een conducted to identify those characteristics that favour the freezability of spermatozoa , –. The ultimate goal of these studies is twofold: on the one hand, to be able to know in advance the sperm cryoability of a male, and on the other hand, the design of individualized cryopreservation protocols aimed at maximizing the performance of this technique. Both goals support the idea that sperm cryosurvival can be under genetic control .
In this study, the use of Manchega sheep breed has been motivated by low fertility rates derived from the use of frozen semen in artificial insemination in sheep, compared to those obtained from the use of cooled semen. Benefits resulting from the use of frozen semen are clear. However, the poor ability of ram spermatozoa to resist the freezing-thawing process – and, therefore, the low performance derived from its use are responsible of the low dissemination of this technique in the breeding programs of numerous sheep breeds, as is the case of the Manchega sheep breed. This fact together with the individual variability above-mentioned has resulted in a growing interest in the characterization of the sperm cryoability of a male and the development of individual-specific protocols of sperm cryopreservation.
Here we present a novel approach to characterize the sperm cryoresistence of a male. We use morphometry of sperm heads to monitor changes that occur during cryopreservation. As other authors, we examine spermatozoa before and after cryopreservation, but instead of considering these stages in a static way and perform comparison among them, we propose to consider them all together and define patterns of response. In this study, males were first assigned to a group based on the changes that occur on head morphometry of spermatozoa during cryopreservation. Then, we examined the differences among these groups and hypothesize on the causes of this differential response to cryopreservation.
Among the sperm head parameters measured, only those that better characterize the three ejaculate groups were considered in the results.
In this study, three different patterns of response were observed on males throughout the cryopreservation process (Figure 1). From the 38 males used in this study, 44.7% showed the response pattern 1, 26.3% showed the response pattern 2, and 28.9% showed the response pattern 3. Sperm head dimensions among ejaculates were quite similar for each male with coefficients of variation within a male (between ejaculates) lower than 10 (ranging from 1.14 to 9.30) for all the sperm head parameters. Males always showed the same pattern of response to cryopreservation regardless of the ejaculate.
Data plotted correspond to three males, each one showing a different response pattern. p2a: perimeter to area (Perimeter2/4·π·Area).
The males grouped under the “response pattern 1” showed no significant changes in their sperm head dimensions during cryopreservation (Table 1). Changes on sperm dimension were close to 0 and quite similar between stages of the crypreservation process.
The males characterized as “response pattern 2” showed a progressive decrease in their sperm head dimensions throughout the cryopreservation process. Changes in sperm head dimensions from fresh to prefreezed and from prefreezed to thawed semen were quite similar, being the change from fresh to thawed semen the highest (Table 1). This did not apply to the p2a (perimeter to area) shape factor because in the transition from fresh to prefreezed stage there was an increase (prefreezing) and then decrease (postthawing) to levels below those observed in fresh semen.
Finally, males belonging to the “response pattern 3” showed an important decrease of their sperm head dimension from fresh to prefreezed semen, with minimal changes at thawing (Table 1). For these males, total changes on sperm head dimensions took place mainly during the prefreezing stage and these changes were close to 0 from prefreeze to thawing. As for males with response pattern 2, sperm head p2a initially increase on average and then decrease at thawing.