Cellular production of a de novo membrane cytochrome.

Heme-containing integral membrane proteins are at the heart of many bioenergetic complexes and electron transport chains. The importance of these electron relay hubs across biology has inspired the design of de novo proteins that recreate their core features within robust, versatile, and tractable protein folds. To this end, we report here the computational design and in-cell production of a minimal diheme membrane cytochrome which successfully integrates into the cellular membrane of live bacteria. This synthetic construct emulates a four-helix bundle found in modern respiratory complexes but has no sequence homology to any polypeptide sequence found in nature. The two b-type hemes, which appear to be recruited from the endogenous heme pool, have distinct split redox potentials with values close to those of natural membrane-spanning cytochromes. The purified protein can engage in rapid biomimetic electron transport with small molecules, with other redox proteins, and with biologically relevant diffusive electron carriers. We thus report an artificial membrane metalloprotein with the potential to serve as a functional electron transfer module in both synthetic protocells and living systems.

An expandable, modular de novo protein platform for precision redox engineering.

The electron-conducting circuitry of life represents an as-yet untapped resource of exquisite, nanoscale biomolecular engineering. Here, we report the characterization and structure of a de novo diheme "maquette" protein, 4D2, which we subsequently use to create an expanded, modular platform for heme protein design. A well-folded monoheme variant was created by computational redesign, which was then utilized for the experimental validation of continuum electrostatic redox potential calculations. This demonstrates how fundamental biophysical properties can be predicted and fine-tuned. 4D2 was then extended into a tetraheme helical bundle, representing a 7 nm molecular wire. Despite a molecular weight of only 24 kDa, electron cryomicroscopy illustrated a remarkable level of detail, indicating the positioning of the secondary structure and the heme cofactors. This robust, expressible, highly thermostable and readily designable modular platform presents a valuable resource for redox protein design and the future construction of artificial electron-conducting circuitry.

A de novo peroxidase is also a promiscuous yet stereoselective carbene transferase.

By constructing an in vivo-assembled, catalytically proficient peroxidase, C45, we have recently demonstrated the catalytic potential of simple, de novo-designed heme proteins. Here, we show that C45's enzymatic activity extends to the efficient and stereoselective intermolecular transfer of carbenes to olefins, heterocycles, aldehydes, and amines. Not only is this a report of carbene transferase activity in a completely de novo protein, but also of enzyme-catalyzed ring expansion of aromatic heterocycles via carbene transfer by any enzyme.

Structure and Dynamics of Three Escherichia coli NfsB Nitro-Reductase Mutants Selected for Enhanced Activity with the Cancer Prodrug CB1954.

Escherichia coli NfsB has been studied extensively for its potential for cancer gene therapy by reducing the prodrug CB1954 to a cytotoxic derivative. We have previously made several mutants with enhanced activity for the prodrug and characterised their activity in vitro and in vivo. Here, we determine the X-ray structure of our most active triple and double mutants to date, T41Q/N71S/F124T and T41L/N71S. The two mutant proteins have lower redox potentials than wild-type NfsB, and the mutations have lowered activity with NADH so that, in contrast to the wild-type enzyme, the reduction of the enzyme by NADH, rather than the reaction with CB1954, has a slower maximum rate. The structure of the triple mutant shows the interaction between Q41 and T124, explaining the synergy between these two mutations. Based on these structures, we selected mutants with even higher activity. The most active one contains T41Q/N71S/F124T/M127V, in which the additional M127V mutation enlarges a small channel to the active site. Molecular dynamics simulations show that the mutations or reduction of the FMN cofactors of the protein has little effect on its dynamics and that the largest backbone fluctuations occur at residues that flank the active site, contributing towards its broad substrate range.

The structures of E. coli NfsA bound to the antibiotic nitrofurantoin; to 1,4-benzoquinone and to FMN.

