Animal performance and environmental efficiency of cool- and warm-season annual grazing systems.

Annual forage crops can provide short-term grazing between crop rotations or can be interseeded into perennial pastures to increase forage quality and productivity. They also provide an opportunity to increase the economic and environmental sustainability of grazing systems. Cool-season annual forage crops provide high-quality, abundant forage biomass when forage availability from perennial forage species is lacking, reducing the need for stored feeds during the winter months. For example, ADG of 1.5 kg have been reported using small grains alone and in mixtures with annual ryegrass (Lolium multiflorum Lam.) while maintaining an average stocking rate of 3.5 animals/ha. No-till (NT) establishment has been shown to be as effective as conventional tillage for establishing small grain pastures. Stocker performance during the fall was not affected by tillage treatment, but during the spring grazeout, BW gain per hectare was 8% greater in NT pastures. An in vitro study showed that daily production of CH4 was 84% lower, respectively, in turnip (Brassica rapa L.) and rapeseed (B. napus L.) diets compared with annual ryegrass. Warm-season annuals are frequently used during the summer forage slump when perennial pasture growth and quality are reduced. Research has shown that brown mid-rib sorghum × sudangrass (BMR SSG; Sorghum bicolor L. × S. arundinaceous Desv.) and pearl millet (PM; Pennisteum glaucum L.R. Br.) with crabgrass (Digitaria sanguinalis (L.) Scop.) tended to have greater ADG (0.98 kg) than sorghum × sudangrass or peal millet alone (0.85 kg). However, non-BMR and BMR SSG tended to have greater gains per hectare than PM or PM + crabgrass (246, 226, 181, and 188 kg/ha, respectively). Feeding of brown mid-rib sorghum × sudangrass reduced daily production of CH4 and CH4 per gram of NDF fed by 66% and 50%, respectively, compared with a perennial cool-season forage in continuous culture. Cool- and warm-season annual pastures not only provide increased animal gains, but also increase soil cover and in vitro data suggest that annual forages (i.e., brassicas and warm-season annual grasses) decrease enteric CH4 emissions. Establishment method, grazing management, and weather conditions all play important roles in the productivity and environmental impact of these systems. A more complete life cycle analysis is needed to better characterize how management and climatic conditions impact the long-term economic and environmental sustainability of grazing annuals.

Stockpiled Tifton 85 bermudagrass for backgrounding stocker cattle.

A 2-yr grazing trial was conducted to evaluate the use of conserved forage type, stockpiled Tifton 85 bermudagrass ( L.; T85) or T85 hay, and supplement type for stocker cattle during the fall forage deficit in the southeastern United States. The study site consisted of six 0.75-ha paddocks of stockpiled T85 for grazing and six 0.20-ha drylot paddocks for feeding T85 hay. Steers ( = 5 per paddock; mean initial BW for both yr, 263 ± 30 kg) were randomly assigned to paddocks on November 11, 2014 and October 28, 2015 in yr 1 and yr 2, respectively. Treatments included T85 hay or stockpile (control), T85 hay or stockpile plus 0.2 kg cottonseed meal and 0.7 kg soybean hulls∙steer∙d (SUPPL1), or 0.45 kg cottonseed meal and 0.45 kg soybean hulls∙steer∙d (SUPPL2). Polytape fencing was used for frontal grazing stockpiled T85 paddocks to allocate a 3- to 4-d allotment of forage DM for the animals based on available forage mass and steer DMI requirements. There were no differences among treatments ( > 0.10) in mean pregrazing forage mass across sampling dates (5,099 kg DM/ha and 7,998 kg DM/ha in yr 1 and yr 2, respectively), forage allowance (1.9 kg DM/kg steer BW and 3.0 kg DM/kg steer BW in yr 1 and yr 2, respectively) and forage utilization (84% and 88% in yr 1 and yr 2, respectively). No differences ( > 0.10) were detected for nutritive value parameters CP, ADF, NDF, and TDN among stockpiled T85 treatments in both yr. However, there were differences across sampling dates ( < 0.0001) for stockpiled T85 such that forage quality generally declined as the grazing season progressed in both yr. Steers fed T85 hay had greater final BW and ADG ( = 0.018) than those grazing stockpiled T85. Mean final BW and ADG of steers grazing T85 stockpile or fed hay with no supplementation was less ( = 0.020) than those receiving supplementation. Steers grazing stockpiled T85 had decreased ADG in the second half of the grazing season in both yr of the study, which is a result of declining forage nutritive value as the stockpiling season progressed. Results indicate that stockpiled T85 supplemented with CP and energy can support stocker cattle at a maintenance level, but to achieve a target gain of 0.9 kg/day would require a greater level of supplementation to overcome decreasing forage nutritive value dynamics.

