cess article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Education and Science Council of Castilla-La Mancha (PCC08-0105-8042). Manuel Ramón was supported by the DOC-INIA program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Understanding the switching of phenotypes in response to environmental changes is at the forefront of biological research in diverse areas of study . One of these areas is focused on the study of cell response to low temperatures. Cell cryopreservation has become an indispensable tool in biology. Biological materials can be safely kept and used after a very long period of time. For the particular case of spermatozoa, cryopreservation is used in livestock management and in the conservation of wild and domestic species, as a complementary tool for managing live animals and preserving their genetic diversity.
Several studies have been conducted to understand the changes that occur during cryopreservation of spermatozoa and develop a protocol to ensure a successful storage (see  for references and general discussion). These studies have focused on different traits such as sperm motility and morphometry, integrity of sperm membranes or DNA status among others –. As a result of these studies, standard cryopreservation protocols have been developed for many species, both domestic and wild.
One important problem for standardizing sperm cryopreservation protocols is that spermatozoa from different individuals exhibit significant different responses to the same freezing treatment , –. Males can be classified into “bad” and “good” freezers depending on the ability of their spermatozoa to resist to the cryopreservation process. Many studies have been 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