Changes to Extender, Cryoprotective Medium, and In Vitro Fertilization Improve Zebrafish Sperm Cryopreservation

Abstract

Sperm cryopreservation is a highly efficient method for preserving genetic resources. It extends the reproductive period of males and significantly reduces costs normally associated with maintenance of live animal colonies. However, previous zebrafish (Danio rerio) cryopreservation methods have produced variable outcomes and low post-thaw fertilization rates. To improve post-thaw fertilization rates after cryopreservation, we developed a new extender and cryoprotective medium (CPM), introduced quality assessment (QA), determined the optimal cooling rate, and improved the post-thaw in vitro fertilization process. We found that the hypertonic extender E400 preserved motility of sperm held on ice for at least 6 h. We implemented QA by measuring sperm cell densities with a NanoDrop spectrophotometer and sperm motility with computer-assisted sperm analysis (CASA). We developed a CPM, RMMB, which contains raffinose, skim milk, methanol, and bicine buffer. Post-thaw motility indicated that the optimal cooling rate in two types of cryogenic vials was between 10 and 15°C/min. Test thaws from this method produced average motility of 20% ± 13% and an average post-thaw fertilization rate of 68% ± 16%.

Keywords: aquatic; biomedical; gene banking; genetic repository; model organism; resource center.

FIG. 1.

Sperm samples diluted in E400 can be held as long as 6 h on ice without significant loss of motility. Pooled sperm was collected and maintained in E400 extender as collected (1.7 × 10⁹ cells/mL) or further diluted 3.4-fold with E400 (to 5 × 10⁸ cells/mL) and held on ice for as long as 6 h. A portion of sperm from each group was cryopreserved hourly. Prefreeze and post-thaw sperm motility were analyzed for each time point. Percent motility of 1.7 × 10⁹ and 5 × 10⁸ cells/mL diluted sperm cells was plotted against storage time. The average percent motility of 1.7 × 10⁹ cells/mL sperm samples decreased over time (prefreeze, black triangles; post-thaw, white triangles). In contrast, sperm diluted to 5 × 10⁸ cells/mL with E400 (prefreeze, black circles; post-thaw, white circles) maintained motility for up to 6 h on ice. Data values labeled (a) were not significantly different for motilities of 5 × 10⁸ or 1.7 × 10⁹ cells/mL prefreeze (black) and post-thaw (white) samples during the first 3 h of storage on ice. However, all data values labeled (b) were significantly different between 5 × 10⁸ and 1.7 × 10⁹ cells/mL test groups between 4- and 6-h storage on ice, except for diluted post-thaw samples (white circles), which had comparable or greater motility than 1.7 × 10⁹ cells/mL prefreeze samples (black triangles). Two-way ANOVA post hoc statistics was calculated using Sidak's multiple comparison test. p < 0.001, n = 3 for prefreeze data; n = 9 for post-thaw data. ANOVA, analysis of variance.

FIG. 2.

Sperm cells were held in RMMB CPM for at least 5 min without significant impact of solution toxicity on cell motility. Average post-thaw motility of sperm incubated with RMMB CPM at room temperature for various amounts of time before freezing is shown. The 60-min control was incubated for 60 min (in E400 without RMMB) at 25°C before adding RMMB and freezing with a 0.5-min equilibration time. Motility was assessd post-thawing, and an ordinary post hoc one-way ANOVA was performed using Tukey's multiple comparison test to the mean of each column with every other column. n = 27 for each of the test and control groups. No significant difference was found between control and 0.5–10 min test groups (labeled a). The average motility rate for the time points 15–60 min (labeled b, c, d, and e) was statistically significantly different to any of the average motilities labeled (a). The adjusted p-values for the control group versus 0.5, 1, 2, and 5 min test groups were 0.49<p < 0.9996, whereas p = 0.001 for control versus 10 min, and p < 0.0001 for control versus 15, 30, 45, and 60 min test groups. CPM, cryoprotective medium.

FIG. 3.

Controlled-rate cooling rates and resulting post-thaw motility can be reproduced with dry ice in two types of cryogenic vials. (A) Dark gray bars: post-thaw motility (mean ± SD) of sperm frozen in 0.5-mL matrix vials in a CRF. Cooling rates ranged from 5 to 35°C/min. An ordinary post hoc one-way ANOVA was performed using Tukey's multiple test to compare the mean of each column against every other column. The optimal cooling rate was identified as 10 to 15°C/min (labeled a). At these freeze rates, post-thaw motility was 74% ± 10% and 76% ± 5% (respectively, each n = 9), and these averages were not significantly different (p = 0.9998). However, all average post-thaw motilities of sperm frozen at rates of 5°C/min and between 20 and 35°C/min were significantly different (and lower). Light gray bars: post-thaw motility of sperm frozen in dry ice using 0.5-mL matrix vials (−14.1°C/min; 81% ± 2% motility, n = 9) and 2-mL Corning cryovials (−16.1°C/min; 74% ± 4% motility, n = 9). (B) Dry ice freezing vial assemblies. Cryogenic vial with sperm sample is placed in a 15-mL conical tube on top of an empty spacer vial of the same kind. (Left) 0.5-mL Matrix sample vial on top of an empty spacer Matrix vial with cap. (Right) 2-mL Corning cryogenic vials on top of an empty 2-mL spacer vial without cap. CRF, controlled-rate freezer; SD, standard deviation.

FIG. 4.

ZIRC test thaws suggest that successive implementation of protocol changes resulted in a stepwise increase in post-thaw fertilization rates. The cryopreservation and in vitro fertilization protocols utilized were: Draper and Moens (2004–2008, n = 1475); ZIRC 1 (2009, n = 591): Draper–Moens freeze protocol with reduced IVF volume and no mixing of sperm and eggs; ZIRC 2 (2012, n = 334): cryopreserved using E400, RMMB, and optimized freeze rate, same IVF protocol as ZIRC 1; and ZIRC 3 (2014, n = 173): same as ZIRC 2, but osmolality of fertilization solution was raised (Table 2). Post-thaw fertilization rates were obtained and averaged, and an ordinary post hoc one-way ANOVA was performed using Tukey's multiple test to compare the mean of each column against the average of every other column. 0.0001 > p > 0.00001; the averages of every column were significantly different from every other column. Cohen's d (size effect) was determined for all comparisons: a versus b, d = 1.29, b versus c, d = 1.0; c versus d, d = 0.93; a versus d, d = 3.59; a versus c, d = 2.53; a versus d, d = 3.59). IVF, in vitro fertilization; ZIRC, Zebrafish International Resource Center.