Dietary peptide-specific antibodies against interleukin-4 differentially alter systemic immune cell responses during Eimeria challenge with minimal impacts on the cecal microbiota.

Eimeria spp. induce host interleukin (IL)-4 production, a potent immune regulator, during coccidiosis to evade immune responses. Dietary anti-IL-4 may preserve bird performance during challenge; however, specific mechanisms have not been investigated. Study objectives were to develop peptide-specific anti-IL-4 antibodies and evaluate immune cell profiles and the cecal microbiota during Eimeria challenge. Four candidate IL-4 peptides were selected based on antigenicity and location. Hens were injected with conjugated peptide or carrier-only control (3/injection), eggs were collected post-vaccination and yolks were pooled by peptide before freeze-drying. On d 0, 300 Ross 708 broilers were placed in floor pens (10/pen) and assigned to 5 diets consisting of basal diet + 2% egg yolk powder containing antibodies against 1 of 4 target peptides or carrier-only control for 14-d starter and grower periods (28 d total). Baseline blood and cecal contents were collected on d 14 (6 birds/diet) before half the remainder were inoculated with 10X Coccivac-B52 (Merck Animal Health, Kenilworth, NJ). Body weight (BW) and feed intake (FI) were recorded weekly and blood and cecal samples were collected at 3, 7, and 14 d post-inoculation (pi; 3/treatment). Immune cell profiles in peripheral blood mononuclear cells (PBMC) were evaluated flow cytometrically and cecal microbial communities determined by 16S/18S rRNA gene amplicon sequencing. Data were log-transformed when necessary and analyzed with diet, Eimeria, and timepoint fixed effects plus associated interactions (SAS 9.4; P ≤ 0.05). Anti-IL-4 did not alter baseline performance but generally increased PBMC Bu-1+ B cells 38.0 to 55.4% (P < 0.0001). Eimeria challenge reduced FI and BWG 16.1 and 30.3%, respectively, regardless of diet (P < 0.0001) with only birds fed peptide 4 antibodies not recovering feed conversion by d 28. Minimal diet-associated cecal microbiota changes were observed, indicating that anti-IL-4 effects were likely host-specific. Eimeria-challenged birds fed peptide 3 antibodies displayed minimal immune cell fluctuations compared to unchallenged counterparts, suggesting these antibodies potentially modulated intestinal immune responses to minimize systemic requirements, making them good candidates for further research.

Basal diet composition contributes to differential performance, intestinal health, and immunological responses to a microalgae-based feed ingredient in broiler chickens.

Novel feed ingredients may improve poultry health, but functionality of these ingredients may vary across basal diet formulations. This study evaluated a proprietary algae ingredient's effects on broiler performance, intestinal health, systemic immunity, and metabolic/immune kinotypes between corn- or wheat-based diets. Ross 308 broilers were housed in 80 floor pens (14 birds/pen) and assigned to 1 of 4 corn or wheat-based diets ± 0.175% algae ingredient for 42 d. At the end of each 14 d starter, grower, and finisher period, 10 birds/treatment were euthanized for tissue collection to assess intestinal histomorphology, systemic immune cell populations by flow cytometry and kinotypes by peptide arrays. On d 28 and 29, forty-three birds/treatment underwent a 12 h feed restriction challenge followed by a fluorescein isothiocyanate-dextran intestinal permeability assay. For the entire 42 d study, wheat-based diets improved feed conversion rate (FCR) by 5 points compared to corn-based diets (P < 0.0001). Performance benefits related to algae inclusion were diet dependent, with algae inclusion improving 42 d FCR by 6 points only in corn-based diets (P = 0.006). Birds fed wheat-based diets had reduced splenic monocyte/macrophage, CD1.1+, and T cell populations in the first 14 d (P < 0.0001) and reduced serum fluorescence on d 28/29 (P = 0.0002). Algae inclusion in the corn-based diet increased villus height in the duodenum on d 28 and jejunum on d 42, while reducing splenic CD3+CD8α+ cytotoxic T cells 13.4 to 27.5% compared to the corn-based control at the same timepoints (P < 0.0001). Kinome results showed a significant innate immune toll-like receptor (TLR) response via MyD88 at d 14 in the small intestine of birds fed corn-based diets with algae that shifted to a more growth factor and adaptive immune-oriented response by d 42. Concurrent with immune changes, signaling changes indicative of lipid metabolism in the small intestine, ceca, and liver were seen in birds fed the corn-based diet with algae. The observed differential responses to basal diet composition and algae inclusion emphasize the need to comparatively evaluate feed ingredients in various diet formulations.

Dietary vasodilator and vitamin C/L-arginine/choline blend improve broiler feed efficiency during finishing and reduce woody breast severity at 6 and 7 wks.

