Studies On The Fertilization Biology Of Misgurnus Anguillicaudatus

Misgurnus anguillicaudatus, belonging to the order of Cypriniformes, family Cobitinae, genus Misgurnus, is widely distributed in the main streams, tributaries,reservoirs, lakes, ditches and rice field water in China, except the area of western plateau. It is famous for its tender and delicious meat, and it has been known as “a ginseng in water” for a long period in China for its nutritious meat and medicinal value.M. anguillicaudatus is not just an important freshwater aquaculture species in China,but also a high quality aquatic product of Chinese foreign trade export. The economic benefit is considerable. However, the fertilization biology of M. anguillicaudatus is unclear.Healthy adult male and female loaches, with well development were selected for the parent fish in this study. These fish were bought from the Dianjiang Aquatic Station of Chongqing. After the artificial spawning, dry insemination was used for the artificial fertilization. Fertilized eggs of different time were collected, and the structures of testis and mature eggs, the entry of the sperm into egg, the cortical reaction, the migration and the integration of male and female pronucleus of M. anguillicaudatus, were investigated by using histological and zymological methods, scanning electron microscopy and transmission electron microscopy. The main results are as follows:The testis of M. anguillicaudatus was consisted of head, mid-piece and tail, which are organized as a lobular structure, without seminal vesicle. The heads of most sperms were in a sphere like, and few were pear-shaped. The diameter of sperms is(1.877±0.071) μm. No acrosome was observed at anterior end of the nucleus. The nucleus can be divided into two types: with and without nuclear vesicles, which may be due to the different maturity degree of sperms. There was a shallow implantation fossa,with a depth of about 1/4 of the length of head, at the caudal-lateral end of the nucleus.The mid-piece was short and be consisted of the centriolar complex and the asymmetric sleeve. The centriolar complex contained proximal centriole and basal body that were arranged like a “T”. The main structure of the tail was axoneme, which was structured as a typical “9+2”. No lateral fins in the sperm tail.The mature egg of M. anguillicaudatus was global and demersal, with the diameter of about 0.90 mm. The egg envelope, with relatively uniform small pores on the surface,was connected to the plasma membranes and belonged to primary envelop. The egg envelope comprised radiate zona, which was divided into three gradations based on the electron density, and the attached membrane. There was a single eddy-like and clockwise micropyle on the animal pole. The eddy-like shape might be beneficial to attract sperms and brought them into micropylar tube. The micropyle, consisted of vestibule and micropylar tube, was deeply sunken with a short tunnel. The vestibule was funnel-like, with a depth of(15.038±1.347) μm, and the largest diameter of(39.245±3.845) μm. The micropylar tube was(2.694±0.573) μm in length and the external diameter of micropylar tube was(3.639±0.587) μm. But the internal diameter of micropylar tube was(2.286±0.364) μm, which was similar to the size of a sperm.Thus we inferred that, only one sperm can be penetrated through the micropylar at one time. The cortical alveoli were the metabolic waste which was accumulated during the process of growth and development. According to the characteristies and morphological of contents, the cortical alveoli could be divided into three types.Under the water temperature of 20-21℃, at 0 s after insemination, the ova were attached to a large number of sperms. The globe-like first polar bodies were appearred around some vestibules. The surface of some first polar bodies had a part of bulge. Two globes with different size were appeared near some micropyles. All micropylar tubes were open. Some first polar bodies were connected to outside of the concave plasma membranes, which was near the micropylar tubes of mature eggs. These first polar bodies were oval in majority and globe in minority. Sperms were just entering micropylar tubes or had entered ova at 3s after insemination. At 5-8 s after insemination,the sperm star light was observed, the cortical reaction started and most micropylar tubes were plugged by floccule or the external diameter of micropylar tubes became narrowed. At 10-20 s after insemination, the perivitelline space and fertilization cone were observed. At 25-30 s after insemination, the fertilization cone got bigger. At 35-50 s after insemination, spermaster expanded, cortical reaction was obvious, and the firstpolar body could be observed in the perivitelline space. At 55-70 s after insemination,the fertilization cone was outward, and the cortical reaction reached the the climactic phase at about 60 s after insemination. At 70 s after insemination, chromosomes had started to move towards two poles, and the fertilization cone became gradually broke down at 80-110 s after insemination. At 120 s after insemination, only a small amount of cortical alveoli were still releasing contents and a few micropylar tubes were open. At150 s after insemination, all micropylar tubes were closed. At 6-8 min after insemination, the second polar body was observed, while very few detained first polar bodies were still observed not expelled to the outside of egg envelope at 6 min after insemination. At 20-25 min after insemination, male and female prnucleus were fused.At 25-30 min after insemination, zygote was inmetaphase of first mitosis. At 40-45 min after insemination, the first mitosis finished and two cells then formed.M. anguillicaudatus was monospermy, and the mechanism of preventing polyspermy was mainly the following three steps.(1) Only one sperm was permitted to enter the internal diameter of micropylar tube at one time.(2) After the first sperm entered the oocyte cytoplasm, the micropylar tube was plugged by floccule or the external diameter of micropylar tube became narrowed.(3) The contents released from the cortical alveoli to the perivitelline space by ortical reaction could harden the egg envelope to prevent polyspermy.The presence of the first polar body had no effects on the discharge of the second polar body in early stage of the fertilization process of M. anguillicaudatus. After the expel, the first polar body could still be split into two polar bodies. All micropylar tubes were closed at 150 s after insemination, thus we proposed that, the fertilization process of M. anguillicaudatus eggs can be completed in 150 s after entering to water.