Transcriptomic Study on the Lungs of Broilers with Ascites Syndrome.
The main results reproduced, with only marginal, non-material deviations.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓Same input data as the authors
- ✓Reported values are derivable from the shared data
- ✓The central claim held under reproduction
- 🟡Reported values were only indirectly comparable
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡The deviation was non-trivial in magnitude
- 🟡Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
STRONG PARTIAL reproduction (described well enough; mostly 1:1 on the core pipeline). RNA-seq of broiler lung, ascites(C) vs normal(N), n=3 each (PRJNA758269; data is paired-end 2x151 despite paper/ENA saying single-end). Core DESeq2 result reproduced closely: 1516 DEGs (661 up/855 down) vs reported 1442 (614/828), +3-8%; per-sample mapping 92.7-94.1% vs reported 92.44-93.31% (within ~1.5%); top up-gene FABP4 reproduced EXACTLY (padj 8.4e-20 vs 9.6e-20); GRIK1 confirmed as a top-2 down DEG. Substitutions (all documented, forced by tool/infra limits not analysis choice): STAR 2.7.11b for TopHat2 v2.1.1 (which HUNG on the PE data); fastp 'clean reads'; Ensembl-105 GO + a propagate-and-count script for GOATOOLS+majorbio. Diverging secondary claims: GO 'regulation of response to stimulus' present but 232 not 676 DEGs and not the dominant term (denser proprietary annotation in paper); named up-gene panel 3/8 significant (3 more up-trending but borderline n.s.); KEGG (majorbio) NOT attempted. NOT a drop: code tool (GOATOOLS, P16) + open SRA data both resolvable and run.
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Assessment versions
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v1 current initial assessment Score 71assessed: 2026-06-22 ⛓ 938ee990b20e
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Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-22
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-22no human curator yet
- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: sonnetSince the lung is the primary target organ of broiler ascites syndrome (AS) but its transcriptome had not been studied, the study tests whether RNA-seq of lung tissue can reveal differentially expressed genes and pathways underlying AS pathogenesis.
- ★ 1442 genes were differentially expressed in AS broiler lung vs normal lung (614 up-regulated, 828 down-regulated) finding
- ★ KEGG enrichment showed cell adhesion molecules (CAMs) as the most significantly enriched pathway, possibly reflecting endothelial dysfunction or vascular injury finding
- ★ HIF-1, NF-kappa B, and p53 signaling pathways are enriched in AS lung tissue and implicated in hypoxia-induced pulmonary arterial hypertension mechanism
- ★ Up-regulated genes FABP4, APLN, EIF2AK4, HMOX1, MMP9, THBS1, TLR4, BCL2 and down-regulated genes APELA, FGF7, WNT5A, CDK6, IL7, IL7R, APLNR are candidate genes relevant to pulmonary hypertension in AS finding
- ★ A multifactorial cold-stress and high-energy/fat-diet protocol successfully induced an ascites syndrome model in broilers, confirmed by AHI>0.29, HCT>36%, and pericardial/abdominal effusion method
- qRT-PCR of six selected genes (FABP4, APELA, FGF7, WNT5A, CDK6, APLNR) validated RNA-seq differential expression direction and significance method
- Multiple metabolic pathways (amino sugar/nucleotide sugar metabolism, alanine/aspartate/glutamate metabolism, glycine/serine/threonine metabolism) are enriched, indicating abnormal lung metabolism in AS finding
- Lung histopathology in AS broilers shows pulmonary capillary thickening, respiratory capillary atrophy, narrowed lung atrium, congestion, edema, and lymphocyte infiltration finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| RNA sequencing (RNA-seq) | lung tissue, broiler chickens (Arbor Acres) | ascites syndrome model (cold stress + high-fat/high-energy diet + NaCl) | differentially expressed genes | Illumina Novaseq 6000; TopHat2 alignment; DESeq2 for DEG analysis |
| GO functional annotation and KEGG pathway enrichment analysis | lung tissue DEGs, broiler chickens | none (bioinformatic analysis of RNA-seq data) | enriched GO terms and KEGG pathways | GOATOOLS; KEGG database (majorbio cloud platform) |
