53 Vitrification of canine epididymal spermatozoa in semen straws: effects of volumes on post-thaw motility

Vitrification is an emerging method of cryopreservation used with assisted reproductive technologies. We evaluated the effects of pellet size and sample volume during straw-in-straw vitrification on post-thaw motility parameters. After overnight refrigerated storage of canine testes postorchiectomy, sperm samples were extracted by slicing the cauda epididymis in phosphate-buffered saline (PBS), centrifuged, and transferred to Tris citrate sucrose extender containing 20% egg yolk. Resuspended samples were cooled to 4°C for 1 to 2 h before vitrification involving three experiments: pellets (experiment 1), or different straw-in-straw volumes (experiments 2 and 3). In experiment 1, 33-μL samples (n = 4) were dropped in LN₂ from a height of 11 cm, which resulted in pellets of different sizes that were stored in cryovials; then ~10- or ~20-μL pellets were thawed individually by dropping them in a microcentrifuge tube prefilled with warm PBS. In experiments 2 and 3 (n = 8 each), four (20, 60, 100, and 140 μL) and five more refined (30, 40, 50, 60, and 70 μL) volumes were evaluated. Specific volumes were loaded into 0.25-mL straws, which were then sealed, placed in 0.5-mL straws, and then vitrified by directly plunging in LN₂. Experiment 3 was designed because the 60-μL group supported better post-thaw motility in experiment 2. Samples were thawed in a water bath at 38.5°C, diluted 3× in PBS and motility evaluated using IVOSII CASA system. Data were analysed using t-test (experiment 1) and one-way ANOVA (experiments 2 and 3) using IBM SPSS version 26. In experiment 1, vitrification in larger pellets (~20 μL) at least numerically resulted in better post-thaw motility parameters than the smaller pellets (total motility: 9.1 vs 6.3%; P = 0.81; total progressive motility: 2.3 vs 0.7%, P = 0.05; VAP: 91.5 vs 91.0 um/s, P = 0.86; straightness: 93.7 vs 88.9%), P = 0.15). In experiment 2, the highest post-thaw motility parameters were observed in the 60-μL group with a moderate treatment effect (P = 0.07, Table 1). In experiment 3, volume had little effect on the post-thaw motility parameters (P > 0.81). Our observations indicate that straw-in-straw vitrification of spermatozoa in volumes greater than 30 μL supports better post-thaw motility than vitrification in smaller volumes. This counteracts the dogma that vitrification of cells in the smallest volumes supports better cryosurvival.

We acknowledge The National Institutes of Health (T35OD010432) for funding this research and Tuskegee Veterinary Scholars for student support.