What does this research mean for the field?

Post-ovulatory aging in Atlantic halibut eggs significantly reduces fertilization success and normal development, with critical molecular pathways failing after 4 hours post-ovulation. Novelty: ClaimNovelty.NOVEL_FINDING. Consensus alignment: ConsensusAlignment.CHALLENGES_CONSENSUS.

What question did this study set out to answer?

The aim is to determine how post-ovulatory aging affects fertilization success and gene expression in Atlantic halibut eggs.

February 23, 2026SHILAP Revista de lepidopterología0 citationsOpen Access

The effects of post-ovulatory aging on fertilization success, survival, hatching performance and gene expression in Atlantic halibut (Hippoglossus hippoglossus) eggs

NNNils NiepagenNorwegian University of Science and Technology FBFrancesca BertoliniAnimal, Food and Health Sciences EKElin KjørsvikNorwegian University of Science and Technology

Key Points

The aim is to determine how post-ovulatory aging affects fertilization success and gene expression in Atlantic halibut eggs.
Fertilized eggs from five females were stored at different time points post-ovulation.
Endpoints measured included fertilization rates, morphological development, and survival rates.
RNA sequencing was conducted at various developmental stages to assess gene expression changes.
Fertilization rates decreased from approximately 82% to 30% over 12 hours post-ovulation.
Normal morphology at the 8-cell stage remained stable until 2 hours, then declined significantly afterward.
Significant reductions in normal development at 50 days post-fertilization occurred after 4 hours, with severe impacts observed by 6 and 12 hours.

Abstract

Introduction Post-ovulatory aging reduces Atlantic halibut ( Hippoglossus hippoglossus ) egg quality, but the practical time window and the stage at which developmental programs fail are not well defined. We quantified performance loss across storage times and mapped when molecular pathways break down during embryogenesis. Methods Eggs from five females were fertilized at t0, t1, t2, t4, t6, and t12 after in vitro storage at 6 °C and reared in a small-scale system. Endpoints were fertilization, normal 8-cell (8C) morphology, normal development at 50dd, survival to hatching, and hatching success. Mixed effects beta regression and Kaplan Meier analyses tested time effects. One batch underwent RNA seq at unfertilized, 8C, blastula (BL), and 50dd egg stages. Results Fertilization declined from about 82 percent at t0 to about 30 percent at t12. Normal 8C morphology was unchanged to t2, then lower at t6 and t12. Normal 50dd morphology dropped after t4 and was significantly reduced at t6 (p = 0.023) and t12 (p 0.001). Survival varied among batches but was consistently worst at t12. Transcriptomics pinpointed BL stage as the main failure window: from t4 onward, genes for RNA metabolism and ribosome biogenesis, germ layer and system development, Wnt signaling, and cell migration were down regulated, while maternal pools at unfertilized and 8C were largely unchanged. At 50dd, rRNA biogenesis and mitochondrial organization were up regulated and morphogenesis related terms were suppressed; organizer and patterning genes such as gsc , ved , and vox declined with aging. Advancing stripping time by 10–13 hours relative to fixed hatchery protocols substantially improved egg quality, with fertilization rates of 77–87% and normal 50dd development of 55–96%. Discussion The data support an operational window of at most 4 to 6 hours post stripping to preserve competence and establish normal 50dd morphology as a practical quality marker. Mechanistically, post-ovulatory aging acts primarily by stage-specific suppression of programs required for axis formation and organogenesis at BL stage.

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