s on their sperm head dimensions during cryopreservation (response patterns 2 and 3) showed significant differences in sperm quality at thawing but were characterized by a similar sperm head morphometry in fresh semen. And those males with response patterns 1 and 3 showed significant differences in fresh semen but resulted in quite similar quality after thawing. Therefore, it is important to know that the sperm dimensions have changed, but also how these changes occur and at which time of the cryopreservation process.
Cryopreservation involves major changes in sperm cell environment, and their success to survive this process will depend largely on their ability to respond to these changes within the finite time period allowed by the protocol . The responsiveness of spermatozoa to osmotic challenge and their ability to regulate cell volume is a characteristic closely related to cryopreservability . Five distinct processing steps can be recognized in relation with changes in cellular volume : extension and cooling, cryoprotectant addition and packaging, freezing, storage and thawing, each having its special relationship to membrane structure function and cell metabolism. Cooling is known to alter the physical properties of all cell membranes –. The second step demands a large volume change and a rapid recovery –. The addition of cryoprotectant leads to osmotically driven egress of intracellular water, followed by a slower return to the originally volume as the cryoprotectant enters. Freezing alters membrane structure and volume over a very short time period . Extracellular ice crystallization results in very large increases of all other solutes in the remaining liquid and the outward movement of water in response to high concentrations. Finally, thawing requires membrane recovery and cellular expansion.
The changes that occur in sperm volume during cryopreservation and their mechanisms allow us to hypothesize about sperm behavior in term of patterns of response. One of the patterns of response identified in this study has been characterized by maintaining a relatively constant sperm head morphometry throughout the cryopreservation (response pattern 1). This would not imply the absence of changes in sperm shape during cryopreservation, but that changes could occur rapidly at each stage. Males showing this response pattern have been characterized by smaller sperm head dimensions in fresh semen and have showed an intermediate sperm quality at thawing. As we pointed out above, many authors have associated this sperm shape with good freezability. In this study, sperm quality at thawing has been higher for those males that have undergone the change on head dimensions mainly during cooling (response pattern 3). It is possible that spermatozoa from these males have responded in a better way to the structural changes resulting from the addition of cryoprotectant. The membrane structure function would not have been impaired during cooling allowing the movement of water and cryoprotectant in an efficient manner. Thus, sperm from these males would be less susceptible to damage from freezing and thawing. Finally, we have identified a third pattern of response, in which sperm have changed their morphometry in a progressive way (response pattern 2). The males having this response pattern have been characterized by large sperm head dimensions on fresh semen and small dimensions and low quality on thawed semen. Two hypotheses could support the behavior of this sperm: that the sperm membrane structure has been significantly damaged during cooling so the exchange of water and cryoprotectant has occurred in a suboptimal way, or that given the characteristics of their cell membranes, more time is needed to complete the exchange. As a result, spermatozoa would not have responded appropriately to changes in their environment during freezing and thawing, leading to a decrease of quality on thawed semen. We think it would be of utmost importance to further study the cryopreservation process in males that have showed a progressive change on their s