b8e40f145d255fa8151ae6477525c39b fishes-11-00392.pdf 59c63e3c3e0548e783c6d0943bc5bdf4044112d7 fishes-11-00392.pdf 7d1ce91fc78a5f74f3c0cd615cdc0e7180fe0013be822f363d5e7053c47381fe fishes-11-00392.pdf Title: Developmental Stage Shapes Acute Transcriptional Responses to Operational Chemical Euthanasia Formulations in Zebrafish Embryos and Larvae Subject: Chemical euthanasia protocols are routinely applied across zebrafish developmental stages, but the effects of formulation, time exposure, and developmental stage remain unclear and could introduce variability in transcriptional results and interfere with interlaboratory reproducibility of molecular endpoints. Wild-type AB zebrafish were exposed at three developmental windows (6 hpf, gastrulation; 30 hpf, pharyngula; 100 hpf, pre-feeding larva) to three operational euthanasia formulations (clove oil 0.1% v/v, tricaine/MS-222 1 g/L, or lidocaine 1 g/L), for 3, 10, or 25 min. Expression of ten genes covering immediate-early activation, cellular stress, metabolism, neuroendocrine signalling, apoptosis, and inflammation was quantified by qPCR. Expression responses were analysed using a three-way factorial framework including the developmental phase, formulation, and exposure time. The developmental phase explained substantial developmental context in several genes. The Phase Treatment interaction was significant after global FDR correction for nine of ten genes, whereas treatment main effects did not provide a stable cross-stage ranking of formulations. Descriptively, gene expression varied across formulations and stages, with several Phase III markers showing positive shifts, but no operational formulation showed universally consistent effects after multiple-testing correction, while effect-size magnitudes should be interpreted cautiously. Acute transcriptional responses to operational chemical euthanasia formulations in zebrafish embryos and larvae were context-dependent. Developmental stage, rather than a universal formulation effect, emerged as the most consistent modulator. Because euthanasia efficacy and non-recovery were not experimental endpoints, these transcriptional data do not identify a best formulation for each developmental phase. These results support stage-aware reporting of euthanasia conditions. Keywords: Danio rerio; euthanasia exposure; MS-222; clove oil; lidocaine; developmental stage; gene expression Author: Juan Ramos, Juan Carlos Balasch, Lluis Tort and Ali Reza Khansari Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Wed Jul 1 11:02:58 2026 CEST ModDate: Wed Jul 1 11:23:49 2026 CEST Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1476627 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- GHNBIW+URWPalladioL-Bold Type 1 Custom yes yes yes 103 0 QMDDXF+CMSY10 Type 1 Builtin yes yes yes 104 0 YYCOVL+URWPalladioL-Roma Type 1 Custom yes yes yes 105 0 KALPRK+URWPalladioL-Ital Type 1 Custom yes yes yes 106 0 MIKRSI+VnURWPalladioL Type 1 Custom yes yes yes 107 0 YTNLDA+EURM10 Type 1 Builtin yes yes yes 189 0 DUJUUF+PazoMath-Italic Type 1 Builtin yes yes yes 190 0 CIDFont+F2 CID TrueType Identity-H yes no yes 216 0 KXEOLC+EURB10 Type 1 Builtin yes yes yes 345 0 WBVKDJ+URWPalladioL-BoldItal Type 1 Custom yes yes yes 346 0 CIDFont+F2 CID TrueType Identity-H yes no yes 241 0 CIDFont+F7 CID TrueType Identity-H yes no yes 242 0 CIDFont+F4 CID TrueType Identity-H yes no yes 243 0 CIDFont+F3 CID TrueType Identity-H yes no yes 280 0 CIDFont+F2 CID TrueType Identity-H yes no yes 281 0 CIDFont+F6 CID TrueType Identity-H yes no yes 282 0 CIDFont+F3 CID TrueType Identity-H yes no yes 329 0 CIDFont+F2 CID TrueType Identity-H yes no yes 330 0 CIDFont+F6 CID TrueType Identity-H yes no yes 331 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2026-09-05 03:56:58 CEST RepresentationInformation: fishes-11-00392.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2026-09-04 14:01:47 CEST Size: 1476627 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 649 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Materials and Methods Children: Item: Title: Zebrafish Husbandry and Embryo Production Item: Title: Euthanasia Formulations and Exposure Item: Title: Experimental Design Item: Title: RNA Extraction, Quality Control and qPCR Item: Title: Data Processing and Statistical Analysis Item: Title: Results Children: Item: Title: Dataset Structure and Quality Item: Title: Phase Treatment Interactions Item: Title: Descriptive Transcriptional Profiles Item: Title: Discussion Item: Title: Conclusions Item: Title: References Info: Title: Developmental Stage Shapes Acute Transcriptional Responses to Operational Chemical Euthanasia Formulations in Zebrafish Embryos and Larvae Author: Juan Ramos, Juan Carlos Balasch, Lluis Tort and Ali Reza Khansari Subject: Chemical euthanasia protocols are routinely applied across zebrafish developmental stages, but the effects of formulation, time exposure, and developmental stage remain unclear and could introduce variability in transcriptional results and interfere with interlaboratory reproducibility of molecular endpoints. Wild-type AB zebrafish were exposed at three developmental windows (6 hpf, gastrulation; 30 hpf, pharyngula; 100 hpf, pre-feeding larva) to three operational euthanasia formulations (clove oil 0.1% v/v, tricaine/MS-222 1 g/L, or lidocaine 1 g/L), for 3, 10, or 25 min. Expression of ten genes covering immediate-early activation, cellular stress, metabolism, neuroendocrine signalling, apoptosis, and inflammation was quantified by qPCR. Expression responses were analysed using a three-way factorial framework including the developmental phase, formulation, and exposure time. The developmental phase explained substantial developmental context in several genes. The Phase Treatment interaction was significant after global FDR correction for nine of ten genes, whereas treatment main effects did not provide a stable cross-stage ranking of formulations. Descriptively, gene expression varied across formulations and stages, with several Phase III markers showing positive shifts, but no operational formulation showed universally consistent effects after multiple-testing correction, while effect-size magnitudes should be interpreted cautiously. Acute transcriptional responses to operational chemical euthanasia formulations in zebrafish embryos and larvae were context-dependent. Developmental stage, rather than a universal formulation effect, emerged as the most consistent modulator. Because euthanasia efficacy and non-recovery were not experimental endpoints, these transcriptional data do not identify a best formulation for each developmental phase. These results support stage-aware reporting of euthanasia conditions. 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