Mdf u32v

The fish were divided into a control group (fish without hormone stimulation) and two groups with hormonally stimulated fish. Five females were collected shortly after catching as a blind (control—beginning group) sample of the cadmium level in muscle and ovary. Twenty maturing (after hormonal treatment) female European eels were subjected to a cadmium (Cd) content analysis. Before sampling, all the fish handling was conducted in a state of anesthesia (egg collection) or euthanasia (tissue sampling) using MS-222 (Finquell, Redmond, WA, USA). Sample tissues (muscle, ovary, and in some cases eggs) were taken after ten weeks of the experiment from freshly captured specimens (n = 13) and from females (which start to ovulate) after 15 weeks of hormonal stimulation (n = 7). Eggs were stripped manually and they (unfertilized) were analysed for cadmium content from seven females after spawning. The sample tissues were stored in an ultra-low temperature freezer (Sanyo, MDF-U32V; Japan) at −80 °C until analysis. Fish and ovaries were weighed (to calculate gonadosomatic index (GSI) (%): 100 × gonad weight (g)/fish weight (g)) or fish and stripping eggs (to calculate pseudo-gonadosomatic index (PGSI) (%): 100 × egg weight (g)/fish weight (g)).

The dried powder (2 kg) was extracted with n-hexane to remove fatty substances, and then sequential extraction was performed with 100% dichloromethane, 100% methanol, and 70% aqueous methanol using a temperature-controlled orbital shaker (Figure 2). The extract was filtered, and the filtrate was concentrated and dried by means of rotary evaporation (Heidolph, Hei-Vap, Schwabach, Germany) under reduced pressure (800 millibar) at 40 °C to afford semisolid crude extracts. Dried extracts thus obtained were stored at −18 °C in an upright ultralow freezer (Sanyo, MDF-U32V, Osaka, Japan) for future experiments.

Using a temperature-controlled orbital shaker, the powdered plant material was extracted with n-hexane to remove non-polar compounds like fat, followed by successive extractions with dichloromethane, methanol, and 70% aqueous methanol. The extracts were evaporated at 40 °C in a rotary evaporator (Heidolph, Schwabach, Germany) to produce semi-solid extracts and stored at 4 °C in an upright ultralow freezer (Sanyo, MDF-U32V, Osaka, Japan) for further investigations.