107 COMPARISON OF PRIMATE SPERM CRYOPRESERVATION PROTOCOLS: POST-THAW SPERM RECOVERY AND HYPERACTIVATION IN CULTURE

Abstract

Cryopreservation of spermatozoa provides material for gene banking of genetically valuable males and offers convenience for in vitro fertilization (IVF). In addition, cryobanking of spermatozoa allows less frequent ejaculation collections from males. The present study compared the effectiveness of several published techniques in non-human primates to find the most efficient one for rhesus macaque (Macaca mulatta) semen cryopreservation. The effectiveness of each method was assessed by longevity (post-thaw motility % and duration) and ability to hyperactivate in culture in response to chemical activators (caffeine, dbcAMP) needed for rhesus sperm capacitation (Boatman and Bavister 1984 J. Reprod. Fertil. 71, 357-366). The ability to hyperactivate provides a reasonable assessment of the fertilizing capability of spermatozoa prior to performing IVF; the latter was impractical for this study, given the large number of treatments and endpoints. Spermatozoa were collected via electroejaculation from one male on three occasions to avoid confounding treatments with male effects. Each ejaculate was divided into one of four treatment groups for cryopreservation: Method A (Seier et al. 1993 J. Med. Primatol. 22, 355-359); Method B (Wei et al. 2000); Method C (Sanchez-Partida et al. 2000, Biol. Reprod. 63, 1092-1097); and Method D (Isachenko et al. 2005 Reprod. Biomed. Online 10, 350-354). Protocols were followed according to each published technique. Upon thawing, each sample was split into different incubation conditions: 37°C, 5% CO₂ in air or room temperature for 0-24 h. One dose of activators was used according to standard protocol. Statistical analyses of motility rates were performed using 2 × 2 G tests (Sokal and Rohlf 1981 Biometry. New York: W. H. Freeman Co.) to determine significance. Samples cryopreserved using method D did not survive the method (motility = 0) and were not included in the statistical analysis. Methods A-C all demonstrated reasonable post-thaw motility recovery rates (68%, 73%, and 62%, respectively) and underwent capacitation within 30 min of exposure to activators. Sperm motility decreased over time in culture within each treatment at 37°C. However, spermatozoa in Method A were significantly less motile at 4 and 24 h than those in Methods B and C, and Method B spermatozoa were significantly less motile at 24 h than those in Method C. Sperm motility also decreased over time in samples incubated at room temperature, with motility of sperm in Method A motility being significantly less at 24 h than that of sperm in Methods B and C. Method C best preserved motility over time regardless of temperature of incubation upon thawing. Overall, incubation at room temperature preserved motility better than incubation at 37°C. Methods A-C yielded satisfactory post-thaw recovery of progressively motile spermatozoa despite the various differences among their protocols. For long-term use of each sample, however, it would be beneficial to incubate spermatozoa at room temperature after using Method C. This technique appears to be more appropriate for gene banking rhesus semen, and applying this protocol would allow more efficient usage of each semen sample, potentially providing for multiple IVF cases over a 24-h period.

This work was supported by NIH Grant RR15395.