| quantitative real-time PCR (qRT-PCR) | lung tissue, broiler chickens | ascites syndrome model | expression of 6 selected genes (2^-ΔΔCt method) to validate RNA-seq | 2×T5 Fast qPCR Mix, real-time thermal cycler |
| histopathology (hematoxylin-eosin staining) | left lung tissue, broiler chickens | ascites syndrome model | lung tissue morphology (vascular wall, capillaries, inflammation) | Leica DM4000B microscope; KD2508 paraffin slicer |
| routine blood analysis | whole blood, broiler chickens | ascites syndrome model | RBC, hemoglobin (HB), hematocrit (HCT), WBC, lymphocytes (LYM), PLT, PCT | automatic blood analyzer (Shenzhen Pukang Electronic Co.) |
| gross/anatomical and cardiac index measurement | heart, lung, whole body, broiler chickens | ascites syndrome model | body weight, ascites heart index (AHI = RV/TV), lung organ coefficient | — |
- – 1442 DEGs identified in AS vs normal lung tissue (614 up-regulated, 828 down-regulated) p-adjust<0.05, |log2FC|>=1
- – Cell adhesion molecules (CAMs) pathway was the most significantly enriched KEGG pathway
- – HIF-1 signaling pathway enriched among DEGs
- – qRT-PCR confirmed FABP4 up-regulated and APELA, FGF7, WNT5A, CDK6, APLNR down-regulated in AS group, consistent with RNA-seq p<0.01
- – AHI, average body weight, and pulmonary organ coefficient significantly differed between AS and normal groups p<0.01
- ▲ RBC, HB, HCT, WBC, and LYM significantly differed between AS and normal groups p<0.01
- – Mapping rate of RNA-seq reads to reference genome exceeded 65% (actual 92.44–93.31% across samples) 92.44-93.31%
- – Regulation of response to stimulus was the most dominant enriched GO biological process term 676 DEGs
- count 1442 total DEGs (614 up, 828 down) (differential gene expression, AS vs normal lung, p-adjust<0.05, |log2FC|>=1)
- count 676 DEGs (genes in top enriched GO term 'regulation of response to stimulus')
- pvalue p < 0.01 (differences in body weight, AHI, pulmonary organ coefficient between AS and normal groups)
- pvalue p < 0.01 (differences in RBC, HB, HCT, WBC, LYM between AS and normal groups)
- pvalue p < 0.01 (qRT-PCR validation of FABP4, APELA, FGF7, WNT5A, CDK6, APLNR expression differences)
- other AHI (RV/TV) > 0.29 (criterion for ascites model selection)
- other HCT > 36% (criterion for ascites model selection)
- other mapping rate 92.44-93.31% (RNA-seq read alignment to reference genome per sample (C1-C3, N1-N3))
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The study compared lung transcriptomes of ascites syndrome (AS) broilers (n=3) and healthy broilers (n=3) using RNA sequencing analyzed with DESeq2, applying a negative-binomial model and correcting p-values with the Benjamini-Hochberg method to define differentially expressed genes (padj<0.05, |log2FC|≥1), followed by GO and KEGG enrichment analyses of the DEGs. Physiological/hematological parameters (body weight, AHI, lung organ coefficient, blood counts) were compared between treatment groups and across ages using two-factor (two-way) ANOVA in SPSS 17.0, and qRT-PCR validation of six selected genes was analyzed in GraphPad Prism v9.0. p<0.05 was used as the significance threshold throughout.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| DESeq2 (negative-binomial generalized linear model, Wald-type test) with Benjamini-Hochberg FDR correction | Differential gene expression between AS (group C) and normal (group N) lung transcriptomes | 3 biological replicates per group (3 AS, 3 normal broilers) | not stated |
| Two-way (two-factor) ANOVA | Ascites-related parameters (average body weight, AHI, pulmonary organ coefficient; Table 4) and blood routine indices (RBC, HB, HCT, WBC, LYM, PLT, PCT; Table 5) across treatment group and age (15/25/35 days), including main effects and interaction | not explicitly stated (multiple broilers sampled per group at each age) | not stated |
| Statistical comparison in GraphPad Prism (specific test not named) applied to 2^-ΔΔCt qRT-PCR values | Validation of 6 selected DEGs (FABP4, APELA, FGF7, WNT5A, CDK6, APLNR) between group C and group N | not explicitly stated (likely the same n=3 per group used for RNA-seq) | not stated |
