Studies On The Genetic Characteristics Of Yellow Catfish “Huangyou No.1” And Improvement Of Female Reproductive

Yellow catfish(Pelteobagrus fulvidraco) is one of the economic important fish species in the freshwater aquaculture.The growth rate of male yellow catfish is 1.5-2 times greater than females.Therefore,the Chinese scientists have developed a technical route for the breeding of all-male fish,which is assisted by X-and Ychromosome linked markers.All-male yellow catfish has improved the economic benefits of aquaculture and promoted the rapid development of the yellow catfish industry.In recent years,the breeding lines of parent super-male fish degraded after multiple generations of self-crossing,and the inbred lines showed a tendency of weakening in growth and disease resistance.Therefore,it is necessary to improve the variety of all-male yellow catfish or develop new varieties of yellow catfish.The yellow catfish harvested from Liangzi Lake and bred in 3 consecutive generations was selected as the female parent,and the darkbarbel catfish(Pelteobagrus vachelli) collected from the Yangtze River and bred in 2 consecutive generations was selected as the male parent.We obtained the F1 generation of hybrid yellow catfish(P.fulvidraco ♀ × P.vachelli ♂) by using artificial reproduction,named the novel variety “Huangyou No.1”.In this study,we aimed to systematically compare the morphological and genetic characteristics of yellow catfish and “Huangyou No.1”,and to resolve the problems meeting in the reproduction and breeding process of female yellow catfish.A comparative analysis of morphology and gonad development of 10-month-,22-month-and 34-month-old yellow catfish and “Huangyou No.1” was conducted in the same culture condition.The “Huangyou No.1” has a growth advantage to yellow catfish.In the 22-month-and 34-month-old yellow catfish,male weight is about 2 times of the female.However,sexual size dimorphism was greatly reduced in “Huangyou No.1”.On the basis of the gonadal anatomy analysis,we observed completely degenerated ovarian with a fine line structure and no egg production in female “Huangyou No.1”.Therefore,the female “Huangyou No.1” is completely sterile.Moreover,the testes of male “Huangyou No.1” were transparent and degenerated.The spermatogenic cyst was almost empty and had no sperm production in 10-month-old male “Huangyou No.1” by H&E staining of testis sections,while a few sperms were observed in the spermatogenic cyst of 22-month-and 34-month-old male “Huangyou No.1”.Compared to yellow catfish,the number of sperm was too small and the activity was very low in “Huangyou No.1” by computer assisted spermatozoa analysis system(CASA).In addition,22-month-old male “Huangyou No.1” do not have reproductive capacity.The novel variety “Huangyou No.1” shows hybrid advantage in improving growth performance and infertility,and has the potential to promote the development of yellow catfish industry.In order to explore the genetic mechanism of hybrid sterility,we analyzed the early gonad development of yellow catfish and “Huangyou No.1”.The results of H&E staining showed that the ovary of yellow catfish developed normally with oocytes at different stages including 20-dph-(days post hatching),2-month-and 8-month-old.However,the ovary of “Huangyou No.1” was degenerated without oogonia at 20 dph,and completely degenerated at 2-month-old and 8-month-old.At 40 dph,both the yellow catfish and “Huangyou No.1” testes showed a small number of spermatogonia.At 2-month-and 8-moth-old,the “Huangyou No.1” testes were degenerated.The results of semi-quantitative RT-PCR indicated that the expression of four germ cell marker genes,such as vasa,piwi1,dnd and dazl were obviously down-regulated in “Huangyou No.1”,and the expression levels of gonadal somatic cell development-related genes,including wnt4,esr2 and foxl2 in ovary,as well as dmrt1,star and cyp17a1 in testis,were also decreased significantly in “Huangyou No.1”,compared with yellow catfish.In addition,the Vasa protein expression in gonads was also reduced in “Huangyou No.1” as revealed by the immunofluorescence assay.In the testes of “Huangyou No.1”,both the expression of Sycp3,a meiosis marker expressed in spermatocytes,and PH3,a mitosis marker of cell proliferation,were obviously reduced.Interestingly,at 4-cell-stage,the germline-specific genes,such as vasa,dnd,piwi1 and dazl were reduced in “Huangyou No.1”.Moreover,vasa transcript in the cleavage furrows of “Huangyou No.1” embryos was also reduced compared to yellow catfish.In summary,those results indicated the diminutive and infertile gonads were found in the “Huangyou No.1”,accompanied with the decreasing expression of maternal m RNA and impaired mitosis and meiosis of germ cells.Multiple repeat spawners make large contributions to long-term population stability and aquaculture breeding programs.A high percentage of female yellow catfish died for spawning failure or incomplete spawning after artificial spawning by traditional synthetic hormones including human chorionic gonadotropin(hCG),luteinizing hormone releasing hormone(LHRH) and domperidone(DOM).The present study was designed to compare the efficacy of different combinations of exogenous hormones for inducing ovulation in yellow catfish using hCG,LHRH,DOM and carp pituitary extraction(CPE).We found an optimal strategy for exogenous hormones administration,the mixture of LHRH/CPE for the first injection and LHRH/CPE/DOM for the second injection,could greatly improve the rates of spawning success,weight of ovulated eggs and survival rate after spawning.Interestingly,a population of female yellow catfish with defective reproductive duct could not spawn and showed high mortality after induced by a combination of h CG/LHRH/DOM,whereas a synergistic combination of hCG,LHRH,DOM and CPE could efficiently induce spawning and reduce mortality in the defective yellow catfish,in which a significant decrease of Vitellin and E2 levels.Altogether,our findings provide an effective combination of exogenous hormones to improve spawning and post-spawning survival of female yellow catfish.The mesenteric fat deposition(MFD) in the female parent is the common problem of yellow catfish in most breeding farms.Since the quality of fry is closely related to the quality of female parent,four populations of female parent were selected to evaluate the effects of MFD on the reproductive performance of yellow catfish,including group 1,a wild population of yellow catfish;group 2,a cultured population with low MFD that was carefully cultivated and fed with animal protein such as surimi;group 3 and 4,two cultured populations with high MFD that was fed with artificial expanded feed.Our results showed that there was a negative correlation between mesenteric fat index(MFI) and gonad somatic index(GSI).The results of artificial propagation indicated that there were no significant differences in the spawning rate,fertilization rate and number of fry produced per female between group 1 and group 2,which were significantly higher than those in group 3 and group 4.In addition,the fry malformation rate of group 1 and 2 was significantly lower than that of group 3 and 4.The serum Luteinizing hormone(LH) and Vitellogenin(VTG) levels decreased with the increase of MFD.On the contrary,the contents of lipid and glycogen in livers and eggs increased with the increase of MFD.In conclusion,our results show that excessive MFD in female yellow catfish have a great negative impact on its physiological and reproductive performance,and suggest that reducing MFD could significantly improve reproductive performance,which provides a clue for breeding high-quality parents and improving fry quality of yellow catfish.All together,our results showed that the growth rate of hybrid yellow catfish "Huangyou No.1" was significantly higher than that of yellow catfish,and it had the characteristics of hybrid sterility,which was mainly caused by the defects in germ cell and gonad development.We found an optimal strategy for exogenous hormones administration to improve the reproductive performance and enhance the oviposition rate and postnatal-survival rate of the female parent yellow catfish,whose reproductive performance could also been further improved by reducing MFD.These studies provide ideas for cultivating high-quality parents and improving the quality of fries of yellow catfish.