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We hypothesized that a strategy employing tissue-specific endothelial cells (EC) might facilitate the identification of tissue- or organ-specific vascular functions of ubiquitous metabolites. An unbiased approach was employed to identify water-soluble small molecules with mitogenic activity on choroidal EC. We identified adenosine diphosphate (ADP) as a candidate, following biochemical purification from mouse EL4 lymphoma extracts. ADP stimulated the growth of bovine choroidal EC (BCEC) and other bovine or human eye-derived EC. ADP induced rapid phosphorylation of extracellular signal-regulated kinase in a dose- and time-dependent manner. ADP-induced BCEC proliferation could be blocked by pretreatment with specific antagonists of the purinergic receptor P2Y1 but not with a vascular endothelial growth factor (VEGF) inhibitor, indicating that the EC mitogenic effects of ADP are not mediated by stimulation of the VEGF pathway. Intravitreal administration of ADP expanded the neovascular area in a mouse model of choroidal neovascularization. Single-cell transcriptomics from human choroidal datasets show the expression of P2RY1, but not other ADP receptors, in EC with a pattern similar to VEGFR2. Although ADP has been reported to be a growth inhibitor for vascular EC, here we describe its growth-stimulating effects for BCEC and other eye-derived EC.

Individual grazing patterns among cattle can contribute to sustainability of land use, however, little is known about the consistency of these grazing patterns. To address this knowledge gap, fifty Angus x Hereford cows were observed in repeated assays: A management assay (handling procedure, narrow chute, hydraulic squeeze), a social-feed trade-off assay (SFTA; choice between social mates and feed item), and novel approach assay (choice between social mates and feed item with novel pattern). The same cattle were tracked with GPS collars over two grazing seasons (June-August 2021 and 2022) and average grazing-related metrics (e.g., elevation used, distance traveled) were calculated within each season. Cows with a more passive response in the chute were found at higher elevation (p = 0.017), further from water (p = 0.043), and closer to supplement sites (p = 0.029). Cows that had higher latency to supplement in the SFTA traveled shorter distances on rangeland (p = 0.035). Thus, there was some evidence that cows with a more passive response to isolation and management had more optimal grazing patterns (grazed underutilized areas of the range at higher elevations and further from water sources). Selecting cattle with more optimal foraging patterns based on observable behaviors during handling and isolation could improve the sustainability of rangeland grazing.

We expected mitigation of the hypophagic effects of urea (U) with a coated urea (CU) product that aimed to partially shift urea supply to the post-ruminal gastrointestinal tract. Ruminal release and post-ruminal digestibility of CU was evaluated in vitro, followed by a randomised complete block experiment (54 Holstein-Friesian cows; 177 ± 72 days in milk). Soybean meal (SBM) was partially (PR) or fully (FR) replaced on an isonitrogenous basis by beet pulp and U or CU. Urea sources were included at 12 (U-PR, CU-PR) and 19 (U-FR, CU-FR) g/kg dietary dry matter (DM). Hypophagic effects were similar for U-PR and CU-PR (-11% vs. -7%), and for U-FR and CU-FR (-13% vs. -12%) compared with SBM (average 25.8 kg DM intake/d). Compared with SBM, U-PR and CU-PR reduced yields of milk (-8%) and protein (-12%), U-PR reduced yield of fat (-9%) and fat- and protein-corrected-milk (FPCM; -9%), and CU-PR tended to reduce FPCM yield (-5%). Compared with SBM, U-FR and CU-FR respectively reduced yields of milk (-21%, -22%), protein (-25%, -26%), fat (both -14%), lactose (-20%, -21%), and FPCM (-17%, -19%), and lowered N (-15%, -12%) and feed (-8%, trend, -9%) efficiency. Human-edible protein efficiency approximately doubled with U-PR and CU-PR and approximately tripled with U-FR and CU-FR compared with SBM. Milk composition and plasma urea concentration were similar between U and CU, except for a trend for a greater plasma urea concentration with U-PR compared with CU-PR. Dry matter intake patterns differed for CU-PR compared with U-PR and for CU-FR compared with U-FR, suggesting effects of urea release rate or location on feeding behaviour. Overall, replacing SBM with U or CU reduced DM intake and milk production and affected nutrient efficiencies. Coated urea influenced DM intake pattern but did not affect total DM intake or milk production compared with U.

