Studies On Sperm Of Different Ploidy Penetration Into Egg And Fertilizated Cytology In Oncorhynchus Mykiss

Rainbow trout (Oncorhynchus mykiss, Walbaum) is affiliated to Osteichthyes, Salmoniformes, Salmonidae, Salmoninae, Oncorhynchus. It originates in the western Pacific coast of North America and was one of famous and expensive cold water species which begin to the earliest artificial breeding and was the most widely distributed in the world. It has of cultivated history of fifty years inducted into china in1959, but most of cultivated technology of Trout was inducted from abroad. So the basic research on Rainbow trout still has important theoretical directive significance.This paper carried out studies on scanning electron microscope observation of tetraploid sperm penetration into diploid Rainbow trout eggs, the whole process of fertilization cytology of diploid Rainbow trout eggs and triploid Rainbow trout sperm, confirmation of sampling time point of fertilization rate. It designed to provide a theoretical basis and technical guidance for artificial propagation, fry rearing, genetic and breeding, for improving fertilization and hatching rate on cold water fishes such as Rainbow trout.The result showed that the tetraploid rainbow trout sperm is typical flagellar structure which composed of three parts that were head, neck and tail; It has no acrosome but has a palpable neck; long diameter and short diameter of head part is respectively3.22±0.21μm and2.88±0.22μm. The micropylar apparatus of diploid eggs located in the centre of funnel depression surrounded circular projection in animal pole of egg membrane and composed by two parts of fertilized vestibule and long fertilized aperture pipeline.The vestibule was shallow, flat and open, approximate to circular.and outside diameter is33.1±0.16μm. The fertilized aperture pipeline was deep, its export is circular and outside diameter is6.67±0.19μm; spiral crest ring of its inner wall extended to the bottom and inside diameter. The micropylar apparatus was closed and has no floc cover before dry fertilization but no Injecting water; The micropylar apparatus still was closed and no sperm reached to fertilization vestibule after sperms and eggs meeting1s、3s、5s、10s. At1s after dry fertilization and injecting water, micropylar apparatus located in the surface of egg membrane opened but relatively small where no sperm reached. At5s after fertilization, most of sperms attached to eggs membrane and the largest number centralized nearby of micropylar apparatus; a few of sperms of strong vitality arrived the fertilized aperture across cyclic annular apophysis and sperm head contact with penetration sites while sperm tail swung in fertilization vestibule. At10s after fertilization, head of sperm around micropylar apparatus pointed to export but far from micropylar apparatus are respectively different. At20s after fertilization, several sperms enter into fertilized aperture pipeline in turn and its heads individually completely disappear in micropylar apparatus inside. At30s after fertilization, heads of all sperm which arrive to micropylar apparatus completely enter into fertilized aperture pipeline but its tails stay outside of hole. At40s after fertilization, Late sperms are blocked by scattered tiles fragments of fertilized aperture pipeline outside. At50s after fertilization, fertilized hole is partly blocked by scattered tiles fragments which produce fusion. At60s after fertilization, fertilized hole is completely blocked by "fertilization bolt" which is formed scattered tiles fragments; sperm heads already enter into hole but the tails stay outside of fertilization bolt but yet have no degradation.The whole process of fertilization cytology was observed on sperm of triploid Rainbow trout penetration into egg of diploid Rainbow trout by method of tissue slice. The results showed:hybrid embryos can successfully complete fertilization process and fertilization procedure is identical with most of bony fish which contains recognition and fusion of plasma membrane of sperm and egg, formation of the male pronucleus, the second maturation division of oocyte, expelling the second polar, formation of the female pronucleus, fusion of male and female pronucleus forming a zygote nuclei which turns into the first cleavage. Hybrid embryos didn’t gradually developed normally after the first cleavage such as fertilization rate will fall, cleavage didn’t enter into next step normally, a large number of hybrid embryos dead after eyeing stage but the whole is death after membrane rupture and before feeding. The study suggests that reason of causing this situation has basically two kinds:chromosome distribution was nonuniform that leaded to non-homologous chromosome occur complicated synapsis and parts of homologous chromosome matched obstacles that caused low fertilization rate.②The chromosome aneuploidy may cause nucleus and cytoplast of hybrid embryos was incompatible, gene expression regulation disorder. Hybrid embryos can’t develop normally, can’t complete transition from endogenous nutrition supplied by maternal yolk to exogenous nutrition that all finally result in most of hybrid embryos dead in the process of development and all of hybrid series completely dead after rupture of membrane before feeding.Fertilization rate of diploid rainbow trout, was surveyed through two kinds of methods with differential solution and Bouin’s liquid fixed and stripped membrane, the result showed that:the sampling time of fertilization rate is Cleavage stage and the optimal time is morula stage. Two kinds of methods have no significant difference (P>0.05), but method of differential solution was more fast more convenient and was more suitable for promotion of Production practice.This study firstly resear ched appearance and structure of micropylar apparatus on diploid Rainbow trout, time subsequence of sperms on tetraploid Rainbow trout penetration into eggs, mechanism of monospermy and single precision fertilization; and described the whole process of fertilization cytology on sperm of triploid Rainbow trout penetration into diploid Rainbow trout eggs; and determined accurate time point and rapid method of fertilization rate surveyed through two kinds of methods with differential solution and Bouin’s liquid fixed and stripped membrane. The research provided basic dates of Rainbow trout investigation and at the same time provided important parameter and theoretical evidence for further study.