Silkworm Resource Division, Institute of Genetic Resources, Kyushu University Graduate School of Bioresources and Bioenvironmental Science, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan.
Genetic Resources Conservation Research Unit, Genetic Resources Center, National Institute of Agrobiological Sciences, Tsukuba, 305-8602, Japan.
Cryobiology. 2020 Aug;95:9-14. doi: 10.1016/j.cryobiol.2020.06.016. Epub 2020 Jul 1.
Successful cryopreservation of the important silkworm bioresource, Bombyx mori, is essential. In this study, we aimed for successful cryopreservation using vitrification of silkworm embryos. Furthermore, the embryos were assessed for the most appropriate sampling stage. We found that vitrified embryos developed to the serosa ingestion stage when they were vitrified at embryonic stage 24-25. The most suitable stage for vitrification was around a 5-10 h period when the tracheal fibers were elongating in stage 25. None of the vitrified embryos developed into larvae, although some did develop to the pre-hatching stage. From histological analysis, we found that several small cracks formed on the cuticle covering the hypodermis in the vitrified embryos. Additionally, the midgut epithelium was detached from the midgut wall and mixed with the yolk in the midgut lumen. We speculate that the vitrified embryos died from a rapid loss of body water from the small cracks formed in the cuticle. We also suggest that the vitrified embryos may have resulted in dysfunction of the midgut.
成功地冷冻保存重要的家蚕生物资源是至关重要的。在本研究中，我们旨在通过家蚕胚胎的玻璃化冷冻来实现成功的冷冻保存。此外，还评估了胚胎的最佳取样阶段。我们发现，在家蚕胚胎 24-25 期进行玻璃化冷冻时，玻璃化冷冻的胚胎可以发育到原肠胚期。最适合玻璃化的阶段是在 25 期，此时气管纤维伸长，大约有 5-10 小时的时间。虽然有些胚胎发育到预孵化阶段，但没有一个玻璃化的胚胎发育成幼虫。从组织学分析中，我们发现玻璃化冷冻的胚胎的真皮覆盖物上形成了几个小裂缝。此外，中肠上皮与中肠壁分离，并与中肠腔中的卵黄混合。我们推测，玻璃化冷冻的胚胎因真皮上形成的小裂缝导致体内水分迅速流失而死亡。我们还认为，玻璃化冷冻的胚胎可能导致中肠功能障碍。
