Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Summary

The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Additionally, the repeatability of the method in five different zebrafish strains was tested.

Abstract

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

Introduction

Novel trends in science and biotechnology have led to the creation of thousands of new mutant lines of mice, Drosophila, zebrafish and other species used as model organisms in biomedical and other sciences¹. Furthermore, as new technologies are developed and become available, the numbers of mutant lines steadily increase². This leads to the necessity for a safe storage of genetic resources beyond the common practices of keeping breeding colonies. As a method which enables safe storage of genetic resources for an indefinite period of time, cryopreservation offers many advantages such as extension of reproductive ....

Protocol

All methods described here have been approved by the Hungarian Animal Welfare Law.

1. Reagent Preparation

1. Stock solutions
   1. Prepare 1 M trehalose by adding 0.378 g of trehalose dihydrate into 1 mL of dH₂O. Mix well until it dissolves completely.
   2. Prepare 1 M sucrose by adding 0.342 g of sucrose into 1 mL of dH₂O. Mix well until it dissolves completely.
   3. Prepare 1 M HEPES by adding 0.238 g of HEPES to 1 mL of dH₂O. Mix well until it dissolves completely.
   4. Prepare 20 mg/mL collagenase (290 U/mg) by adding 100 mg to 5 mL of PBS. Mix thoroughly until ....

Results

On average, the number of early-stage germ cells isolated from a single fresh zebrafish testis varied between 40,000 and 200,000 cells depending on the size of the fish. When digesting fresh zebrafish testes in all 5 strains, early-germ cells were not the only cells present in the cell suspensions (Figure 2). Beside the early-stage germ cells, numerous spermatozoa were found as well. On the other hand, there were far less spermatozoa after digestion of cryopr.......

Discussion

The main purpose of this study was to adapt the needle immersed ultra-rapid cooling procedure developed for avian and mammalian species^10,11,12 to the cryopreservation of fish testis (zebrafish as a model organism). Most of the previous studies regarding cryopreservation of zebrafish genetic resources were primarily focused on cryopreservation of zebrafish sperm^2,3,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Leibovitz media (L-15)	Sigma-Aldrich	L1518	Supplemented with L-glutamine
Fetal bovine serum (FBS)	Sigma-Aldrich	F9665
Tricaine methanesulfonate (MS-222)	Sigma-Aldrich	E10521
HEPES	Sigma-Aldrich	H3375
Sucrose	Acros Organics	57-50-1
Trehalose	Acros Organics	99-20-7	Dihydrate
Methanol	Reanal	20740-0-08-65
Propylene glycol	Reanal	08860-1-08-65
Dimethyl sulfoxide	Reanal	00190-1-01-65
Collagenase	Gibco	9001-12-1
Trypsin	Sigma-Aldrich	T8003
DNase I	Panreac AppliChem	A3778
Trypan blue	Sigma-Aldrich	T6146
Phospate buffered saline (PBS)	Sigma-Aldrich	P4417	Tablets for preparation of 200 ml PBS solution