Figure 3.

Storage effect on sperm responsiveness to capacitation stimuli in relation to the presence of sperm with cytoplasmic droplets. (A) Responsive spermatozoa in different media after 24 h or 96 h semen storage for samples with ≤15% of spermatozoa with cytoplasmic droplets (n = 66 boars) or >15% of spermatozoa with cytoplasmic droplets (n = 12 boars). Responsiveness was calculated based on the propidium iodide- and Fluo-3-low sperm population as the difference (Δ) between the values at the beginning (3 min) and after 60 min incubation (Δ3–60 min = 3 min–60 min) in a capacitating medium with 15 mM bicarbonate and 2 mM calcium (Tyr_BicCa) or in non-capacitating variants with either 2 mM calcium (Tyr_Ca) or 1 mM EGTA (Tyr_Control). * indicates significant differences between the sample groups (p < 0.05). # indicates significant difference for a given group between storage times (p < 0.05). (B) The specific response to bicarbonate was calculated as the difference (Δ) in response to a full capacitating medium (Tyr_BicCa) within 60 min minus the response to a calcium-containing control medium (Tyr_Ca) within 60 min. An asterisk indicates significant differences between the sample groups (p < 0.05). A hash indicates significant differences for a given group between storage times (p < 0.05). (C,D) Dot plot of the responsive sperm population after 24 h storage (B) or 96 h storage (C) in relation to the percentage of sperm with cytoplasmic droplets (proximal and distal) within a sample. Orange dots are samples with >15% of spermatozoa with cytoplasmic droplets (n = 12 boars).