Lipid peroxidation, assessed with BODIPY-C11, increases after cryopreservation of stallion spermatozoa, is stallion-dependent and is related to apoptotic-like changes

Abstract

Lipid peroxidation (LPO) of stallion spermatozoa was assessed in fresh semen and in samples of the same ejaculates after freezing and thawing. Particular attention was paid to individual differences in the susceptibility to LPO and its possible relationship with freezability. Innate levels of LPO were very low in fresh spermatozoa but increased after thawing, a change that was largely stallion-dependent. The level of LPO in fresh spermatozoa was not correlated with that of the thawed spermatozoa. Negative correlations existed between LPO and intact membranes post-thaw (rZK0.789, P!0.001), and also between LPO and spermatozoa with high mitochondrial membrane potential (Djm) post-thaw (rZK0.689, P!0.001). LPO was also highly and significantly correlated with caspase activity. The correlation between caspase activity in ethidium positive cells and LPO was rZ0.772, P!0.001. This LPO is unlikely to represent, per se, a sign of cryopreservation-induced injury, but it is apparently capable of triggering 'apoptotic-like changes' that could result in the sub-lethal cryodamage often seen among surviving spermatozoa.

Toxicity of glycerol for the stallion spermatozoa: Effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential

“…Also this may explain the lack of lipid peroxidation in our experiment, but another possible explanation relies in the ability of glycerol to scavenge the hydroxyl radical [40]. The sperm mitochondria have been characterized as the main source of reactive oxygen species in the spermatozoa [41,42], and also the sperm structures more sensitive to the damage induced by cryopreservation [25,26,[43][44][45], but glycerol toxicity does not seem to be a major factor involved in mitochondrial damage. Finally we studied the activity of caspases 3, 7 and 8, however none of the glycerol concentrations studied resulted in a significant increase in any of the caspases studied.…”

“…Early sperm membrane changes and viability were determined as described in Peña et al [24] with modifications for adaptation to the equine species [25,26] In brief, one mL of sperm suspension (5 x 10 6 spermatozoa/mL) was loaded with 3 µL of Yo-Pro-1 (25 µM) and one µL of Ethidium Homodimer-1 (1.167 mM) (Molecular Probes Europe), which was -after thorough mixing-incubated at 37 ºC in the dark for 16 min. This staining distinguishes four sperm subpopulations.…”

Fatty acids and plasmalogens of the phospholipids of the sperm membranes and their relation with the post-thaw quality of stallion spermatozoa

“…Lipid peroxidation (LPO) was measured using the probe BODIPY 581/591 -C 11 (Molecular Probes, Leiden, The Netherlands) as previously described in our laboratory [5]. In brief, a suspension of 2x10 6 spermatozoa was loaded with the probe at a final concentration of 2 M.…”

“…Such variability is often ascribed to the fact that most stallions have been selected by performance and phenotype, and not for sperm quality either directly or indirectly. The physiological and biochemical reasons behind this variability are being slowly disclosed [2], and recent attempts include the predictive value of several markers for successful freeezability of a given ejaculate [2][3][4][5][6][7]. In many species including horses, peroxidation of lipids of the plasma membrane (lipid peroxidation, LPO) has been claimed to be a major factor involved in sperm quality after thawing [5,8,9].…”

“…The physiological and biochemical reasons behind this variability are being slowly disclosed [2], and recent attempts include the predictive value of several markers for successful freeezability of a given ejaculate [2][3][4][5][6][7]. In many species including horses, peroxidation of lipids of the plasma membrane (lipid peroxidation, LPO) has been claimed to be a major factor involved in sperm quality after thawing [5,8,9]. The particular susceptibility of the sperm plasma membrane to peroxidative damage is due to a high cellular content of polyunsaturated fatty acids as well as their deficiency in protective enzymes, consequence of spermatozoa loosing most of their cytoplasm during spermiogenesis.…”