Frozen spermatozoa compositions and uses thereof
View Patent ↗The present invention provides a composition for freezing or freeze-drying spermatozoa or sperm heads thereof, wherein the composition enables the spermatozoa or sperm heads thereof to maintain chromosome integrity at a temperature of about +4° C. to about −200° C. Also provided is a container containing the composition. The present invention further provides a method for maintaining chromosome integrity of spermatozoa or sperm heads during freezing or freeze-drying. The present invention also provides methods for freezing or freeze-drying spermatozoa to obtain at least one spermatozoan capable of fertilizing an oocyte to produce a live offspring. Also provided are frozen or freeze-dried spermatozoa produced by these methods, and containers containing the frozen or freeze-dried spermatozoa. Finally, the present invention provides methods for producing a live mammalian offspring from an oocyte fertilized with a rehydrated freeze-dried (or thawed or freeze-thawed) spermatozoan.
1. A composition for freezing or freeze-drying spermatozoa or sperm heads thereof, comprising a buffered medium, an ion-chelating agent, and spermatozoa or sperm heads thereof, wherein the composition enables the spermatozoa or sperm heads to maintain chromosome integrity at a temperature of about +4° C. to about −200° C., and the pH of the composition is between 8.0 and 8.6.
2. The composition of claim 1 , wherein the pH of the composition is between 8.2 and 8.4.
3. A composition for freezing or freeze-drying spermatozoa or sperm heads thereof, comprising a buffered medium, an ion-chelating agent, and spermatozoa or sperm heads thereof, wherein the composition enables the spermatozoa or sperm heads to maintain chromosome integrity at a temperature of about +4° C. to about −200° C. and the composition is freeze-dried.
4. The composition of claim 3 , wherein the composition's moisture level is less than about 5%.
5. A method for maintaining chromosome integrity of spermatozoa or sperm beads in a composition during freezing or freeze-drying, comprising adding an ion-chelating agent to the composition prior to freezing or freeze-drying, wherein the pH of the composition is between 8.0 and 8.6.
6. The method of claim 5 , wherein the pH of the composition is between 8.2 and 8.4.
7. A method for maintaining chromosome integrity of spermatozoa or sperm heads in a composition during freezing or freeze-drying, comprising adding an ion-chelating agent to the composition prior to freezing or freeze-drying, wherein the composition is freeze-dried.
8. The method of claim 7 , wherein the composition's moisture level is less than about 5%.
9. A method for freezing spermatozoa to obtain at least one spermatozoan capable of fertilizing an oocyte to produce a live offspring, comprising the steps of:
(a) collecting live spermatozoa;
(b) suspending the spermatozoa in a composition comprising a buffered medium and an ion-chelating agent to produce a suspension of spermatozoa; and
(c) freezing the suspension of spermatozoa, to produce a frozen suspension, and further comprising the step of demembranating the spermatozoa, prior to the freezing step, to produce demembranated spermatozoa heads.
10. A method for freeze-drying spermatozoa to obtain at least one spermatozoan capable of fertilizing an oocyte to produce a live offspring, comprising the steps of:
(a) collecting live spermatozoa;
(b) suspending the spermatozoa in a composition comprising a buffered medium and an ion-chelating agent, to produce a suspension of spermatozoa;
(c) freezing the suspension of spermatozoa, to produce a frozen suspension; and
(d) drying the frozen suspension of spermatozoa to a moisture level of less than 1%, to produce freeze-dried spermatozoa.
11. The method of claim 10 , wherein the spermatozoa are collected from a vertebrate.
12. The method of claim 10 , further comprising the step of demembranating the spermatozoa, prior to the freezing step, to produce demembranated spermatozoa heads.
13. The method of claim 10 , wherein the buffered medium is a physiological suspension medium.
14. The method of claim 10 , wherein the buffered medium is Tris-HCl buffer.
15. The method of claim 10 , wherein the ion-chelating agent is EGTA.
16. The method of claim 10 , wherein the freezing step is carried out at −196° C. for 10 minutes.
17. The method of claim 10 , further comprising the step of:
(e) rehydrating the frozen suspension of spermatozoa to produce rebydrated spermatozoa, wherein at least one rehydrated spermatozoan is capable of fertilizing an oocyte to produce a live offspring.
18. The method of claim 17 , further comprising the step of storing the freeze-dried spermatozoa for a period of time prior to the rehydrating step.
19. The method of claim 18 , wherein the freeze-died spermatozoa are stored at ambient temperature.
20. The method of claim 18 , wherein the freeze-dried spermatozoa are stored at about 4° C.
21. The method of claim 18 , wherein the freeze-dried sperm are stored at or below −20° C.
22. The method of claim 18 , wherein the period of time is up to about three months.
23. The method of claim 18 , wherein the period of time is up to one year.
24. The method of claim 18 , wherein the period of time is greater than one year.
25. Freeze-dried spermatozoa produced by the method of claim 10 .
26. A container containing freeze-dried spermatozoa produced by the method of claim 10 .