Fig. 6

Sperm and midpiece object-based phasor fingerprints detected an increase in OXPHOS metabolism with age. Phasor fingerprints A and B of PWD spermatozoa from old males are shifted toward bound NAD(P)H when compared to young males. Sperm cells from old (3 males, 102 cells) and young (3 males, 45 cells) males were generated manually. Midpieces from old (3 males, 1040 objects) and young (3 males, 1003 objects) mice were segmented using U-Net automatically. See Additional file 8: Fig. S8 for head and principal piece metabolic fingerprints. Object-based phasor plots in C and D depict spermatozoa treated with 50 μM H₂O₂ and untreated controls. Sperm cells from H₂O₂-treated (2 males, 46 cells) and control (2 males, 43 cells) males were generated manually. Midpieces from H₂O₂-treated (2 males, 945 objects) and control (2 males, 949 objects) sperm were segmented using U-Net automatically. Treatment of sperm from young males with H₂O₂ resulted in a phasor shift toward bound NAD(P)H, indicating increased OXPHOS metabolism. The treatment of sperm with 2-DG also induced an increase in OXPHOS activity (Additional file 9: Fig. S9)