Concentrate levels and \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e affect rumen fluid-associated bacteria numbers in dairy heifers

Total viable rumenbacteria counts through the use of colony-unit forming assays lack accuracy because they only include culturable bacteria capable of initiating cell division. Thus, bacterial counts can be underestimated. The use of fluorescent characteristics of cell membranes allows flow cytometry to enumerate and distinguish dead from live bacteria cells. The objective of this experiment was to investigate the viable and total ruminal bacteria counts when 3 levels of forage:concentrate in diets were fed at restricted levels with the addition of Saccharomycescerevisiae (YC). Three cannulated post-pubertal Holstein heifers (age 18 ± 1.0 months) were fed corn silage (CS)-based diets in a 3-period (35 d) Latin square design. Heifers were fed the diets for 21 d with no yeast addition, followed by 14 d where yeast culture (YC) was added (1 g/kg as-fed basis); (Yea-Sacc1026, Alltech, Inc., Nicholasville, KY). A low concentrate (LC) TMR (80% CS, 20% concentrate; 12.4% CP, 35% NDF), a medium concentrate (MC) TMR (60% CS, 40% concentrate; 12.3% CP. 28% NDF), and a high concentrate (HC) TMR (40% CS, 60% concentrate; 12.6% CP, 25% NDF), were fed once per day. Rumenfluid was sampled − 2, 0, 2, 4, 6, 8, 10, 12 h after feeding. Samples were immediately stained with fluorescent dyes using the BacLight kit (Molecular Probes Inc., Eugene, OR) and analyzed with a Coulter XL-MCL single laser flow cytometer. Mean rumen viable bacteria counts linearly increased among treatments (4.96, 4.78, 6.73 × 1011 ± 0.53 × 1011 cells/ml; P = 0.02) for LC, MC and HC respectively, and YC addition increased number of viable bacteria cells (P \u3c 0.01). Total and viable bacteria counts decreased for the first 2 h after feeding then increased 4 h post-feeding. Dietary concentratelevel and YC can alter rumenbacteria counts as measured by this method

Effectiveness of Two Ruminally Protected Methionine Sources for Lactating Dairy Cows

Two sources of ruminally protected methionine were tested for their ability to provide available methionine to lactating dairy cattle. Based on milk protein yield and milk protein percent, NTP-1401 (an unreleased product from Novus International, Inc., St. Charles, MO) and Smartamine (Adisseo, αretta, GA) provided similar amounts of available methionine to the cows. These two products led to different methioninerelated compounds appearing in blood plasma, suggesting that they contained different methionine precursors

Immune responses in lactating Holstein cows supplemented with Cu, Mn, and Zn as sulfates or methionine hydroxy analogue chelates

The aim of this study was to compare effects of inorganic sulfate versus chelated forms of supplemental Cu, Mn, and Zn on milk production, plasma and milk mineral concentrations, neutrophil activity, and antibody titer response to a model vaccination. Holstein cows (n = 25) were assigned in 2 cohorts based on calving date to a 12-wk randomized complete block design study. The first cohort consisted of 17 cows that had greater days in milk (DIM; mean of 77 DIM at the start of the trial) than the second cohort of 8 cows (32 DIM at the start of the trial). Diets were formulated to supplement 100% of National Research Council requirements of Cu, Mn, and Zn by either inorganic trace minerals (ITM) in sulfate forms or chelated trace minerals (CTM) supplied as metal methionine hydroxy analog chelates, without accounting for trace mineral contribution from other dietary ingredients. Intake and milk production were recorded daily. Milk composition was measured weekly, and milk Cu, Mn, and Zn were determined at wk 0 and 8. Plasma Cu and Zn concentrations and neutrophil activity were measured at wk 0, 4, 8, and 12. Neutrophil activity was measured by in vitro assays of chemotaxis, phagocytosis, and reactive oxygen species production. A rabies vaccination was administered at wk 8, and vaccine titer response at wk 12 was measured by both rapid fluorescent focus inhibition test and ELISA. Analyzed dietary Cu was 21 and 23 mg/kg, Mn was 42 and 46 mg/kg, and Zn was 73 and 94 mg/ kg for the ITM and CTM diets, respectively. No effect of treatment was observed on milk production, milk composition, or plasma minerals. Dry matter intake was reduced for CTM compared with ITM cows, but this was largely explained by differences in body weight between treatments. Milk Cu concentration was greater for CTM than ITM cows, but this effect was limited to the earlier DIM cohort of cows and was most pronounced for multiparous compared with primiparous cows. Measures of neutrophil function were unaffected by treatment except for an enhancement in neutrophil phagocytosis with the CTM treatment found for the later DIM cohort of cows only. Rabies antibody titer in CTM cows was 2.8 fold that of ITM cows as measured by ELISA, with a trend for the rapid fluorescent focus inhibition test. Supplementation of Cu, Mn, and Zn as chelated sources may enhance immune response of early lactation dairy cows compared with cows supplemented with inorganic sources