NfsA is a dimeric flavoprotein that catalyses the reduction in nitroaromatics and quinones by NADPH. This reduction is required for the activity of nitrofuran antibiotics. The crystal structure of free Escherichia coli NfsA and several homologues have been determined previously, but there is no structure of the enzyme with ligands. We present here crystal structures of oxidised E. coli NfsA in the presence of several ligands, including the antibiotic nitrofurantoin. Nitrofurantoin binds with the furan ring, rather than the nitro group that is reduced, near the N5 of the FMN. Molecular dynamics simulations show that this orientation is only favourable in the oxidised enzyme, while potentiometry suggests that little semiquinone is formed in the free protein. This suggests that the reduction occurs by direct hydride transfer from FMNH- to nitrofurantoin bound in the reverse orientation to that in the crystal structure. We present a model of nitrofurantoin bound to reduced NfsA in a viable hydride transfer orientation. The substrate 1,4-benzoquinone and the product hydroquinone are positioned close to the FMN N5 in the respective crystal structures with NfsA, suitable for reaction, but are mobile within the active site. The structure with a second FMN, bound as a ligand, shows that a mobile loop in the free protein forms a phosphate-binding pocket. NfsA is specific for NADPH and a similar conformational change, forming a phosphate-binding pocket, is likely to also occur with the natural cofactor.

Decorated networks of native proteins: nanomaterials with tunable mesoscopic domain size.

Natural and artificial proteins with designer properties and functionalities offer unparalleled opportunity for functional nanoarchitectures formed through self-assembly. However, to exploit this potential we need to design the system such that assembly results in desired architecture forms while avoiding denaturation and therefore retaining protein functionality. Here we address this challenge with a model system of fluorescent proteins. By manipulating self-assembly using techniques inspired by soft matter where interactions between the components are controlled to yield the desired structure, we have developed a methodology to assemble networks of proteins of one species which we can decorate with another, whose coverage we can tune. Consequently, the interfaces between domains of each component can also be tuned, with potential applications for example in energy - or electron - transfer. Our model system of eGFP and mCherry with tuneable interactions reveals control over domain sizes in the resulting networks.

Protein-polymer mixtures in the colloid limit: Aggregation, sedimentation, and crystallization.

While proteins have been treated as particles with a spherically symmetric interaction, of course in reality, the situation is rather more complex. A simple step toward higher complexity is to treat the proteins as non-spherical particles and that is the approach we pursue here. We investigate the phase behavior of the enhanced green fluorescent protein (eGFP) under the addition of a non-adsorbing polymer, polyethylene glycol. From small angle x-ray scattering, we infer that the eGFP undergoes dimerization and we treat the dimers as spherocylinders with aspect ratio L/D - 1 = 1.05. Despite the complex nature of the proteins, we find that the phase behavior is similar to that of hard spherocylinders with an ideal polymer depletant, exhibiting aggregation and, in a small region of the phase diagram, crystallization. By comparing our measurements of the onset of aggregation with predictions for hard colloids and ideal polymers [S. V. Savenko and M. Dijkstra, J. Chem. Phys. 124, 234902 (2006) and Lo Verso et al., Phys. Rev. E 73, 061407 (2006)], we find good agreement, which suggests that the behavior of the eGFP is consistent with that of hard spherocylinders and ideal polymers.

Effects of peripartal yeast culture supplementation on lactation performance, blood biomarkers, rumen fermentation, and rumen bacteria species in dairy cows.