Woody breast has become a considerable economic concern to the poultry industry. This myopathy presents rigid, pale breasts characterized by replacement of lean muscle protein with connective tissue, a result of hypoxia and oxidative stress in a metabolically starved muscle with inadequate circulation. Hence, the objectives were to supplement broiler diets with ingredients specifically aimed to improve circulation and oxidative status. About 1,344 male Ross 708 broilers were assigned to 1 of 4 diets: 1) a basal diet (control), 2) basal diet plus a blend of 0.2% supplemental L-arginine, 0.17% choline bitartrate, and 0.03% vitamin C (blend), 3) 0.1% vasodilator ingredient (vasodilator), or 4) 0.02% Astaxanthin ingredient (AsX). At d 14, 28, 42, and 49, performance outcomes were collected on all birds and serum from 16 broilers/diet (n = 64) was analyzed for creatine kinase and myoglobin. Once weekly beginning on d 28, a subset of 192 broilers were measured for breast width. On d 42 and 49, breast fillets from 16 broilers/diet (n = 64) were palpated for woody breast severity, weighed, and analyzed for compression force at 1-day postmortem and water-holding capacity at 2-day postmortem. mRNA was isolated from 15 breast fillets/timepoint for qPCR quantification of myogenic gene expression. Data were analyzed using Proc Mixed (SAS Version 9.4) with the fixed effect of diet. Feed conversion ratio was improved in the blend and vasodilator-fed birds d 42 to 49, each by over 2 points (P < 0.05). Breast width was increased in the control on d 42 compared to the vasodilator and AsX-fed broilers (P < 0.05). At d 42, there were 12% greater normal fillets in blend diet-fed birds and 13% more normal scores in vasodilator-fed birds at d 49 compared to the control. At d 49, myogenin expression was upregulated in the AsX diet compared to blend and control diets (P < 0.05), and muscle regulatory factor-4 expression was increased by 6.5% in the vasodilator diet compared to the blend and AsX diets (P < 0.05). Blend and vasodilator diets simultaneously improved feed efficiency in birds approaching market weight while reducing woody breast severity.

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

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.

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.

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.

Evaluation of a high-protein DDGS product in broiler chickens: performance, nitrogen-corrected apparent metabolisable energy, and standardised ileal amino acid digestibility.

1. New production processes and additional uses for corn co-products have increased the availability of distiller's dried grains with solubles (DDGS) with varying energy and amino acid digestibility, for use in poultry feed. The objective of this study was to determine the performance, N-corrected metabolisable energy (AMEn), and amino acid (AA) digestibility of a 34% CP (as fed) high-protein DDGS (HP-DDGS) included in poultry diets for Cobb 500 broiler chickens.2. A total of 832 Cobb 500 broilers were randomly assigned to four dietary treatments containing 5% conventional DDGS (CV-DDGS) as a control or 10%, 15% and 20% HP-DDGS and fed for 42 d. After the performance trial, 240 birds from the original 832 were selected for a concurrent AMEn and AA digestibility experiment consisting of two AMEn diets and two AA diets.3. Birds fed diets containing 15% and 20% HP DDGS gained less weight than birds fed the CV-DDGS (P < 0.05) but did not differ in feed intake (FI), and therefore had a less efficient FCR than the control (P < 0.05). The AMEn of HP-DDGS was determined to be 11.4 MJ/kg. The standardised ileal amino acid digestibility (SIAAD) of the essential amino acids Lys and Met were determined to be 80.9% and 88.6%, respectively.4. HP-DDGS can be included in broiler diets up to 10% without any negative impact on performance or requiring supplemental Lys and Arg. The results from the AA digestibility study indicated that HP-DDGS could be a good source of digestible Lys.

Oil source and peroxidation status interactively affect growth performance and oxidative status in broilers from 4 to 25 d of age.

The objectives of this experiment were to evaluate the effect of oil source and peroxidation status on broiler performance and oxidative stress. Broilers (initial BW 85.1 ± 7.8 g) were allotted to 40 cages with 5 birds per cage in a completely randomized design. The 4 × 2 factorial arrangement of treatments consisted of oil source (palm oil, soybean oil, flaxseed oil, and fish oil) and peroxidation status (fresh or peroxidized). Broilers were fed experimental diets for 20 d to measure growth performance; on day 21 of the experiment, plasma and liver samples were harvested for analysis of oxidative stress including thiobarbituric acid reactive substances (TBARS), protein carbonyls (PC), 8-hydroxy-2'-deoxyguanosine (8- OH-2dG), glutathione peroxidase activity (GPx) and superoxide dismutase and catalase (CAT). An interaction occurred between oil source and peroxidation status where broilers fed peroxidized oils had reduced ADFI, ADG, G:F, and plasma GPx in all oil sources except for fish oil (P ≤ 0.04). Plasma 8-OH-2dG was increased by feeding peroxidized oils (P = 0.01). An interaction occurred in liver TBARS where broilers fed peroxidized palm oil had greater liver TBARS compared to fresh palm oil (P = 0.09). An interaction was noted for liver PC where broilers fed palm, flaxseed, and fish oil had similar liver PC regardless of peroxidation status, while broilers fed peroxidized soybean oil had increased liver PC compared to the fresh soybean oil diet (P = 0.04). Oil source affected plasma TBARS and 8-OH-2dG (P = 0.01), plasma PC (P = 0.09), liver 8-OH-2dG (P = 0.08), and liver CAT (P = 0.02). Correlations between oil composition with growth performance and oxidative stress markers imply that oil UFA:SFA, p-anisidine value, DDE, total polar compounds, and polymerized triglycerides should be measured as an indicator of oil quality, with growth performance being correlated to plasma TBARS, PC, and GPx. In conclusion, the degree of unsaturation and peroxidation status of dietary oils affected growth performance and markers of oxidative stress in poultry.

