Then, the hybrid combinations of 2n × 4n were created. Similarly, semen from the male fish was extruded from the genital pores of both sides, collected in centrifuge tubes, and diluted 100-fold in Kurokura solution (750 mg NaCl, 20 mg CaCl 2, 20 mg NaHCO 3, and 20 mg KCl dissolved in 100 ml distilled water). After 12 h, the abdomens of the females were gently pressed to discharge eggs, which were collected into 9 cm culture dishes. The loaches were subsequently injected with human chorionic gonadotropin (injection doses: females 20–25 IU g −1 males 10–12.5 IU g −1). For artificially induced spawning and insemination, progenitors were chosen from well-developed diploid male (2n = 50) and natural tetraploid female loaches (4n = 100).
China, and reared in aquaria (25 ± 1 ☌) in the HZAU laboratories. Wild-adult diploid male and tetraploid female loaches were collected from Wuhan City, Hubei Province, P.R.
The findings provide insights that can be applied to the development of triploid loach aquaculture. To elucidate the mechanisms of abortion in the hybrid triploid loach, in this study, histological observations were performed and transcriptomes were analyzed of both the early hybrid triploid loach and diploid controls at different developmental stages. Despite research on the reproductive capacity and cytogenetics of hybrid triploid loaches, their early fertility mechanism and concomitant molecular mechanisms remain unknown. Other individuals had either small and immature gonads or none at all. While some individuals developed gonads and produced a small number of either eggs or sperm, these gametes could not be fertilized. Their studies on the chromosomal stability and epigenetics of hybrid triploid loaches showed that sexuality varied among individuals. They created an intraspecific hybrid triploid by crossing diploid and tetraploid loaches, combining hybrid and triploid breeding. (2010, 2011) clarified that the natural tetraploid loach is a genetic tetraploid (4n = 100) which produces normal 2n eggs and 2n sperm. Loach species with five different ploidy levels were found in the natural waters of China, including diploids, triploids, tetraploids, pentaploids, and hexaploids. Natural triploids and tetraploids were first found among loaches in Japan. Such methods have been deemed the best way to realize the industrialization of triploid fish production. Yet, stable hybrid triploid loaches can be efficiently created by crossing tetraploids with normal diploids. Indeed, while inductions and cultivations are carried out annually, they require expensive equipment and toxic drugs, which result in high production costs. However, no currently available triploid induction method results in 100% triploids. Therefore, creating triploid loaches is not only of economic value but also promotes the further development of the loach aquaculture. Triploid fish are characterized by fast growth, high-quality meat, high edibility, high resistance to disease, and sterility-these characteristics are especially important in aquaculture. The rapid development of the loach breeding industry has made it desirable to cultivate high-quality loach breeds. Colloquially, it is referred to as "ginseng in water" because its meat is not only delicious but also nutritious, making it an important freshwater aquaculture product of China for both local and export markets. Misgurnus anguillicaudatus is taxonomically a member of the Misgurnus genus of the Cobitidae family in the Cypriniformes order. The findings laid a foundation for further exploration of their molecular inhibition mechanism in hybrid triploid loaches. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of DEGs, two pathways were identified that are closely related to the early fertility mechanism at 50dph: the estrogen signaling pathway and steroid biosynthesis. To explore its fertility mechanism, through transcriptome analyses of triploid offspring and diploid controls at four periods, 10 differentially expressed genes related to the early fertility mechanism were identified: amh, hormad1, rec8, h2b, plvap, zp3, h2a, nrb0b1, ddx4, and esr2. The histological observations of triploid offspring and diploid controls at 4 days post-hatching (dph), 15dph, 22dph, and 50dph showed that most of the hybrid triploid loaches were abortive in the early gonad differentiation process. In this study, a natural tetraploid loach (4n = 100) (♀) was crossed with a diploid loach (2n = 50) (♂), thus creating the hybrid triploid loach (3n = 75). As a new freshwater aquaculture product, triploid loaches ( Misgurnus anguillicaudatus) are characterized by fast growth, high-quality meat, high edibility, high resistance to disease, and sterility.
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