Vertebrates differ over 100,000-fold in responses to pro-inflammatory agonists such as bacterial lipopolysaccharide (LPS), complicating use of animal models to study human sepsis or inflammatory disorders. We compared transcriptomes of resting and LPS-exposed blood from six LPS-sensitive species (rabbit, pig, sheep, cow, chimpanzee, human) and four LPS-resilient species (mice, rats, baboon, rhesus), as well as plasma proteomes and lipidomes. Unexpectedly, at baseline, sensitive species already had enhanced expression of LPS-responsive genes relative to resilient species. After LPS stimulation, maximally different genes in resilient species included genes that detoxify LPS, diminish bacterial growth, discriminate sepsis from SIRS, and play roles in autophagy and apoptosis. The findings reveal the molecular landscape of species differences in inflammation. This may inform better selection of species for pre-clinical models and could lead to new therapeutic strategies that mimic mechanisms in inflammation-resilient species to limit inflammation without causing immunosuppression.

The high emissions intensity of terrestrial animal source food (TASF) and projected increasing demand in low- and middle-income countries (LMIC) have spurred interest in the development of animal-free alternatives and manufactured food items that aim to substitute for meat, milk, and eggs with the promise of reduced environmental impact of producing food. The developing world is the source of 75% of global emissions from ruminants and will house 86% of the worlds human population by 2050. The adoption of cost-effective, genetic, feed and nutrition practices, and improving livestock health in LMIC are seen as the most promising interventions to reduce emissions resulting from projected increased TASF demand though 2050. Genetic improvement is a particularly attractive approach to productivity enhancements, as such improvements are permanent and cumulative. Alternative proteins may play a role in addressing demand for affordable sources of nutrient-dense foods, however, price will be a major factor influencing adoption given 3.1 billion people globally (42%) were currently unable to afford a healthy diet in 2021. Additionally, there is currently a mismatch between the location of alternative protein companies, and both projected increased TASF demand and emissions. To date, the vast majority (>81%) of these companies are based in high-income countries. The sustainability implications of replacing TASF with alternative proteins at scale needs to consider not only environmental metrics but also the wider economic and social sustainability impacts, given the essential role that livestock play in the livelihoods and food security of approximately 1.3 billion people.

The ruminant livestock sector considerably contributes to global greenhouse gas emissions. This study investigates the effectiveness of pelleted bromoform-containing seaweed (Asparagopsis taxiformis) (Brominata) as an enteric methane (CH4) inhibitor in grazing beef cattle. The primary objective was to assess the impact of this antimethanogenic additive on enteric CH4 emissions under real-world farm conditions. Twenty-four beef steers, crossbreeds of Wagyu and Angus, with an average liveweight of 399 ± 21.7 kg, were allocated to two treatment groups: Control and Brominata. These animals underwent regular weigh-ins every 14 d, and measurements of CH4, carbon dioxide (CO2), and hydrogen (H2) emissions were conducted using the GreenFeed system. Statistical analysis was conducted using SAS 9.4, wherein the model incorporated fixed effects for treatment, time, their interaction, and a covariate, while accounting for animal variations as a random effect within each phase. Three phases of bromoform intake were identified: a 3-wk ramp-up phase, a 3-wk optimal phase, and a 2-wk decreasing phase. No differences were observed between the weekly initial and final liveweight, average daily gain, and predicted dry matter intake. During optimal and decreasing phases, average enteric CH4 emissions were significantly reduced in steers that received Brominata supplementation compared to those without supplementation (115 vs. 185 g/d, respectively). Additionally, both groups had similar CO2 emissions (6.8 vs. 7.2 kg/d), while H2 emissions were lower in the control group (3.4 vs. 1.8 g/d). The findings suggest that pelleted bromoform-containing feed additive has the potential to reduce enteric CH4 emissions from grazing beef cattle.