Comparative omega-3 fatty acid enrichment of egg yolks from first-cycle laying hens fed flaxseed oil or ground flaxseed.

When laying hen diets are enriched with omega-3 polyunsaturated fatty acids to generate value-added eggs for human consumption markets, concentrations of alpha-linolenic (ALA), eicosapentaenoic (EPA), and docosahexaenoic acids (DHA) in the yolk can reach 250 mg/50 g whole egg. Flaxseed, a rich source of ALA, is commonly used for omega-3 enrichment; however, the impact of dietary flaxseed source (extracted oil vs. milled seed) on fatty acid transfer to egg yolk in laying hens is unknown. Therefore, transfer of ALA, EPA, and DHA into egg yolk from extracted flaxseed oil or milled flaxseed was evaluated in Hy-Line W-36 laying hens over an 8-week feeding period (25 to 33 wk old). Hens (n = 132) were randomly housed with 3 birds/cage (4 replicates/treatment) for each of the 11 treatment groups. Diets were isocaloric and consisted of a control diet, 5 flaxseed oil diets (0.5, 1.0, 2.0, 3.0, or 5.0% flaxseed oil), and 5 milled flaxseed diets (calculated flaxseed oil concentration from milled flaxseed 0.5, 1.0, 2.0, 3.0, 5.0%). Increasing dietary concentrations of flaxseed oil and milled flaxseed resulted in increased ALA, EPA, and DHA concentration in egg yolk, and fatty acid deposition from flaxseed oil was 2 times greater compared to milled flaxseed when fed at the same dietary inclusions (P < 0.01). Egg yolk EPA and DHA concentrations were not different due to oil or milled source (P = 0.21); however, increasing dietary inclusion rates of flaxseed oil from either source increased yolk EPA and DHA (P < 0.01). Hens fed either flaxseed oil or milled flaxseed resulted in reduced BW change as dietary concentrations increased (P = 0.02). Feed efficiency increased as flaxseed oil increased in concentration, while feeding milled flaxseed decreased feed efficiency (P = 0.01). Analysis of the nitrogen corrected apparent metabolizable energy of flaxseed oil resulted in 7,488 kcal/kg on an as-fed basis. Dietary flaxseed oil improved feed efficiency and increased ALA deposition into yolk compared to a milled source, demonstrating flaxseed oil to be a viable alternative for ALA egg enrichment.

Effects of long-term supplementation of laying hens with high concentrations of cholecalciferol on performance and egg quality.

There is current interest in increasing human vitamin D dietary intake without having to modify human eating habits. One method to increase human dietary vitamin D intake is to generate eggs with increased concentrations of vitamin D through high-concentration vitamin D feeding in the diets of laying hens. Although eggs can be produced with high concentrations of vitamin D, the consequences of these diets on hen performance and egg quality have not been validated. The objective of this research is to quantify the effects of high concentrations of cholecalciferol (D3) on laying hen performance and egg quality. Hy-Line W36 laying hens were placed on 1 of 5 experimental diets for 40 wk: 1) control (contained 2,200 IU of D3/kg of diet), 2) control + 7,500 IU of D3/kg of diet (9,700 IU of D3/kg of diet total), 3) control + 15,000 IU of D3/kg of diet (17,200 IU of D3/kg of diet total), 4) control + 22,500 IU of D3/kg of diet (24,700 IU of D3/kg of diet total), and 5) control + 100,000 IU of D3/kg of diet (102,200 IU of D3/kg of diet total). Egg production and hen mortality were monitored daily. Feed intake was determined weekly. Eggs were collected at predetermined points throughout the 40-wk period (19 to 58 wk of bird age) for assessment of egg weight, egg component weights, Haugh unit, yolk color score, specific gravity, egg mass, and feed efficiency. There were no consistent differences among the dietary treatments over the experimental period. Hens supplemented with up to 102,200 IU of D3/kg of diet resulted in no significant reductions in egg production, feed intake, feed efficiency, egg component weights, yolk color, Haugh units, and specific gravity in comparison with the control-fed hens (P > 0.05). These data suggest the addition of cholecalciferol to the diet of the laying hen at concentrations up to 102,200 IU of D3/kg of diet had no consistent negative effects on laying hen performance or egg quality.