The association between selective dry cow therapy, milking routine and dry cow management practices with somatic cell count in early lactation cows from 21 commercial grazing dairy herds.

This observational study aimed to explore the association of farmer-driven selective dry cow therapy (DCT), milking routine and dry cow management practices with somatic cell count (SCC) in early lactation cows from 21 commercial dairy herds. Milking routine practices evaluated referred to cow preparation for milking, in-lactation mastitis management and recording. Dry cow management practices related to dry cow environment and cleaning, dry-off procedure, milk cessation strategy and calving environment. Data from 2,016 multiparous cows in 21 commercial spring-calving grazing dairy herds were available for the study. Herd owners self-reported DCT (the assignment and administration of DCT was at the discretion of the herd owners with no involvement from the research team), management practices during milking and the dry period. Cow-level last test-day SCC records in 2020 [range = 105 to 285 d in milk (DIM)] and first test-day records in 2021 (range = 5 to 60 DIM) were obtained from milk recording databases. Quarter-level milk sampling was carried out on all cows in late lactation of 2020 (range = 240 to 261 DIM) for bacterial culturing. Bacteriological results were used to define cows with intramammary infection (IMI) when ≥ 1 quarter sample resulted in bacterial growth and there were no contaminated samples from that cow. Mixed model analyses were used to evaluate the association of selective DCT, milking routine and dry cow management practices with cows' first test-day log 10 SCC (logSCC) in 2021. At dry-off in 2020, 47.6% of the cows were administered an internal teat sealant alone (ITS) while 52.4% were administered an antibiotic plus an internal teat sealant (AB+ITS). The mean herd-level percentage of cows with IMI was 19.7% (range = 9.8% to 39.5%); Staphylococcus aureus accounted for the majority of cow-level IMI (89.9%, 357/397). Between herds, the proportion of cows administered ITS ranged from 17.7% (14/79; in a herd with an IMI prevalence of 10.1%) to 86.8% (66/76; in a herd with an IMI prevalence of 27.6%). In total, 11.8% (105/889) and 29.8% (292/980) of cows that were administered ITS or AB+ITS had an IMI in late lactation 2020, respectively. The mean untransformed SCC at the last test-day in 2020 of cows administered ITS and AB+ITS was 55,000 and 197,200 cells/mL, respectively. The statistical analysis showed a significant interaction between selective DCT and milk yield at last test-day in 2020; cows with a milk yield of 15 kg and administered ITS had a 0.1 higher (untransformed SCC of 19,000 cells/mL higher) first test-day logSCC compared with cows administered AB+ITS. Additionally, greater parity, IMI in late lactation, higher log SCC at the last test-day in 2020 and longer dry periods were associated with higher logSCC at the first test-day in 2021. The current study identified cow- and herd-level management practices that could aid dairy farmers in improving the outcome of selective DCT and decrease early lactation SCC.

Structure and Mechanisms of Assembly-Line Polyketide Synthases.

Three decades of studies on the multifunctional 6-deoxyerythronolide B synthase have laid a foundation for understanding the chemistry and evolution of polyketide antibiotic biosynthesis by a large family of versatile enzymatic assembly lines. Recent progress in applying chemical and structural biology tools to this prototypical assembly-line polyketide synthase (PKS) and related systems has highlighted several features of their catalytic cycles and associated protein dynamics. There is compelling evidence that multiple mechanisms have evolved in this enzyme family to channel growing polyketide chains along uniquely defined sequences of 10-100 active sites, each of which is used only once in the overall catalytic cycle of an assembly-line PKS. Looking forward, one anticipates major advances in our understanding of the mechanisms by which the free energy of a repetitive Claisen-like reaction is harnessed to guide the growing polyketide chain along the assembly line in a manner that is kinetically robust yet evolutionarily adaptable.