BACKGROUND/OBJECTIVES: Lactoferrin (Lf) is an iron-binding glycoprotein with multiple bioactivities, including promotion of cell proliferation and differentiation, immunomodulation, and antimicrobial activity. Lf, a basic glycoprotein, can bind to α-lactalbumin (α-Lac), an acidic whey protein. The current study aimed to evaluate whether Lf forms protein complexes with α-Lac and proteins/peptides from whey protein hydrolysate (WPH) and nonfat bovine milk powder (MP) and whether forming protein complexes influences resistance to gastrointestinal digestion and affects the bioactivities of Lf in human intestinal epithelial cells (HIECs and differentiated Caco-2 cells). METHODS: Lf was blended with α-Lac, WPH, or MP. Assays were conducted to evaluate the bioactivities of proteins (Lf, α-Lac, WPH, or MP) and Lf-protein blends on HIECs and Caco-2 cells. RESULTS: (1) Lf forms complexes with α-Lac and proteins/peptides from WPH and MP; (2) compared with Lf alone, complexed Lf shows greater resistance to in vitro digestion; (3) forming protein complexes does not affect Lfs binding to the Lf receptor or its uptake by HIECs; and (4) forming protein complexes does not impact Lfs bioactivities, including the promotion of cell proliferation and differentiation, reduction of cell permeability by upregulating tight-junction proteins, immune modulation through the regulation of IL-18, inhibition of enteropathogenic Escherichia coli growth, and modulation of immune responses to EPEC infection. CONCLUSIONS: Lf forms complexes with α-Lac and other milk proteins/peptides from WPH and MP in protein blends, and forming complexes does not affect the functionalities of Lf.

The cation-independent mannose 6-phosphate receptor (CI-MPR) is clinically significant in the treatment of patients with lysosomal storage diseases because it functions in the biogenesis of lysosomes by transporting mannose 6-phosphate (M6P)-containing lysosomal enzymes to endosomal compartments. CI-MPR is multifunctional and modulates embryonic growth and fetal size by downregulating circulating levels of the peptide hormone insulin-like growth factor 2 (IGF2). The extracellular region of CI-MPR comprises 15 homologous domains with binding sites for M6P-containing ligands located in domains 3, 5, 9, and 15, whereas IGF2 interacts with residues in domain 11. How a particular ligand affects the receptor's conformation or its ability to bind other ligands remains poorly understood. To address these questions, we purified a soluble form of the receptor from newborn calf serum, carried out glycoproteomics to define the N-glycans at its 19 potential glycosylation sites, probed its ability to bind lysosomal enzymes in the presence and absence of IGF2 using surface plasmon resonance, and assessed its conformation in the presence and absence of IGF2 by negative-staining electron microscopy and hydroxyl radical protein footprinting studies. Together, our findings support the hypothesis that IGF2 acts as an allosteric inhibitor of lysosomal enzyme binding by inducing global conformational changes of CI-MPR.

Providencia alcalifaciens is a Gram-negative bacterium found in various water and land environments and organisms, including insects and mammals. Some P. alcalifaciens strains encode gene homologs of virulence factors found in pathogenic Enterobacterales members, such as Salmonella enterica serovar Typhimurium and Shigella flexneri. Whether these genes are pathogenic determinants in P. alcalifaciens is not known. In this study, we investigated P. alcalifaciens-host interactions at the cellular level, focusing on the role of two type III secretion systems (T3SS) belonging to the Inv-Mxi/Spa family. T3SS1b is widespread in Providencia spp. and encoded on the chromosome. A large plasmid that is present in a subset of P. alcalifaciens strains, primarily isolated from diarrheal patients, encodes for T3SS1a. We show that P. alcalifaciens 205/92 is internalized into eukaryotic cells, lyses its internalization vacuole, and proliferates in the cytosol. This triggers caspase-4-dependent inflammasome responses in gut epithelial cells. The requirement for the T3SS1a in entry, vacuole lysis, and cytosolic proliferation is host cell type-specific, playing a more prominent role in intestinal epithelial cells than in macrophages or insect cells. In a bovine ligated intestinal loop model, P. alcalifaciens colonizes the intestinal mucosa and induces mild epithelial damage with negligible fluid accumulation in a T3SS1a- and T3SS1b-independent manner. However, T3SS1b was required for the rapid killing of Drosophila melanogaster. We propose that the acquisition of two T3SS has allowed P. alcalifaciens to diversify its host range, from a highly virulent pathogen of insects to an opportunistic gastrointestinal pathogen of animals.

