d 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 lower-right corner of graphs indicate to which pattern each male was assigned. p2a: perimeter to area (Perimeter2/4·π·Area).
We acknowledge E.R.S Roldan for helpful discussions. Manuel Ramón is supported by the DOC-INIA program.
Conceived and designed the experiments: MR JJG. Performed the experiments: MR MDPG PJR MCE OGÁ AMM LAL AJS MRFS JJG. Analyzed the data: MR. Contributed reagents/materials/analysis tools: MR MDPG AJS MRFS JJG. Wrote the paper: MR MDPG PJR MCE OGÁ AMM LAL AJS MRFS JJG.
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