Feeding yeast culture fermentation products has been associated with improved feed intake and milk yield in transition dairy cows. These improvements in performance have been further described in terms of rumen characteristics, metabolic profile, and immune response. The objective of this study was to evaluate the effects of a commercial yeast culture product (YC; Culture Classic HD, Phibro Animal Health) on performance, blood biomarkers, rumen fermentation, and rumen bacterial population in dairy cows from -30 to 50 d in milk (DIM). Forty Holstein dairy cows were enrolled in a randomized complete block design from -30 to 50 DIM and blocked according to expected calving day, parity, previous milk yield, and genetic merit. At -30 DIM, cows were assigned to either a basal diet plus 114 g/d of ground corn (control; n = 20) or a basal diet plus 100 g/d of ground corn and 14 g/d of YC (n = 20), fed as a top-dress. Cows received the same close-up diet from 30 d prepartum until calving [1.39 Mcal/kg of dry matter (DM) and 12.3% crude protein (CP)] and lactation diet from calving to 50 DIM (1.60 Mcal/kg of DM and 15.6% CP). Blood samples and rumen fluid were collected at various time points from -30 to 50 d relative to calving. Cows fed YC compared with control showed a trend for increased energy-corrected milk (+3.2 kg/d). Lower somatic cell counts were observed in YC cows than in control. We detected a treatment × time interaction in nonesterified fatty acids (NEFA) that could be attributed to a trend for greater NEFA in YC cows than control at 7 DIM, followed by lower NEFA in YC cows than control at 14 and 30 DIM. In the rumen, YC contributed to mild changes in rumen fermentation, mainly increasing postpartal valerate while decreasing prepartal isovalerate. This was accompanied by alterations in rumen microbiota, including a greater abundance of cellulolytic (Fibrobacter succinogenes) and lactate-utilizing bacteria (Megasphaera elsdenii). These results describe the potential benefits of supplementing yeast culture during the late pregnancy through early lactation, at least in terms of rumen environment and performance.

Effects of yeast culture supplementation on lactation performance and rumen fermentation profile and microbial abundance in mid-lactation Holstein dairy cows.

The continuous trend for a narrowing margin between feed cost and milk prices across dairy farms in the United States highlights the need to improve and maintain feed efficiency. Yeast culture products are alternative supplements that have been evaluated in terms of milk performance and feed efficiency; however, less is known about their potential effects on altering rumen microbial populations and consequently rumen fermentation. Therefore, the objective of this study was to evaluate the effect of yeast culture supplementation on lactation performance, rumen fermentation profile, and abundance of major species of ruminal bacteria in lactating dairy cows. Forty mid-lactation Holstein dairy cows (121 ± 43 days in milk; mean ± standard deviation; 32 multiparous and 8 primiparous) were used in a randomized complete block design with a 7-d adaptation period followed by a 60-d treatment period. Cows were blocked by parity, days in milk, and previous lactation milk yield and assigned to a basal total mixed ration (TMR; 1.6 Mcal/kg of dry matter, 14.6% crude protein, 21.5% starch, and 38.4% neutral detergent fiber) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.). Treatments were top-dressed over the TMR once a day. Cows were individually fed 1 × /d throughout the trial. Blood and rumen fluid samples were collected in a subset of cows (n = 10/treatment) at 0, 30, and 60 d of the treatment period. Rumen fluid sampled via esophageal tubing was analyzed for ammonia-N, volatile fatty acids (VFA), and ruminal bacteria populations via quantitative PCR amplification of 16S ribosomal DNA genes. Milk yield was not affected by treatment effects. Energy balance was lower in YC cows than CON, which was partially explain by the trend for lower dry matter intake as % body weight in YC cows than CON. Cows fed YC had greater overall ruminal pH and greater total VFA (mM) at 60 d of treatment period. There was a contrasting greater molar proportion of isovalerate and lower acetate proportion in YC-fed cows compared with CON cows. Although the ruminal abundance of specific fiber-digesting bacteria, including Eubacterium ruminantium and Ruminococcus flavefaciens, was increased in YC cows, others such as Fibrobacter succinogenes were decreased. The abundance of amylolytic bacteria such as Ruminobacter amylophilus and Succinimonas amylolytica were decreased in YC cows than CON. Our results indicate that the yeast culture supplementation seems to promote some specific fiber-digesting bacteria while decreasing amylolytic bacteria, which might have partially promoted more neutral rumen pH, greater total VFA, and isovalerate.

Controlling Protein Nanocage Assembly with Hydrostatic Pressure.

