Artificial insemination using cryopreserved sperm in the silkworm, Bombyx mori

We report in this paper that female moths artificially inseminated with cryopreserved sperm (−196°C) could oviposit eggs when the sperm was preserved for 356 days, and that the fertilization rate and the number of eggs laid were almost equivalent to those obtained in normally mated moths. The optimal cooling rate for sperm freezing was 5–65°C/min for maintaining a high fertility of sperm. The simple and reliable method of cryopreservation was to put the semen first in a deep freezer at −80°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 very small number of eggs of normal color. The result shows that there was a competitive fertilization of sperm between the two strains of the silkworm, and that sperm fertility was reduced to a considerable extent by freezing at −196°C. These results may contribute not only to basic studies on fertilization in Lepidoptera but also to the development of long-term preservation procedure of genetic resources by using cryopreserved sperm of Bombyx mori.