Post-ovulatory aging effects on Atlantic halibut (Hippoglossus hippoglossus) egg transcriptome across developmental stages
Post-ovulatory aging effects on Atlantic halibut (Hippoglossus hippoglossus) egg transcriptome across developmental stages
Eggs were collected from a single female Atlantic halibut (Hippoglossus hippoglossus) during the reproductive spawning season at Nordic Halibut AS (Midsund, Norway). The scheduled stripping time was advanced by 12 hours compared to the commercial hatchery timing to ensure eggs were collected closer to ovulation and minimize pre-existing post-ovulatory aging. Following collection, eggs were transferred into a dry bowl, gently mixed to reduce inhomogeneity, and evenly distributed into six storage containers. Each container was filled to the top and sealed with plastic film to minimize air exposure. One fraction (t0) was fertilized immediately, while the remaining five fractions were stored at 6°C and fertilized after 1, 2, 4, 6, and 12 hours of storage (t1, t2, t4, t6, t12). Fertilization was performed using cryopreserved milt. Following fertilization, eggs were left to harden for 30 minutes, rinsed in seawater, and transferred into 800 mL cell culture flasks containing seawater with 0.25 mg/L terramycin. Incubators were maintained at 6°C in darkness. RNA samples were collected in triplicate at four developmental stages: unfertilized eggs (UF), 8-cell stage (8C, ~14 hours post-fertilization), blastula stage (BL, ~48 hours post-fertilization), and 50 degree-days (50dd, ~200 hours post-fertilization). Samples were flash-frozen in liquid nitrogen and stored for subsequent RNA extraction. Total RNA was extracted from pools of 10 eggs per sample using the RNeasy Universal Plus Mini Kit (Qiagen) following the manufacturer's instructions. RNA concentration was measured with a NanoDrop spectrophotometer, and RNA integrity (RIN) was assessed on an Agilent 2100 Bioanalyzer, yielding mean RIN = 9.8 ± 0.24 mRNA libraries were prepared by Novogene Co. (Beijing, China) using their standard Illumina TruSeq RNA Sample Preparation protocol, following manufacturer guidelines for paired-end sequencing. Libraries were quality-checked by agarose gel electrophoresis and Bioanalyzer QC prior to sequencing