Cryopreservation of sperm
First Claim
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1. A method of providing sperm comprising:
- a) cooling a sample which includes sperm to a first temperature sufficient to protect sperm from glycerol toxicity, at a rate sufficiently slow that the metabolic rate of sperm is decreased, to provide a cooled sperm sample;
b) adding a solution comprising glycerol to the cooled sperm sample; and
c) freezing said cooled sperm sample to second temperature for a sufficient period of time to equilibrate glycerol and sperm to thereby provide a frozen sperm sample, such that the sperm is preserved.
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Abstract
The invention features methods of cryopreserving sperm. The methods include providing a sample of sperm; cooling the sperm to a first temperature; maintaining the sperm sample at the first temperature; cooling the sperm to a second temperature; maintaining the sperm at the second temperature; and storing the sperm at a third temperature. These methods can be used to cryopreserve sperm from mammals, e.g., transgenic mammals and can be to preserve sperm for subsequent artificial insemination or in vitro fertilization.
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Citations
26 Claims
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1. A method of providing sperm comprising:
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a) cooling a sample which includes sperm to a first temperature sufficient to protect sperm from glycerol toxicity, at a rate sufficiently slow that the metabolic rate of sperm is decreased, to provide a cooled sperm sample;
b) adding a solution comprising glycerol to the cooled sperm sample; and
c) freezing said cooled sperm sample to second temperature for a sufficient period of time to equilibrate glycerol and sperm to thereby provide a frozen sperm sample, such that the sperm is preserved. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26)
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13. A method of preserving sperm comprising:
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a) combining sperm with a first cryoprotectant buffer;
b) cooling said sperm to a first temperature between about 2°
C. to about 10°
C. at a rate sufficiently slow that the metabolic rate of sperm is decreased to produce cooled sperm;
c) freezing the cooled sperm at a second temperature between about −
60°
C. to about −
90°
C.; and
d) storing the frozen sperm in liquid nitrogen.
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24. A method of providing sperm comprising:
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a) providing a sample comprising sperm;
b) isolating sperm from the sample;
c) combining said isolated sperm with a first cryoprotectant buffer which lacks glycerol;
d) cooling said sperm to a first temperature of about 2°
C. to about 8°
C. at a rate of about 0.2°
C. to 0.5°
C. per minute to produce cooled sperm;
e) adding a second cryoprotectant buffer which contains glycerol;
f) maintaining the cooled sperm at the first temperature for a duration of about 4 hours to about 21 hours;
g) freezing the cooled sperm at a second temperature of about −
60°
C. to about −
90°
C. for a time of between about 10 minutes to about 15 minutes;
h) storing the frozen sperm at a temperature of between about −
180°
to about −
220°
C. for a desired time period; and
i) thawing said sperm, to thereby provide sperm.
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Specification