Detection of “Apoptosis‐Like” Changes During the Cryopreservation Process in Equine Sperm

Abstract

The kinematics of the appearance of apoptotic markers was studied by flow cytometry and immunoblot assays in equine spermatozoa subjected to freezing and thawing. Caspase activity, low mitochondrial membrane potential, and increases in sperm membrane permeability were observed in all of the phases of the cryopreservation procedure. Freezing and thawing caused an increase in membrane permeability and changes in the pattern of caspase activity; decreases in mitochondrial membrane potential were observed after centrifugation and cooling to 4uC and after freezing and thawing. It is proposed that sperm mitochondria may be directly involved in the subtle damage that is present in most spermatozoa surviving freezing and thawing.

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.…”

“…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. Since cryopreservation [26,46] induces activation of caspases, we also studied the effect of a hyperosmotic and a hyposmotic shock in caspase activation. Only exposition to high osmolality induced a significant increase in the percentage of dead (ethidium+) spermatozoa showing caspase activity.…”

“…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

“…Early sperm membrane changes and viability were determined as described in Peña et al [11], with modifications for adaptation to the equine species [2]. In brief, one mL of sperm suspension (5 x 10 6 spermatozoa/mL) from freshly ejaculated or post-thawed spermatozoa was loaded with 3 L of YO-PRO-1 (25 M) and one L of Ethidium Homodimer-1 (1.167 mM) (Molecular Probes Europe, Leiden, The Netherlands), which was -after thorough mixing-incubated at 37 ºC in the dark for 16 min.…”

“…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].…”