The ultrafast photochemical reaction mechanism, transient spectra, and transition kinetics of the human blue cone visual pigment have been recorded at room temperature. Ultrafast time-resolved absorption spectroscopy revealed the progressive formation and decay of several metastable photo-intermediates, corresponding to the Batho to Meta-II photo-intermediates previously observed with bovine rhodopsin and human green cone opsin, on the picosecond to millisecond timescales following pulsed excitation. The experimental data reveal several interesting similarities and differences between the photobleaching sequences of bovine rhodopsin, human green cone opsin, and human blue cone opsin. While Meta-II formation kinetics are comparable between bovine rhodopsin and blue cone opsin, the transition kinetics of earlier photo-intermediates and qualitative characteristics of the Meta-I to Meta-II transition are more similar for blue cone opsin and green cone opsin. Additionally, the blue cone photo-intermediate spectra exhibit a high degree of overlap with uniquely small spectral shifts. The observed variation in Meta-II formation kinetics between rod and cone visual pigments is explained based on key structural differences.

Background/Objectives: Human milk is the optimal source of nutrition and protection against infection for infants. If breastfeeding is not possible, standard and hydrolyzed infant formulas (IF) are an alternative. Extensively hydrolyzed IFs (eHFs) contain bioactive peptides, but their activities have rarely been evaluated. The aim of this study was to characterize and compare the bioactive peptide profiles of different eHFs and standard IFs before and after in vitro digestion. Methods: Two forms, liquid and powder, of intact protein formula (iPF) and eHF were subjected to in vitro gastrointestinal digestion, mimicking a young infants gut (age 0-4 months) and an older infants gut (>6 months). Bioactive peptides of in vitro digested and undigested formulas were analysed with Liquid Chromatography-Mass Spectrometry (LC-MS). Results: In all samples, a variety of peptides with potential bioactive properties were found. Immuno-regulatory peptides, followed by antimicrobial and antioxidative peptides were most frequent, as were peptides promoting wound healing, increasing mucin secretion, regulating cholesterol metabolism, and preventing bacterial infection. Peptides typically found in yoghurt and colostrum were identified in some formula samples. Conclusions: The high amounts of bioactive peptides with various properties in eHFs and iPFs indicate a possible contribution to infection protection, healthy gut microbiomes, and immunological development of infants. eHFs showed similar compositions of bioactive peptides to iPFs, with intermittently increased peptide variety and quantity.

Bacillary hemoglobinuria (BH) is an infectious disease, mostly affecting cattle, caused by Clostridium haemolyticum (C. novyi type D), with acute hepatic necrosis and intravascular hemolysis. Cattle are typically predisposed to BH by liver injury caused by Fasciola hepatica, although cases have been reported in cattle without evidence of this parasite. Here we describe a cluster of 14 BH cases from 7 counties in north-central to central Missouri submitted to a veterinary diagnostic laboratory between December 2020 and April 2023. Postmortem examination in all cases revealed hemoglobinuria and acute hepatic necrosis with large numbers of gram-positive bacilli with terminal-to-subterminal spores. Flukes, fluke ova, and/or fluke pigment consistent with Fascioloides magna were identified in 12 of 14 cases. Sequences of the nuclear ribosomal internal transcribed spacer 1 (ITS1) from one fluke had 100% identity to F. magna. C. novyi was detected by fluorescent antibody testing of liver impression smears (11 of 12 cases) and by immunohistochemistry of liver sections (7 of 7 cases). PCR on formalin-fixed, paraffin-embedded tissues amplified the C. haemolyticum beta toxin gene in each of the 7 cases tested. To our knowledge, a confirmed cluster of BH associated with F. magna has not been reported previously in cattle.

