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We explore the possibility of characterizing sperm cells without the need to stain them using spectral and fluorescence lifetime analyses after multi-photon excitation in an insect model. The autofluorescence emission spectrum of sperm of the common bedbug,  Cimex lectularius , was consistent with the presence of flavins and NAD(P)H. The mean fluorescence lifetimes showed smaller variation in sperm extracted from the male (tau m,  τ  m  = 1.54–1.84 ns) than in that extracted from the female sperm storage organ (tau m,  τ  m  = 1.26–2.00 ns). The fluorescence lifetime histograms revealed four peaks. These peaks (0.18, 0.92, 2.50 and 3.80 ns) suggest the presence of NAD(P)H and flavins and show that sperm metabolism can be characterized using fluorescence lifetime imaging. The difference in fluorescence lifetime variation between the sexes is consistent with the notion that female animals alter the metabolism of sperm cells during storage. It is not consistent, however, with the idea that sperm metabolism represents a sexually selected character that provides females with information about the male genotype.

Sperm metabolism is altered during storage by female insects: evidence from two-photon autofluorescence lifetime measurements in bedbugs