Eclipse e 2000

UV radiation was used to inactivate nuclear DNA in the rainbow trout spermatozoa. The portion of 0.375 mL of semen was diluted in 15 mL (40×) of seminal plasma. Then, 15.375 mL of diluted milt was exposed to the UV radiation for 11 min. During irradiation, the diluted milt was placed on the magnetic stirrer 20 cm under the UV-C lamp (30 W). After UV treatment, 1 µL sample of the milt mixed with 49 µL of sperm-activating medium (SAM) (154 mM NaCl and 1 mM Ca²⁺, buffered to pH 9.0 with 20 mM Tris + 30 mM glycine) [16 (link)] was checked under the microscope (Nikon Eclipse E 2000) to confirm the motility of irradiated spermatozoa.
For gynogenetic activation, approximately 980 eggs from each female were inseminated (activated) separately using irradiated sperm. About 3 min after activation, the eggs were washed with the hatchery water. To double haploid sets of maternal chromosome and to produce four stocks of gynogenetic doubled haploids (Fg1–4), gynogenetically activated eggs were exhibited to high hydrostatic pressure (HHP) shock (9500 PSI) 350 min after insemination. HHP shock lasted 3 min and was performed using TRC-APV electric/hydraulic apparatus (TRC Hydraulic Inc. Dieppe, Canada). Eggs were incubated in three replicates (Fg1–4) and in two replicates (Fc1–4) at 10 °C under routine conditions in the Department of Salmonid Research, Rutki.

Rainbow trout utilized as gamete donors originated from the spring spawning broodstock raised in the Department of Salmonid Research, Inland Fisheries Institute in Olsztyn, Rutki, Poland. The number of dams and sires used in the research was determined to meet the requirements for the statistical analysis and it was based on the previous studies concerning the chromosomes set manipulations in fish [12 (link),13 (link),15 (link)]. Semen was taken from four males, pooled and kept in the plastic box. The motility of the spermatozoa was analyzed under microscope (Nikon Eclipse E 2000). Sperm was stored in the fridge until use. Four randomly selected females (F1–4) were stripped and the eggs were collected into separate plastic containers. After the quality examination, the eggs were kept at 10 °C until further manipulations. Fin clips from each gamete donors were stored in 96% ethanol for the molecular analysis. Approximately 240 eggs from each female were inseminated using untreated spermatozoa to establish control groups (Fc1–4) for the gynogenetic variants of the experiment.