A high-throughput method for zebrafish sperm cryopreservation and in vitro fertilization

Area of Science:

• Aquatic biology
• Reproductive biology
• Cryobiology

Background:

• Zebrafish are vital model organisms in biological research.
• Efficient cryopreservation of zebrafish sperm is essential for maintaining genetic resources and facilitating research.
• Existing methods, like the Harvey method, can be improved for uniformity and efficiency.

Purpose of the Study:

• To present an adapted and improved protocol for zebrafish sperm cryopreservation.
• To enhance sample uniformity and streamline the cryopreservation procedure.
• To ensure long-term stability of sperm fertility after cryopreservation.

Main Methods:

• Adaptation of the Harvey method for zebrafish sperm cryopreservation.
• Normalization of sperm volumes using non-cryoprotectant freezing media.
• Storage of cryopreserved sperm in cryovials instead of capillary tubes.
• Controlled rates of sperm freezing and thawing (delta degrees C/time).

Main Results:

• The adapted protocol streamlines the procedure and increases sample uniformity.
• Cryopreserved zebrafish sperm achieved an average fertility rate of 25%.
• This fertility rate remained stable over multiple years.

Conclusions:

• The modified cryopreservation protocol offers a more uniform and efficient method for preserving zebrafish sperm.
• The protocol ensures reliable and stable fertility, supporting long-term genetic resource management.
• This method is practical for large-scale sperm banking, with teams of two able to process 100 males in approximately 2 hours.