Natural mating with sterile triploid males after artificial insemination recapitulates manual sperm mixing to restore the fertility of cryopreserved semen in Bombyx mori.

Silkworm sperm exist as two types: eupyrene and apyrene. Apyrene sperm lack nuclei and are incapable of fertilization. However, they play a crucial role in facilitating fertilization by assisting nucleated sperm in migrating from the bursa copulatrix to the spermatheca. Additionally, apyrene sperm exhibit lower cryotolerance compared with eupyrene sperm, resulting in significantly reduced fertility following cryopreservation. Conventionally, the fertility of cryopreserved semen has been restored by mixing it with semen from sterile triploid males, which contains functional apyrene sperm and nonfunctional eupyrene sperm. However, collecting semen from triploid males is labor-intensive. To reduce this effort, we investigated whether mating with triploid males could improve the fertility of cryopreserved semen without artificial mixing with semen from triploid males. The results of this study demonstrated that females mated with triploid males and artificially inseminated with cryopreserved semen produced more fertilized eggs compared with unmated females. Furthermore, females mated with triploid males after artificial insemination (AI) with cryopreserved semen laid more fertilized eggs and exhibited a significantly higher fertilization rate than those mated before AI with cryopreserved semen. Notably, injecting 5 μL of cryopreserved semen produced more fertilized eggs than 10 μL when females were mated with triploid males after AI. These findings suggest that mating with triploid males eliminates the need for semen collection and permits AI with smaller volumes of cryopreserved semen.