为了探寻三角帆蚌胚胎体外培养方法，本实验以解剖获得的雌雄配子为材料，使用与三角帆蚌体液等渗的平衡盐溶液(BSS)进行人工体外受精和体外培养，观察各阶段胚胎的形态特征，并分析计算不同胚胎发育阶段的生物学零度和有效积温。结果显示，在温度为(25±1) °C的BSS培养条件下，受精卵发育至第二极体排放、二细胞期、四细胞期、八细胞期、十六细胞期和桑葚期的时间分别为1.8 、2.8 、5.6 、10.5 、13.9和17.3 h，胚胎最长发育至桑葚期后停止发育。另外，池塘平均水温分别为26.5、28.1和29.2 °C时，三角帆蚌胚胎发育至成熟钩介幼虫分别需要10、9和8 d，计算得出三角帆蚌胚胎发育的生物学零度为14.81 °C，根据生物学零度计算胚胎发育至卵裂期、囊胚期、原肠期、膜内钩介幼虫期和成熟钩介幼虫的有效积温分别为12.95、25.99、42.27、69.21和118.14 °C×d。研究结果初步尝试对三角帆蚌受精卵进行体外培养，并根据胚胎发育水温和时间获得三角帆蚌早期胚胎发育的生物学零度和有效积温，可为突破三角帆蚌全人工繁育技术奠定基础，对淡水贝类现代生物育种技术研发和种质资源保护具有重要意义。

Hyriopsis cumingii is a unique freshwater breeding pearl mussel in our country. It has the reproductive biology characteristics that the fertilized eggs develop to the larvae in gills. The unique reproductive biology characteristics make the offspring produced by the mussels random in the process of artificial breeding and restrict the application of modern biological breeding techniques to mussels such as triploid induction, gynogenesis and gene editing. In this study, in order to do research on development of H. cumingii embryos and explore the in vitro culture of its early embryos, male and female gametes obtained by anatomy were used as materials for in vitro fertilization and in vitro culture with isoosmotic balance salt solution (BSS) suitable for freshwater mussels. And the morphological characteristics of each stage of embryo were observed with the required time for development recorded. Meanwhile, the embryos in its nurturing pouch of outer gill were continuously observed and the water temperatures were recorded day and night at the same time, then we calculated the embryonic developmental biological zero point and examined the reliability of the biological zero point. After that, effective accumulated temperature of each embryonic development stage was calculated according to the biological zero point confirmed. Results show that when the water temperature is (25 ± 1) °C, the anatomy of the sperms and eggs were mixed after diluting by BSS saline and fertilized eggs could develop to morula stage. The fertilized eggs developed to polar body emissions at 1.8 h, developed to 2-cell stage at 2.8 h, developed to 4-cell stage at 5.6 h, developed to 8-cell stage at 10.5 h, developed to 16-cell stage at 13.9 h, and developed to morula stage at 17.3 h after observing under optical microscope. And then embryos did not change, and some of embryos were deformed. The morphological characteristics of embryos at each stage under in vitro culture condition were similar to those in the gills. Meanwhile, when the average water temperature in the pond was 26.54 °C, 28.08 °C and 29.51 °C, it took respectively 10 d, 9 d and 8 d for embryos to develop into mature glochidium. And the biological zero point for embryonic development of H. cumingii was 14.81 °C which was confirmed reliable after examining. The effective accumulated temperature were 12.95 °C×d for embryos developing to cleavage stage, 25.99 °C×d for embryos developing to blastocyst stage, 42.27 °C×d for embryos developing to gastrulation stage, 69.21 °C×d for embryos developing to glochidium stage and 118.14 °C×d for embryos developing to cleavage stage mature mlochidium stage, respectively, according to the biological zero point calculated previously. The study tried to cultivate H. cumingii embryos under in vitro condition, which confirmed the feasibility to cultivate H. cumingii embryos under in vitro condition, and then we analyzed and calculated the biological zero point and effective accumulated temperature of early embryonic development. The results of this study can provide reference for the researches on artificial breeding and development of modern biological genetic breeding technology of H. cumingii.

S966.22⁺1

国家重点研发计划(2018YFD0901406)；现代农业产业技术体系专项(CARS-49)；国家自然科学基金(31872565)；江苏省苏北科技专项(SZ-SQ2019045)