Bobeck, Elizabeth

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Mitochondrial and Glycolytic Capacity of Peripheral Blood Mononuclear Cells Isolated From Diverse Poultry Genetic Lines: Optimization and Assessment

2022-01-28 , Lamont, Susan , Bobeck, Elizabeth , Bobeck, Elizabeth A. , Animal Science

Cellular metabolic preference is a culmination of environment, nutrition, genetics, and individual variation in poultry. The Seahorse XFe24 analyzer was used to generate foundational immune cellular metabolic data in layer, broiler, and legacy genetic strains using fresh chicken peripheral blood mononuclear cells (PBMCs). Baseline mitochondrial respiration [oxygen consumption rate (OCR)] and glycolytic activity [extracellular acidification rate (ECAR)] were determined in modern commercial laying hen (Bovans White) and broiler (Ross 308) lines, as well as the highly inbred lines of Iowa State University (L8, Fayoumi M-15.2, Spanish, Ghs-6), partially inbred broiler line, and advanced intercrosses of broiler by Fayoumi M-15.2 and broiler by Leghorn lines. Commercial broiler vs. Bovans layer and unvaccinated vs. vaccinated Bovans layer immune cell metabolic potential were compared following an in-assay pathway inhibitor challenge. Titrations consistently showed that optimal PBMC density in laying hens and broilers was 3 million cells per well monolayer. Assay media substrate titrations identified 25 mM glucose, 1 mM glutamine, and 1 mM sodium pyruvate as the optimal concentration for layer PBMCs. Pathway inhibitor injection titrations in Bovans layers and broilers showed that 0.5 μM carbonyl cyanide-4 phenylhydrazone (FCCP) and 1 μM oligomycin were optimal. Baseline OCR and ECAR were significantly affected by genetic line of bird (p < 0.05), with the dual-purpose, L8 inbred line showing the highest OCR (mean 680 pmol/min) and the partially inbred broiler line showing the greatest ECAR (mean 74 mpH/min). ECAR metabolic potential tended to be greater in modern layers than broilers (p < 0.10), indicating increased ability to utilize the glycolytic pathway to produce energy. OCR was significantly higher in vaccinated than unvaccinated hens (p < 0.05), while baseline ECAR values were significantly lower in vaccinated Bovans laying hens, showing increased oxidative capacity in activated immune cells. These baseline data indicate that different genetic strains of birds utilized the mitochondrial respiration pathway differently and that modern commercial lines may have reduced immune cell metabolic capacity compared with legacy lines due to intense selection for production traits. Furthermore, the Seahorse assay demonstrated the ability to detect differences in cellular metabolism between genetic lines and immune status of chickens.

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Algae-based feed ingredient protects intestinal health during Eimeria challenge and alters systemic immune responses with differential outcomes observed during acute feed restriction

2021-09-01 , Fries-Craft, K. , Meyer, M. M. , Bobeck, Elizabeth , Bobeck, Elizabeth , Animal Science

Compounds in microalgae-derived feed ingredients in poultry diets may improve intestinal physiology and immunity to protect against damage induced by physiological and pathogen challenges, but mechanisms are examined sparingly. The study objective was to evaluate changes to intestinal morphology, permeability, and systemic immunity in broilers fed a proprietary microalgae ingredient during 2 separate challenge studies. In study 1, two replicate 28 d battery cage trials used 200 Ross 308 broilers each (n = 400) fed a control diet ± 0.175% algae ingredient. Half of the birds were subjected to a 12 h feed restriction challenge and fluorescein isothiocyanate dextran (FITC-D) intestinal permeability assay on d 28. Study 2 used 800 broilers randomly assigned to the same dietary treatments and housed in floor pens for 42 d. At d 14, intestine and spleen samples were collected from 10 birds/ diet. Half of the remainder was orally inoculated with 10X Coccivac-B52 vaccine in a 2 × 2 factorial treatment design (diet and Eimeria inoculation). The FITC-D assay was conducted at 1, 3, 7, and 14 d post-inoculation (pi) while intestinal and spleen samples were collected at 3, 7, 14, and 28 dpi for histomorphology and flow cytometric immune cell assessment. Study 1 validated intestinal leakage via FITC-D absorbance induced by feed restriction but showed no algae-associated protective effects. In study 2, algae preserved intestinal integrity during coccidiosis (P = 0.04) and simultaneously protected jejunal villus height as early as 7dpi (P < 0.0001), whereas intestinal damage resolution in control birds did not occur until 14 dpi. Algae inclusion increased splenic T cells in unchallenged broilers at d 14 by 29.6% vs. control (P < 0.0001), specifically γδ T cell populations, without impacting performance (P < 0.03). During Eimeria challenge, splenic T cells in algae-fed birds did not show evidence of recruitment to peripheral tissues, while control birds showed a 16.7% reduction compared to their uninoculated counterparts from 3 to 7 dpi (P < 0.0001). This evidence suggests the algae ingredient altered the immune response in a manner that reduced recruitment from secondary lymphoid organs in addition to protecting intestinal physiology.

