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.