Controlling the assembly and disassembly of nanoscale protein cages for the capture and internalization of protein or non-proteinaceous components is fundamentally important to a diverse range of bionanotechnological applications. Here, we study the reversible, pressure-induced dissociation of a natural protein nanocage, E. coli bacterioferritin (Bfr), using synchrotron radiation small-angle X-ray scattering (SAXS) and circular dichroism (CD). We demonstrate that hydrostatic pressures of 450 MPa are sufficient to completely dissociate the Bfr 24-mer into protein dimers, and the reversibility and kinetics of the reassembly process can be controlled by selecting appropriate buffer conditions. We also demonstrate that the heme B prosthetic group present at the subunit dimer interface influences the stability and pressure lability of the cage, despite its location being discrete from the interdimer interface that is key to cage assembly. This indicates a major cage-stabilizing role for heme within this family of ferritins.

Trimethylamine-N-oxide is present in human follicular fluid and is a negative predictor of embryo quality.

STUDY QUESTION: Are levels of trimethylamine-N-oxide (TMAO) in human follicular fluid (FF) related to IVF outcomes? SUMMARY ANSWER: Higher levels of TMAO are a negative predictor of oocyte fertilization and embryo quality. WHAT IS KNOWN ALREADY: TMAO is a metabolic product of dietary choline and l-carnitine produced via subsequent enzymatic modifications by the intestinal microbiota and hepatocytes. TMAO promotes inflammatory and oxidative stress pathways and has been characterized as a causative biomarker for the development of cardiometabolic disease. STUDY DESIGN, SIZE, DURATION: For the present cross-sectional study, samples (FF and plasma) from 431 modified natural cycle (MNC)-IVF cycles of 132 patients were collected prospectively between October 2014 and March 2018 in a single academic medical center. PARTICIPANTS/MATERIALS, SETTING, METHODS: TMAO and its precursors (choline, l-carnitine and gamma-butyrobetaine) were measured by ultra-high-performance liquid chromatography/mass spectrometry in (i) matched FF and plasma from 63 MNC-IVF cycles, in order to compare metabolite levels in the two matrices and (ii) FF from 232 MNC-IVF cycles in which only one oocyte was retrieved at follicular puncture. The association between metabolite levels and oocyte fertilization, embryo fragmentation percentage, embryo quality and the occurrence of pregnancy was analyzed using multilevel generalized estimating equations with adjustment for patient and cycle characteristics. MAIN RESULTS AND THE ROLE OF CHANCE: The level of choline was higher in FF as compared to matched plasma (P < 0.001). Conversely, the levels of TMAO and gamma-butyrobetaine were lower in FF as compared to plasma (P = 0.001 and P = 0.075, respectively). For all metabolites, there was a positive correlation between FF and plasma levels. Finally, levels of TMAO and its gut-derived precursor gamma-butyrobetaine were lower in FF from oocytes that underwent normal fertilization (TMAO: odds ratio [OR] 0.66 [0.49-0.90], P = 0.008 per 1.0-µmol/L increase; gamma-butyrobetaine: OR 0.77 [0.60-1.00], P = 0.047 per 0.1-µmol/L increase) and developed into top-quality embryos (TMAO: OR 0.56 [0.42-0.76], P < 0.001 per 1.0-µmol/L increase; gamma-butyrobetaine: OR 0.79 [0.62-1.00], P = 0.050 per 0.1-µmol/L increase) than in FF from oocytes of suboptimal development. LIMITATIONS, REASONS FOR CAUTION: The individual contributions of diet, gut bacteria and liver to the metabolite pools have not been quantified in this analysis. WIDER IMPLICATIONS OF THE FINDINGS: More research on the contribution of diet and the effect of gut bacteria on FF TMAO is warranted. Since TMAO integrates diet, microbiota and genetic setup of the person, our results indicate potential important clinical implications for its use as biomarker for lifestyle interventions to improve fertility. STUDY FUNDING/COMPETING INTEREST(S): No external funding was received for this project. The Department of Obstetrics and Gynecology of the University Medical Center Groningen received an unrestricted educational grant of Ferring Pharmaceutical BV, the Netherlands. The authors have no other conflicts of interest. TRIAL REGISTRATION NUMBER: Netherlands Trial Register number NTR4409.

