The aim of this study was to evaluate the generation of hydrogen peroxide (H2O2) and membrane lipid peroxidation of subfertile spermatozoa stored at 5°C for 24 h. Semen samples, collected from 5 subfertile stallions (≤40% of conception rate), were diluted in a skim-based extender (BotuSemen; Botupharma) and stored in a passive transport container at 5°C over a period of 24 h. Sperm motility was determined by computer-assisted semen analysis (CASA; IVOS 12, Hamilton Thorne Inc., Beverly, MA, USA) for total motility (TM), progressive motility (PM), and rapid sperm (RAP). Plasma membrane integrity, generation of H2O2, and membrane lipid peroxidation were determined by flow cytometry (LSRFortessa cell analyzer, BD Biosciences, Franklin Lakes, NJ, USA). For evaluation of acrosome and plasma membrane integrity, samples were stained with Hoechst 33342 dye (H33342; Molecular Probes, Eugene, OR, USA), iodide propidium (IP; Sigma, St. Louis, MO, USA), and fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA; Sigma). For the assessment of generation of H2O2, 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma), H33342, and IP were added to the sperm suspension. For the assessment of sperm lipid peroxidation, samples were stained with C11-BODIPY581/591 (Molecular Probes), H33342, and IP. Samples were evaluated after semen collection (0 h) and after 24 h of cooled storage. A total of 10 000 gated events were analyzed per sample by flow cytometry. The green fluorescence (FL1) was collected through a 580-nm band-pass filter and the red fluorescence (FL3) through a 635-nm band-pass filter. Statistical analysis was performed using GraphPad Prism version 4.03 (2005; GraphPad Software Inc., La Jolla, CA, USA), through paired t-test to identify the significant differences (P ≤ 0.05). In general, sperm motility parameters of TM and RAP, and acrosome and plasma membrane integrity significantly decreased after 24 h of refrigeration at 5°C (P ≤ 0.05). Interestingly, no differences were found in PM at 0 and 24 h (P ≥ 0.05). The percentage of sperm cells with high generation of H2O2 did not differ at 0 and 24 h (P ≥ 0.05), whereas the percentage of sperm cells with membrane peroxidation increased (0.56 ± 0.3 v. 2.24 ± 1.3; P ≤ 0.05) at these periods. The percentage of viable sperm cells with low generation of H2O2 had a significant decrease from 22.6 ± 11.2 at 0 h to 0.08 ± 0.1 after 24 h of storage (P ≤ 0.05), although no differences were found in the percentage of viable sperm cells without membrane peroxidation (P ≥ 0.05). In conclusion, the cooled storage of subfertile spermatozoa for 24 h drastically decreased the number of viable spermatozoa with low generation of H2O2 and increased the percentage of membrane lipid peroxidation, which is related to the decrease in sperm motility and increase in dead sperm. These results make it difficult to use refrigerated semen of subfertile stallions with poor semen quality in commercial breeding programs.
São Paulo Research Foundation (FAPESP) is acknowledged for supporting this research.
Cooling of equine semen at 16°C for 36 hours with addition of different glutathione concentrations