Biological Demands and Toxicity of Isoprenoid Precursors in Bacillus Subtilis Through Cell-Permeant Analogs of Isopentenyl Pyrophosphate and Dimethylallyl Pyrophosphate.

Bacterial isoprenoids are necessary for many biological processes, including maintaining membrane integrity, facilitating intercellular communication, and preventing oxidative damage. All bacterial isoprenoids are biosynthesized from two five carbon structural isomers, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are cell impermeant. Herein, we demonstrate exogenous delivery of IPP and DMAPP into Bacillus subtilis by utilizing a self-immolative ester (SIE)-caging approach. We initially evaluated native B. subtilis esterase activity, which revealed a preference for short straight chain esters. We then examined the viability of the SIE-caging approach in B. subtilis and demonstrate that the released caging groups are well tolerated and the released IPP and DMAPP are bioavailable, such that isoprenoid biosynthesis can be rescued in the presence of pathway inhibitors. We further show that IPP and DMAPP are both toxic and inhibit growth of B. subtilis at the same concentration. Lastly, we establish the optimal ratio of IPP to DMAPP (5 : 1) for B. subtilis growth and find that, surprisingly, DMAPP alone is insufficient to rescue isoprenoid biosynthesis under high concentrations of fosmidomycin. These findings showcase the potential of the SIE-caging approach in B. subtilis and promise to both aid in novel isoprenoid discovery and to inform metabolic engineering efforts in bacteria.

Calculations of Absolute Free Energies, Enthalpies, and Entropies for Drug Binding.

Computer simulation methods can aid in the rational design of drugs aimed at a specific target, typically a protein. The affinity of a drug for its target is given by the free energy of binding. Binding can be further characterized by the enthalpy and entropy changes in the process. Methods exist to determine exact free energies, enthalpies, and entropies that are dependent only on the quality of the potential model and adequate sampling of conformational degrees of freedom. Entropy and enthalpy are roughly an order of magnitude more difficult to calculate than the free energy. This project combines a replica exchange method for enhanced sampling, designed to be efficient for protein-sized systems, with free energy calculations. This approach, replica exchange with dynamical scaling (REDS), uses two conventional simulations at different temperatures so that the entropy can be found from the temperature dependence of the free energy. A third replica is placed between them, with a modified Hamiltonian that allows it to span the temperature range of the conventional replicas. REDS provides temperature-dependent data and aids in sampling. It is applied to the bromodomain-containing protein 4 (BRD4) system. We find that for the force fields used, the free energies are accurate but the entropies and enthalpies are not, with the entropic contribution being too positive. Reproducing the entropy and enthalpy of binding appears to be a more stringent test of the force fields than reproducing the free energy.

The Effect of Maternal Probiotic or Synbiotic Supplementation on Sow and Offspring Gastrointestinal Microbiota, Health, and Performance.

The increasing prevalence of antimicrobial-resistant pathogens has prompted the reduction in antibiotic and antimicrobial use in commercial pig production. This has led to increased research efforts to identify alternative dietary interventions to support the health and development of the pig. The crucial role of the GIT microbiota in animal health and performance is becoming increasingly evident. Hence, promoting an improved GIT microbiota, particularly the pioneer microbiota in the young pig, is a fundamental focus. Recent research has indicated that the sow's GIT microbiota is a significant contributor to the development of the offspring's microbiota. Thus, dietary manipulation of the sow's microbiota with probiotics or synbiotics, before farrowing and during lactation, is a compelling area of exploration. This review aims to identify the potential health benefits of maternal probiotic or synbiotic supplementation to both the sow and her offspring and to explore their possible modes of action. Finally, the results of maternal sow probiotic and synbiotic supplementation studies are collated and summarized. Maternal probiotic or synbiotic supplementation offers an effective strategy to modulate the sow's microbiota and thereby enhance the formation of a health-promoting pioneer microbiota in the offspring. In addition, this strategy can potentially reduce oxidative stress and inflammation in the sow and her offspring, enhance the immune potential of the milk, the immune system development in the offspring, and the sow's feed intake during lactation. Although many studies have used probiotics in the maternal sow diet, the most effective probiotic or probiotic blends remain unclear. To this extent, further direct comparative investigations using different probiotics are warranted to advance the current understanding in this area. Moreover, the number of investigations supplementing synbiotics in the maternal sow diet is limited and is an area where further exploration is warranted.