Fiber digestion kinetics and protein degradability characteristics of stockpiled Tifton 85 bermudagrass.

A 2-yr study was conducted to determine effects of N fertilization level on fiber digestion kinetics and protein degradability characteristics of stockpiled Tifton 85 bermudagrass (T85). Six 0.76-ha pastures of stockpiled T85 were cut to a 10-cm stubble height on August 1 of each yr and fertilized with 56 (56N), 112 (112N), or 168 (168N) kg N/ha (2 pastures/treatment). Fiber digestion kinetics included the 72-hr potential extent of NDF digestion (PED), rate of NDF digestion, and lag time. In yr 1 and 2, PED decreased over the stockpile season. Rates of NDF digestion did not differ ( > 0.05) among N fertilization treatments in either yr. In yr 1, rate of NDF digestion was greatest ( < 0.05) in October/November, and decreased beginning in December. In yr 2, rate of NDF digestion decreased ( < 0.05) in January compared with November, but digestion rates were similar for November and January 21 sampling dates. Lag time was greater ( < 0.05) for the 112N than 56N and 168N treatments, and increased ( < 0.05) across sampling dates in yr 1. In yr 2, lag time increased ( < 0.05) from 9.0 to 17.7 h across the season. In yr 1 and 2, a negative correlation ( < 0.05) between forage lignin concentration and both PED ( = -0.91 and -0.87 in yr 1 and 2, respectively) and rate of NDF digestion ( = -0.60 and -0.25 in yr 1 and 2, respectively) was observed. There was a trend ( = 0.06) for lignin concentration to be positively correlated with lag time ( = 0.39) in yr 1, and a strong relationship was observed in yr 2 ( = 0.91; < 0.05). The RDP fraction as a % of CP was ≥ 90% throughout both years. Concentration of RDP (% of total DM) decreased across the stockpiling season through January in yr 1 and 2. Results suggest that kinetic parameters of NDF digestion in stockpiled T85 were influenced more by temporal changes over the stockpile season than by N fertilization level. Supplement formulations based on kinetic parameters of fiber digestion may require periodic adjustment to insure that energy-yielding components of NDF are sufficient to meet animal requirements throughout the stockpile season. The CP fraction in stockpiled T85 contains sufficient RDP to support fibrolytic activity and growth of ruminal microorganisms throughout the stockpile season. Toward the latter end of the season, supplementation with sources of digestible fiber and RDP could be expected to increase MP supply to the host animal.

Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics.

BACKGROUND: Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification. RESULTS: Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies. CONCLUSION: The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings.

Calcium ion binding regions in C-reactive protein: location and regulation of conformational changes.

C-reactive protein (CRP) is a pentameric acute phase serum protein composed of identical 206 amino acid subunits that associate by non-covalent bonds. The biological activities ascribed to CRP are initiated by binding ligands via the single PC-binding site within each subunit. CRP binding to PC requires a conformational change in the intact pentraxin triggered by the binding of two free Ca2+ ions per subunit. Residues 134-148 of each subunit were previously implicated by indirect measures as one of the Ca(2+)-binding sites. In this study, 45Ca2+ autoradiography revealed that fragments of CRP of 6.5 and 16 kDa generated by proteolysis between residues 146 and 147 bind Ca2+ indicating that a second Ca(2+)-binding site is located within the C-terminal 60 amino acids. Synthetic peptides corresponding to residues 134-148 and 152-176 both bound 45Ca2+ in equilibrium dialysis experiments with a Kd = 5.2 x 10(-4) and 1.7 x 10(-4) M, respectively. The addition of Ca2+ to peptide 152-176 induced a shift in the CD-spectra between 210 and 230 nm. Rabbit anti-peptide 152-176 antibody (Ab) inhibited the availability of an epitope within the PC-binding site of CRP recognized by mAb EA4-1. Reactivity of CRP with both anti-peptide 134-148 mAb and anti-peptide 152-176 Ab enhanced the expression of the PC-binding site epitope. The results suggest that the two distinct Ca(2+)-binding sites within each CRP subunit are composed of residues 134-148 and 152-176 and that these two nearly adjacent sites cooperate to exert an allosteric change in conformation allowing access to the PC-binding site.

Cell attachment peptide of C-reactive protein: critical amino acids and minimum length.