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Lipid Source and Peroxidation Status Alter Immune Cell Recruitment in Broiler Chicken Ileum

2020-12-09 , Fries-Craft, Krysten , Bobeck, Elizabeth , Meyer, Meaghan , Lindblom, Stephanie , Kerr, Brain , Bobeck, Elizabeth , Animal Science

Background: Restaurant oil in poultry diets increases energy content, reduces production costs, and promotes sustainability within the food supply chain. However, variable oil composition and heating temperatures among restaurant oil sources can impact broiler chicken health due to heat-induced lipid modifications.

Objectives: A 21-d experiment was conducted to evaluate ileal morphology, liver cytokine gene expression, and ileal immune cell populations in broilers fed control or peroxidized lipids with varying chain and saturation characteristics.

Methods: Day-old broilers were housed in battery cages (5 birds per cage) and fed diets containing 5% control or peroxidized oils. Eight diets were randomly assigned in a 4 × 2 factorial arrangement consisting of oil source (palm, soybean, flaxseed, or fish) and peroxidation status (control or peroxidized). At day 21, samples were collected for ileal histomorphology [villus height (VH), crypt depth (CrD), and the VH:CrD ratio], and liver cytokine expression (qPCR). Ileum cytokine expression and T-cell markers were analyzed by RNAscope in situ hybridization (ISH). Data were analyzed as a mixed model (SAS 9.4) with fixed effects of lipid source, peroxidation, and lipid × peroxidation interaction.

Results: CD3+ T-cells in the ileum decreased 16.2% due to peroxidation (P = 0.001) with 30.3% reductions observed in birds fed peroxidized flaxseed oil (P = 0.01). Peroxidation increased IL6+ and IL1B+ cells by 62.0% and 40.3%, respectively (P = 0.01). Soybean oil increased IFNG+ cells by 55.1% compared with palm oil, regardless of peroxidation status (P = 0.007). Lipid source and peroxidation did not alter ileal histomorphology or liver cytokine expression.

Conclusions: Lipid peroxidation increased ileal IL1B and IL6 in broiler chickens, whereas soybean oil diets increased IFNG. Generally, peroxidation decreased overall CD3+ T-cell populations, suggesting impaired T-cell presence or recruitment. These results identify potential immunomodulatory lipid profiles in restaurant oil while supporting RNAscope-ISH as a method to describe avian tissue-level immune responses.

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Development and Validation of Broiler Welfare Assessment Methods for Research and On-farm Audits

2020-01-01 , Bobeck, Elizabeth , Johnson, Anna , Bobeck, Elizabeth , Johnson, Anna , Animal Science

Required auditing of on-farm broiler welfare in the U.S. has increased; however, a lack of validated tools exists for assessment of enrichment. National Chicken Council (NCC) guidelines were used on a subset of 300 Ross 308 broilers out of 1200 to validate and adapt welfare measures. Half of the broilers were exposed to environmental enrichment, hence these measures were used to evaluate the enrichment within the context of behavior and welfare, although the nature of the enrichment is not described in detail here as the aim is to serve solely as a description and validation of methods using a subset of example data. Birds were recorded in repeated 4-min periods to quantify behavior and walking distance. Outcomes were categorized to improve auditing and make recommendations to producers and researchers. Bone mineral density, content, and breaking strength were successful in determining numerical differences. Quantifying lameness using an enclosed walkway and measuring footpad dermatitis weekly are recommended on-farm. We recommend including additional measures not required by the NCC: monitoring breast condition in the flock and including a behavior component with a scoring ethogram.

