Evaluation of Techniques for the Cryopreservation of Washed Spermatozoa: Comparisons between Ham's F-10 and TEST-Yolk Media

The objective of this study was to develop new techniques for the cryopreservation of washed spermatozoa. Two media (Ham's F-10 and nonthermoprecipitated TEST-yolk buffer [NT-TYB]) containing 7% (v/v) glycerol were compared to semen cryopreservation by adding glycerol directly to the semen. Twenty four men collected a semen specimen each after 4 days of sexual abstinence via the use of a semen collection device at intercourse. Specimens were assessed for volume (ml), count (×106), percentage and grade of motility, morphology (% normal) and acrosomal status (% intact acrosomes). Each ejaculate was split into 3 aliquots (Aliquots 1 to 3) and processed for freezing. Aliquot 1 was prepared for cryopreservation by adding glycerol (7% [v/v] final concentration) directly via a dropwise mode. Aliquot 2 and 3 were diluted 1 : 1 (v/v) with Ham's F-10 and NT-TYB, respectively. Aliquots 2 and 3 were then centrifuged (400×g for 10 minutes) and resuspended into the corresponding media containing 7% (v/v) glycerol to complete the sperm wash procedure. All aliquots were frozen in 0.5 ml french straws. Sperm specimens were frozen in liquid nitrogen (LN2) vapor from +23°C to −68°C at a slow rate (2.3°C/minute), after which the specimens were plunged directly into LN2 and stored for 30 days. The quality of the spermatozoa were monitored throughout each step of the overall procedure by measuring the motility characteristics of the spermatozoa. Straws corresponding to each aliquot were thawed in a water bath at 37°C for 2 minutes, followed by assessment of sperm motility and acrosomal status. The percentage of motility after thawing was 31.6±5.6%, 32.8±1.8% and 37.3±1.9% in Aliquots 1 to 3, respectively. Similarly, the grade of motility was 2.4±0.2, 2.6±0.1 and 3.0±0.1 in Aliquots 1 to 3, respectively. The acrosomal status (% intact acrosomes) in Aliquots 1 to 3 was 41.2±2.6, 43.1±3.6 and 51.6±4.5, respectively. The results suggest that the characteristics of spermatozoa washed and frozen in NT-TYB (Aliquot 3) were improved over those spermatozoa prepared via direct addition of glycerol to the semen (Aliquot 1) or by using Ham's F-10 (Aliquot 2). The most significant reduction noted during freezing was in the loss of acrosomal integrity. The results obtained in this study point out that washed spermatozoa can be cryopreserved with some success and that the recovered spermatozoa could be used for intrauterine insemination in an artificial insemination program using husband's or