The Effect of Prebiotic Supplements on the Gastrointestinal Microbiota and Associated Health Parameters in Pigs.

Establishing a balanced and diverse microbiota in the GIT of pigs is crucial for optimizing health and performance throughout the production cycle. The post-weaning period is a critical phase, as it is often associated with dysbiosis, intestinal dysfunction and poor performance. Traditionally, intestinal dysfunctions associated with weaning have been alleviated using antibiotics and/or antimicrobials. However, increasing concerns regarding the prevalence of antimicrobial-resistant bacteria has prompted an industry-wide drive towards identifying natural sustainable dietary alternatives. Modulating the microbiota through dietary intervention can improve animal health by increasing the production of health-promoting metabolites associated with the improved microbiota, while limiting the establishment and proliferation of pathogenic bacteria. Prebiotics are a class of bioactive compounds that resist digestion by gastrointestinal enzymes, but which can still be utilized by beneficial microbes within the GIT. Prebiotics are a substrate for these beneficial microbes and therefore enhance their proliferation and abundance, leading to the increased production of health-promoting metabolites and suppression of pathogenic proliferation in the GIT. There are a vast range of prebiotics, including carbohydrates such as non-digestible oligosaccharides, beta-glucans, resistant starch, and inulin. Furthermore, the definition of a prebiotic has recently expanded to include novel prebiotics such as peptides and amino acids. A novel class of -biotics, referred to as "stimbiotics", was recently suggested. This bioactive group has microbiota-modulating capabilities and promotes increases in short-chain fatty acid (SCFA) production in a disproportionally greater manner than if they were merely substrates for bacterial fermentation. The aim of this review is to characterize the different prebiotics, detail the current understating of stimbiotics, and outline how supplementation to pigs at different stages of development and production can potentially modulate the GIT microbiota and subsequently improve the health and performance of animals.

Exploring the Differential Expression of a Set of Key Genes Involved in the Regulation and Functioning of the Stomach in the Post-Weaned Pig.

Despite playing a key role in digestion, there is only a broad characterization of the spatiotemporal development of the three glandular regions of the stomach (cardiac, fundic and pyloric) in the weaned pig. Hence, the objective of this experiment was to explore the differential expression (DE) of a panel of key genes within the three glandular regions of the stomach. Eight pigs were sacrificed at d 8 post-weaning, and three mucosal samples were collected from each stomach's glandular regions. The expression of a panel of genes were measured using QPCR. The true cardiac gland region was characterized by increased expression of PIGR, OLFM4, CXCL8 and MUC2 relative to the two other regions (p < 0.05). The fundic gland region was characterized by increased expression of ATP4A, CLIC6, KCNQ1, HRH2, AQP4, HDC, CCKBR, CHIA, PGA5, GHRL and MBOAT4 compared to the two other regions (p < 0.05). The pyloric gland region was characterized by exclusive expression of GAST (p < 0.05). A transition region between the cardiac and fundic region (cardiac-to-oxyntic transition) was observed with a gene expression signature that resembles a cross of the signatures found in the two regions. In conclusion, unique gene expression signatures were identifiable in each of the glandular regions, with a cardiac-to-oxyntic transition region clearly identifiable in the post-weaned pigs' stomachs.

Evaluation of test-day milk somatic cell count to predict intramammary infection in late lactation grazing dairy cows.

