采用紫外线(UV)对冻存的花鲈精子进行照射使其遗传物质失活,作为异源精子诱导源,与漠斑牙鲆卵子进行"授精",可以诱导漠斑牙鲆卵进行雌核发育。结合静水压处理,抑制第二极体排出,成功获得了漠斑牙鲆雌核发育二倍体鱼苗。实验表明,同源精子和异源精子均可在紫外线照射遗传失活后诱导漠斑牙鲆卵雌核发育,经实验筛选出异源精子诱导漠斑牙鲆雌核发育的最佳条件为花鲈冻存精子采用80mJ/cm2的紫外线照射,然后与漠斑牙鲆卵子进行授精,培育水温18℃条件下,受精后4~5min,施以65MPa的静水压休克处理6min,可有效诱导漠斑牙鲆卵的雌核发育。采用形态学、流式细胞仪DNA含量分析和微卫星标记技术对雌核发育鱼苗进行了分析,证明了雌核发育鱼苗为雌核发育二倍体。本文首次报道了采用异源冷冻精子诱导漠斑牙鲆鱼卵进行雌核发育的技术方法,为漠斑牙鲆性别控制和遗传改良提供了技术手段。
Female southern flounder( Paralichthys lethostigma) grow larger than males.Therefore,all-female production will maximize profit potential for the culture of this species.It may be effective to develop protocols to produce all-female southern flounder through induction of meiotic gynogenesis with the cryopreserved heterologous sperm of Lateolabrax japonicas.To test methods for inducing diploid gynogenesis in southern flounder using heterogenous sperm,the UV was used to inactivate sperm and pressure shock was used to prevent extrusion of the second polar body.The results of experiments showed that gynogenetic diploid can be induced by inactivated heterogenous sperm because that hybrid would die before hatching.Diploid gynogenesis was induced by activating egg development with UV irradiated sperm( 80 mJ/cm2) 4-5 min after fertilization ( 18 ℃) ,and then 65 MPa pressure treatment for 6 min.Flow cytometry and microsatellite DNA analysis were used to demonstrate that the gynogenetic fry are diploidy.These results indicate that the use of UV irradiated sperm from Lateolabrax japonicas for activation of flounder eggs and pressure shock for polar body retention is an effective method to produce gynogenetic diploid in southern flounder.