Ethanol Stimulation of Microglia Release Increases ERK1/2-Dependent Neuronal cPLA2 Activity in Immature Cultured Cortical Preparations.

Ethanol consumption typically begins during adolescence and is associated with age-dependent responses and maladaptive neuronal consequences. Our previous work established the role of a putative signaling cascade involving cytoplasmic phospholipase A2 (cPLA2), arachidonic acid (AA) and novel protein kinase C isoforms in adolescent hypnotic sensitivity. The current study aimed to further delineate this pathway by ascertaining the cellular specificity as well as the upstream activators of cPLA2 using an immature cultured cortical preparation. A threefold increase in cPLA2 was detected within 2 min of 100 mM ethanol exposure as measured by phosphorylation of serine 505 (Ser505). Increases in cPLA2 activity were further observed to be primarily confined to neuronal cells. Increases in the number of neurons co-expressing cPLA2 Ser505 phosphorylation were prevented by preincubation with an ERK1/2 inhibitor, but not P38 MAPK inhibition. Finally, conditioned media studies were used to determine whether glial cells were involved in the ethanol-induced neuronal cPLA2 activity. Rapid increases in neuronal cPLA2 activity appears to be initiated through ethanol stimulated microglial, but not astrocytic releasable factors. Taken together, these data extend the proposed signaling cascade involved in developmental ethanol responding.

Symposium review: Colostrum management and calf nutrition for profitable and sustainable dairy farms.

Multistate dairy management research project NC-2042, which is part of the National Institute of Food and Agriculture, has devoted an ongoing objective to calf and heifer nutrition and management. Within this objective, colostrum research has been a priority due to continued opportunities to improve this area on US dairy farms. Research has focused on heating colostrum to reduce bacterial populations and pathogens while increasing IgG absorption. Research also identified other proteins that are reduced when heating colostrum. Studies indicated an apparent upper limit to IgG absorption from colostrum fed to calves. Additional studies have shed light on absorption of IgG from colostrum replacers and evaluated the use of lactoferrin and sodium bicarbonate in both maternal colostrum and colostrum replacers. Milk replacer formulation, feeding strategies, and the effect of calfhood nutrition on future performance have also been researched. Finally, water quality and its effect on calves and heifers have been studied. This review focuses on research done by multistate research project NC-2042 member states in the area of colostrum and calf nutrition and management.

Symposium review: Strategies to improve the efficiency and profitability of heifer raising.

Regional Research Project NC-2042 has a main objective to study calf and heifer nutrition. Within this objective, feeding the postweaned heifer is considered a major priority to improve the profitability and sustainability of US dairy farms. Through optimizing nutrient utilization by precision feeding, using alternative feeds, high-fiber diets, and feed additives, this research group has worked to enhance dairy heifer nutrition. Research has focused on precision feeding heifers and incorporating high- and low-fiber diets into this system of feeding. This is accomplished by meeting the nutrient needs of the heifer for a desired rate of growth while enhancing total-tract nutrient digestibility, reducing waste and improving profitability. High-fiber forages have been studied as a means of controlling ad libitum dry matter intakes and thus weight gain in heifers. These results provide producers with a means of feeding heifers while reducing costs. Similarly, utilizing alternative feedstuffs in heifer diets has also been a major research area for this group including comprehensive research on distillers co-products, and new protein sources such as camelina and carinata meals. Results indicated that these products can be satisfactorily incorporated into heifer diets. Studying feed additives has also been a function of the research group. Research with Ascophyllum nodosum and cinnamaldehyde indicated that calves find these additives unpalatable and that supplementing cinnamaldehyde to postweaned heifers showed no benefit. However, sodium butyrate and yeast supplementation proved to be beneficial in the growth and feed efficiency of heifers. Research from this group has an effect on heifer feeding, resulting in new information that can aid in the sustainability of dairy farms. This review will focus on the area of postweaned heifer nutrition.

Altered rumen fermentation patterns in lactating dairy cows supplemented with phytochemicals improve milk production and efficiency.

