Ultra-rapid freezing of severe oligospermic samples using cryoloops

Abstract

Objective: To determine feasibility of ultra-rapid freezing oligospermic samples using cryoloops. Design: Laboratory study Materials/Methods: Sperm motility following exposures to different freezing solutions (F1 and F2) diluted to 4 separate concentrations with test yolk buffer (TYB) was assessed (N=10). Four solutions were further evaluated for changes in motility over 4 min. The two optimal solutions were evaluated as cryoprotectants (C1, C2) during ultra-rapid freezing. Aliquots from 14 semen samples were mixed with C1, C2, C3 (TYB + 12% (v/v) glycerol), and C4 (seminal plasma), placed on cryoloops and directly submerged into liquid nitrogen or placed into liquid nitrogen vapor for 5 min prior to submersion. After freezing, sperm were thawed in TYB (C1–4) or seminal plasma (C4) and motility and viability were assessed. Using the optimal cryoprotectant (C3), post-thaw motility was compared between ultra-rapid and slow-rate freezing (N=10) Results: Sperm motility in F1 1:1 dilution was 77% +/− 2.4% (mean +/− S.E.); 3:1 dilution 51% +/− 4.3%; and 4:1 dilution 39% +/− 5.7% while that in F2 1:1 dilution was 41% +/− 6.3%; remaining tested solutions all had motilities <20% (p <0.05 for all dilutions when compared to initial samples). Sperm motility assessed after 4 min were optimal in F1 1:1 dilution (66% +/− 8.1%) and F1 3:1 dilution (49% +/− 4.4%). These solutions were selected as C1 and C2, respectively. Prior to ultra-rapid freezing, mean sperm motility and viability was 54% +/− 3.4% and 66% +/− 3.8%, respectively. Regardless of tested solutions, post-thaw motility of sperm submerged directly into liquid nitrogen was 0%. Of samples initially suspended in liquid nitrogen vapor, post-thaw sperm motility was 5% +/− 1.9% (C1); 0% (C2); 40% +/− 3.2% (C3); 16% +/− 3.6% and 21% +/− 4.7% (C4 resuspended in seminal plasma or TYB, respectively). Viability of thawed sperm was 4% +/− 2.0% (C1); 0% (C2); 17% +/− 3.9% and 21% +/− 4.7% (C4 resuspended in seminal plasma or TYB, respectively). Viability stain could not be performed on sperm frozen in C3 because of reagent incompatibility. Post-thaw sperm motility of semen frozen with C3 by ultra-rapid means (45 +/− 3.4%) and conventional means (45 +/− 3.1%) were similar (p = 0.95) but less than unfrozen samples (55 +/− 22%; p <0.05) Conclusions: Ultra-rapid freezing of small numbers of sperm (∼100) using cryoloops is feasible. Of cryoprotectants tested, the best post-thaw motility is seen when freezing in TYB + 12% (v/v) glycerol and thawing in TYB alone. Initial post-thaw sperm motility after ultra-rapid freezing with this cryoprotectant is comparable with conventional slow-rate freezing. Considering the comparable results, one can appreciate the potential impact of a technique that takes 5 min compared to conventional slow-rate freezing which takes approximately 1.5 hours. Supported by: None.