r on sperm head dimension throughout the cryopreservation process, the five sperm head morphometry parameters described above were used. However, we focused mainly on three sperm head measures, the sperm head length, sperm head area, and the p2a sperm head factor when displaying these differences. These three parameters have been shown to be sufficient to describe the dimensions of the sperm head in a precise way . Coefficients of variation (CV) among ejaculates were calculated to evaluated the within-male (between ejaculate of the same male) variability.
For the recognition of the different patterns of response, an initial graphical exploration of the sperm population head dimensions throughout the cryopreservation process were performed. Scatter plots of the sperm head area against the sperm head length and p2a were drawn for each male and each stage of the cryopreservation process (see Figures S1 and S2). Three different patterns of response were observed. Males were assigned to one of these patterns based on the following rules: if no significant differences were observed among the stages of the cryopreservation process (fresh, prefrezee and post-thaw) for the 3 sperm head morphometry parameters listed above males were assigned to response pattern 1; if sperm morphometry significantly differed for the three stages, males were assigned to response pattern 2; and if head morphometry in fresh semen was significant different from the other to stages, being the latter similar, males were assigned to response pattern 3. For the particular case in which a male did not meet these requirements for the three morphometric parameters, it was assigned to the closest group. For that, graphical visualization of data was considered as well (see Figures S1 and S2).
Once males were assigned to one of the different pattern of response, multiple comparisons between different stages of the cryopreservation process within each response pattern, and between different responses patterns within each cryopreservation stage were carried out. This analysis allowed us to examine if males showing different patterns were significantly different for all stages or only at a certain stages. The analysis was performed by using a mixed-effects model that account for male variability (random effect). For multiple comparison analyses, Holm’s correction was applied. When comparing semen quality among different group of males throughout the conservation process, we also calculated the standardized percentages defined as the ratio between values at freezing stage compared to values at the initial stage (fresh semen). This allows us to evaluate changes during cryopreservation more accurately.
To examine relatedness between males, additive relationships coefficients were estimated. All males included in this study were from the same generation (contemporaneous). A pedigree consisting of 391,691 individuals of Manchega sheep breed were used to construct the additive genetic relationship matrix, A. Within relationship coefficients were calculated as the average of additive genetic relationship among all males belonging to the same group. Between relationships coefficient was calculated as the average of additive genetic relationship of males of one group with those males belonging to the other two groups.
All statistical were performed using the R (2.15.0) software package (R Development Core Team 2012) .
Patterns of response to cryopreservation I. Relationship between sperm head area and length for the 38 males used in this study throughout the cryopreservation process: fresh semen (red points), pre-freezing (yellow points) and post-thawing (blue points). Numbers within a circle in the lower-right corner of graphs indicate to which pattern each male was assigned.
Patterns of response to cryopreservation II. Relationship between sperm head area and p2a relationship for the 38 males used in this study throughout the cryopreservation process: fresh semen (red points), pre-freezing (yellow points) and post-thawing (blue points). Numbers within a circle in the lowe