Tannins and other phytochemicals are known to improve RUP in the diet by binding protein and then limiting ruminal degradation, which may improve milk yield and milk protein synthesis. The objective of this study was to evaluate the effects of dietary phytochemicals (tannins and Capsicum species) as rumen modifiers on production parameters and milk efficiency in dairy cows. Twenty-four multiparous Holstein cows (96 ± 16 d in milk; mean ± standard deviation) were used in a replicated 3 × 3 Latin square design balanced to measure carryover effects. Cows were blocked according to days in milk, milk production, and body weight and randomly assigned to 1 of 3 groups (n = 8/group). Each group was assigned to a unique treatment sequence across the 3 periods in the Latin square. The experiment consisted of a 14-d covariate period and three 30-d treatment periods. Cows received a basal diet supplemented with soybean meal pellets (SB) as the control diet, phytochemicals (RUM; Rumiviv, CCPA, Janzé, France) pelleted with soybean meal, or expeller soybean meal (ESBM; SoyPlus, West Central Soy, Ralston, IA). Milk production and dry matter intake during the last 4 d of each period were used for statistical analysis. Blood and rumen fluid samples were collected on d 27 of each period. Rumen fluid was analyzed for ammonia N and volatile fatty acids as well as ruminal bacteria via quantitative PCR amplification of 16S ribosomal DNA genes. Greater milk yield (37.9 vs. 36 kg/d), energy-corrected milk (39.7 vs 37.1 kg/d), and protein yield (1.15 vs. 1.08 kg/d) were observed in RUM compared with SB, but these parameters were similar between RUM and ESBM. Concentrations of total volatile fatty acids (118.1 vs. 101.5 mM) were greater in RUM in comparison to SB and ESBM diets. Cows fed RUM had greater ß-hydroxybutyrate (0.49 vs. 0.42 mmol/L) than SB and ESBM. Selenomonas ruminantium, Succinimonas amylolytica, and Streptococcus bovis in rumen fluid were lower in RUM fed cows in comparison to SB and ESBM. Increased total volatile fatty acids and lower ruminal abundance of bacteria associated with low feed efficiency in RUM cows can partially explain the improvements observed in milk yield and milk efficiency. Overall, these data suggest that feeding a combination of tannin mixture and Capsicum can significantly affect rumen fermentation characteristics via partial manipulation of rumen microbiota, and these effects were reflected in improved milk production and efficiency.

Growth performance, nutrient utilization, and health of dairy calves supplemented with condensed whey solubles.

Our objective was to evaluate growth performance, nutrient utilization, and health of calves supplemented with condensed whey solubles (CWS). Twenty-four Holstein calves (2 d old) in hutches were used in a 12-wk randomized complete block design study. Calves were blocked by birth date and sex. Treatments were (1) control (CON) with no supplement and (2) 50 g/d of CWS. Preweaning CWS was fed with milk and postweaning CWS was top-dressed on starter pellets. Calves were fed 2.83 L of pasteurized milk 2×/d during wk 1 to 5, 1×/d in wk 6, and weaned at d 42. Calves had ad libitum access to starter pellets and water. Individual intakes of milk and starter pellets were measured daily. Fecal scores (0 = firm, 3 = watery) and respiratory scores (healthy ≤3, sick ≥5), calculated from the sum of scores for rectal temperature, cough, ocular, and nasal discharge, were recorded daily. Body weights (BW), frame growth, and jugular blood samples were taken once every week at 3 h after the morning feeding. Fecal grab samples were collected in wk 12 for analysis of apparent total-tract digestibility. Data were analyzed using MIXED procedures of SAS 9.4 (SAS Institute Inc., Cary, NC) with repeated measures. Total dry matter intake was greater in calves fed CWS compared with CON. Calves fed CWS tended to have greater BW during the postweaning period. Gain:feed, average daily gains, withers heights, and concentrations of plasma urea nitrogen were not different. Concentrations of plasma ß-hydroxybutyrate were greater in calves fed CWS compared with CON. Serum glucose concentrations were greater in calves fed CON during the preweaning stage, but similar concentrations were found between treatments during the postweaning stage. Rumen pH, ammonia-N, and total volatile fatty acids were similar between treatments. The acetate:propionate ratio was greater in calves fed CWS. The apparent total-tract digestion of nutrients was similar; however, CON tended to have greater digestion of neutral detergent fiber. Fecal scores were not different, but a treatment by week interaction was observed. Supplementing CWS improved starter intake, postweaning BW, ß-hydroxybutyrate, fecal scores, and maintained frame growth.

