Method for cryoconservation of Zebrafish sperm

• US 6,465,169 B2
• Filed: 12/19/2000
• Issued: 10/15/2002
• Est. Priority Date: 01/14/2000
• Status: Expired due to Term

• Current Assignee: Artemis Pharmaceuticals GmbH
• Original Assignee: Artemis Pharmaceuticals GmbH
• Inventors: Walderich, Brigitte, Nordin, Renate
• Primary Examiner(s): Marx, Irene
• Assistant Examiner(s): Afremova, Vera
• Application Number:
  US09/741,282
• Publication Number:
  US 20010023060A1
• Time in Patent Office: 665 Days
• Field of Search: 435/374, 435/325, 435/2
• US Class Current: 435/2
• CPC Class Codes: A01K 61/00 Culture of aquatic animals receptacles for live fish, e.g. aquaria, A01K63/00
• CPC Class Codes: A01K 61/10 of fish
• CPC Class Codes: A01K 67/02 Breeding vertebrates
• CPC Class Codes: A61D 19/02 for artificial insemination
• CPC Class Codes: A61D 19/025 with means for controlling the temperature, e.g. heating or cooling
• CPC Class Codes: Y02A 40/81 Aquaculture, e.g. of fish

Abstract

A method for cryoconservation of Zebrafish sperm, and Zebrafish sperm obtained by said method.

Claims

1. A method for cryoconservation of Zebrafish sperm that comprises (a) removing the testes from the body cavity of a male Zebrafish;

(b) isolating sperm from the testes by flushing the testes isolated in step (a) with a cryoprotectant using a micropipette to yield a sperm suspension having a volume of less than 30 μ

l per fish; and

(c) freezing said sperm suspension.

2. The method of claim 1, wherein prior to step (a) the male Zebrafish is anaesthetized, dried, and/or decapitated.

3. The method of claim 1, wherein the amount of cryoprotectant used in step (b) is from 10 to 25 μ

l.

4. The method according to claim 3, wherein the amount of cryprotectant used in step (b) is from 17 to 22 μ

l.

5. The method of claim 1, wherein the cryoprotectant is a modified 1×

Ginzburg-Ringer-solution containing a polar organic solvent.

6. The method of claim 1, wherein the temperature of the cryoprotectant is from 0 to 10°

C.

7. The method of claim 6, wherein the temperature of the cryoprotectant is from 2 to 5°

C.

8. The method of claim 5, wherein the modified 1×

Ginzburg-Ringer-solution contains 5 to 20 vol. % of the polar organic solvent and optionally contains 5 to 25 wt.-% of a concentrated protein.

9. The method of claim 8, wherein the modified 1×

Ginzburg-Ringer-solution contains 10 to 17 vol. % of the polar organic solvent and optionally contains 10 to 20 wt.-% of a concentrated protein.

10. The method of claim 5, wherein the polar organic solvent is selected from methanol, DMSO, and ethanol.

11. The method of claim 10, wherein the polar solvent is methanol.

12. The method of claim 8, wherein the concentrated protein is selected from milk powder, soya and bovine serum albumin.

13. The method of claim 12, wherein the concentrated protein is milk powder.

14. The method according to claim 1, wherein prior to step (c) the sperm suspension is divided into 2 to 5 portions.

15. The method of claim 14, wherein the sperm suspension is divided into 5 μ

l portions.

16. The method of claim 14, wherein the portions are kept in glass capillaries.

17. The method according to claim 1, where in step (c) the sperm suspension is kept at −

70°

C. (±

10°

C.) for at least 30 minutes and is then cooled down to the temperature of liquid nitrogen.

18. The method of claim 1 wherein the micropipette is a 10 μ

l cristal tip.