Use of selective dry cow antimicrobial therapy requires to precisely differentiate cows with an intramammary infection (IMI) from uninfected cows close to drying-off to enable treatment allocation. Milk somatic cell count (SCC) is an indicator of an inflammatory response in the mammary gland and is usually associated with IMI. However, SCC can also be influenced by cow-level variables such as milk yield, lactation number and stage of lactation. In recent years, predictive algorithms have been developed to differentiate cows with IMI from cows without IMI based on SCC data. The objective of this observational study was to explore the association between SCC and subclinical IMI, taking cognizance of cow-level predictors on Irish seasonal spring calving, pasture-based systems. Additionally, the optimal test-day SCC cut-point (maximized sensitivity and specificity) for IMI diagnosis was determined. A total of 2,074 cows, across 21 spring calving dairy herds with an average monthly milk weighted bulk tank SCC of ≤200,000 cells/mL were enrolled in the study. Quarter-level milk sampling was carried out on all cows in late lactation (interquartile range = 240-261 d in milk) for bacteriological culturing. Bacteriological results were used to define cows with IMI, when ≥1 quarter sample resulted in bacterial growth. Cow-level test-day SCC records were provided by the herd owners. The ability of the average, maximum and last test-day SCC to predict infection were compared using receiver operator curves. Predictive logistic regression models tested included parity (primiparous or multiparous), yield at last test-day and a standardized count of high SCC test-days. In total, 18.7% of cows were classified as having an IMI, with first parity cows having a higher proportion of IMI (29.3%) compared with multiparous cows (16.1%). Staphylococcus aureus accounted for the majority of these infections. The last test-day SCC was the best predictor of infection with the highest area under the curve. The inclusions of parity, yield at last test-day, and a standardized count of high SCC test-days as predictors did not significantly improve the ability of last test-day SCC to predict IMI. The cut-point for last test-day SCC which maximized sensitivity and specificity was 64,975 cells/mL. This study indicates that in Irish seasonal pasture-based dairy herds, with low bulk tank SCC control programs, the last test-day SCC (interquartile range days in milk = 221-240) is the best predictor of IMI in late lactation.

Discovery and Characterization of Antibody Probes of Module 2 of the 6-Deoxyerythronolide B Synthase.

Fragment antigen-binding domains of antibodies (Fabs) are powerful probes of structure-function relationships of assembly line polyketide synthases (PKSs). We report the discovery and characterization of Fabs interrogating the structure and function of the ketosynthase-acyltransferase (KS-AT) core of Module 2 of the 6-deoxyerythronolide B synthase (DEBS). Two Fabs (AC2 and BB1) were identified to potently inhibit the catalytic activity of Module 2. Both AC2 and BB1 were found to modulate ACP-mediated reactions catalyzed by this module, albeit by distinct mechanisms. AC2 primarily affects the rate (kcat), whereas BB1 increases the KM of an ACP-mediated reaction. A third Fab, AA5, binds to the KS-AT fragment of DEBS Module 2 without altering either parameter; it is phenotypically reminiscent of a previously characterized Fab, 1B2, shown to principally recognize the N-terminal helical docking domain of DEBS Module 3. Crystal structures of AA5 and 1B2 bound to the KS-AT fragment of Module 2 were solved to 2.70 and 2.65 Å resolution, respectively, and revealed entirely distinct recognition features of the two antibodies. The new tools and insights reported here pave the way toward advancing our understanding of the structure-function relationships of DEBS Module 2, arguably the most well-studied module of an assembly line PKS.

Membrane Permeant Analogs for Independent Cellular Introduction of the Terpene Precursors Isopentenyl- and Dimethylallyl-Pyrophosphate.

Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are the central five-carbon precursors to all terpenes. Despite their significance, exogenous, independent delivery of IPP and DMAPP to cells is impossible as the negatively charged pyrophosphate makes these molecules membrane impermeant. Herein, we demonstrate a facile method to circumvent this challenge through esterification of the ß-phosphate with two self-immolative esters (SIEs) that neutralize the negatively charged pyrophosphate to yield membrane-permeant analogs of IPP and DMAPP. Following cellular incorporation, general esterase activity initiates cleavage of the SIEs, resulting in traceless release of IPP and DMAPP for metabolic utilization. Addition of the synthesized IPP and DMAPP precursor analogs rescued cell growth of glioblastoma (U-87MG) cancer cells concurrently treated with the HMG-CoA reductase inhibitor pitavastatin, which otherwise abrogates cell growth via blocking production of IPP and DMAPP. This work demonstrates a new application of a prodrug strategy to incorporate a metabolic intermediate and promises to enable future interrogation of the distinct biological roles of IPP and DMAPP.

In vivo visualization and molecular targeting of the cardiac conduction system.

Accidental injury to the cardiac conduction system (CCS), a network of specialized cells embedded within the heart and indistinguishable from the surrounding heart muscle tissue, is a major complication in cardiac surgeries. Here, we addressed this unmet need by engineering targeted antibody-dye conjugates directed against the CCS, allowing for the visualization of the CCS in vivo following a single intravenous injection in mice. These optical imaging tools showed high sensitivity, specificity, and resolution, with no adverse effects on CCS function. Further, with the goal of creating a viable prototype for human use, we generated a fully human monoclonal Fab that similarly targets the CCS with high specificity. We demonstrate that, when conjugated to an alternative cargo, this Fab can also be used to modulate CCS biology in vivo, providing a proof of principle for targeted cardiac therapeutics. Finally, in performing differential gene expression analyses of the entire murine CCS at single-cell resolution, we uncovered and validated a suite of additional cell surface markers that can be used to molecularly target the distinct subcomponents of the CCS, each prone to distinct life-threatening arrhythmias. These findings lay the foundation for translational approaches targeting the CCS for visualization and therapy in cardiothoracic surgery, cardiac imaging, and arrhythmia management.

Cyclic Disulfide Liposomes for Membrane Functionalization and Cellular Delivery.

Liposomes are effective therapeutic delivery nanocarriers due to their ability to encapsulate and enhance the pharmacokinetic properties of a wide range of therapeutics. Two primary areas in which improvement is needed for liposomal drug delivery is to enhance the ability to infiltrate cells and to facilitate derivatization of the liposome surface. Herein, we report a liposome platform incorporating a cyclic disulfide lipid (CDL) for the dual purpose of enhancing cell entry and functionalizing the liposome membrane through thiol-disulfide exchange. In order to accomplish this, CDL-1 and CDL-2, composed of lipoic acid (LA) or asparagusic acid (AA) appended to a lipid scaffold, were designed and synthesized. A fluorescence-based microplate immobilization assay was implemented to show that these compounds enable convenient membrane decoration through reaction with thiol-functionalized small molecules. Additionally, fluorescence microscopy experiments indicated dramatic enhancements in cellular delivery when CDLs were incorporated within liposomes. These results demonstrate that multifunctional CDLs serve as an exciting liposome system for surface decoration and enhanced cellular delivery.

Reactive Oxygen Species (ROS) Activated Liposomal Cell Delivery using a Boronate-Caged Guanidine Lipid.

We report boronate-caged guanidine-lipid 1 that activates liposomes for cellular delivery only upon uncaging of this compound by reactive oxygen species (ROS) to produce cationic lipid products. These liposomes are designed to mimic the exceptional cell delivery properties of cell-penetrating peptides (CPPs), while the inclusion of the boronate cage is designed to enhance selectivity such that cell entry will only be activated in the presence of ROS. Boronate uncaging by hydrogen peroxide was verified by mass spectrometry and zeta potential (ZP) measurements. A microplate-based fluorescence assay was developed to study the ROS-mediated vesicle interactions between 1-liposomes and anionic membranes, which were further elucidated via dynamic light scattering (DLS) analysis. Cellular delivery studies utilizing fluorescence microscopy demonstrated significant enhancements in cellular delivery only when 1-liposomes were incubated with hydrogen peroxide. Our results showcase that lipid 1 exhibits strong potential as an ROS-responsive liposomal platform for targeted drug delivery applications.

