the omasal fluid flow measurement (16.4 h), protozoal generation time was approximated much more closely using the particulate than the fluid passage rate from the rumen (generation times of 15.7 and 7.5 h, respectively). There seems to be minimal selective retention of protozoal genera in the rumen in dairy cattle fed every 2 h. Data support the validity of the omasal sampling technique under our conditions.

Integration of ruminal metabolism in dairy cattle.

An important objective is to identify nutrients or dietary factors that are most critical for advancing our knowledge of, and improving our ability to predict, milk protein production. The Dairy NRC (2001) model is sensitive to prediction of microbial protein synthesis, which is among the most important component of models integrating requirement and corresponding supply of metabolizable protein or amino acids. There are a variety of important considerations when assessing appropriate use of microbial marker methodology. Statistical formulas and examples are included to document and explain limitations in using a calibration equation from a source publication to predict duodenal flow of purine bases from measured urinary purine derivatives in a future study, and an improved approach was derived. Sources of specific carbohydrate rumen-degraded protein components probably explain microbial interactions and differences among studies. Changes in microbial populations might explain the variation in ruminal outflow of biohydrogenation intermediates that modify milk fat secretion. Finally, microbial protein synthesis can be better integrated with the production of volatile fatty acids, which do not necessarily reflect volatile fatty acid molar proportions in the rumen. The gut and splanchnic tissues metabolize varying amounts of volatile fatty acids, and propionate has important hormonal responses influencing milk protein percentage. Integration of ruminal metabolism with that in the mammary and peripheral tissues can be improved to increase the efficiency of conversion of dietary nutrients into milk components for more efficient milk production with decreased environmental impact.

Enzyme immunohistochemistry with mono- and polyclonal antibodies in the pathological diagnosis of systemic bovine mycoses.

To improve the immunohistopathological diagnosis of systemic bovine mycoses, we have evaluated the utility of antifungal polyclonal and monoclonal antibodies, and peroxidase and alkaline phosphatase staining techniques. A rabbit polyclonal antibody to mannan from Candida albicans was specific for candidosis. The diagnosis of aspergillosis was accomplished using a rat monoclonal antibody to the galactofuran side chains of Aspergillus galactomannan. A murine monoclonal antibody reacting with weakly Con-A binding 41 and 46 kDa somatic antigens from Absidia corymbifera was used for immunostaining of zygomycetic hyphae. Peroxidase antiperoxidase (PAP) and alkaline phosphatase antialkaline phosphatase (APAAP) complexes were visualized using aminoethylcarbazole and fast red substrates. A green staining of PAP reactions with dioctyl sulfosuccinate sodium and 3,3',5,5'-tetramethylbenzidine (DONS/TMB) was effective for the demonstration of fungi in dual and triple infections. Tissue sections of experimentally infected mice were used to determine the sensitivity and specificity of the antibodies. Tissues obtained from 161 bovine mycotic lesions previously studied by indirect immunofluorescence staining were further evaluated using the three antibodies. In all of 45 lesions solely affected by aspergillosis and in three solely affected by candidosis the diagnoses were confirmed by the new evaluation. In 85 of 96 cases of single infections with zygomycetes the diagnosis was confirmed, while none of the antibodies reacted with fungal elements in the remaining 11 lesions. Aspergillus hyphae were detected in all three lesions with dual aspergillosis and zygomycosis, whereas zygomycetic material was confirmed in only two of these cases. A mixed infection of candidosis and zygomycosis in a lymph node was confirmed too. In 13 cases in which a diagnosis had not hitherto been obtained, aspergillosis and zygomycosis were recorded each in three cases.

The occurrence of fungi in bovine tissues in relation to portals of entry and environmental factors.

The occurrence of fungi in tissue specimens from 72 cattle was examined by culture, histopathology and indirect immunofluorescence staining (IIF). Groups of 12 animals each had been fed either concentrate or roughage and had been housed either in tie stalls, on slatted floors or on deep bedding. Specimens were obtained from the lung, omasum and Peyer's patches of the ileum and corresponding lymph nodes. Both hyphae and spores were made visible by IIF and by combination of IIF, morphology and conventional staining it was possible to differentiate between aspergilli and zygomycetes. In the lungs, aspergilli were detected at the same rate by morphology and IIF, whereas zygomycetes were found nearly twice as often by IIF than by culture. Fungi in pulmonary tissue were most frequent in cattle tied or kept on deep bedding (P < 0.01) as assessed by IIF. Within lymph nodes only spores were found, and Aspergillus fumigatus was the predominant species. Lesions devoid of fungi, especially ulcerations, were observed on the edges of the largest omasal laminae with a notable preference for the aboral third. The localization and histopathology suggested a reflux of acid abomasal contents to be the pathogenic principle. Granulomas with centrally located plant material were found more frequently in cattle fed roughage than in cattle fed concentrate (P = 0.01) and were differentiated from mycotic granulomas.

Ruminant forestomach and abomasal mucormycosis under rumen acidosis.

Spores of Absidia corymbifera were inoculated orally into sheep with ruminal acidosis produced by feeding barley. Lesions, which developed in forestomachs of all four inoculated cases, included desquamation of superficial layers of the mucosae and focal necrosis from lamina propria to muscular layers. Granulomatous lesions were in the submucosa of three sheep. Lesions in the abomasum (two sheep) included focal necrosis, diffuse hemorrhages, and infiltration of neutrophils. All lesions were accompanied by mycotic proliferation. These results show that A. corymbifera can invade forestomach mucosae through degenerate epithelium resulting from ruminal acidosis.