perm head dimensions. Confirmation that the membrane structure of spermatozoa would require more time to complete the water and cryoprotectant exchange during cooling would allow the design of specific freezing protocols for each type of male.
This study has allowed us to evaluate whether different ejaculates of a male had the same sperm head dimensions and if these ejaculates showed the same pattern of response throughout the cryopreservation process. Results have showed that, in fresh semen, all sperm head morphometric parameters were quite similar for the different ejaculates of a male, being the coefficients of variation of low magnitude. Also, males have always showed the same pattern of response to cryopreservation for the three ejaculates assessed. The low variability on sperm head dimensions between ejaculates of a male has already been stated for this breed. Thus, Maroto-Morales et al.  have reported low CV’s for all the sperm head dimensions. These authors have presented the important role of sperm head morphometry on the characterization of spermatozoa of a male given its low intra-male variability, and have supported the genetic determinism of the sperm morphometry pointed out by other authors , . To further explore this idea, we examined the degree of relatedness (obtained from the additive relationship matrix) between males showing the same (within-) and different (between-) response patterns. Results have showed that additive relationship coefficients for males belonging to the same group were twice larger than those among groups of response patterns. Thus, males who share the same pattern of response were found to be more closely related to each other than with those showing different responses. This result supports the idea of a genetic control of sperm cryoresistance.
In conclusion, the present study shows that sperm head morphometry is a key feature to be included in conservation and breeding programs. The assessment of response patterns suggested here allows us to characterize the sperm cryoresistance of a male. Since this pattern of response has resulted to be a constant feature of a male, this gives greater prominence to the idea of designing specific freezing protocols to each animal.
Materials and Methods
A total of 38 adult males from Manchega dairy sheep breed were used in this study. Males were kept at the Regional Centre of Animal Selection and Reproduction (CERSYRA, Spain) at the same environmental conditions. Age of males ranged from 1.5 to 3 years. All males were maintained in the same conditions of housing and feeding. Males were trained for semen collection by artificial vagina and maintained a regimen of regular collection (twice per week). These semen samples are mainly used for artificial insemination as a part of the breeding program of the Manchega breed.
The study was approved by the “Comité de Ética en Investigación de la Universidad de Castilla-La Mancha”. All animal handling was done following Spanish Animal Protection Regulation RD1201/2005, which conforms to European Union Regulation 2003/65.
Sperm Collection and Cryopreservation
Semen was collected from males from October to November, coinciding with the breeding season. Each male was collected three times. After collection, sperm quality was evaluated. Traits assessed were: percentage of motile spermatozoa, quality of spermatozoa movement, percentage of spermatozoa with intact acrosomes and percentage of live spermatozoa.
To conduct the sperm quality assessment, 5 µl of fresh semen were diluted on 1500 µl of phosphate-buffered saline (PBS; pH 7.5, 310 mOsm/kg) with 0.5% bovine serum albumin. Sperm motility and quality of movement were evaluated by placing 10 µl of diluted sperm between a pre-warmed slide and a 22 mm×22 mm coverslip and examining it at ×100 magnification under phase contrast optics. The percentage of motile sperm was estimated subjectively with values ranging from 0%, when no motile spermatozoa were observed, and 100%, when all spermatozoa were moving. Quality of sperm movement was also estimated subjectively 