Artificial insemination using cryopreserved sperm in the silkworm, Bombyx mori

We report in this paper that female moths artificially inseminated with cry opreserved sperm (-196 degrees C) could oviposit eggs when the sperm was pr eserved for 356 days, and that the fertilization rate and the number of egg s laid were almost equivalent to those obtained in normally mated moths. Th e optimal cooling rate for sperm freezing was 5-65 degrees C/min for mainta ining a high fertility of sperm. The simple and reliable method of cryopres ervation was to put the semen first in a deep freezer at -80 degrees C and thereafter put them in liquid nitrogen. When female moths of 'white 2' egg- color mutant strain were inseminated with a mixture of frozen-thawed sperm from males of normal-colored egg strain and non-frozen sperm from males of the 'white 2', female moths deposited a majority of 'white 2' eggs and a ve ry small number of eggs of normal color. The result shows that then was a c ompetitive fertilization of sperm between the two strains of the silkworm, and that sperm fertility was reduced to a considerable extent by freezing a t - 196 degrees C. These results may contribute not only to basic studies o n fertilization in Lepidoptera but also to the development of long-term pre servation procedure of genetic resources by using cryopreserved sperm of Bo mbyx mori. (C) 2000 Elsevier Science Ltd. All rights reserved.