Fig 1.
Analysis of flow cytometry fourfold stain for simultaneous assessment of plasma, acrosomal and mitochondrial membranes of boar sperm: an epifluorescence view.
(A) Side scatter (SSC) x forward scatter (FSC) dot plot showing the “Sperm + cell debris” gate. (B) Hoechst 33342 histogram originating from the “Sperm + cell debris” gate to exclude non-cellular particles by a DNA probe, Hoechst 33342. (C) Propidium iodide histogram based on Hoechst 33342 for evaluating sperm cell membrane integrity. IM gate showing spermatozoa with intact plasma membranes and the DM gate showing damaged sperm plasma membranes. (D) Dot plot from the IM gate for analyzing mitochondrial membrane potential (axis y; JC-1 orange) and acrosome integrity (axis x; PSA-FITC), as represented by each quadrant of expected sperm cells. (E) Dot plot from the DM gate for analyzing mitochondrial membrane potential (axis y; JC-1 orange) and acrosome integrity (axis x; PSA-FITC), as represented by each quadrant of expected sperm cells.
Table 1.
Differentiation of stain type regards sperm compartment evaluated.
Fig 2.
Linear regression and correlation.
(A) Linear regression between treatments of the sperm population with plasma and acrosomal membrane integrity and high mitochondrial membrane potential (IPIAH). (B) Correlation between sperm IPIAH and total motility.
Fig 3.
Graphic representation of all semen treatment dynamics analyzed using three graphs: a histogram that represents the plasma membrane integrity and two dot plots for analyzing mitochondrial membrane potential (axis y) and acrosome integrity (axis x). The dot plot with green stain represents intact plasma sperm membranes (from the IP histogram gate–plasma membrane integrity), and the dot plot with red coloration represents damaged plasma sperm membranes (from the DP histogram gate–damaged plasma membranes). IPIAH–simultaneous plasma and acrosome membrane integrity and high mitochondrial membrane potential (Δψm), IPIAL—simultaneous plasma and acrosome acrosomal integrity and low Δψm, IPRAH—simultaneous plasma membrane integrity, reacted acrosome and high Δψm, IPRAL—simultaneous plasma membrane integrity, reacted acrosome and low Δψm, DPIAH—simultaneous damaged plasma membrane, acrosome integrity and high Δψm, DPIAL—simultaneous damaged plasma membrane, acrosome integrity and low Δψm, DPRAH—simultaneous damaged plasma membrane, reacted acrosome and high Δψm, DPRAL—simultaneous damaged plasma membrane, reacted acrosome and low Δψm.
Table 2.
Mean ± standard error of different cell category obtained from different stain type, in bases of percentage (%) of raw diluted semen.
Fig 4.
Effect of treatments on computer-assisted sperm analysis (CASA).
(A) Total and progressive motility; (B) VCL—curvilinear velocity, VSL—straight-line velocity, VAP—average path velocity; C) LIN—linearity, STR—straightness; (D) ALH—amplitude of lateral head displacement, BCF—beat cross frequency and HIPER—hypermotility. CT—control; CS—centrifuged and suspended in autologous seminal plasma (SP); CW—centrifuged and withdrawn SP; CWSP—CW containing autologous seminal plasma. Different letters represent a significant difference (p < 0.05). CT—control; CS—centrifuged and suspended in autologous seminal plasma (SP); CW—centrifuged and withdrawn SP; CWSP—CW containing autologous seminal plasma. Different letters in the same row represent a difference (p < 0.05) between treatments at the same time.
Fig 5.
Effect of time on computer-assisted sperm analysis (CASA).
(A) Total and progressive motility; (B) VCL—curvilinear velocity, VSL—straight-line velocity, VAP—average path velocity; (C) LIN—linearity, (D) ALH—amplitude of lateral head displacement, BCF—beat cross frequency and HIPER—hypermotility. Different letters represent a significant difference (p < 0.05).
Table 3.
Mean ± standard error of time vs. treatment interaction for straightness.
Fig 6.
Effects of treatment (A) and time (B) on plasma and acrosomal membrane integrity and mitochondrial membrane potential. CT—control; CS—centrifuged and suspended in autologous seminal plasma (SP); CW—centrifuged and withdrawn SP; CWSP—CW containing autologous seminal plasma. IPIAH—plasma and acrosome membrane integrity and high Δψm. Different letters represent a significant difference (p < 0.05).
Fig 7.
Tyrosine phosphorylation on the surface of spermatozoa.
CT—control; CS—centrifuged and suspended in autologous seminal plasma (SP); CW—centrifuged and withdrawn SP; CWSP—CW containing autologous seminal plasma. Different letters represent a significant difference (p < 0.05).