2 phenoxyethanol

In this study, 55 adult female Amur sturgeons were used. Sturgeons were raised outdoors in river or spring water under natural day length at Nanae Fresh-Water Station (Nanae, Hokkaido, Japan), Bifuka Sturgeon Museum (Bifuka, Hokkaido, Japan), or Shimizugawa Trout Farm (Hachimantai, Iwate, Japan). Final oocyte maturation and ovulation were induced by an LHRHa (Sigma-Aldrich, St. Louis, MO, USA) priming injection (2 μg/kg body weight) and a high-dose LHRHa injection (50 μg/kg body weight), 24 h after the priming injection.
Ovarian fragments were obtained by biopsy from females #1–26 anesthetized with approximately 0.01% of 2-phenoxyethanol (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) immediately before the priming injection, 24 h after the priming injection, and 8 h after the high-dose injection (Table 1). In addition to the above sampling schedule, females #1–4 were routinely biopsied the year prior to the administration of the priming injection. The samples were stored in Ringer’s solution modified for the sturgeons (111.2 mM NaCl, 3.4 mM KCl, 2.7 mM CaCl₂, and 23.8 mM NaHCO₃; pH 7.4) at 4 °C until the start of the experiment.

Body weight and length of zebrafish from the three populations were measured on designated days 0, 10, 20, and 30 throughout this study. Before measurements, zebrafish were anesthetized with 2-phenoxyethanol (Wako, Tokyo, Japan) at 1:1000 dilution for 15 s. Total body length was measured from the head to the end of the body. Once the measurement was finished, zebrafish were put back into fresh water. Body mass index (BMI) was calculated as weight/length².

Embryos were injected with TL2-mRNA pairs (150 ng/μL each; Fig. 5b) and the F₀ adult fish obtained were mated with each other in the rearing/spawning facility. The F₁ or later generations were subjected to genotyping analysis as follows: fish were anaesthetised with 300–500 ppm of 2-phenoxyethanol (Wako Pure Chemical Industries) in seawater. After tagging with Visible Implant Elastomer (Northwest Marine Technology, Inc.), the caudal fins were cut with a sharp scalpel and fish were quickly returned to a 0.2- or 0.5-tonne tank and reared until their genotypes were identified. To detect indel mutations, HMA was performed and the amplicons containing the target site were sequenced. Fish that were homozygous for the MSTN-2 null mutation were selected and their offspring reared.