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

Abstract

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.

Determination of Sperm Concentration for Small-Bodied Biomedical Model Fishes by Use of Microspectrophotometry

“…For morphology, zebrafish and medaka sperm have round heads, and Xiphophorus sperm have elongated spindle-shaped heads, and the sperm are usually packaged into sperm bundles. For motility activation, zebrafish sperm respond to hypotonic solutions ( 150 mOsmol=kg), 19 Xiphophorus respond to iso-osmotic solutions (*300 mOsmol=kg), 20 and medaka sperm respond to a wide osmolality range from hypotonic (20 mOsmol=kg) to hypertonic (600 mOsmol=kg) solutions. 21 In addition to accounting for these variations in biological sample characteristics, considerations must be made for the techniques used to handle samples, and procedural factors that can greatly affect the accuracy and reproducibility of measurements.…”

“…Samples were obtained by crushing the testes to suspend the sperm in a volume (mL) of 5-10 times the testis weight (mg) using Hanks' balanced salt solution at 300 mOsmol=kg osmolality (HBSS 300). [19][20][21] For stripping of live males, the anesthetized fish were placed on a holder (made by cutting a slice in a piece of foam rubber) with the belly facing up, and gentle pressure was applied from each side of the belly in the direction toward the anal opening while sperm were collected using a micropipette. The collected sperm samples were placed into a microcentrifuge tube with 20 mL of HBSS 300 (*10-20 times of the sperm volume), and the fish was returned to aquarium water for recovery, where they were allowed to recover for at least 2 weeks before subsequent sperm collection.…”

A Procedure-Spanning Analysis of Plasma Membrane Integrity for Assessment of Cell Viability in Sperm Cryopreservation of ZebrafishDanio rerio

“…4 Sperm cryopreservation has been studied in zebrafish since the 1980s, and effective protocols have been developed and applied for recovery of mutant lines, longterm germplasm preservation, and artificial fertilization. [5][6][7][8][9][10][11][12] In these published protocols, the extenders for dilution of sperm samples included Ginsberg's buffer, 13 Hanks' balanced salt solution (HBSS), 5,14 and balanced Tris buffer (75 mM NaCl, 70 mM KCl, 2 mM CaCl2, 1 mM MgSO4, and 20 mM Tris) with or without addition of skim milk, fetal bovine serum, glucose, or other sugars. The cryoprotectants used include methanol, N,N-dimethyl acetamide (DMA), and N,N-dimethyl formamide (DMF) at concentrations of 8%-10%.…”

“…Once motility is activated, sperm usually lose motility and fertility after a few minutes (e.g., most external fertilization fishes) or several days (e.g., viviparous fishes). 5,[15][16][17][18][19] For zebrafish, sperm are activated by hypotonic solutions and motility can persist for only a few minutes. 5,20 After mixing with extenders, sperm cells undergo osmotic exchange with the extenders resulting in changes to the plasma membrane (e.g., expansion or shrinkage).…”

“…5,[15][16][17][18][19] For zebrafish, sperm are activated by hypotonic solutions and motility can persist for only a few minutes. 5,20 After mixing with extenders, sperm cells undergo osmotic exchange with the extenders resulting in changes to the plasma membrane (e.g., expansion or shrinkage). In addition, sperm suspended in extender can be chilled to reduce sperm metabolism and increase longevity for shipping or other applications, and thus refrigerated storage conditions can affect sperm viability.…”