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Laser Environmental Enrichment and Spirulina Algae Improve Broiler Growth Performance and Alter Myogenic Gene Expression and pectoralis major Dimensions

2021-12-14 , Johnson, Anna , Bobeck, Elizabeth , Bobeck, Elizabeth A. , Animal Science

Sustainability in poultry production is evident in efforts to reduce inputs and a focus on bird welfare and livability. Dietary protein alternatives to traditional sources such as soybean meal aim to meet or exceed efficiency benchmarks and be cost-effective. Environmental enrichment encouraging activity may reduce the occurrence of the predominant breast muscle myopathy, woody breast (WB); interventions to minimize muscle damage and economic loss have yet to be established. The study objectives were to maintain or improve broiler performance and breast quality through environmental enrichment and partially replacing dietary soybean meal with Spirulina. Twelve hundred Ross 708 broilers were randomly assigned to enrichment (LASER; laser enrichment, or CON; no laser enrichment) and diet (algae; 2.5% Spirulina algae, or control) in a 2 × 2 factorial design for 49 days. The same 70 randomly selected birds were examined for contact dermatitis wk 1–6. Breast width was measured weekly on 200 growing broilers beginning on d22. On d42 and 49 slaughter, WB score was assigned using a tactile 0–3 scale and the right breast filet was weighed (n = 200). RNA isolated from 30 breast muscle samples each at d42 and 49 was analyzed using real-time qPCR. Laser enrichment increased body weight at all timepoints (d49: 0.148 kg, P < 0.001). Feed conversion ratio was improved in LASER-enriched birds by 3 points in the starter period (P = 0.003). Breast width was increased at all timepoints in LASER-enriched birds compared to CON (d49: 0.47 cm, P < 0.001). Algae inclusion increased body weight at d28 (0.059 kg, P = 0.005). At d42, 12% more LASER-enriched WB scores were 0 (normal) compared to CON, and at d49, 15% more enriched scores were 0. At d42, 5% more algae-fed broiler scores were 0 compared to control. LASER-enriched broiler breast tissue showed upregulated expression of myogenin, muscle regulatory factor 4, insulin-like growth factor 1, and myostatin compared to CON (P < 0.01). Both laser enrichment and algae inclusion improved broiler performance without negatively impacting environmental or physiological outcomes. LASER enrichment decreased severity of WB score and positively shifted myogenic gene expression in the breast muscle at slaughter.

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Comparison of Naturally Occurring vs. Experimental Infection of Staphylococcus aureus Septicemia in Laying Hens in Two Different Age Groups

2021-06-01 , Meyer, Meaghan , Bobeck, Elizabeth , Sato, Yuko , Bobeck, Elizabeth , El-Gazzar, Mohamed , Animal Science , Veterinary Diagnostic and Production Animal Medicine

In April and November of 2018, multiple commercial laying hen flocks within the same company presented with a sharp increase in mortality and drop in egg production that persisted for several days. These flocks showed striking necropsy lesions consistent with systemic infection and responded to antimicrobial treatment in the feed. Staphylococcus aureus (SA) was the most frequently isolated organism from multiple tissues including comb and wattle lesions, lungs, liver, ovary, spleen, and bone marrow. Given such an uncommon presentation of SA, which is known as a secondary opportunistic pathogen, a challenge study was conducted to evaluate its role in these disease outbreaks. In the present study, laying hens of two ages (22 and 96 wk) were inoculated with SA via three routes: oral gavage, subcutaneous (SC) injection, and intravenous (IV) injection. Both young and old hens in the IV group showed a significant increase in body temperature and drop in body weight; however, the clinical signs observed in the naturally occurring outbreaks were not present. SA was reisolated at multiple time points postchallenge from all challenge groups except the negative control group. While the SC group showed localized necrosis at the injection site, microscopic changes were different from changes observed in birds from the natural outbreaks. Despite observed initial differences in route and age, the SA challenge strain was not capable of reproducing the disease on its own. The results of this study indicate that SA may have played a role in the increased mortality, clinical signs, and necropsy lesions reported with the naturally occurring outbreaks. However, SA should still be considered as a secondary opportunistic pathogen. Other factors that could have caused the initial insult are stress, immunosuppression, or other primary infectious agents. The results of this study may aid veterinary diagnosticians, clinicians, and all poultry professionals to include SA in their differentials list as a secondary opportunistic pathogen in similar cases. This is an uncommon presentation and further field observations and clinical studies are needed to better elucidate the pathogenesis of this disease, which will in turn help to prevent future outbreaks.

