Preliminary Studies On Reproductive Biology Of Sinibrama Taeniatus

Sinibrama taeniatus is a kind of rare and small economical fish in the upper Yangtze River.This fish tends to propagate in the torrent shoal,and the breeding environment has been seriously damaged due to the large-scale hydropower development.S taeniatus was listed as a key proliferation and release target by Sichuan Province,therefor,researches on the reproductive biology is imminent.Members of our laboratory have been domesticating this kind of fish collected from Meishan,Sichuan since 2016,and then have basically mastered its breeding methods.We selected healthy adult fish from domesticated S taeniatus,and took gonads from different periods for germ cell research.In May 2017,we purchased more than 1,000 S taeniatus from a fisherman in Meishan.After two weeks,600 healthy adult fish were selected for unified breeding.From June 2017 to August 2018,10 male and female gonads were taken each month as experimental materials for the study of annual changes in gonads.This study focuses on the basic data required for artificial reproduction of S taeniatus,using gross anatomy and tissue sectioning techniques to observe the development of germ cells and intra-annual gonadal development.And then we used Software such as Image-Pro Plus,SPSS,Origin,Adobe Illustrator to measure,analyze,and make graph.At the same time,the type of spawning was verified using artificial oxytocin.The main results and conclusions are as follows:1.The characteristics and laws of oogenesis in S taeniatus were clarified.we observed and described the microstructure of its oogenesis process which is divided into five phases: oogonium(phase ?),single-layer follicular cell(phase ?),yolk vesicle(phase ?),yolk filled(phase ?),and mature oocyte(phase ?).Results from paraffin section revealed that oogonium could be distinguished as two types according to their microscopic characteristic: oogonium at early stage and mitosis.In the former,there was a large central nucleolus in the nucleus,while in the latter,the large central nucleoli disappeared,and chromatin was scattered in the nucleus.Meanwhile,the early primary oocyte was divided into the phase ?,and several small nucleoli appeared on the edge of the nuclear membrane,which was regarded as the sign of the beginning of little growth period.At the same time,we classify the most basophilic oocytes as mid-phase II,and the yolk nuclei appeared at this stage.The yolk vesicles carried a large amount of yolk material to the center of the cell in the middle and late phase ?.There were also a large number of eosinophilic yolk particles in the perinuclear region where the yolk vesicles hadn't yet reached.We hold the opinion that the yolk particles appearing in the perinuclear region should be endogenous yolk.After entering the phase ?,the yolk vesicles began to decrease for cell growth.This early accumulation of yolk material allowed the oocyte to grow rapidly while the yolk material fills the cytoplasm quickly,saving time for oogenesis maturation.After the oocyte was mature,the area of the cortical vesicles was exhibited as lumps or particulates,and was dyed orange by hematoxylineosin staining.Therefore,in the presence of a large number of oocytes entering phase III,more nutrition was needed for female fish and the oocytes can be fertilized by the late phase IV.2.The characteristics and laws of spermatogenesis in S taeniatus were clarified.The results showed that the volume of germ cells gradually decreased with the strengthen of cell basophilia,and the nuclear-to-mass ratio index was increased in the process of spermatogenesis.Spermatogonia cells could be distinguished as two types by their microscopic and ultrastructuralcharacteristic.The volume of type A spermatogonia was significantly smaller than that of type B spermatogonia(P < 0.05).Type A spermatogonia can be observed with large central nucleoli and clear nuclear membrane,while type B spermatogonia have scattered basophils in the nucleus,and the nucleoluslike bodies outside the nucleus were significantly reduced.The incomplete division of cytoplasm dated from the spermatogonia cell and the intercellular cytoplasm bridge occurred to contact adjacent cells which would disappear in the late stage of spermiogenesis,ensuring the synchronous development of spermatogenic cells in the same seminal vesicle.The basal body and proximal centriole were observed to be perpendicular to each other during spermiogenesis,and the centriolar appendage arose in the late stage and disappeared with proximal centriole before sperm maturation The head of sperms of S.taeniatus were nearly round or slightly irregular with small nuclear vesicles,undeveloped nuclear pocket,symmetrical lateral fin and no acrosome;moreover,the axoneme of sperm was "9 + 2" duplex microtubule.3.Clarified the rule of annual changes of gonads in S taeniatus under the culture conditionThe results showed that the shape of female was generally larger than that of male(P < 0.01).The gonadosomatic index of female peaked twice in a year,respectively in May and September.The peak spawning season was May-June in spring and SeptemberNovember in autumn.Histological observations showed that there was always a part of female ovaries in stage IV from April to October.Ovaries of most females were still in stage ? after the spring breeding climax.There were still a few degenerated oocytes and a large number of early oocytes in the ovaries after spawning,which could mature and lay eggs under proper conditions.Most of females' ovaries appeared to be in stage ? during winter.Male fish was mature earlier than females,and the gonadosomatic index remained at a high level from April to October.At this time,most of them are in stage ?,and their testis can produce sperms for more than half a year.In general,oocytes in the ovaries of this kind of fish didn't develop synchronously.In the middle and late stages of development,the testis of S.taeniatus also showed obvious asynchronism.The standard deviation of gonadosomatic index both the male and female fish were larger before and after reproductive period.These results indicate that there is a time difference between ovarian and testicular development in both seasons.In a long period of time,the fish lay eggs in batches.Also,the same female can mature and lay eggs many times within a year.Conclusion: Through this study,we have clarified the basic rules of germ cell development in S taeniatus,especially the annual variation of gonads under artificial farming conditions.And it was clear that the fish can achieve the conditions of oxytocin production under cultivating conditions.The artificial reproduction can be realized,which laid a foundation for the full artificial reproduction of the fish.And it was found that the fish may spawn several times a year,which provided important clues for further exploration of the breeding technology of this fish.