The cryopreservation process has an important impact on sperm structure and physiology. The negative effects have been mainly observed on the plasma membrane, which is directly stabilized by the cytoskeleton. Since cytoskeleton proteins are osmosensitive and thermosensitive, the aim of this study was to evaluate the damage caused to the bull sperm cytoskeleton by cryopreservation (freezing-thawing). Fresh and frozen-thawed bull semen samples were exposed to a treatment with the neutral detergent Brij 36-T. Electron microscopy evidenced important damages at the sperm perinuclear theca after the protein extraction protocol; the perinuclear theca was partially solubilized, the perinuclear theca substructure disappeared in the cryopreserved samples. Furthermore, the sperm head's shape was significantly altered on the cryopreserved samples. Fluorescence analysis showed a decrease of the intensity of actin and dystrobrevin on the frozen-thawed samples. Western blot assays revealed a stronger signal for actin and β-dystrobrevin in the frozen-thawed sperm samples than in the fresh ones. Our results suggest that the cryopreservation process highly alters the sperm cytoskeleton stability, causing its proteins to become more fragile and therefore more susceptible to be extracted.
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