David R. Mertens

pH dynamics and bacterial community composition in the rumen of lactating dairy cows

The influence of pH dynamics on ruminal bacterial community composition was studied in 8 ruminally cannulated Holstein cows fitted with indwelling electrodes that recorded pH at 10-min intervals over a 54-h period. Cows were fed a silage-based total mixed ration supplemented with monensin. Ruminal samples were collected each day just before feeding and at 3 and 6h after feeding. Solid and liquid phases were separated at collection, and extracted DNA was subjected to PCR amplification followed by automated ribosomal intergenic spacer analysis (ARISA). Although cows displayed widely different pH profiles (mean pH=6.11 to 6.51, diurnal pH range=0.45 to 1.39), correspondence analysis of the ARISA profiles revealed that 6 of the 8 cows showed very similar bacterial community compositions. The 2 cows having substantially different community compositions had intermediate mean pH values (6.30 and 6.33) and intermediate diurnal pH ranges (averaging 0.89 and 0.81 pH units). Fortuitously, these 2 cows alone also displayed milk fat depression, along with markedly higher ruminal populations of 1 bacterial operational taxonomic unit (OTU) and reduced populations of another ARISA amplicon. Cloning and sequencing of the elevated OTU revealed phylogenetic similarity to Megasphaera elsdenii, a species reportedly associated with milk fat depression. The higher populations of both M. elsdenii and OTU246 in these 2 cows were confirmed using quantitative real-time PCR (qPCR) with species-specific primers, and the fraction of total bacterial rDNA copies contributed by these 2 taxa were very highly correlated within individual cows. By contrast, the fraction of total bacterial rDNA copies contributed by Streptococcus bovis and genus Ruminococcus, 2 taxa expected to respond to ruminal pH, did not differ among cows (mean= <0.01 and 10.6%, respectively, of rRNA gene copies, determined by qPCR). The data indicate that cows with widely differing pH profiles can have similar ruminal bacterial community compositions, and that milk fat depression can occur at intermediate ruminal pH. The results support recent reports that milk fat depression is associated with shifts in bacterial community composition in rumine and is specifically related to the relative abundance of Megasphaera elsdenii.

Shifts in bacterial community composition in the rumen of lactating dairy cows under milk fat-depressing conditions

Eighteen ruminally cannulated dairy cattle were fed a series of diets (in 28-d periods) designed to elicit different degrees of milk fat depression (MFD) for the purpose of relating MFD to ruminal bacterial populations. Cows were fed a TMR containing 25% starch (DM basis) supplied as corn silage, a slowly fermented starch (SFS treatment, period 1), then switched to a TMR containing 27% starch, much of it supplied as ground high-moisture corn, a rapidly fermented starch (RFS treatment, period 2). In period 3, the RFS diet was amended with 13.6 mg of monensin/kg of DM (RFS/Mon treatment), and in period 4, the cows were returned to the RFS diet without monensin (RFS/Post treatment). Effect of both starch source and monensin on milk fat percentage varied by cow, and cluster analysis identified 4 pairs of cows having distinct milk fat patterns. Archived ruminal liquors and solids from the 4 pairs were processed to isolate bacterial DNA, which was subjected to automated ribosomal intergenic spacer analysis followed by correspondence analysis to visualize bacterial community composition (BCC). One pair of cows (S-responsive) showed MFD on RFS feeding, but displayed no additional MFD upon monensin feeding and a fat rebound upon monensin withdrawal. The second pair of cows (M-responsive) showed no MFD upon switch from the SFS diet to the RFS diet, but displayed strong MFD upon monensin feeding and no recovery after monensin withdrawal. Both groups displayed major shifts in BCC upon dietary shifts, including dietary shifts that both did and did not change milk fat production. The third pair of cows (SM-responsive) displayed reduction of milk fat on both RFS and RFS/Mon diets, and fat returned to the levels on the RFS diet upon monensin withdrawal; these cows showed a more gradual shift in BCC in response to both starch source and monensin. The fourth pair of cows (nonresponsive) did not display changes in milk fat percentage with dietary treatment and showed only minor shifts in BCC with dietary treatment. Regardless of milk fat response, BCC did not reassemble its original state upon monensin withdrawal, though the difference was strongest in M-responsive cows. One amplicon length (representing a single bacterial species) was elevated in most, but not all, MFD-susceptible (S-, M-, or SM-responsive) cows relative to milk fat-nonresponsive cows, whereas 2 amplicon lengths displayed reduced abundance under MFD conditions. Overall, this study demonstrates an association between MFD and wholesale shifts of microbial communities in the rumen.