Human C-reactive protein (CRP) is an acute phase blood component that accumulates at sites of tissue damage and necrosis and is degraded by neutrophils to biologically active peptides. A dodecapeptide composed of amino acids 27-38 of CRP mediates cell attachment in vitro. This peptide was designated the cell-binding peptide (CB-Pep) of CRP. Characterization of the interaction between fibroblasts and modified synthetic peptides with sequential deletions from either the N-terminus or C-terminus revealed that the minimal sequence for cell attachment or inhibition of cell attachment to the CB-Pep was Phe-Thr-Val-Cys-Leu, which corresponds to residues 33-37 within each of the five 206 amino acid subunits of CRP. The pentapeptide by itself mediated cell attachment. Substitutions for each residue within the CB-Pep indicated that the critical residues for activity were Phe-33 and Thr-34. This cell-binding pentapeptide represents a recognition motif for cell adhesion not found in other proteins.

A cell attachment peptide from human C-reactive protein.

The serum acute phase reactant, C-reactive protein (CRP), is selectively deposited at sites of tissue damage and degraded by neutrophils into biologically active peptides. A synthetic peptide corresponding to residues 27-38 present in each of the five identical subunits of CRP mediated cell attachment activity in vitro. Although the CRP-derived peptide contains a Tuftsin (TKPR)-like sequence at its amino-terminus, the Tuftsin tetrapeptide itself, as well as several synthetic peptides of CRP, failed to inhibit the cell-attachment activity to the CRP-derived peptide. Peptides containing the sequences responsible for the cell attachment activity of the extracellular matrix proteins, fibronectin (Fn) and laminin, failed to inhibit the CRP-derived peptide cell attachment activity. However, the addition of the RGDS and RGDSPASSLP cell-binding peptides of Fn to cells enhanced attachment to the active peptide from CRP. In the converse experiment, the cell-binding peptide of CRP did not influence cell attachment to Fn or laminin. A peptide corresponding to the same stretch of amino acid residues within the homologous Pentraxin, serum amyloid P-component (SAP), displayed nearly identical cell-attachment activity. Several monoclonal antibodies (mAb) specific for the CRP-derived cell-binding peptide neutralized its cell-attachment activity. These mAbs reacted with intact CRP and neutralized the cell-binding activity of CRP itself. The findings suggest that a peptide with cell-binding activity could be generated from the breakdown of CRP and then contribute directly to cellular events leading to tissue repair.

Monoclonal antibodies to the calcium-binding region peptide of human C-reactive protein alter its conformation.

Five mouse mAb were generated against a synthetic peptide corresponding to the proposed Ca(2+)-binding region of human C-reactive protein (CRP). The peptide consists of amino acids 134 to 148 and possesses a calmodulin Ca(2+)-binding sequence. The mAb reacted with a surface epitope(s) on native, intact CRP as well as the closely related pentraxin protein, serum amyloid P-component. Three of the 5 mAb inhibited the Ca(2+)-dependent phosphorylcholine-(PC) binding activity of CRP, but did not bind to the PC-binding region itself. Four of the five mAb also inhibited the recognition of an epitope in the PC-binding site of CRP. Four of the mAb partially, or completely, protected CRP from selective cleavage by pronase between residues 146 and 147. The findings suggest that the Ca(2+)-binding region is on the surface of CRP, has substantial flexibility, and is probably responsible for the allosteric effects of Ca2+ ions on CRP.

A synthetic peptide corresponding to the phosphorylcholine (PC)-binding region of human C-reactive protein possesses the TEPC-15 myeloma PC-idiotype.

C-reactive protein (CRP) is a major acute phase reactant in most mammalian species. CRP molecules from all species display Ca2(+)-dependent binding to phosphorylcholine (PC). The conserved PC-binding region of CRP corresponds to amino acids 51-66 within the human CRP sequence. A synthetic peptide composed of residues 47-63 of human CRP was previously shown to possess PC binding activity. The charged amino acids at positions 57, 58, 60, and 62 of this synthetic peptide were critical for PC-binding based on lower binding activity of synthetic peptides containing uncharged residues at these positions. The PC-binding peptide was used to generate mouse mAb that were tested for reactivity with intact CRP and with the TEPC-15 (T-15) mouse myeloma protein that also binds PC. The PC-binding peptide of CRP was recognized by two mAb specific for the T-15 Id. One of the mAb generated against the PC-binding peptide of CRP (IID6.2) recognized an epitope on the T-15 protein that was also recognized by the near-binding site-specific mAb (F6) to the T-15 PC-Id. Binding of IID6.2 to T-15 myeloma protein was not inhibited by PC and did not require Ca2+; however, binding was inhibited by the synthetic PC-binding peptide itself. Recognition of synthetic peptides containing uncharged amino acid substitutions by mAb F6 and IID6.2 was greatly reduced indicating that the shared epitope on T-15 and CRP was composed of similar charged residues. Therefore, CRP displays the same idiotope as an antibody that shares its specificity for the hapten, PC.