Photoswitchable Phase Separation and Oligonucleotide Trafficking in DNA Coacervate Microdroplets.

Coacervate microdroplets produced by liquid-liquid phase separation have been used as synthetic protocells that mimic the dynamical organization of membrane-free organelles in living systems. Achieving spatiotemporal control over droplet condensation and disassembly remains challenging. Herein, we describe the formation and photoswitchable behavior of light-responsive coacervate droplets prepared from mixtures of double-stranded DNA and an azobenzene cation. The droplets disassemble and reassemble under UV and blue light, respectively, due to azobenzene trans/cis photoisomerisation. Sequestration and release of captured oligonucleotides follow the dynamics of phase separation such that light-activated transfer, mixing, hybridization, and trafficking of the oligonucleotides can be controlled in binary populations of the droplets. Our results open perspectives for the spatiotemporal control of DNA coacervates and provide a step towards the dynamic regulation of synthetic protocells.

Evaluation of carinata meal as a feedstuff for growing dairy heifers: Effects on growth performance, rumen fermentation, and total-tract digestibility of nutrients.

Our objective was to determine the effects of feeding carinata meal (CRM) compared with distillers dried grains with solubles (DDGS) on growth performance, rumen fermentation, and nutrient utilization in peripubertal dairy heifers. A 16-wk randomized block design experiment with 24 Holstein heifers [6.6 ± 0.7 mo and 218 ± 27 kg of body weight (BW)] was conducted. Treatments diets were (1) 10% cold-pressed CRM and (2) 10% DDGS on a dry matter (DM) basis. The remainder of the diets consisted of grass hay, ground corn, soybean meal, and mineral mix. Diets were formulated to be isonitrogenous and isocaloric. Heifers were individually fed using a Calan gate feeding system, and the rations were limit-fed at 2.65% of BW on a DM basis to target a 0.8 kg/d of average daily gain. Heifers were weighed every 2 wk and the ration amount offered was adjusted accordingly. Frame sizes, BW, and body condition scores were measured 2 d every 2 wk throughout the study. During wk 12 and 16, rumen fluid samples were collected via esophageal tubing for pH, ammonia N, and volatile fatty acid analyses. In wk 16, fecal grab samples were collected for apparent total-tract digestibility estimation. Heifer DM intake, BW, average daily gain, and gain:feed were similar between treatments. No differences were observed between treatments in frame measurements or body condition scores. Rumen pH tended to be greater in CRM compared with DDGS. Rumen ammonia N and total volatile fatty acid concentration were not different between treatments. Apparent total-tract digestibility of DM, neutral detergent fiber, and acid detergent fiber were decreased in CRM compared with DDGS. A tendency was detected for reduced organic matter digestibility in CRM. No difference was observed between treatments for crude protein total-tract digestibility. However, these differences in total-tract nutrient digestibility were not large enough to influence growth performance. Overall, results demonstrated that growing heifers can be limit-fed diets with 10% CRM and maintain growth performance compared with a control diet containing 10% DDGS.

Growth performance, rumen fermentation, nutrient utilization, and metabolic profile of dairy heifers limit-fed distillers dried grains with ad libitum forage.

