Comparison of the TBARS Assay and BODIPY C₁₁ Probes for Assessing Lipid Peroxidation in Red Deer Spermatozoa

Abstract

Several methods are used to measure lipid peroxidation (LPO) in spermatozoa. The objective of this study was comparing the thiobarbituric acid reactive species (TBARS) method and the BODIPY 581/591 C(11) (B581) and BODIPY 665/676 C(11) (B665) fluorescent probes to measure induced peroxidative damage in thawed epididymal spermatozoa from Iberian red deer. Samples from three males were thawed, pooled, diluted in PBS, incubated at room temperature and assessed at 0, 3, 6 and 24 h under different experimental conditions: Control, hydrogen peroxide (H(2)O(2) ) 0.1 mM or 1 mM, or tert-butyl hydroperoxide (TBH) 0.1 mM or 1 mM. LPO was assessed by the TBARS assay [malondialdehyde (MDA) detection] and by the fluorescence probes B581 and B665 (microplate fluorimeter and flow cytometry). Increasing MDA levels were only detectable at 1 mM of TBH or H(2)O(2). Both fluorescence probes, measured with fluorometer, detected significant increases of LPO with time in all treatments, except Control. Flow cytometry allowed for higher sensitivity, with both probes showing a significant linear relationship of increasing LPO with time for all oxidizing treatments (p < 0.001). All methods showed a good agreement, except TBARS, and flow cytometry showed the highest repeatability. Our results show that both B581 and B665 might be used for LPO analysis in Iberian red deer epididymal spermatozoa, together with fluorometry or flow cytometry. Yet, the TBARS method offered comparatively limited sensitivity, and further research must determine the source of that limitation.

Probes and Techniques for Sperm Evaluation by Flow Cytometry

“…BODIPY ® 665/676 is a similar probe emitting red fluorescence, which we have tested in red deer spermatozoa (Martínez‐Pastor et al. 2009a), finding similar results when comparing to BODIPY ® 581/591 C 11 (Domínguez‐Rebolledo et al. 2009b).…”

“…Oxidant attack on membranes results in lipid peroxidation, negatively affecting sperm quality. BODIPY ® 581/591 C 11 has been used to evaluate lipid peroxidation using flow cytometry analysis of spermatozoa [bull: (Brouwers and Gadella 2003); red deer: (Domínguez‐Rebolledo et al. 2009b); stallion: (Ortega Ferrusola et al.…”

Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art

“…Among these, BODIPY became popular in andrology. [153][154][155][156][157] BODIPY-based probes have high-quantum yield and high sensitivity of fluorescence, and the appearance of BODIPY-labeled spermatozoa can be regarded as an indication of physiologically relevant exposure of phospholipids to ROS. 83 However, it is also assumed that FC-sperm sorting generates lipid pre-oxidation leading to oxidative stress, although there is no direct evidence on this prediction.…”

Response of Thawed Epidi dymal Red Deer Spermatozoa to Increasing Concentrations of Hydrogen Peroxide, and Importance of Individual Male Variability

“…For instance, Alvarez and Storey (1989) could increase LPO and loss of motility in human and mouse sperm by adding H 2 O 2 (1 and 5 m m ), whereas the same concentrations of H 2 O 2 were insufficient to induce LPO in rabbit sperm. Similarly, we could detect an increase in LPO using the TBARS technique in deer spermatozoa after incubating with 1 m m H 2 O 2 , but we could not detect a significant increase when applying 100 μ m H 2 O 2 (Dominguez‐Rebolledo et al. 2010a,b).…”

“…Reactive oxygen species can be also detrimental to sperm DNA integrity (Baumber et al. 2003; Dominguez‐Rebolledo et al. 2010a,b).…”