Chemistry: molecular biology and microbiology – Maintaining blood or sperm in a physiologically active state...
Reexamination Certificate
2000-12-19
2002-10-15
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Maintaining blood or sperm in a physiologically active state...
C435S374000, C435S325000
Reexamination Certificate
active
06465169
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention provides a method for cryoconservation of Zebrafish sperm, and samples of Zebrafish sperm obtainable by said method.
Zebrafish sperm can be prepared by two different methods for cryoconsevation and In Vitro Fertilization (IVF) of Zebrafish eggs:
1. Sperm can be squeezed directly from male fish and frozen in cryoprotectant.
2. Sperm can be isolated from prepared fish testis and frozen in cryoprotectant. (Ransom & Zon, Methods in Cell Biology, Vol. 60, p. 365-372, (1999)). Said method comprises homogenizing the testis by using a Kontes pellet pestle.
Method 1 is established and functions, however, it has several disadvantages:
About 50% of all fish give no sperm sample by using the squeezing procedure.
Such fish can be squeezed again after 2 weeks: This needs space and time.
The recovered sperm has a volume of 0.5-2 &mgr;l, resulting in only one aliquot of sperm per squeezing, i.e. the procedure has to be repeated several times.
The squeezed sperm can differ very much in quality yielding fertilization rates between 0-50%.
Method 2 has been described to function in a protocol obtained from Ransom & Zon, 1999, however we have not been able to reproduce it. This method would have several advantages in comparison to the squeezing method:
Sperm samples can be obtained from every single fish.
Several sperm aliquots can be frozen from one testis preparation: time and space saving.
SUMMARY OF THE INVENTION
Starting from the method of Ranson & Zon, (1999), a reliable method to freeze Zebrafish sperm prepared from testis was established. This method gives at least 2 sperm samples from one testis preparation. The new method can be done very quickly: 5 minutes per fish are needed for the sperm preparation. As demonstrated in 23 independent experiments, fertilization rates between 10-50% were observed, using fish between 4 and 12 months old.
The present invention thus provides a method for cryoconservation of Zebrafish sperm comprising
(a) removing the testis from the body cavity of a male Zebrafish;
(b) rinsing the testis isolated in step (a) with a cryoprotectant to yield a sperm suspension: and
(c) freezing said sperm suspension,
wherein the amount of cryoprotectant used in step (b) is such that the volume of the sperm suspension be less than 30 &mgr;l.
The present invention further provides samples of Zebrafish sperm obtained by the above method.
REFERENCES:
Harvey et al. Cryopreservation of zebra fish spermatozoa using methanol. Can. J. Zool. 1982. 60:1867-1870.*
Aoki et al. Cryopreservation of medaka spermatozoa. Zoological Science. 1997. 14;641-644.*
F.G.Ransom, et al.; Collection, Storage, and Use of Zebrafish Sperm; Methods of Cell Biology, vol. 60; 1999; pp. 365-372.
Nordin Renate
Walderich Brigitte
Afremova Vera
Artemis Pharmaceuticals GmbH
Brunelle Jan P.
Elson Sarah
Marx Irene
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