| GO/KEGG enrichment analysis (GOATOOLS; KEGG database), underlying statistical test not explicitly named | Functional annotation and pathway enrichment of the 1442 DEGs | na (based on the DEG list, not sample n) | not stated |
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Differential expression between the two groups was assessed with DESeq2 using a negative-binomial model and BH-FDR correction.↳ Could also: edgeR or limma-voom — These are widely used alternative count-based frameworks for RNA-seq differential expression that could also be applied to this design and are often compared for concordance, especially with small replicate numbers.
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Multiple physiological and hematological variables (Tables 4 and 5) were each tested separately with two-way ANOVA for treatment, age, and their interaction.↳ Could also: A multivariate approach such as MANOVA, or applying a multiple-testing correction (e.g., Bonferroni or Benjamini-Hochberg) across the panel of traits — Testing many related outcome variables in parallel is a setting where a joint multivariate test or a family-wise correction can also be used to account for the number of comparisons being made.
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qRT-PCR validation of six genes was analyzed in GraphPad Prism, with the specific test not named in the text.↳ Could also: An unpaired Student's t-test or, if normality of ΔΔCt values is not assumed, a Mann-Whitney U test, along with reporting exact p-values — Explicitly naming the test and reporting exact p-values (rather than only p<0.05/p<0.01 thresholds) can make the comparison easier to interpret and reproduce.
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RNA-seq comparisons were based on 3 biological replicates per group.↳ Could also: Increasing biological replicate number where feasible, or reporting a power/sample-size rationale — Additional replicates or a stated power justification can also be used to support the stability of fold-change estimates in small-n transcriptomic comparisons, complementing DESeq2's built-in shrinkage approach.
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Variability of measured parameters in Tables 4-6 is not described in the retrieved text (e.g., as SD, SEM, or CI).↳ Could also: Reporting means with SD (for describing the observed sample) or 95% confidence intervals (for inference about the population) — Either measure would also let readers judge the spread of the data and the precision of the estimated group differences alongside the p-values.
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Histological assessment of lung sections (Figure 2) was performed by observation without a stated blinding procedure.↳ Could also: Blinded scoring of histological features by an independent assessor, potentially with a semi-quantitative scoring system — Blinded or scored assessment is a standard complementary approach that can also be used to support qualitative histopathological descriptions.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Assessments & scoring basis
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An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
Strong partial reproduction. The DESeq2 core is essentially 1:1 against the public SRA data — 1516 vs 1442 total DEGs (+5.1%), up/down splits within 3-8%, per-sample mapping within ~1.5%, and FABP4 reproduced almost exactly (padj 8.38e-20 vs 9.59e-20), with GRIK1 confirmed a top-2 down DEG. The deviations are on our (forced) methodology side and the authors' proprietary-tooling side, not fabrication: STAR substituted for the hung TopHat2, and the GO/KEGG enrichment relied on the closed majorbio/eggNOG stack (denser annotation → 676 vs 232 GO DEGs; KEGG not runnable). Severity is moderate and confined to secondary enrichment claims while magnitude and direction of the central signature hold, so overall solid-with-explainable-deviations (yellow).
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Reproduction footprint
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