Cryopreservation and/or thawing induce many changes in mammalian spermatozoa . Diminished motility and membrane changes, including sperm capacitation or acrosomal reaction, are some of the main forms of damage brought out by cryopreservation. Cryopreservation definitely affects sperm viability.
Necrosis and apoptosis are two forms of cell death. Necrosis results from injury and affects large numbers of cells, causing cell swelling and membrane rupture; in contrast, apoptosis is physiologically programmed cell death that affects single cells without any related inflammation in the surrounding tissue [2, 3]. Apoptosis is a complex phenomenon that can be divided into three phases: induction, execution, and degradation. Mitochondria are known to play a central role during the execution phase. After induction of apoptosis, mitochondrial pores are opened, characterized by decreased mitochondrial membrane potential (m). Opening of mitochondrial pores leads to the release of proapoptotic factors from the mitochondria . In the cytoplasmic compartment, the proapoptotic factors—for example, different proteases related to the caspases family (cysteine proteases with aspartate specificity)—are subsequently activated, leading to the degradation phase. During this phase, changes at both the cell surface and the nucleus occur. Phosphatidylserine (PS), ordinarily sequestered in the plasma membrane inner leaflet, appears in the outer leaflet, where it triggers noninflammatory phagocytic recognition of the apoptotic cell . In the apoptotic cells, internucleosomal cleavage of DNA by specific endonucleases produces 180-base pair DNA fragments .
Normal spermatogenesis depends on the efficiency of apoptosis. Approximately 25–75% of germ cells degenerate and die in the adult mammal testis. Spontaneous apoptosis has been clearly observed in rat testis seminiferous epithelium, affecting spermatogonia, spermatocytes, and spermatids .
Using electron microscopy, ejaculated spermatozoa have been shown to exhibit certain characteristics of apoptotic somatic cells: DNA fragmentation and chromatin condensation, lobulation of the acrosome membrane, and mitochondrial distention [7, 8]. Furthermore, apoptotic spermatids and apoptotic corpses phagocytosed by macrophages can be observed in ejaculated sperm . In 1993, Gorczyca et al.  first observed spermatozoa with DNA fragmentation, analogous to apoptosis in somatic cells. They hypothesized that this phenomenon would inactivate abnormal ejaculated spermatozoa, which could be dangerous. Failure to remove these defective germ cells efficiently during spermatogenesis would result in high numbers of abnormal sperm cells in the semen and consequent low fertility.
The pathway of such putative apoptosis in sperm cells is the subject of controversy. Weil et al. , using in situ immunofluorescence, defended the hypothesis of a caspase-independent pathway on the basis of the observation that less than 8% of dead cells expressed caspase-3. In contrast, Weng et al.  concluded in favor of caspase-dependent apoptosis after studying caspase enzymatic activity by fluorometry and immunoblot assay.
Few studies have focused on the effects of cryopreservation on apoptotic manifestations in sperm cells. They have shown that cryopreservation is associated with induction of membrane PS translocation in human [12–14], boar , and bull [16, 17] sperm cells. This membrane modification is not correlated with DNA fragmentation in human  or in bull  sperm or with free-radical production in human sperm .
We therefore compared the expression of various apoptosis markers before and after cryopreservation-thawing. As a single assay is not sufficient to assess apoptosis, various probes specific to the main apoptotic targets were studied. First, all the techniques were validated on the human myeloid leukemia cell line U937 because induction and visualization of apoptosis in this cell line is easy and abundantly documented [18–21].
We here present our results concerning apoptosis markers, which shed light on the effects of cryopreservation on bovine spermatozoa. This multiparametric study provided evidence that a phenomenon, similar to the apoptosis observed in somatic cells, with the exception of nucleus changes, is induced in ejaculated bovine spermatozoa by cryopreservation and/or thawing.