17α methyltestosterone

All steroid standards were purchased as pure powders from Steraloids (Newport, RI, USA). 111
Methanol, methyl tert-butyl ether (TBME), ethyl acetate, 17α-methyltestosterone, dithioerythritol, 112 N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and synthetic urine were provided by 113 Sigma-Aldrich (Milan, Italy). β-glucuronidase from Escherichia Coli was purchased from Roche 114 Life Science (Indianapolis, IN, USA). Ultra-pure water was obtained using a Milli-Q® UF-Plus 115 apparatus (Millipore, Bedford, MA, USA). Solid-phase extraction (SPE) C-18 endcapped cartridges 116 were from UCT Technologies (Bristol, PA, USA). 117
Standards solutions were prepared in methanol at the concentration of 1 mg/mL. Then, two working 118 solution mixtures were prepared by dilution (MIX I = 3 µg/mL, MIX II = 100 µg/mL, internal 119 standard solutions = 10 µg/mL). Two internal standards were used: testosterone-D 3 for the 120 quantification of mix I; 17α-methyltestosterone for mix II (Table 1). 121

Stock solution of 17α-methyltestosterone (Sigma Chemical co., USA) was formulated by dissolving the 17 α-MT in ethanol at a concentration of 1mg/ml for egg immersion purpose which was safely maintained at dark cool place because it is photosensitive (Haniffa et al., 2004) . Various hormone concentrations (HC) were prepared by adding 1.5, 3, 4.5 and 6ml to 10 liters of water to obtain 17 α-MT concentrations of 150, 300, 450 and 600 ug/L (Piferrer and Donaldson, 1992) (Table 1).

Diets were prepared by mixing an appropriate amount of finely grounded ingredients at predetermined levels in an electric mixer for 10 minutes. Later, fish oil was gradually added while mixing continually for a further five minutes. Subsequently, to prepare the dough 10-15% water was added. Diet was prepared using a linear formulation process (Lovell, 1991) . The methyl testosterone diet was prepared by dissolving 60 mg and 70 mg 17α-methyl testosterone (Sigma) in 500 ml of 95% ethanol which was later mixed with 1 kg of the prepared feed dough and then converted into pellets (3mm). Dried feed was kept in the refrigerator until use. Diets with natural and were prepared by using fresh common carp testes of more than one-year-old fish. Testes were separated from other muscle groups and blood vessels before being cut into small pieces and then placed in Petri dishes for sun drying. The dried testes were then ground with a grinder and sieved through a sieve with a mesh size of 60m. After that, 70 and 80% of powdered testes were mixed with feed and then placed in the refrigerator until use (Table 1).

All steroid standards were purchased as pure powders from Steraloids (Newport, RI, USA); DHT was provided by LGC Promochem (Milan, Italy). Methanol, methyl tert-butyl ether (TBME), ethyl acetate, 17α-methyltestosterone, dithioerythritol and N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) were provided by Sigma-Aldrich (Milan, Italy). β-glucuronidase from Escherichia Coli was purchased from Roche Life Science (Indianapolis, IN, USA). Ultra-pure water was obtained using a Milli-Q® UF-Plus apparatus (Millipore, Bedford, MA, USA).
Standards solutions were prepared in Methanol at the concentration of 1 mg/mL. Then, three working solution mixtures were prepared by dilution. Two internal standards were used: testosterone-d 3 served for the quantification of all the target analytes, with the exception of A and Etio which were quantified by 17α-methyltestosterone. The list of the studied steroids, together with details about each working solution are reported in Table 1. All standard solutions were stored at 10°C until used.