Sexing by genotype
Upon completion of the observer trials, all trout were euthanised with an overdose of benzocaine solution. Genomic DNA was extracted from adipose fin clips using a NucleoSpin™ Tissue kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) following the manufacturer’s instructions; quality controlled with spectrophotometry (NanoDrop, ThermoFisher Scientific, Waltham, MA, USA); and quantified fluorometrically (Qubit 2.0, ThermoFisher Scientific, Waltham, MA, USA). Extracted DNA was diluted to 20 ng μL-1.
To determine the sex of each trout, following a modification of established protocols (Anglès d’Auriac et al., 2014; Lavender et al., 2024), duplex PCR amplified the male-specific Y-chromosome gene,sdY (forward primer: CCC AGC ACT GTT TTC TTG TCT CA; reverse primer: CTT AAA ACC ACT CCA CCC TCC AT), using the 18S gene as a positive amplification control (forward primer: GTY CGA AGA CGA TCA GAT ACC GT; reverse primer: CCG CAT AAC TAG TTA GCA TGC CG). PCR was performed using 3 μL of DNA with 0.3 μL of each sdY primer, 0.075 μL of each 18S primer, 7.5 μL of Qiagen Multiplex PCR mix containing 3 mM MgCl2, HotStarTaq DNA polymerase and proprietary buffer (Qiagen N.V., Hilden, Germany), and 2.2 μL nuclease-free H2O. Thermal cycling consisted of initialisation for 15 minutes at 95 °C, followed by 35 amplification cycles of 30 seconds at 94 °C, 90 seconds at 63 °C and 90 seconds at 72 °C, with a final extension for 10 minutes at 72 °C. The resulting PCR products were visualised with 2 % agarose gel electrophoresis.