Mitochondrial Fission and Fusion in Tumor Progression to Metastasis.

Mitochondria are highly dynamic organelles which can change their shape, via processes termed fission and fusion, in order to adapt to different environmental and developmental contexts. Due to the importance of these processes in maintaining a physiologically healthy pool of mitochondria, aberrant cycles of fission/fusion are often seen in pathological contexts. In this review we will discuss how dysregulated fission and fusion promote tumor progression. We focus on the molecular mechanisms involved in fission and fusion, discussing how altered mitochondrial fission and fusion change tumor cell growth, metabolism, motility, and invasion and, finally how changes to these tumor-cell intrinsic phenotypes directly and indirectly impact tumor progression to metastasis. Although this is an emerging field of investigation, the current consensus is that mitochondrial fission positively influences metastatic potential in a broad variety of tumor types. As mitochondria are now being investigated as vulnerable targets in a variety of cancer types, we underscore the importance of their dynamic nature in potentiating tumor progression.

Structure and mechanism for iterative amide N-methylation in the biosynthesis of channel-forming peptide cytotoxins.

SignificanceThe channel-forming proteusins are bacterial helical peptides that allow permeation of positively charged ions to influence membrane potential and cellular physiology. We biochemically characterize the effect of two critical posttranslational modifications on the secondary structure of the peptide substrate. We determine how a methyl group can be added to the side chains of D-Asn residues in a peptide substrate and show how flanking residues influence selectivity. These studies should foster the development of small-molecule peptide ion channels as therapeutics.

MIRO2 Regulates Prostate Cancer Cell Growth via GCN1-Dependent Stress Signaling.

There is a continued need to identify novel therapeutic targets to prevent the mortality associated with prostate cancer. In this context, mitochondrial Rho GTPase 2 (MIRO2) mRNA was upregulated in metastatic prostate cancer compared with localized tumors, and higher MIRO2 levels were correlated with poor patient survival. Using human cell lines that represent androgen-independent or -sensitive prostate cancer, we showed that MIRO2 depletion impaired cell growth, colony formation, and tumor growth in mice. Network analysis of MIRO2's binding partners identified metabolism and cellular responses to extracellular stimuli as top overrepresented pathways. The top hit on our screen, General Control Nonderepressible 1 (GCN1), was overexpressed in prostate cancer, and interacted with MIRO2 in prostate cancer cell lines and in primary prostate cancer cells. Functional analysis of MIRO2 mutations present in patients with prostate cancer led to the identification of MIRO2 159L, which increased GCN1 binding. Importantly, MIRO2 was necessary for efficient GCN1-mediated GCN2 kinase signaling and induction of the transcription factor activating transcription factor 4 (ATF4) levels. Further, MIRO2's effect on regulating prostate cancer cell growth was mediated by ATF4. Finally, levels of activated GCN2 and ATF4 were correlated with MIRO2 expression in prostate cancer xenografts. Both MIRO2 and activated GCN2 levels were higher in hypoxic areas of prostate cancer xenografts. Overall, we propose that targeting the MIRO2-GCN1 axis may be a valuable strategy to halt prostate cancer growth. IMPLICATIONS: MIRO2/GCN1/GCN2 constitute a novel mitochondrial signaling pathway that controls androgen-independent and androgen-sensitive prostate cancer cell growth.

Internal teat sealants alone or in combination with antibiotics at dry-off - the effect on udder health in dairy cows in five commercial herds.