The objective of this study was to determine the effects of feeding a corn- and soybean-product-based concentrate mix or distillers dried grains with solubles concentrate mix with ad libitum grass hay to dairy heifers. A 16-wk randomized complete block design study was conducted using 24 heifers [18 Holstein and 6 Brown Swiss; 219 ± 2 d of age (±standard deviation); 230 ± 4 kg of body weight] to evaluate the effect of diet on dry matter intake (DMI), growth performance, rumen fermentation, metabolic profile, and nutrient digestibility. Treatments were (1) corn and soybean product concentrate mix, and (2) distillers-dried-grains-with-solubles-based concentrate mix (DDG). Both concentrate mixes were limit-fed at 0.8% of body weight and grass hay was offered ad libitum. Heifers were individually fed using Calan gates and orts were recorded daily at feeding. Heifers were weighed every 2 wk and ration concentrate mix offered was adjusted accordingly. Frame measurements and body condition score were recorded every 2 wk. Rumen fluid was collected via esophageal tubing during wk 12 and 16 for pH, ammonia N, and volatile fatty acid analysis. Jugular blood samples were collected every 4 wk for metabolite and metabolic hormone analysis. Total-tract digestibility of nutrients was evaluated during wk 16 by fecal grab sampling. No treatment by week interactions were observed for any of the growth measurements and growth measurements and DMI did not differ between treatments. A treatment by time interaction was observed for rumen butyrate percentage with heifers fed DDG having a greater percentage. Total volatile fatty acid concentration, acetate molar percentage, and acetate:propionate decreased with the DDG treatment, whereas propionate molar percentage increased. No treatment by week interactions were observed for any of the metabolites or metabolic hormones measured. A tendency was observed for glucose and plasma urea nitrogen concentration to decrease with DDG. Plasma cholesterol and insulin increased with DDG. Results demonstrated that limit-feeding heifers DDG at 0.8% of body weight with ad libitum grass hay maintained growth performance, average daily gain, DMI, and gain:feed, with shifts in the metabolic profile compared with the corn and soybean product concentrate mix.

Growth performance and health of dairy calves given water treated with a reverse osmosis system compared with municipal city water.

Our objective was to determine effects of drinking reverse osmosis water (RW) versus municipal city water (MW) on growth, nutrient utilization, and health scores of calves. Twenty-four Holstein calves (14 females, 10 males; 2 d old; 44.6 ± 6.10 kg of body weight), housed in individual hutches, were used in a 10-wk randomized complete block design study. Calves were blocked by birthdate and sex. Treatments were RW (Culligan Water Filtration System, Brookings, SD) versus MW (Brookings Municipal Utilities, Brookings, SD) that contained 13 and 387 mg/L of total dissolved solids, respectively. Milk replacer (28% crude protein; 18% fat) was fed twice daily during wk 1 through 5 and then once daily during wk 6. At each feeding, 0.45 kg of dry milk replacer was mixed with 2.83 L of the respective water type according to treatment. Calves were fed water, respective to treatment, and calf starter pellets ad libitum throughout the study. All intakes were recorded daily. Daily total respiratory scores (healthy ≤3, sick ≥5) were calculated from the sum of scores for rectal temperature, cough, and ocular and nasal discharges. Fecal consistency scores (0 = firm, 3 = watery) were also recorded daily. Body weights and frame growth were measured 2 d every 2 wk and jugular blood samples were collected 1 d every 2 wk at 3 h after the morning feeding. Fecal grab samples were collected 5 times per day for 3 d during wk 10 for analysis of apparent total-tract digestibility of nutrients. We found treatment by week interactions for dry matter intake and gain-to-feed ratio, and total dry matter intake increased more during the study for RW than MW. Gain-to-feed ratio was greater during the first few weeks of the feeding period for RW compared with MW and then comparable during the rest of study. Water intake was less in RW than MW, potentially indicating more efficient water use by calves. Frame growth, body weight, average daily gain, serum glucose, plasma urea nitrogen, ß-hydroxybutyrate, total-tract digestibility of dry matter, and crude protein were similar. Fecal scores tended to be lower (firmer) in calves on RW, with an interaction by time. Respiratory scores decreased during the weaning period when calves drank RW. Results demonstrated calves drinking RW had similar growth and improved health scores with treatment by time interactions compared with MW.