Characterization of rumen microbiota in lactating Holstein cows fed molasses versus corn grain at two levels of rumen-degradable protein

We evaluated the influence of diets differing in non-fiber carbohydrates and rumen-degradable protein (RDP) levels on changes in the ruminal bacterial populations in lactating Holstein cows. In all, 12 ruminally cannulated cows were assigned to diets with high or low RDP levels. Within each RDP level, molasses was substituted for corn grain at a concentration of 0%, 5.25%, or 10.5% of diet dry matter in a replicated 3 × 3 Latin square design with 28-day periods. Liquid and solid rumen digesta fractions collected at the end of each period underwent 16S rRNA gene sequencing to identify operational taxonomic units and were analyzed for short-chain fatty acids. Protein degradability affected 6 bacterial genera, whereas carbohydrate alteration impacted 13 genera (p < 0.05). Of the 30 genera with the highest relative abundance, 26 differed by digesta fraction (p < 0.05), with Bacteroidetes genera showing a greater abundance in solids and Firmicutes genera demonstrating a greater prevalence in liquids. Regarding relative abundances, with increasing molasses, Succiniclasticum decreased in liquid (p < 0.05), and CF231, YRC22, Clostridium, Desulfovibrio, BF311, and Oscillospira increased in solids (p < 0.05). In contrast, at higher RDP levels, Succiniclasticum increased while YRC22 and Pseudobutyrivibrio decreased in solids (p < 0.05). Genera with abundances found to be correlated with fermentation products in the liquid included Shuttleworthia, Treponema, Lachnospira, and Schwartzia, which typically have lower relative abundances, showing strong positive correlations with molar proportions (mol%) of propionate, butyrate, and valerate (p < 0.05), and negative correlations with pH and acetate mol% (p < 0.05). Fibrobacter was positively correlated with lactate mol% (p < 0.05). Butyrate mol% exhibited a quadratic increase as molasses increased (p = 0.017), and lactate mol% rose with increased RDP levels (p = 0.042). No treatment effects were detected for pH propionate and valerate mol%; however, we observed a tendency (p = 0.075) for a quadratic effect of molasses treatment on the mol% of acetate. These findings substantiate the pivotal role of diet in shaping rumen microbiota and metabolism, elucidating a nuanced relationship between dietary components, bacterial community structure, and metabolic output. This offers a more detailed understanding of rumen function and the potential for high-precision dietary management in lactating cows

Genome-Wide Modeling of Transcription Preinitiation Complex Disassembly Mechanisms using ChIP-chip Data

