The higher ability of AFPIII to protect spermatozoa during the freezing-thawing procedure could be the result of the above reported mechanism or/and to a better sinergy between AFPIII and DMSO. The observed decrease in protein abundance may be due either to degradation following freezing-thawing stress, or leakage of proteins from spermatozoa to the extracellular medium, as reported in human, boar and bull sperm, while the observed increase of some protein spots could be due to one or more post-translation modifications following the cryopreservation procedure, as we demonstrated in gilthead sea bream spermatozoa or be a consequence of the freezing/thawing procedure and/or exposure to cryoprotectants on the regulation of mRNA translation, since, as it has been demonstrated in mammals, spermatozoa are not transcriptionally and translationally dormant cells. In the present study the protein profiles were obtained by using both isolated flagella and head plasma membranes of sea bream spermatozoa. In our previous paper the protein profile was obtained by using proteins extracted from the whole spermatozoa of sea bass. Due to the different starting samples and also to differences in sample preparation it is very difficult to compare the effect of the cryopreservation procedure on the detected protein markers obtained in the present and in our previous work. Among the six identified proteins spot 7Fl was an Alcohol dehydrogenase class-III. Spot 10Fl was identified as Glyceraldehyde 3-phosphate dehydrogenase. This protein is expressed in sperm at specific stages of spermiogenesis and can still be detected in mature spermatozoa of vertebrates. Variations in the expression of GAPDH block the progressive motility of spermatozoa. Thus, it can be hypothesized that the observed decrease of sperm motility after the freezing-thawing procedure could be attributed, at least partially, to the reduction of GAPDH expression. Protein spot 11Fl matched with the cytosolic malate dehydrogenase thermolabile form, which was previously found in the midpiece of ram, boar and buffalo spermatozoa. Interestingly, ADHIII, GAPDH and MDH show as common feature to be linked to the bioenergetic system of the cell. NADH+H+ is a product of both the ADHIII and GAPDH activities. In mammalian spermatozoa the transfer of reduced equivalents from the cytosol to the mitochondria occurs by the Malate-Aspartate shuttle, in which two isoforms of MDH, cytosolic and mitochondrial, are operative. By this shuttle, the hydrogen ions of the cofactor NADH produced in the cytosol can reach the electron transport chain in the mitochondria, and generate ATP by the oxydative phosphorylation system. Note that motility of fresh spermatozoa mainly depends on sperm ATP synthesized by mitochondrial OXPHOS. Therefore, the observed reduction in GAPDH and MDH expression in cryopreserved sperm may contribute to the reduced sperm motility observed after freezing-thawing procedure.