AZOMITE DTB in Eimeria-challenged broilers: growth, antioxidant status, intestinal health, and digestibility

Objective

  • Assess the effects of a dacitic tuff breccia (DTB; AZOMITE) on performance, antioxidant defense, intestinal health, nutrient digestibility, and inflammatory responses in broilers mildly challenged with Eimeria spp.
  • Specific focus: growth performance, GI permeability, intestinal morphology, tight junction/mucin gene expression, antioxidant enzymes (GPx, SOD) and glutathione status (GSH, GSSG), inflammatory cytokines (IL-10, IFN-γ, IL-1β, TNF-α), apparent ileal digestibility (AID) of crude protein (CP) and dry matter (DM), ileal digestible energy (IDE).
  • Hypothesis: DTB supplementation mitigates Eimeria-induced oxidative stress and inflammation and improves nutrient utilization and growth during and after challenge.

Experimental Design

  • Subjects: 600 one-day-old Cobb 500 male broiler chicks.
  • Design: randomized complete block design (RCBD) with 5 treatments and 10 replicates (12 birds per replicate cage).
  • Treatments:
    • UC: unchallenged control (0% DTB)
    • CC: challenged control (0% DTB)
    • DTB0.125: challenged + 0.125% DTB
    • DTB0.25: challenged + 0.25% DTB
    • DTB0.50: challenged + 0.50% DTB
  • Challenge: On day 14, CC and all DTB-fed groups gavaged orally with mixed Eimeria spp. (approximately 12,500 E. maxima, 12,500 E. tenella, 62,500 E. acervulina per 1 mL). UC received water only.
  • Duration: 1–26 days of age.
  • Housing: 50 cages (0.04 m2 per bird); ad libitum feed and water; trough feeder and drinker.
  • Ethics: study approved by IACUC, University of Georgia.

Diet Formulation and DTB Details

  • DTB product: AZOMITE feed-grit (dacitic rhyolitic tuff breccia), hydrated sodium calcium aluminosilicate, containing >70 minerals and trace minerals (e.g., Fe, Mg, Mn, Se, Zn, Cu, rare earth elements).
  • DTB inclusion levels: 0.125%, 0.25%, 0.50% in starter (0–14 d) and grower (14–26 d) diets as a replacement for ground corn.
  • Control diet: 0% DTB (with TiO2 at 0.3% as an indigestible marker).
  • Diet composition (as-fed basis): starter and grower formulations detailed in Table 1 of the study; metabolizable energy and nutrient contents adjusted to meet Cobb 500 specs.
  • Rationale for DTB: DTB minerals serve as cofactors for antioxidant enzymes (e.g., selenium, copper, manganese for GPx and SOD) and may modulate immune function and nutrient digestibility, potentially mitigating coccidial-induced oxidative stress and inflammation.
  • TiO2 marker and digestibility methodology: TiO2 added as indigestible marker to calculate AID and IDE.

Data Collected and Time Points

  • Growth performance: ADG, ADFI, GF measured at 0–14 d (prechallenge), 14–20 d (challenge), 20–26 d (recovery), and 0–26 d (overall).
  • Gastrointestinal permeability: FITC-d assay performed on day 19 (5 dpi); serum FITC-d concentration measured (excitation 485 nm, emission 528 nm).
  • Gene expression: on day 20 (6 dpi), proximal jejunal and cecal tonsil samples collected for qRT-PCR. Targets included IL-10, IFN-γ, IL-1β in cecal tonsil; Occludin, Claudin-1 (CLDN-1), Mucin-2 (Muc-2) in jejunum. GAPDH used as endogenous control. Relative expression calculated by 2^(-ΔΔCt) using UC mean as calibrator.
  • Histomorphology: on day 20 (6 dpi), sections from duodenum, jejunum, ileum stained with H&E; measurements of villus height (VH) and crypt depth (CD) for 3 villi/crypts per slide; V:C ratio computed.
  • Antioxidant system: liver GPx and SOD activities and concentrations of GSH and GSSG measured on days 20 (6 dpi) and 26 (12 dpi); GSH:GSSG ratio also calculated. Assays followed manufacturer instructions; protein quantification for standardization.
  • Apparent ileal digestibility (AID) and IDE: ileal digesta collected on day 20 (6 dpi) from 4 birds per replicate (40 birds/treatment); digesta pooled by replicate; TiO2 content measured; CP, DM, and IDE calculated using equations below.