Effect of monensin feeding and withdrawal on populations of individual bacterial species in the rumen of lactating dairy cows fed high-starch rations

Real-time polymerase chain reaction (PCR) was used to quantify 16 procaryotic taxa in the rumina of two lactating dairy cows following supply and subsequent withdrawal of the feed additive monensin (13.9xa0mg/kg of diet dry matter) in a high-starch, silage-based ration. PCR was conducted on DNA from rumen samples collected 6xa0h post feeding on two successive days before monensin supplementation, after 30xa0days of monensin supplementation, and at six weekly intervals after monensin withdrawal. Mean values of relative population size (RPS, the percent of bacterial 16S rRNA copy number) for genus Prevotella increased (Pu2009<u20090.05) from 41.8% without monensin to 49.2% with monensin and declined to 42.5% after monensin withdrawal. Mean RPS values for two biohydrogenating species (Megasphaera elsdenii and Butyrivibrio fibrisolvens) were low (<0.4%) and declined several-fold in response to monensin. Mean RPS values for the biohydrogenating species Eubacterium ruminantium, four cellulolytic species, four starch- or dextrin-fermenting species, and Domain Archaea were not altered (Pu2009>u20090.10) upon monensin feeding or withdrawal. The data suggest that monensin in high-starch diets does not suppress populations of classical ruminal Gram-positive bacteria or the availability of H2, though it may affect bacteria involved in biohydrogenation of lipids that regulate bovine mammary lipogenesis.

Effect of reduced ferulate-mediated lignin/arabinoxylan cross-linking in corn silage on feed intake, digestibility, and milk production1

Cross-linking of lignin to arabinoxylan by ferulates limits in vitro rumen digestibility of grass cell walls. The effect of ferulate cross-linking on dry matter intake (DMI), milk production, and in vivo digestibility was investigated in ad libitum and restricted-intake digestion trials with lambs, and in a dairy cow performance trial using the low-ferulate sfe corn mutant. Silages of 5 inbred corn lines were fed: W23, 2 W23sfe lines (M04-4 and M04-21), B73, and B73bm3. As expected, the W23sfe silages contained fewer ferulate ether cross-links and B73bm3 silage had a lower lignin concentration than the respective genetic controls. Silages were fed as the sole ingredient to 4 lambs per silage treatment. Lambs were confined to metabolism crates and fed ad libitum for a 12-d adaptation period followed by a 5-d collection period of feed refusals and feces. Immediately following the ad libitum feeding trial, silage offered was limited to 2% of body weight. After a 2-d adaptation to restricted feeding, feed refusals and feces were collected for 5 d. Seventy Holstein cows were blocked by lactation, days in milk, body weight, and milk production and assigned to total mixed ration diets based on the 5 corn silages. Diets were fed for 28 d and data were collected on weekly DMI and milk production and composition. Fecal grab samples were collected during the last week of the lactation trial for estimation of feed digestibility using acid-insoluble ash as a marker. Silage, total mixed ration, feed refusals, and fecal samples were analyzed for crude protein, starch, neutral detergent fiber (NDF), cell wall polysaccharides, and lignin. The W23sfe silages resulted in lower DMI in the ad libitum trial than the W23 silage, but DMI did not differ in the restricted trial. No differences were observed for NDF or cell wall polysaccharide digestibility by lambs with restricted feeding, but the amount of NDF digested daily increased for lambs fed the M04-21 W23sfe silage ad libitum. Lambs were less selective against NDF and lignin when offered W23sfe silages. The B73bm3 silage did not affect DMI or digestibility of cell walls at the restricted feeding level, but total daily NDF digested was greater at ad libitum intake. Intake, milk production, and cell wall digestibility were greater for cows fed diets containing W23sfe silages than for those fed W23 silage. Although milk production was greater for the B73bm3 diet, DMI and cell wall digestibility were not altered. Cows were less selective against cell wall material when fed both W23sfe and B73bm3 silages. Reduced ferulate cross-linking in sfe corn silage is a new genetic mechanism for improving milk production.