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Host immunity and the colon microbiota of mice infected with Citrobacter rodentium are beneficially modulated by lipid-soluble extract from late-cutting alfalfa in the early stages of infection

2020-07-16 , Fries-Craft, Krysten , Bobeck, Elizabeth , Schmitz-Esser, Stephan , Bobeck, Elizabeth , Schmitz-Esser, Stephan , Animal Science , Microbiology

Alfalfa is a forage legume commonly associated with ruminant livestock production that may be a potential source of health-promoting phytochemicals. Anecdotal evidence from producers suggests that later cuttings of alfalfa may be more beneficial to non-ruminants; however, published literature varies greatly in measured outcomes, supplement form, and cutting. The objective of this study was to measure body weight, average daily feed intake, host immunity, and the colon microbiota composition in mice fed hay, aqueous, and chloroform extracts of early (1st) and late (5th) cutting alfalfa before and after challenge with Citrobacter rodentium. Prior to inoculation, alfalfa supplementation did not have a significant impact on body weight or feed intake, but 5th cutting alfalfa was shown to improve body weight at 5- and 6-days post-infection compared to 1st cutting alfalfa (P = 0.02 and 0.01). Combined with the observation that both chloroform extracts improved mouse body weight compared to control diets in later stages of C. rodentium infection led to detailed analyses of the immune system and colon microbiota in mice fed 1st and 5th cutting chloroform extracts. Immediately following inoculation, 5th cutting chloroform extracts significantly reduced the relative abundance of C. rodentium (P = 0.02) and did not display the early lymphocyte recruitment observed in 1st cutting extract. In later timepoints, both chloroform extracts maintained lower splenic B-cell and macrophage populations while increasing the relative abundance of potentially beneficially genera such as Turicibacter (P = 0.02). At 21dpi, only 5th cutting chloroform extracts increased the relative abundance of beneficial Akkermansia compared to the control diet (P = 0.02). These results suggest that lipid soluble compounds enriched in late-cutting alfalfa modulate pathogen colonization and early immune responses to Citrobacter rodentium, contributing to protective effects on body weight.

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Laser Enrichment Device Stimulates Broiler Laser-Following Behavior While Increasing Individual Bird Locomotion and Pen-Wide Movement

2021-11-29 , Johnson, Anna , Bobeck, Elizabeth , Bobeck, Elizabeth A. , Animal Science

Genetic selection for fast growth rate and high breast muscle yield in modern broilers has unintended effects on animal welfare and behavior, namely in terms of inactivity and leg disorders. We hypothesized that exercise stimulated through environmental enrichment could positively stimulate pen-wide activity and improve bird welfare. The study objectives were to implement a laser enrichment device to motivate active and feeding behaviors throughout the pen. Twelve hundred Ross 708 broilers were randomly assigned to enrichment (LASER; laser enrichment, or CON; no laser enrichment) for 49 d. Seventy focal birds were randomly assigned to 14 video-recorded pens for behavioral analysis, including focal bird home pen behavior and walking distance. Pen-wide activity was also measured during the 4-min laser periods, four times daily, d0–8, and 1 day weekly, wk 1–6. Focal birds were gait scored wk 1–6, and were euthanized on d42 for tibia bone mineral content, density, and bone breaking strength analysis. Time spent active was increased in LASER-enriched birds compared to CON on wk 3–5 by up to 214% (wk 4), and percent of time at the feeder was increased in LASER-enriched birds by 761% on wk 4 (P < 0.05). Peak percent of birds following the laser (LASER-enriched pens only) was observed on d0 (8.52%). Over wk 1–6, peak laser-following behavior was observed on wk 3 (3.07% of birds). Percent of birds moving during laser periods was increased in LASER-enriched pens on d0, 1, 2, 6, 7, and 8, with a percent increase of 68.7% observed on d1 (P < 0.05). Percent of birds moving (laser-following or not) was increased on wk 1, 3, and 4 in LASER-enriched pens, with an increase of 69.7% observed on wk 4 (P < 0.05). No differences were found in tibia measures. These data indicate that laser enrichment stimulated voluntary locomotion through wk 5 and laser-following behavior through wk 6, and that the relatively small percent of birds actively following the laser stimulated pen-wide movement above the level of the CON through wk 4 on study.

