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

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 signalling, 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.