Trans vaccenic acid (TVA, trans11-18 : 1) and cis9, trans11-CLA (also known as rumenic acid; RA) have received widespread attention as potentially beneficial trans-FA due to their putative health benefits, including anti-diabetic properties. The objective of this study was to determine the effects of beef fat naturally enriched with TVA and RA on parameters related to glucose homoeostasis and associated metabolic markers in diet-induced obese (DIO) mice. Thirty-six male C57BL/6J mice (8 weeks old) were fed for 19 weeks with either a control low-fat diet (CLF), a control high-fat diet (CHF), or a TVA+RA-enriched high-fat diet (EHF). Compared with CLF, feeding either CHF or EHF resulted in adverse metabolic outcomes associated with high-fat diets, including adiposity, impaired glucose control and hepatic steatosis. However, the EHF diet induced a significantly higher liver weight TAG content and elevated plasma alanine transaminase levels compared with the CHF diet. Collectively, the findings from this study suggest that EHF does not improve glucose tolerance and worsens liver steatosis in DIO mice. However, the adverse effects of EHF on the liver could be in part related to the presence of other trans-FA in the enriched beef fat.

Escherichia coli has been attributed to playing a major role in a cascade of events that affect the prevalence and severity of uterine disease in cattle. The objectives of this project were to (i) define the association between the prevalence of specific antimicrobial resistance and virulence factor genes in E. coli with the clinical status related to uterine infection, (ii) identify the genetic relationship between E. coli isolates from cows with diarrhea, with mastitis, and with and without metritis, and (iii) determine the association between the phenotypic and genotypic antimicrobial resistance identified on the E. coli isolated from postpartum cattle. Bacterial isolates (n = 148) were obtained from a larger cross-sectional study. Cows were categorized into one of three clinical groups before enrollment: metritis, cows with purulent discharge, and control cows. For genomic comparison, public genomes (n = 130) from cows with diarrhea, mastitis, and metritis were included in a genome-wide association study, to evaluate differences between the drug classes or the virulence factor category among clinical groups. A distinct E. coli genotype associated with metritis could not be identified. Instead, a high genetic diversity among the isolates from uterine sources was present. A virulence factor previously associated with metritis (fimH) using PCR was not associated with metritis. There was moderate accuracy for whole-genome sequencing to predict phenotypic resistance, which varied depending on the antimicrobial tested. Findings from this study contradict the traditional pathotype classification and the unique intrauterine E. coli genotype associated with metritis in dairy cows.IMPORTANCEMetritis is a common infectious disease in dairy cattle and the second most common reason for treating a cow with antimicrobials. The pathophysiology of the disease is complex and is not completely understood. Specific endometrial pathogenic Escherichia coli have been reported to be adapted to the endometrium and sometimes lead to uterine disease. Unfortunately, the specific genomic details of the endometrial-adapted isolates have not been investigated using enough genomes to represent the genomic diversity of this organism to identify specific virulence genes that are consistently associated with disease development and severity. Results from this study provide key microbial ecological advances by elucidating and challenging accepted concepts for the role of Intrauterine E. coli in metritis in dairy cattle, especially contradicting the existence of a unique intrauterine E. coli genotype associated with metritis in dairy cows, which was not found in our study.

OBJECTIVE: Fibrin sealants are routinely used for intra-articular surgical fixation of cartilage fragments and implants. However, the mechanical properties of fibrin sealants in the context of cartilage repair are unknown. The purpose of this study was to characterize the adhesive and frictional properties of fibrin sealants using an ex vivo model. DESIGN: Native bovine cartilage-bone composites were assembled with a single application of Tisseel or Vistaseal. Composites were tested in tension and lap shear. In addition, the coefficient of friction (COF) was measured in a native cartilage annulus model alone and with minced cartilage. Finally, the effect of a double application of fibrin sealant was evaluated. RESULTS: There were no significant differences in tensile modulus, ultimate tensile strength (UTS), shear modulus, or ultimate shear strength (USS) between the 2 fibrin sealants. Both fibrin sealants demonstrated a UTS and USS of