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Growth performance of male broilers fed ExPress® soybean meal and high-shear dry extruded corn

2021-01-01 , Meyer, Meaghan , Bobeck, Elizabeth , Bobeck, Elizabeth , Animal Science

Soybean meal (SBM), a co-product of soybean oil obtained via solvent or mechanical extrusion, is the key protein source in broiler diet formulations. Mechanical extrusion, or high-temperature processing with high-shear dry extrusion, has demonstrated 1 to 2% increased amino acid digestibility in broilers versus solvent-extracted SBM through reduction of antinutritional factors (ANF). High-shear dry extrusion followed by mechanical pressing using a screw press (extruding-pressing) produces soy oil and partially de-oiled soymeal (ExPress). The extruding-pressing process has been growing in popularity due to improved cost and material efficiency by eliminating the need for solvents or steam dryers in the production of animal feed ingredients. Corn extrusion is likewise linked with increased nutritive value through improved palatability and approximately 20% increased starch gelatinization, resulting in superior growth performance and in one reported case, reduced mortality in broilers. In the present study, high-shear dry extruded corn and ExPress SBM were formulated in combination with conventional corn and solvent-extracted SBM to determine impact on growth performance in male broilers. Extruded corn increased feed intake and ExPress SBM increased weight gain. The diet containing dry extruded corn and ExPress SBM outperformed all other diet combinations, including unprocessed, ground corn and conventional, solventextracted SBM variations. The diet containing unprocessed, ground corn and ExPress SBM had the most efficient overall feed conversion ratio (FCR). The combination of lower feed intake observed with the ground corn ingredient and the improved protein digestibility reflected by superior growth in ExPress SBM made this the ideal formulation, out of the 4 diets examined, in terms of feed efficiency.

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Eggshell and environmental bacteria contribute to the intestinal microbiota of growing chickens

2020-01-01 , Maki, Joel , Bobeck, Elizabeth , Bobeck, Elizabeth , Sylte, Matthew , Looft, Torey , Animal Science , Microbiology

Background: The initial intestinal microbiota acquired from different sources has profound impacts on animal health and productivity. In modern poultry production practices, the source(s) of the establishing microbes and their overall contribution during development of gastrointestinal tract communities are still unclear. Using fertilized eggs from two independent sources, we assessed the impact of eggshell- and environmental-associated microbial communities on the successional processes and bacterial community structure throughout the intestinal tract of chickens for up to 6 weeks post-hatch.

Results: Culturing and sequencing techniques identified a viable, highly diverse population of anaerobic bacteria on the eggshell. The jejunal, ileal, and cecal microbial communities for the egg-only, environment-only, and conventionally raised birds generally displayed similar successional patterns characterized by increasing community richness and evenness over time, with strains of Enterococcus, Romboutsia, and unclassified Lachnospiraceae abundant for all three input groups in both trials. Bacterial community structures differed significantly based on trial and microbiota input with the exception of the egg-exposed and conventional birds in the jejunum at week 1 and the ileum at week 6. Cecal community structures were different based on trial and microbiota input source, and cecal short-chain fatty acid profiles at week 6 highlighted functional differences as well.

Conclusion: We identified distinct intestinal microbial communities and differing cecal short-chain fatty acid profiles between birds exposed to the microbiota associated with either the eggshell or environment, and those of conventionally hatched birds. Our data suggest the eggshell plays an appreciable role in the development of the chicken intestinal microbiota, especially in the jejunum and ileum where the community structure of the eggshell-only birds was similar to the structure of conventionally hatched birds. Our data identify a complex interplay between the eggshell and environmental microbiota during establishment and succession within the chicken gut. Further studies should explore the ability of eggshell- and environment-derived microbes to shape the dynamics of succession and how these communities can be targeted through interventions to promote gut health and mitigate food-borne pathogen colonization in poultry.