A new method for creating androgenetic diploid fish from tetraploid carp sperm and their genetic composition analysis

Abstract

The application of cold shock to inhibit the extrusion of the second polar body of fertilized eggs is one of the main methods to induce triploid in diploid fish. In this study, we originally planned to induce hexaploid by this method in tetraploid carp. However, among the surviving offspring, we observed only 50.7% tetraploid and 49.3% diploid individuals, which could potentially result from either androgenetic diploids or gynogenetic diploids. The absence of irradiation and the high proportion of diploids attracted us. However, the lack of sex-specific markers for tetraploid carp, makes it difficult to determine their origin. Fortunately, we also carried out the cross of yellow carp (Cyprinus carpio yellow var., 2n, ♀) × tetraploid carp (♂) and red crucian carp (Carassius auratus red var., 2n, ♀) × tetraploid carp (♂) with cold shock inhibiting the second meiosis, diploid offspring have also been found, and whether they have undergone gynogenesis or androgenesis can be judged based on phenotype and genotype. In terms of phenotypes, the key phenotypes of two diploids, such as the whisker length, number of lateral scales, and presence of melanin, are consistent with tetraploid carp. In terms of genotypes, through single-copy nuclear gene cloning and fluorescence in situ hybridization (FISH), it is supported that both diploids are androgenetic offspring. In addition, 14.9% of tetraploid offspring were found in red crucian carp (♀) × tetraploid carp (♂) with cold shock inhibiting the second meiosis. By the above-mentioned means of detection, it was found that they were created by inhibiting the extrusion of the second polar body of the fertilized eggs. From this it will be seen that the application of cold shock before the extrusion of the second polar body of fertilized eggs cause not only polyploidy, but also the elimination of both second polar body and female nucleus. Previous studies have found that cold shock can induce lethal androgenetic haploids in diploid fish, but these androgenetic diploid fish produced by tetraploid carp sperm were surviving. We summarize the mechanism behind the above phenomenon, which is mainly related to meiosis occurring at the edge of the cell membrane. Cold shock may disrupt the function of the spindle, resulting in the simultaneous retention or extrusion of female nucleus and second polar body. This study will provide valuable insights into androgenesis, polyploidization, and hybridization in fish genetic breeding.