High-throughput cryopreservation of spermatozoa of blue catfish (Ictalurus furcatus): Establishment of an approach for commercial-scale processing

Abstract

Hybrid catfish created by crossing of female channel catfish (Ictalurus punctatus) and male blue catfish (Ictalurus furcatus) are being used increasingly in foodfish aquaculture because of their fast growth and efficient food conversion. However, the availability of blue catfish males is limited, and their peak spawning is at a different time than that of the channel catfish. As such, cryopreservation of sperm of blue catfish could improve production of hybrid catfish, and has been studied in the laboratory and tested for feasibility in a commercial dairy bull cryopreservation facility. However, an approach for commercially relevant production of cryopreserved blue catfish sperm is still needed. The goal of this study was to develop practical approaches for commercial-scale sperm cryopreservation of blue catfish by use of an automated high-throughput system (MAPI, CryoBioSystem Co.). The objectives were to: 1) refine cooling rate and cryoprotectant concentration, and evaluate their interactions; 2) evaluate the effect of sperm concentration on cryopreservation; 3) refine cryoprotectant concentration based on the highest effective sperm concentration; 4) compare the effect of thawing samples at 20 °C or 40 °C; 5) evaluate the fertility of thawed sperm at a research scale by fertilizing with channel catfish eggs; 6) test the post-thaw motility and fertility of sperm from individual males in a commercial setting, and 7) test for correlation of cryopreservation results with biological indices used for male evaluation. The optimal cooling rate was 5 °C/min (Micro Digitcool, IMV) for high-throughput cryopreservation using CBS high-biosecurity 0.5-ml straws with 10% methanol, and a concentration of 1 × 109 sperm/ml. There was no difference in post-thaw motility when samples were thawed at 20 °C for 40 s or 40 °C for 20 s. After fertilization, the percentage of neurulation (Stage V embryos) was 80 ± 21%, and percentage of embryonic mobility (Stage VI embryo) was 51 ± 22%. There was a significant difference among the neurulation values produced by thawed blue catfish sperm, fresh blue catfish sperm (P = 0.010) and channel catfish sperm (P = 0.023), but not for Stage VI embryos (P ≥ 0.585). Cryopreserved sperm from ten males did not show significant variation in post-thaw motility or fertility at the neurulation stage. This study demonstrates that the protocol established for high-throughput cryopreservation of blue catfish sperm can provide commercially relevant quantities and quality of sperm with stable fertility for hybrid catfish production and provides a model for establishment of commercial-scale approaches for other aquatic species.

3-D printing provides a novel approach for standardization and reproducibility of freezing devices

“…This approach evolves naturally from a standardization and quality assurance approach traditionally used for high-throughput mass production in industry [18]. Individual cryopreservation processes can be standardized or harmonized by systematically reducing the variation at each step.…”

Sperm cryopreservation of silver barb (Barbodes gonionotus): cryoprotectants, cooling rate and storage time on sperm quality

“…Optimal freezing rate is also dependent on factors such as cell type and size, cryoprotectant type and concentration, equilibration time, final temperature prior to plunging in liquid nitrogen, fish species and associated interactions (Viveiros et al 2000;Hu et al 2011). The use of an excessively slow freezing rate caused cell dehydration and solute effect in the cell, while an exceptionally fast freezing rate reduced cellular water diffusion rate and produced ice crystals in the cell (Ohta et al 2001).…”

A numerical study on distributions during cryoprotectant loading caused by laminar flow in a microchannel

“…Although a microdevice may operate at steady state, each cell will move through this device and encounter different extracellular concentrations as a function of time. While there has been a recent push to optimize cryopreservation protocols for survivability and throughput, 2,4,11,18 to optimize CPA loading in microfluidic devices, a more rigorous approach is necessary.…”

“…The application of microfluidics to the CPA loading process also meets the needs cited by researchers in the cryopreservation field, namely, the ability to create highthroughput, readily reproducible, and streamlined processes needed in research and industry. 2,4,6 With a stable of advantages and the decreasing cost of device fabrication, microfluidic devices have the potential to become commonplace in cryopreservation protocols.…”

“…[1][2][3][4][5][6] Cryopreservation is critical to long term storage and off-the-shelf availability. 3,7 Typical cryopreservation protocols aim to remove intracellular water to avoid damaging intracellular ice formation (IIF).…”