A ring test of in vitro neutral detergent fiber digestibility: Analytical variability and sample ranking1

In vitro neutral detergent fiber (NDF) digestibility (NDFD) is an empirical measurement of fiber fermentability by rumen microbes. Variation is inherent in all assays and may be increased as multiple steps or differing procedures are used to assess an empirical measure. The main objective of this study was to evaluate variability within and among laboratories of 30-h NDFD values analyzed in repeated runs. Subsamples of alfalfa (n=4), corn forage (n=5), and grass (n=5) ground to pass a 6-mm screen passed a test for homogeneity. The 14 samples were sent to 10 laboratories on 3 occasions over 12 mo. Laboratories ground the samples and ran 1 to 3 replicates of each sample within fermentation run and analyzed 2 or 3 sets of samples. Laboratories used 1 of 2 NDFD procedures: 8 labs used procedures related to the 1970 Goering and Van Soest (GVS) procedure using fermentation vessels or filter bags, and 2 used a procedure with preincubated inoculum (PInc). Means and standard deviations (SD) of sample replicates within run within laboratory (lab) were evaluated with a statistical model that included lab, run within lab, sample, and lab × sample interaction as factors. All factors affected mean values for 30-h NDFD. The lab × sample effect suggests against a simple lab bias in mean values. The SD ranged from 0.49 to 3.37% NDFD and were influenced by lab and run within lab. The GVS procedure gave greater NDFD values than PInc, with an average difference across all samples of 17% NDFD. Because of the differences between GVS and PInc, we recommend using results in contexts appropriate to each procedure. The 95% probability limits for within-lab repeatability and among-lab reproducibility for GVS mean values were 10.2 and 13.4%, respectively. These percentages describe the span of the range around the mean into which 95% of analytical results for a sample fall for values generated within a lab and among labs. This degree of precision was supported in that the average maximum difference between samples that were not declared different by means separation was 4.4% NDFD. Although the values did not have great precision, GVS labs were able to reliably rank sample data in order of 30-h NDFD (Spearman correlation coefficient = 0.93) with 80% of the rankings correct or off by only 1 ranking. A relative ranking system for NDFD could reduce the effect of within- and among-lab variation in numeric values. Such a system could give a more accurate portrayal of the comparative values of samples than current numeric values imply.

Forage Quality for Ruminants: Plant and Animal Considerations1

Abstract Forage quality is a function of nutrient concentration, intake, nutrient availability, and partitioning of metabolized products within animals. Of these factors, intake potential is the major determinant of production by animals fed forage-based diets; however, it is one of the most difficult aspects of forage quality to determine or predict because variation among animals has a large influence on intake. Physical fill limits intake of forages with high cell wall concentrations when fed to animals with high energy demand. Hence, grasses, with their higher cell wall concentration, typically have lower intake than legumes. Energy availability of forage is also limited by cell wall concentration because cell contents are almost completely digested, whereas forage cell walls are slowly digested. Thus, the proportion of cell walls to cell contents is a major determinant of energy availability in feeds. Protein digestion by ruminants is complex. When crude protein concentration in herbage drops below 7% of dry matter, ruminal fermentation of forages may be limited and protein requirements of animals may not be met. Additionally, inefficient use of protein in high quality forages may limit performance of high producing animals. Usually, only about 25% of the forage protein escapes degradation from the rumen. Efficiency would be improved if a larger portion of forage protein passed from the rumen undegraded so that it can be degraded in the intestines where absorption is more efficient. Another important plant factor influencing forage quality is herbage maturity. Systems are now available for determining maturity of both legumes and grasses that will become more important as aids for predicting forage quality before forages are harvested or grazed. Forage quality is also influenced by the environment in which forages are grown and by soil fertility and these cause year-to-year, seasonal, and geographical variation in forage quality even when herbage is harvested at the same stage of maturity.

Morphology, fiber composition and mean particle diameter relationships in ground barley and oat forages at different ages☆

Abstract Six cuttings (4 and 6 weeks regrowth, 3 harvests per season) of barley and oat were analyzed to evaluate relationships among morphology, fiber composition and mean particle diameter (MPD) of ground forages. Plants were separated into leaf blades, leaf sheaths, stems and inflorescences, ground through a Wiley mill (8 mm), and sieved, and four fractions (screen apertures 1.18 mm (F1), 0.60 (F2), 0.30 (F3) and