In the dairy industry, the dry period has been identified as an area for potential reduction in antibiotic use, as part of a one health approach to preserve antibiotic medicines for human health. The objective of this study was to assess the impact of dry cow treatment on somatic cell count (SCC), intramammary infection (IMI) and milk yield on five commercial Irish dairy herds. A total of 842 cows across five spring calving dairy herds with a monthly bulk tank SCC of < 200 000 cells/mL were recruited for this study. At dry-off, cows which had not exceeded 200 000 cells/mL in the previous lactation were assigned one of two dry-off treatments: internal teat seal (ITS) alone (Lo_TS) or antibiotic plus ITS (Lo_AB + TS). Cows which exceeded 200 000 cells/mL in the previous lactation were treated with antibiotic plus ITS and included in the analysis as a separate group (Hi_AB + TS). Test-day SCC and lactation milk yield records were provided by the herd owners. Quarter milk samples were collected at dry-off, after calving and at mid-lactation for bacteriological culture and quarter SCC analysis. Cow level SCC was available for 789 cows and was log-transformed for the purpose of analysis. Overall, the log SCC of the cows in the Lo_TS group was significantly higher than the cows in Lo_AB + TS group and not statistically different to the cows in the Hi_AB + TS group in the subsequent lactation. However, the response to treatment differed according to the herd studied; the log SCC of the cows in the Lo_TS group in Herds 3, 4 and 5 was not statistically different to the cows in Lo_AB + TS group, whereas in the other two herds, the log SCC was significantly higher in the Lo_TS when compared to the Lo_AB + TS group. There was a significant interaction between dry-off group and herds on SCC and odds of infection in the subsequent lactation. The results of this study suggest that the herd prevalence of IMI may be useful in decision-making regarding the treatment of cows with ITS alone at dry-off to mitigate its impact on udder health.

Mapping the catalytic conformations of an assembly-line polyketide synthase module.

Assembly-line polyketide synthases, such as the 6-deoxyerythronolide B synthase (DEBS), are large enzyme factories prized for their ability to produce specific and complex polyketide products. By channeling protein-tethered substrates across multiple active sites in a defined linear sequence, these enzymes facilitate programmed small-molecule syntheses that could theoretically be harnessed to access countless polyketide product structures. Using cryogenic electron microscopy to study DEBS module 1, we present a structural model describing this substrate-channeling phenomenon. Our 3.2- to 4.3-angstrom-resolution structures of the intact module reveal key domain-domain interfaces and highlight an unexpected module asymmetry. We also present the structure of a product-bound module that shines light on a recently described "turnstile" mechanism for transient gating of active sites along the assembly line.

The effect of concentrate supplement type on milk production, nutrient intake, and total-tract nutrient digestion in mid-lactation, spring-calving dairy cows grazing perennial ryegrass (Lolium perenne L.) pasture.

