Materials and reagents

Biological materials

1. Adult zebrafish males and females

This protocol has been successfully used with the India strain (provided by Dr. Y. Kishimoto, National Institute of Genetics, Japan), the AB strain (provided by Dr. U. Strähle, Karlsruher Institute of Technology, Germany), the RW strain (provided by RIKEN, NBRP zebrafish), and their derivative transgenic and mutant lines. It could be applied to other zebrafish strains. Sexually mature males with a reddish body color and females with a fuller abdomen are suitable for sperm and egg collection, respectively.

2. Adult medaka males and females

This protocol has also been applied to the medaka OK-Cab strain (MT830-NIBB, provided by NBRP medaka), and other medaka strains could be used as well. Sexually mature males and females (4–6-month-old) exhibiting secondary sex characteristics in the anal and dorsal fins are suitable for sperm and egg collection. meioC loss-of-function mutants, in which germ cells arrest at the early stage of spermatogonia [10], can be used as infertile males to mate with wild-type females for the collection of unfertilized eggs.

Reagents

1. L-15 medium, Leibovitz (Sigma, catalog number: L5520)

2. Penicillin-Streptomycin, 5,000 U/mL (Gibco, catalog number: 15070-063)

3. D(+)-Glucose (Wako, catalog number: 041-00595)

4. Fetal bovine serum (FBS), Mexican origin (Sigma, catalog number: 173012, lot number: 0001653205)

5. Bovine serum albumin (BSA), fraction V, cell culture glade (Sigma, catalog number: A9418)

6. HEPES, suitable for cell culture (Sigma, catalog number: H4034)

7. L-(+)-Lactic acid (Sigma, catalog number: L6402-1G)

8. 1 M NaOH (Wako, catalog number: 196-05375)

9. Tricaine (3-amino benzoic acid ethylester) (Sigma, catalog number: A-5040)

10. NaCl (Wako, catalog number: 191-01665)

11. KCl (Wako, catalog number: 160-03555)

12. CaCl₂·2H₂O (Wako, catalog number: 031-00435)

13. MgSO₄·7H₂O (Wako, catalog number: 131-00405)

14. 0.82% methylene blue solution (Japan Pet Design, JAN code: 4975677045615)

15. 70% ethanol (Amakasu Chemical Industry, catalog number: NKH)

16. Bleach solution (Oyalox, JAN code: 4987038730016)

17. NaHCO₃ (Wako, catalog number: 191-01305)

Solutions

1. 1 M HEPES-NaOH pH 7.9 (see Recipes)

2. 5% BSA (see Recipes)

3. 2 M glucose (see Recipes)

4. 1 M L-(+)-lactic acid (see Recipes)

5. 10 mM L-(+)-lactic acid (see Recipes)

6. Sperm preservation medium (see Recipes)

7. 500× tricaine (see Recipes)

8. 1× tricaine (see Recipes)

9. 50× E3 stock solution for zebrafish (see Recipes)

10. 1× E3 for zebrafish (see Recipes)

11. 20× solution A for medaka (see Recipes)

12. Solution B for medaka (see Recipes)

13. Balanced salt solution (BSS) for medaka (see Recipes)

Recipes

1. 1 M HEPES-NaOH pH 7.9

Add 23.83 g of HEPES to ~80 mL of ddH₂O. Dissolve completely. Adjust pH to 7.9 with 1 M NaOH. Make the volume to 100 mL with ddH₂O. Filter sterilize using a 0.2 μm filter, aliquot, and store at -20 °C.

2. 5% BSA

Add 2.5 g of BSA to ~40 mL of ddH₂O in a 50 mL conical tube. Allow the powder to settle without mixing to prevent clumping. Once the powder has swelled, gently mix by rotating the tube, then adjust the volume to 50 mL with ddH₂O. Filter sterilize using a 0.2 µm filter, aliquot, and store at -20 °C.

3. 2 M glucose

Filter sterilize using a 0.2 μm filter and store at 4 °C. Warm up in a water bath if necessary.

