Fig. 2

a Effect of semen processing by SSRT method prior to cryopreservation on post thaw sperm viability in bovine spermatozoa, and representative images of spermatozoa in dead (red) and live state (green). Viability values between SSTR and control straws differ significantly at P < 0.05 (Chi-squared test) for 7 h incubation. b Effect of semen processing by SSRT method prior to cryopreservation on the motility index (VAP × BCF, measured by CASA) of bovine spermatozoa at different incubation time (1 h, 7 h, 24 h). Viability values between SSTR and control straws differ significantly at P < 0.05 (t-test) for 24 h incubation (c) Effect of semen processing by SSRT method prior to cryopreservation on post thaw mitochondrial activity of bovine spermatozoa, and fluorescence photomicrography of sorted spermatozoa stained with JC-1 probe showing orange fluorescence of the sperm midpiece indicating hMMP and green fluorescence indicating low mitochondrial membrane potential. Values between the SSTR and control straws at 1 h and 24 h are not significantly different. Values at 7 h are significantly different at P < 0.01 (t-test). d Effect of semen processing by SSRT method prior to cryopreservation on post thaw DNA integrity measured by TUNEL index (%) that indicates DNA fragmentation. Each spermatozoon was assigned to contain either normal (blue nuclear fluorescence due to Hoechst 33342) or fragmented DNA (green nuclear fluorescence). There are 4 straws analyzed for each treatment (n = 4 per treatment), and every straw was subjected to 4 times CASA measurements, mitochondrial activity, and TUNEL assay. Values differ significantly at P < 0.01 (Chi-squared test)