sistance 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 on a scale of 0–5, where 0 is no motility and 5 is vigorous progressive movement.
Acrosomal integrity after cryopreservation is a well-accepted parameter for estimating sperm function and response to cryopreservation , , especially after relatively long incubation periods to record changes in plasma membrane sensitively . For the assessment of the percentage of spermatozoa with intact acrosomes, 10 µl of sperm suspension in PBS were diluted with 50 µl of a 2% glutaraldehyde in 0.165 M cacodylate/HCl buffer (pH 7.3), and examined by phase-contrast microscopy. A total of 200 spermatozoa were acquired per ejaculate. Percentages of acrosome integrity were recorded.
Sperm viability was assessed in sperm smears stained with eosin-nigrosin. Thus, 5 µl of sperm suspension with PBS and 10 µl eosin-nigrosin solution were mixed on a glass slide placed on a stage at 37°C and 20 s later the mix was smeared and allowed to air-dry. Smears were examined at ×1000 under bright field and 200 spermatozoa per male were examined to evaluate sperm viability. Live spermatozoa were those excluding eosin (from the eosin-nigrosin stain).
Only those sperm samples with a minimum quality (percentage of motile sperm above 80% and quality of movement above 3.5) were cryopreserved, as a way to assure that all sperm samples showed good quality before freezing. If an ejaculate did not meet the requirements of quality was discarded and the male was recollected again to complete the three semen collections. Cryopreservation was performed as described by García-Alvarez et al. . Briefly, fresh semen was diluted with freezing extender Biladyl® (Minitüb, Tiefenbach, Germany) with 20% egg yolk and an osmolarity balanced at 310 mOsm/kg for the Fraction A (non-glycerol fraction). Semen was diluted to 400×106 spermatozoa/ml with Biladyl®, Fraction A, at 30°C. Diluted semen was cooled to 5°C for 2 h and then was further diluted with the same volume of Biladyl®, Fraction B. Sperm samples were allowed to equilibrate at 5°C for 2 h and packed in 0.25 ml plastic