Homozygous Idetification And Histological Observation Of Gonadal Development Of Gynogenetic Haploid Of Japanese Flounder, Paralichthys Olivaceus

Japanese Flounder (Paralichthys olivaceus) is a commercially important marine fish in China, alsocultured along the coastlines of Japan and Korea. Owing to the large body, delicious succulent, highnutritional value and medicinal value, there is a broad market for them. But due to overfishing andpollution of the environment, natural resources faced a big drop which resulted in the contradiction betweensupply and demand.Thus, the artificial breeding becomes necessary. Difference in growth rate between maleand female Japanese Flounder is frequently observed in nature, that is, the growth rate of females issignificantly faster than that in males. Therefore, all-female population construction is of great importance.Meiotic gynogenetic and mitotic gynogenesis are terms that describe different gynogeneticproduction．Homozygous progeny can be produced by inducing mitotic gynogenesis, namely doubledhaploid (DH). Cloned fish then can be produced by the second cycle of gynogenesis in the eggs of DHs.There is clearly great potential for wider use of DH fish and clonal lines in genetic and genomicresearch infishes and in fish breeding programs. Owing to the low yield and survival rate of DH，the gonadal studieshave not been reported yet. Microsatellite markers were used to identify the homozygosity and histologicalstudy was carried out to observe the gonadal development. And then sex ratio and histological features wereanalysed, in order to lay the foundation for the biology of DH fish.Mitotic gynogenetic was induced by activating egg development with UV-irradiated sperm of red seabream Pagrus major.3min afterwards, a pressure shock of650kg/cm~2was initiated at60minpost-activation (at17℃) and maintained for6min, then incubated in seawater.129individuals wereobtained after2years’ cultivation.The results showed that eighty-eight of129individuals were females, accounting for68%of the total;thirty-seven were males, accounting for29%of the total; the remaining four (3%) could not bedistinguished between male and female. Sex ratio is thus1:0.42, while sex ratio of control group was55:45,nearly1:1.Ovarian developmental stages were basically the same(in phase II), but were delayed compared tocontrols(in phaseⅢ), of which the minority showed normal ovarian development. Most ovarian oocytes, on the other hand, showed structural abnormalities in both nucleus and cytoplasm, which can be summarized asseven types according to the morphological structure.We define normal with more than50%and abnormalwith le ovary ss than50%normal oocytes. Six ovaries then were normal,revealing the low fertility of DHfemales.Testis mostly developed but stagnated in Phase Ⅱ, in which the spermatogonia were distributed inspermatocyst with no further differentiation, revealing testis were not sterile. Few normal testis had regularstructure, but was different from that of the controls, which had spermatogonia, primary spermatocytes,secondary spermatocytes, sperm cells and sperm.Yolk nucleus depolymerization process was observed in ovaries, which may be associated with theformation of fat.The minority of testis was found to have few oocytes in DH and controls, namely amphigenetic mosaics.This may be a kind of sex-reversed phenomenon, unrelated with the homozygosity.