The objective of this study was to evaluate the effect of concentrate supplement type on milk production, nutrient intake, and total-tract nutrient digestion in lactating dairy cows grazing mid-season perennial ryegrass (Lolium perenne L.; PRG) pasture. Twelve primiparous (mean ± standard deviation; 95 ± 30 d in milk and 470 ± 43 kg of body weight) and 68 multiparous (99 ± 24 d in milk and 527 ± 64 kg of body weight) lactating dairy cows were blocked based on pre-study milk yield and parity and randomly assigned to 1 of 4 dietary treatments. The 4 dietary treatments were a non-supplemented PRG control (PRG); PRG supplemented with 4.4 kg of dry matter (DM) per cow per day of citrus pulp and 0.067 kg of DM/cow per day of urea (PRG+C); PRG supplemented with 0.8 kg of DM/cow per day of heat-treated soybean meal (PRG+PP); and PRG supplemented with 3.1 kg of DM/cow per day of a combination of heat-treated soybean meal and citrus pulp (PRG+C+PP). The study consisted of a 2-wk adaptation period and a 10-wk period of data collection. Weekly measurements of milk yield, body weight, body condition score, and feeding and rumination time were made. Nutrient intake and total-tract digestibility were measured during wk 6 of the study. A large soil moisture deficit was experienced during the study that probably reduced herbage growth rate and likely altered the chemical composition of the PRG offered when compared with typical mid-season PRG. Total dry matter intake was increased in cows fed PRG+C compared with cows fed PRG and PRG+PP and was similar to cows fed PRG+C+PP (18.0, 15.9, 16.4, and 17.2 ± 0.41 kg of DM/d, respectively). The apparent total-tract neutral detergent fiber digestibility of cows fed the PRG+C diet was lower compared with the PRG and PRG+PP diets and was similar to the PRG+C+PP diet (0.67, 0.70, 0.70, and 0.69 ± 0.01 g/g, respectively). The energy-corrected milk (ECM) yield of cows fed PRG+C+PP was highest (23.7 kg/d), PRG+C was intermediate (22.2 kg/d), and PRG was lowest (20.8 kg/d). Cows fed PRG+PP produced more ECM (22.9 kg/d) compared with cows fed PRG and produced similar ECM compared with cows fed PRG+C and PRG+C+PP diets. The PRG+PP diet increased milk protein yield compared with the PRG diet, tended to increase milk protein yield compared with the PRG+C diet, and was similar to the PRG+C+PP diet. Milk fat concentration and the composition of milk fat were not influenced by treatment. The results demonstrated that, for cows consuming pasture-based diets, increasing metabolizable protein supply allowed higher milk yield as metabolizable protein was more limiting than metabolizable energy. However, due to the large soil moisture deficit experienced during this experiment, caution is recommended when extrapolating these results to cows consuming typical mid-season PRG herbage.

Microbial composition and omasal flows of bacterial, protozoal, and nonmicrobial amino acids in lactating dairy cows fed fresh perennial ryegrass (Lolium perenne L.) not supplemented or supplemented with rolled barley.

The objective of this study was to evaluate the effect of rolled barley supplementation on microbial composition and omasal flows of bacterial, protozoal, and nonmicrobial AA in cows fed fresh perennial ryegrass (Lolium perenne L.; PRG). Ten ruminally cannulated multiparous Holstein cows averaging (mean ± standard deviation) 49 ± 23 d in milk and 513 ± 36 kg of body weight were assigned to 1 of 2 treatments in a switchback design. The treatment diets were PRG only or PRG plus 3.5 kg of dry matter rolled barley (G+RB). The study consisted of three 29-d periods where each period consisted of 21 d of diet adaptation and 8 d of data and sample collection. A double-marker system was used to quantify nutrient flow entering the omasal canal along with 15N-ammonium sulfate to label and measure the microbial and nonmicrobial omasal flow of AA. Overall, rolled barley supplementation had no effect on the AA composition of the omasal liquid-associated and particle-associated bacteria. Rolled barley supplementation affected the AA concentrations of omasal protozoa; however, the differences were nutritionally minor. Particle-associated bacteria AA flow was increased for all AA, except for Trp and Pro, in cows fed the G+RB diet. Rolled barley supplementation had no effect on protozoal AA flow. On average, protozoa accounted for 23% of the microbial essential AA flow, which ranged from 17 to 28% for Trp and Lys, respectively. The flow of all AA in omasal true digesta increased in cows fed the G+RB diet compared with the PRG-only diet, resulting in a 228 g/d increase in total AA flow in cows fed the G+RB diet. This increase in total AA flow in cows fed the G+RB diet was due to an increase in microbial AA flow. Rolled barley supplementation had no effect on nonmicrobial AA flow. The nonmicrobial AA flow modestly contributed to total AA flow, accounting for 15.6% on average. These results indicated that extensive ruminal degradation of PRG AA occurred (83.5%), and we demonstrated that cows consuming PRG-based diets exhibit a large dependence on microbial AA to support metabolizable AA supply. Rolled barley supplementation can increase the omasal flow of microbial AA in cows consuming PRG-based diets. However, further research is required to elucidate if this increased AA supply can support higher milk yield under such dietary conditions.