Sperm viability
Measurements were done only in six-day old virgin males from the control and the 29°C recovery treatment, as especially young males and males from all the other treatments had a low number of sperm in the SV preventing reliable estimates. We dissected out the male reproductive tract, the SVs were isolated and punctured following the procedure above. We stained sperm with SYBR14 ® (1:50 in DMSO) and PI (LIVE/DEAD ® Sperm Viability Kit, ThermoFisher Scientific) following the protocol explained in (Eckel et al., 2017). The temporal decrease in sperm viability of the same male was measured at three different time points: just after the staining (t0), 15 (t15) and 30 (t30) minutes later. With this procedure we can assess sperm quality and future sperm performance (Eckel et al., 2017). The time between staining and taking the first image was approximately 1 min. Pictures were taken under fluorescence (see microscope specifications above). Sperm heads were counted by eye twice once by an observer blind and the second observer non-blind to the treatment codes. As the counts were not significantly different, the mean of both counts was used for the data analysis. Green sperm were considered alive while red and red-green double stained sperm were scored as dead. A total of 21 males per temperature treatment were used.