Male birds were humanely euthanized in accordance with Schedule 1. Live sperm were collected within 20 min from the left seminal glomerus (SG) and analysed using the Sperm Class Analyzer® (Microptic, Barcelona, Spain), following methods described in [23 (link)] (electronic supplementary material).
For the 2015 cohort, the remaining mature sperm (from the distal third portion of the SG) was squeezed into 110 µl of phosphate-buffered saline (PBS) at room temperature (20°C) to avoid activating motility. The sperm suspension was thoroughly aspirated in an eppendorf tube using a pipette and 10 µl was fixed in 90 µl of 5% formalin for sperm concentration and morphology analyses at a later date (see below). The remaining 100 µl was used to quantify ATP content using an ATPlite 300 assay kit (Perkin Elmer, UK) with a modified protocol (described below) allowing sample collection and analysis to be carried out on separate days. ATP was isolated from the sperm suspension by adding 250 µl of PBS, 175 µl of mammalian cell lysis reagent (from ATPlite kit) and incubating at room temperature for 5 min, while mixing with a vortex for 10 s every minute. Samples were centrifuged at 12 000 × g for 2.5 min, and the supernatant was snap-frozen in liquid nitrogen and stored at −80°C until quantification.
For the 2015 cohort, the remaining mature sperm (from the distal third portion of the SG) was squeezed into 110 µl of phosphate-buffered saline (PBS) at room temperature (20°C) to avoid activating motility. The sperm suspension was thoroughly aspirated in an eppendorf tube using a pipette and 10 µl was fixed in 90 µl of 5% formalin for sperm concentration and morphology analyses at a later date (see below). The remaining 100 µl was used to quantify ATP content using an ATPlite 300 assay kit (Perkin Elmer, UK) with a modified protocol (described below) allowing sample collection and analysis to be carried out on separate days. ATP was isolated from the sperm suspension by adding 250 µl of PBS, 175 µl of mammalian cell lysis reagent (from ATPlite kit) and incubating at room temperature for 5 min, while mixing with a vortex for 10 s every minute. Samples were centrifuged at 12 000 × g for 2.5 min, and the supernatant was snap-frozen in liquid nitrogen and stored at −80°C until quantification.
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