Equations and Calculations

  • Apparent ileal digestibility (AID) of a nutrient:
    ext{AID}_{ ext{nutrient}} ( ext{percent}) = 100 imes igg(1 - rac{ ext{NutrientDigesta}}{ ext{NutrientDiet}} imes rac{ ext{TiO2Diet}}{ ext{TiO2Digesta}}igg)
  • Ileal digestible energy (IDE):
    ext{IDE} ( ext{kcal/kg}) = GEDiet - GEDigesta imes rac{ ext{TiO2Diet}}{ ext{TiO2Digesta}}
  • Other measurement details:
    • GF = Gain:Feed ratio; calculated from ADG and ADFI
    • Gene expression: relative to GAPDH control using the 2^(-ΔΔCt) method; mean ΔCt of UC used to calculate ΔΔCt
    • Statistical contrasts: linear and quadratic contrasts performed to account for uneven AZOMITE inclusion levels
    • Significance: P ≤ 0.05; trends discussed for 0.05 < P < 0.10

Statistical Analysis

  • Model: linear mixed model (PROC MIXED SAS v9.4).
  • Fixed effects: treatment (UC, CC, 0.125, 0.25, 0.50% DTB).
  • Random effects: block within RCBD.
  • Post hoc: Tukey’s test for multiple comparisons.
  • Contrasts: linear and quadratic contrasts for AZOMITE dose response.

Results

Growth Performance

  • 0–14 d (prechallenge): ADG and ADFI not different among treatments (P > 0.05).
  • 0–14 d GF: highest in 0.125% DTB; 0.125%, 0.25%, 0.50% DTB all higher than CC and UC (P < 0.001); quadratic with peak at 0.125% DTB (P = 0.004).
  • 14–20 d (challenge): UC had higher ADG, ADFI, GF than all challenged groups (P < 0.001); no dose effect among DTB levels.
  • Challenge effect magnitude: Eimeria reduced ADG by ~52.5%, ADFI by ~25.0%, GF by ~36.9% vs UC.
  • 20–26 d (recovery): ADG higher in UC (trend, P = 0.057); ADFI higher in UC (P < 0.001); GF not different among challenged groups (P = 0.172).
  • Overall (0–26 d): UC higher ADG and ADFI than other groups (P < 0.001); DTB affected GF (P < 0.001) with UC having the highest GF; 0.125% and 0.25% DTB superior to CC (P < 0.001) with a quadratic trend (P = 0.015).
  • Mortality: UC = 2, CC = 9, 0.125% DTB = 10, 0.25% DTB = 10, 0.5% DTB = 6.
  • Overall interpretation: mild coccidial challenge impaired growth and intestinal function; DTB at 0.125–0.25% partially mitigated some performance losses, particularly GF.

Intestinal Permeability

  • 5 dpi FITC-d: challenged groups had higher serum FITC-d than UC (P < 0.001); DTB dose did not differ significantly among challenged groups (P not listed as significant).
  • Tight junction/ mucus gene expression:
    • 20 d (6 dpi): CLDN-1 no difference (P = 0.117). Occludin and Muc-2 were highest in UC; 0.125% DTB downregulated occludin (P = 0.014) and mucin-2 (P < 0.001) vs UC and CC.
    • 26 d (12 dpi): CLDN-1 and Occludin not different overall. Muc-2 lowest in 0.25% and 0.5% DTB; CC downregulated mucin-2 vs UC (P = 0.001). Linear DTB effect: higher DTB associated with lower CLDN-1 (P = 0.017) and Muc-2 (P = 0.011) at 26 d.
  • Interpretation: Eimeria challenge increased intestinal permeability; DTB had limited or dose-dependent effects on tight junction/mucin gene expression, with some downregulation at certain DTB levels.

Antioxidant System and Inflammation Markers

  • SOD activity: no differences at 20 d (P = 0.742).
  • GPx activity: increased in challenged birds, notably with 0.125% DTB (P < 0.001 vs CC/0.5% DTB/UC).
  • GSH concentration: lower in challenged groups; at 6 dpi DTB dose effects observed; at 12 dpi, GSH highest in UC and 0.125% DTB; 0.25% and 0.5% DTB had lower GSH (P = 0.005 at 26 d for overall comparison).
  • GSSG: higher in challenged/dose groups at 20 d with a rising trend; 26 d GSSG not significantly different (P = 0.783).
  • GSH:GSSG ratio: high in controls; lower in challenged groups (P = 0.689 at 20 d; P = 0.110 at 26 d).
  • Inflammation markers (d 20, 6 dpi): IFN-γ and IL-10 upregulated in all challenged treatments vs UC (P = 0.070 for IFN-γ trend; P = 0.038 for IL-10). IL-1β upregulated in 0.125% and 0.5% DTB vs UC (P = 0.001); CC also lower than 0.125%/0.5% DTB in some comparisons. TNF-α did not differ (P = 0.830).
  • Interpretation: Eimeria challenge elevates pro- and anti-inflammatory cytokines; DTB at 0.125% may bolster certain antioxidant responses (GPx) and modulate inflammatory signaling, with complex changes in GSH/GSSG balance depending on dose and time.