OBJECTIVE: The purpose of this study was to determine the in vitro effects of a single exposure of bupivacaine on the mechanical properties of bovine cartilage explants at 3 weeks. DESIGN: Femoral condyle articular cartilage explants were aseptically harvested from juvenile bovine stifle joints before being exposed to chondrogenic medium containing 0.50% (wt/vol) bupivacaine, 0.25% (wt/vol) bupivacaine, or no medication (control) for 1 hour. Explants were then washed and maintained in culture in vitro for 3 weeks before testing. Cell viability, tensile and compressive mechanical properties, histological properties, and biochemical properties were then assessed. RESULTS: Explants exhibited a dose-dependent decrease in mean tensile Youngs modulus with increasing bupivacaine concentration (9.86 MPa in the controls, 6.48 MPa in the 0.25% bupivacaine group [P = 0.048], and 4.72 MPa in the 0.50% bupivacaine group [P = 0.005]). Consistent with these results, collagen content and collagen crosslinking decreased with bupivacaine exposure as measured by mass spectrometry. Compressive properties of the explants were unaffected by bupivacaine exposure. Explants also exhibited a trend toward dose-dependent decreases in viability (51.2% for the controls, 47.3% for the 0.25% bupivacaine-exposed group, and 37.0% for the 0.50% bupivacaine-exposed group [P = 0.072]). CONCLUSIONS: Three weeks after 1-hour bupivacaine exposure, the tensile properties of bovine cartilage explants were significantly decreased, while the compressive properties remained unaffected. These decreases in tensile properties corresponded with reductions in collagen content and crosslinking of collagen fibers. Physicians should be judicious regarding the intra-articular administration of bupivacaine in native joints.

Animal-assisted therapy (AAT) is an integrative model frequently used in conjunction with other modalities such as psychodynamic psychotherapy and cognitive behavioral therapy. The most common AAT model is when a companion animal, such as a dog or cat, is integrated into a therapeutic treatment plan. This model has been studied with a variety of populations and conditions, from treating substance abuse to autism, often with positive results. Organizations that certify therapy animals will sometimes certify several species of animals to work as a therapeutic partner, such as llamas, miniature horses, and rabbits. However, to date there has been little research addressing the therapeutic benefits of animal-assisted interventions with farm animals. Cows have special behavioral traits that allow them to bond with people in a way that is unique to their size and temperament. Using the Human-Animal Interaction Scale (HAIS) as a measurement tool, we examined the behavioral and cognitive traits of cattle who work as therapy animals. Special attention will be paid to the welfare and enrichment benefits of cattle involved in bovine-assisted therapy.

BACKGROUND: As a primary vector of bluetongue virus (BTV) in the US, seasonal abundance and diel flight activity of Culicoides sonorensis has been documented, but few studies have examined how time of host-seeking activity is impacted by environmental factors. This knowledge is essential for interpreting surveillance data and modeling pathogen transmission risk. METHODS: The diel host-seeking activity of C. sonorensis was studied on a California dairy over 3 years using a time-segregated trap baited with CO2. The relationship between environmental variables and diel host-seeking activity (start, peak, and duration of activity) of C. sonorensis was evaluated using multiple linear regression. Fishers exact test and paired-sample z-test were used to evaluate the seasonal difference and parity difference on diel host-seeking activity. RESULTS: Host-seeking by C. sonorensis began and reached an activity peak before sunset at a higher frequency during colder months relative to warmer months. The time that host-seeking activity occurred was associated low and high daily temperature as well as wind speed at sunset. Colder temperatures and a greater diurnal temperature range were associated with an earlier peak in host-seeking. Higher wind speeds at sunset were associated with a delayed peak in host-seeking and a shortened duration of host-seeking. Parous midges reached peak host-seeking activity slightly later than nulliparous midges, possibly because of the need for oviposition by gravid females before returning to host-seeking. CONCLUSIONS: This study demonstrates that during colder months C. sonorensis initiates host-seeking and reaches peak host-seeking activity earlier relative to sunset, often even before sunset, compared to warmer months. Therefore, the commonly used UV light-baited traps are ineffective for midge surveillance before sunset. Based on this study, surveillance methods that do not rely on light trapping would provide a more accurate estimate of host-biting risk across seasons. The association of environmental factors to host-seeking shown in this study can be used to improve modeling or prediction of host-seeking activity. This study identified diurnal temperature range as associated with host-seeking activity, suggesting that Culicoides may respond to a rapidly decreasing temperature by shifting to an earlier host-seeking time, though this association needs further study.