Fig. 6.

Assessment of sperm function based on acrosome reactivity, mitochondrial membrane potential, and DNA integrity. (A) The proportion of capacitation and acrosome-reacted patterns in sorted bovine sperm assessed by CTC assay. Replicates = 7 (unsorted), 12 (sorted). (B) (Top) AR pattern corresponds to acrosome-reacted spermatozoa, (Middle) B pattern corresponds to capacitated and acrosome-intact spermatozoa, and (Bottom) F pattern corresponds to noncapacitated and acrosome-intact spermatozoa. (C) 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. (D) Sperm mitochondrial activity expressed as the ratio of sperm with hMMP 1 and 6 h after sorting. Data shown are mean ± SEM (replicates of 7) for 117–136 sperm per replicate of sorted and unsorted sample per replicate. (E) DNA fragmentation expressed as the mean sperm TUNEL index (%) of sorted and unsorted sperm. Error bars indicate SD. The results are means ± SD of four sorted and four unsorted samples. The total inspected sperm for sorted samples is 1,925, and for unsorted samples the total is 3,994. Different superscript letters in each bar represent significant differences between samples (P < 0.01) detected by a paired t test. (F) Sperm nuclei containing DNA fragmentation are detected by TUNEL assay. Each spermatozoon was assigned to contain either normal (blue nuclear fluorescence due to Hoechst 33342) or fragmented DNA (green nuclear fluorescence). (Magnification: B, 300×; C, 250×; F, 200×.)