Histomorphology and Nutrient Digestibility

  • Histomorphology (d 20):
    • Duodenum CD higher in CC than 0.25% DTB; UC had the lowest CD (P < 0.001).
    • Duodenum VH and V:C highest in UC (P < 0.001); challenged groups did not differ among themselves for VH or V:C.
    • Jejunum: VH highest in UC (P < 0.001); CD similar across treatments; V:C highest in UC (P < 0.001).
    • Ileum: VH and CD not significantly different; V:C not different.
  • Nutrient digestibility:
    • CP AID highest in UC; 0.125% DTB higher than CC and 0.5% DTB (P < 0.001).
    • DM AID highest in UC; challenged groups lower with limited dose effects.
    • IDE highest in UC; no clear differences among challenged groups; overall, a quadratic tendency for CP AID with peak at 0.125% DTB (P = 0.097).
  • Digestibility and IDE interpretation: Eimeria-induced epithelial damage reduces absorptive surface and digestive enzyme expression, lowering VH and transporter function; DTB at low dose (0.125%) can partially mitigate CP digestibility reductions.

Overall Interpretation and Context

  • The mild Eimeria challenge successfully induced intestinal damage and oxidative stress, evidenced by decreased performance, digestibility, and barrier integrity in challenged groups.
  • DTB at 0.125% showed potential to improve antioxidant response (notably GPx activity) and CP digestibility and to support growth during the prechallenge and some recovery periods, with less clear dose-dependent benefits across all measured parameters.
  • Higher DTB levels (0.25%, 0.50%) did not consistently outperform the 0.125% level and sometimes aligned more with the CC group in several metrics, suggesting a possible threshold or carryover effects that warrant further investigation.
  • Mechanistic considerations: DTB minerals can act as cofactors for antioxidant enzymes and may influence gut health and microbiota; reductions in GSH and mucin/occludin expression at higher DTB doses imply a nuanced interaction with oxidative and barrier status under Eimeria challenge.
  • Practical implications: In a low-to-moderate coccidial challenge scenario, 0.125% AZOMITE DTB could offer some advantages in antioxidant defense and CP digestibility, contributing to partial mitigation of Eimeria-induced performance losses. Dose optimization and interactions with other minerals and gut microbiota require further study.

Connections to Foundational Principles and Real-World Relevance

  • Nutrient–immune–oxidative axis: Trace minerals (Se, Cu, Mn) in DTB can support antioxidant enzymes like GPx and SOD, aligning with established roles of trace minerals in poultry oxidative defense.
  • Gut barrier integrity: Tight junctions (Claudin-1, Occludin) and mucin (Muc-2) are essential for preventing enteric leakage; Eimeria disrupts this barrier, increasing permeability; DTB effects on these markers echo broader research on mineral supplements and gut health under enteric challenges.
  • Nutrient digestibility and performance under coccidiosis: Eimeria reduces absorptive surface and transporter expression; minerals that support enzyme cofactors and gut environment can help sustain nutrient use and growth during challenge.
  • Ethical and practical considerations: Use of non-antibiotic nutritional strategies to mitigate disease aligns with public health concerns and regulatory trends toward responsible antibiotic use in poultry.

Key Takeaways

  • Eimeria challenge impaired growth performance, nutrient digestibility, and intestinal barrier function; DTB at 0.125% offered potential benefits in antioxidant response (GPx) and CP digestibility, with modest improvements in GF during the prechallenge period.
  • No clear linear dose-response across all DTB levels; 0.125% DTB often performed better or similarly to higher DTB doses in several metrics, suggesting a possible optimal level around 0.125% under the tested conditions.
  • DTB influenced inflammatory markers, with IL-10 and IFN-γ upregulated by challenge across treatments; IL-1β upregulation was dose-related, especially at 0.125% and 0.5% DTB, indicating nuanced immunomodulation.
  • The study supports potential use of DTB as a mineral-based strategy to mitigate some effects of coccidiosis on oxidative status and nutrient utilization, but further work is needed to clarify mechanisms, dose optimization, and interactions with gut health.

References to Contextual Studies (selected from text)

  • Previous DTB work showing improved feed efficiency and bone characteristics in broilers and laying hens with AZOMITE supplementation.
  • DTB minerals as cofactors for GPx and SOD and potential to enhance oxidative status under various stressors.
  • Eimeria-induced oxidative stress and immune responses; coccidiosis as a risk factor for necrotic enteritis; rationale for nutritional interventions to support gut health during infection.

Notes on Limitations and Future Directions

  • The absence of a strong dose-response to AZOMITE under the specific mild-challenge model suggests the need for exploring a broader range of doses, different challenge severities, and longer-term outcomes.
  • Interactions with other dietary minerals, energy density, and gut microbiota composition warrant additional investigation to understand mechanisms of action.
  • Additional time points for gene expression and barrier function assessments could illuminate dynamic changes across the infection and recovery phases.

End of Notes