4. 1 M L-(+)-lactic acid

Filter sterilize using a 0.2 μm filter. Store at -20 °C.

5. 10 mM L-(+)-lactic acid

Prepare in a sterile condition. Store at -20 °C.

6. Sperm preservation medium

Prepare on the day of sperm collection. Assemble all components in a sterile 1.5 mL tube on a clean bench. Ensure that the medium is prepared under sterile conditions to prevent contamination. The use of penicillin–streptomycin, which is commonly employed in cell culture media, is recommended as an antibiotic. Keep at room temperature (RT) until use.

7. 500× tricaine

Store as 1 mL aliquots at -20 °C.

8. 1× tricaine

Prepare in a 100 mL plastic beaker on the day of use. Mix by agitating the cup gently.

9. 50× E3 stock solution for zebrafish

Dissolve NaCl, KCl, CaCl₂·2H₂O, and MgSO₄·7H₂O in ~800 mL of dH₂O. Adjust pH to 7.2 with 1 M NaOH. Autoclave and store at 4 °C.

10. 1× E3 for zebrafish

Store at RT. Stable for a week.

11. 20× solution A for medaka

Dissolve NaCl, KCl, CaCl₂·2H₂O, and MgSO₄·7H₂O in 1 L of dH₂O. Autoclave and store at 4 °C.

12. Solution B for medaka

Add 2.5 g of NaHCO₃ to ~40 mL of ddH₂O in a 50 mL conical tube. Gently mix by rotating the tube, then adjust the volume to 50 mL with ddH₂O. Filter sterilize using a 0.2 µm filter.

13. Balanced salt solution (BSS) for medaka

Dilute 20× solution A 20-fold with dH₂O, autoclave, and adjust pH to 8.3 by adding solution B. Store at 4 °C.

Laboratory supplies

1. 1.5 mL tube (Watson, catalog number: 131-7155C)

2. Filtration disc with pore size of 0.2 μm (Advantec, catalog number: DISMIC-25CS)

3. 50 mL syringe, slip tip (Terumo, catalog number: SS-50ESZ)

4. 0.6 mL microtube (Watson, catalog number: 130-806C)

5. 1 mL syringe, slip tip (Terumo, catalog number: SS-01T)

6. 5 mL syringe, slip tip (Terumo, catalog number: SS-05SZ)

7. 50 mL syringe, slip tip (Terumo, catalog number: SS-50ESZ)

8. 10 μL tip (Watson, catalog number: 110-207C)

9. 200 μL tip (Watson, catalog number: 110-705C)

10. 1,000 μL tip (Watson, catalog number: 110-706C)

11. 90-mm Petri dish (BMBio, catalog number: BME-SNS0002)

12. Cellulose sponge (Nihon Insole Industry, no catalog number)

13. 100 mL plastic beaker (As One Corp., catalog number: I-100)

14. Plastic container

15. Membrane forceps (Advantec, catalog number: FS-1)

16. Cell counter plate, Burker-Turk (Watson, catalog number: 177-212C)

17. Microscissors (Roboz, model: RS-5803)

18. Tweezers, no. 5 Inox 08 (Dumont, model: 0108-5-PO)

19. Homogenizer pestle for 1.5 mL tube (BIO-BIK, catalog number: 1005-39)

20. Kimwipe (Nippon Paper Crecia, catalog number: S-200)

21. Paper towel (Konya Paper, catalog number: 4952137011014)

22. 24-well plate (Falcon^®, catalog number: 351147)

23. 60-mm Petri dish (Iwaki, catalog number: 1010-060)

Equipment

1. Stereomicroscope with epi-illumination (Olympus, model: SZ61-SZ2-ILST)

2. Stereomicroscope with trans-illumination (Evident, model: SZ61-SZX2-ILTS)

3. Inverted or upright microscope equipped with a 40× objective lens (Nikon, model: Eclipse TE2000-S, or equivalent)

4. Incubator (PHCbi, model: MIR-154-PJ)

5. Timer (any)

Procedure