Preliminary Study On Induction Of Triploid Hybrid Between Japanese Flounder(♀)×spotted Halibut(♂)

Japanese flounder (Paralichthys olivaceus) is an important kind of marineeconomic fish, and it is valuable for its commercial significance. In many recentresearches, triploid fish has been proved to possess reproductive traits of low fertilityor sterility, in addition to its advantages in growth. Thus in recent years, triploids haveserved as ideal receptors in fish germ cells transplantation. After exogenous germ cellsare transplanted, sterility triploid fish can eliminate the interference from endogenousgerm cells. In the present study, the sperm of spotted halibut (Verasper variegatus) wascryopreserved; both hybrid triploids and tetraploids of Japanese flounder （♀）andspotted halibut （♂）were induced by hydrostatic pressure shocks. The optimal inductionconditions were screened, and the triploids were generated for future germ cellstransplantation.1. The sperm of spotted halibut were collected and cryopreserved. Using HBSS asthe cryodilution and15%DMSO as the cryoprotectant was screened to be an effectivecondition. Artificial fertilization was conducted after the sperm recovery and activation,and the fertilization rate could reach (61.00±2.00)%. The motility ratio was detected.The survival time of cryopreserved sperm (150s) was detected to be significantlyshorter than that of fresh sperm (250s) after activation. It was possible that themorphological characteristics of the sperm were injured during cryopreservation, andit caused the lower biological activity.2.Hybrid triploids were generated by fertilizing the gametes of Japanese flounder（♀）and spotted halibut (♂), and by inhibiting the second polar extrusion underhydrostatic pressure shocks. Besides, the optimal conditions were screened. Hydrostaticpressure method was proved to induce the hybrid triploids successfully. The inductionrate was mainly influenced by three factors: the timing after fertilization, the intensityof pressure, and the duration of pressure. The optimal conditions of hydrostatic pressure method were screened: the temperature of sea water was15℃±0.2℃, the initial timeof treatment is3min after fertilization, and inducing6min under70MPa. Under theoptimal conditions, the relative hatching rate was (24.06±1.10)%. Analysis of ploidywas conducted and the triploid rate was (26.67±6.67)%. Chromosome analysisdetected that, the chromosome number in each triploid embryo was71, while it was47in each controlled diploid embryo. Besides, several haploid embryos with24chromosomes were found.3.Hybrid tetraploids were generated under hydrostatic pressure shocks bysuppressing the first cleavage of Japanese flounder （♀）×spotted halibut （♂）zygotes.Results showed that, tetraploids could be generated under conditions of15℃±0.2℃sea water,6min under65MPa, and the treatment began at100-110min after fertilization.But during induction, when the embryos developed to gastrulas, extremely lowhatching rate was caused by massive death of the embryos. Chromosome analysis foundthat chimeras existed among the tetraploid embryos.