Cryopreservation of zebra fish spermatozoa using methanol

Harvey, Brian; Kelley, R. N.; Ashwood-Smith, M.J.

doi:10.1139/z82-242

Cited by 124 publications

(79 citation statements)

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“…In the present study, DMSO had a limited protective effect for zebrafish sperm under the conditions tested (4 and 8% with cooling rates of 10 and 20 °C/min), resulting in low motility of thawed sperm. Methanol and DMA appeared to be suitable cryoprotectants for use with zebrafish sperm, consistent with previous reports [5,8]. However, different cooling rates were required for methanol and DMA to obtain protection for sperm during freezing.…”

Section: Discussionsupporting

confidence: 89%

“…Thus, optimization of cooling rates, specific extenders, containers, and cryoprotectants are important, and this process is usually dictated by the interaction of these parameters. Previous studies of zebrafish sperm were not able to quantify the cooling rate because the freezing was performed by placement of cryovials or capillary tubes on crushed dry ice [5,7,8]. In the present study, the effect of cooling rate with three cryoprotectants was tested using a programmable freezer and standardized plastic straws.…”

Section: Discussionmentioning

confidence: 92%

“…[5]), DMA (8%), DMSO (8%), and methanol (4, and 8%) were chosen for further study as cryoprotectants, and sperm were frozen with cooling rates of 10 and 20 °C/min. Testes from five individual males were used for sperm collection in each experimental group.…”

Section: Experiments Iii: Evaluation Of Motility and Fertility Of Thawmentioning

confidence: 99%

“…Currently, there are three reports of protocols for zebrafish sperm cryopreservation. The first protocol was published more than 20 y ago [5], uses 10% methanol as cryoprotectant. This protocol was later adapted and used by genetics laboratories for recovery of specific strains and lines [6,7]; the fertilization level of frozenthawed sperm was reported to be 28 to 36%.…”

Section: Introductionmentioning

confidence: 99%

“…All three procedures were complicated, and steps such as freezing rate were not quantified, making it difficult to obtain consistent results in other laboratories [8]. Also, these procedures relied on the use of a specific brand of powdered milk in the extender solution [5,7] which is not readily available worldwide, and also impedes the observation of sperm motility and morphology, especially after thawing. Therefore, further investigation was needed to simplify and standardize sperm cryopreservation, and to identify reproducible procedures and containers to provide consistent cooling and thawing profiles, as well as reliable labeling of samples.…”

Section: Introductionmentioning

confidence: 99%

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Development of a simplified and standardized protocol with potential for high-throughput for sperm cryopreservation in zebrafish Danio rerio

et al. 2007

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Sperm cryopreservation offers potential for long-term storage of genetic resources. However, the current protocols for zebrafish Danio rerio are cumbersome and poorly reproducible. Our objective was to facilitate adoption of cryopreservation by streamlining methods from sperm collection through thawing and use. First, sperm activation was evaluated, and motility was completely inhibited when osmolality of the extender was ≥ 295 to 300 mOsmol/kg. To evaluate cryoprotectant toxicity, sperm were incubated with dimethyl sulfoxide (DMSO), N, N-dimethyl acetamide (DMA), methanol, or glycerol at 5, 10, and 15% concentrations. Based on motility, DMSO, DMA, and methanol (≤ 10%) were less toxic; therefore, sperm were cryopreserved using these cryoprotectants at cooling rates of 10 and 20 °C/min. The highest motility (mean ± SD) (35 ± 23%; P ≤ 0.0001) and fertility (13 ± 8%; P ≤ 0.001) in thawed sperm were obtained with the combination of 8% methanol and a cooling rate of 10 °C/min. Further evaluations of 8% methanol and 10 °C/min were performed with males from populations with high (2.05 ± 0.24) and low (1.18 ± 0.12) body condition (P = 0.0001). Motility of thawed sperm from the two populations was 38 ± 16% and 78 ± 10% (P = 0.0001), and fertilization was 6 ± 6% and 33 ± 20% (P = 0.0001). These values were positively related with body condition factor. Overall, this study simplified and standardized sperm cryopreservation, and established a protocol using French straws as a freezing container and an extender without powdered milk. This protocol can be readily adapted for high-throughput application using automated equipment, and motility and fertility comparable to previous reports were obtained. Male variability and sperm quality remain important considerations for future work, especially in mutant and inbred lines.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 92%

Section: Experiments Iii: Evaluation Of Motility and Fertility Of Thawmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%