Apparent occupancy levels of proteins bound to DNA in vivo can now be routinely measured on a genomic scale. A challenge in relating these occupancy levels to assembly mechanisms that are defined with biochemically isolated components lies in the veracity of assumptions made regarding the in vivo system. Assumptions regarding behavior of molecules in vivo can neither be proven true nor false, and thus is necessarily subjective. Nevertheless, within those confines, connecting in vivo protein-DNA interaction observations with defined biochemical mechanisms is an important step towards fully defining and understanding assembly/disassembly mechanisms in vivo. To this end, we have developed a computational program PathCom that models in vivo protein-DNA occupancy data as biochemical mechanisms under the assumption that occupancy levels can be related to binding duration and explicitly defined assembly/disassembly reactions. We exemplify the process with the assembly of the general transcription factors (TBP, TFIIB, TFIIE, TFIIF, TFIIH, and RNA polymerase II) at the genes of the budding yeast Saccharomyces. Within the assumption inherent in the system our modeling suggests that TBP occupancy at promoters is rather transient compared to other general factors, despite the importance of TBP in nucleating assembly of the preinitiation complex. PathCom is suitable for modeling any assembly/disassembly pathway, given that all the proteins (or species) come together to form a complex

Immune responses in lactating Holstein cows supplemented with Cu, Mn, and Zn as sulfates or methionine hydroxy analogue chelates

The aim of this study was to compare effects of inorganic sulfate versus chelated forms of supplemental Cu, Mn, and Zn on milk production, plasma and milk mineral concentrations, neutrophil activity, and antibody titer response to a model vaccination. Holstein cows (n = 25) were assigned in 2 cohorts based on calving date to a 12-wk randomized complete block design study. The first cohort consisted of 17 cows that had greater days in milk (DIM; mean of 77 DIM at the start of the trial) than the second cohort of 8 cows (32 DIM at the start of the trial). Diets were formulated to supplement 100% of National Research Council requirements of Cu, Mn, and Zn by either inorganic trace minerals (ITM) in sulfate forms or chelated trace minerals (CTM) supplied as metal methionine hydroxy analog chelates, without accounting for trace mineral contribution from other dietary ingredients. Intake and milk production were recorded daily. Milk composition was measured weekly, and milk Cu, Mn, and Zn were determined at wk 0 and 8. Plasma Cu and Zn concentrations and neutrophil activity were measured at wk 0, 4, 8, and 12. Neutrophil activity was measured by in vitro assays of chemotaxis, phagocytosis, and reactive oxygen species production. A rabies vaccination was administered at wk 8, and vaccine titer response at wk 12 was measured by both rapid fluorescent focus inhibition test and ELISA. Analyzed dietary Cu was 21 and 23 mg/kg, Mn was 42 and 46 mg/kg, and Zn was 73 and 94 mg/ kg for the ITM and CTM diets, respectively. No effect of treatment was observed on milk production, milk composition, or plasma minerals. Dry matter intake was reduced for CTM compared with ITM cows, but this was largely explained by differences in body weight between treatments. Milk Cu concentration was greater for CTM than ITM cows, but this effect was limited to the earlier DIM cohort of cows and was most pronounced for multiparous compared with primiparous cows. Measures of neutrophil function were unaffected by treatment except for an enhancement in neutrophil phagocytosis with the CTM treatment found for the later DIM cohort of cows only. Rabies antibody titer in CTM cows was 2.8 fold that of ITM cows as measured by ELISA, with a trend for the rapid fluorescent focus inhibition test. Supplementation of Cu, Mn, and Zn as chelated sources may enhance immune response of early lactation dairy cows compared with cows supplemented with inorganic sources

The transcription elongation factor TFIIS is a component of RNA polymerase II preinitiation complexes

In this article, we provide direct evidence that the evolutionarily conserved transcription elongation factor TFIIS functions during preinitiation complex assembly. First, we identified TFIIS in a mass spectrometric screen of RNA polymerase II (Pol II) preinitiation complexes (PICs). Second, we show that the association of TFIIS with a promoter depends on functional PIC components including Mediator and the SAGA complex. Third, we demonstrate that TFIIS is required for efficient formation of active PICs. Using truncation mutants of TFIIS, we find that the Pol II-binding domain is the minimal domain necessary to stimulate PIC assembly. However, efficient formation of active PICs requires both the Pol II-binding domain and the poorly understood N-terminal domain. Importantly, Domain III, which is required for the elongation function of TFIIS, is dispensable during PIC assembly. The results demonstrate that TFIIS is a PIC component that is required for efficient formation and/or stability of the complex