Effects Of Exogenous Cortisol On Oocyte Maturation And Larva Growth Development Of Rainbow Trout(Oncorhynchus Mykiss)

Yunnan is rich in cold water resources and suitable climatic conditions,which is the best ecological environment for rainbow trout（Oncorhynchus mykiss）to grow and breed.As an important part of Yunnan plateau characteristic fishery,O.mykiss culture has a very broad market prospect.However,at present,the excellent characters of cultured female O.mykiss are degraded,the growth is slow,the disease resistance is weakened,the egg quality is not high,the fertilization rate,hatching rate and larval survival rate are low,and so on,still restrict the development of Yunnan O.mykiss culture industry.Studies at home and abroad have found that exogenous cortisol plays an important role in the development and maturation of fish oocytes and the growth and development of fish larvae.Therefore,in this study,Yunnan economic fish O.mykiss was taken as the research object:1)to study the periodic changes of maternal cortisol level and gonadal development of O.mykiss;2)to analyze the effects of different concentrations of exogenous cortisol on oocyte maturation and egg quality of O.mykiss;3)to analyze the effects of different concentrations of exogenous cortisol on the growth and development of O.mykiss larvae.Furthermore,to clarify the regulatory mechanism of exogenous cortisol on ovarian development,maturation and early growth and development of O.mykiss.This paper puts forward a method of using exogenous cortisol to improve the quality of O.mykiss oocytes and the survival rate of larvae,which can provide technical support for solving the problem of domestic O.mykiss reproduction and improving the reproduction rate,and promote the development of O.mykiss industry.1.Periodic changes of maternal cortisol levels and gonadal development in O.mykissIn order to explore the periodic changes of endocrine activity and gonadal development of female O.mykiss in cold water environment at high altitude,the seasonal changes of serum related hormone levels and HPG axis related gene expression of female O.mykiss were measured through 11 months of culture and continuous monthly sampling.The results are as follows:From January to August,the levels of serum estradiol（E₂）,vitellogenin（VTG）,cortisol and thyroid hormone（THs）of O.mykiss increased gradually,and the growth rate of ovary accelerated,but in September,the level of serum cortisol reached the peak,the contents of serum E₂and VTG increased significantly,and the volume of oocytes increased rapidly,indicating that the increase of serum cortisol promoted the development of O.mykiss oocytes.In addition,the expression levels of cgnrh-2,gnrhr and fsh in the brain increased gradually from January to September,and reached the peak from September to November,while from September to November,with the gradual increase of GSI and the continuous decrease of HSI,the expression levels of genes related to liver and gonad increased significantly.The increase of the expression levels of ers and vtg was helpful to increase the content of serum VTG and promote the development of oocytes.Therefore,in the process of regulating oocyte development and maturation,serum hormone levels and gonadal development-related genes change with the season and reach a peak in November,thus promoting oocyte development and maturation.2.Effects of exogenous cortisol on oocyte maturation and egg quality of O.mykissIn this experiment,female O.mykiss at gonadal development stage IV were treated with different concentrations of exogenous cortisol（0,10⁴and 10⁶pg/m L）.After treatment for 1h,3h,6h and 9h,the effects of exogenous cortisol on oocyte maturation and egg quality of O.mykiss were studied.The results are as follows:Exogenous cortisol can affect the maturation and oocyte quality of O.mykiss oocytes through HPG axis:1)after treatment with 10⁶pg/m L cortisol for 9h,the expression levels of cgnrh-2,gnrhr,fsh and lh in O.mykiss brain were significantly increased,and the expression levels of ers and vtg in liver were also significantly increased,indicating that exogenous cortisol stimulates liver synthesis of VTG through gr-mediated ers expression.Similarly,the expression levels of lhr,foxl2,cyp19a1a,11β-HSD,3β-HSD and vtg in the gonads of O.mykiss were also significantly increased,indicating that cortisol may induce gonadal development by activating the expression of aromatase and activate the protective mechanism to prevent excessive deposition of cortisol for self-regulation,thus promoting oocyte maturation.2)the levels of FSH,VTG,E₂and PROG in serum of O.mykiss increased significantly after treatment with 10⁶pg/m L cortisol for 9h,indicating that exogenous cortisol treatment promoted the synthesis and secretion of FSH,VTG,E₂and PROG in serum of O.mykiss,and then had an effect on oocyte development and maturation.3)After treatment with 10⁶pg/m L cortisol for 9h,the radiation band of O.mykiss oocytes thickened,accelerated the separation of oocytes from the follicular membrane,and promoted oocyte maturation and ovulation,but cortisol treatment had no significant effect on the histomorphology of O.mykiss oocytes.Therefore,10⁶pg/m L cortisol affects the level of serum hormones by promoting the expression of genes related to gonadal development,and then affects the quality of oocytes and eggs,but short-term treatment has no significant effect on the histological characteristics of eggs.3.Effects of exogenous cortisol on the growth and development of O.mykiss larvae through maternal transmission.In this experiment,female O.mykiss at stage V of gonadal development were treated by intraperitoneal injection of different concentrations of exogenous cortisol（0,10⁴,10⁶pg/m L）.After a period of treatment,mature oocytes and mature sperm were collected,fertilized eggs were obtained by dry artificial insemination,and hatched in the indoor circulatory system.After the larvae emerged,samples were taken at 7,14,21,28 and 35d.The effects of exogenous cortisol on the growth and development of O.mykiss larvae were analyzed.The results are as follows:After the female of O.mykiss was treated with cortisol,exogenous cortisol could affect the HPT axis,HPI axis and GH/IGF system of O.mykiss larvae through maternal transmission,and then affect the growth and development of O.mykiss larvae:1)with the increase of cortisol concentration and maternal transmission,the expression levels of igf-I,igf-II,gh,ghr1 and ghr2 in O.mykiss larvae increased after treatment with cortisol and 10⁶pg/m L cortisol.The expression levels of ACTHα,CRH,gr1 and gr2 in O.mykiss larvae were significantly higher than those in the control group.After treatment with 10⁶pg/m L cortisol,the expression levels of gnrhr,fsh,lh,cyp19b,erα1,erα2 and erβ1 in O.mykiss larvae increased significantly.Therefore,with the growth of larvae and the increase of cortisol concentration,the expression levels of HPI,HPG and GH/IGF related genes in O.mykiss larvae increased significantly.2)the levels of Gn RH,FSH,GH and cortisol in O.mykiss larvae were significantly increased 21d after hatching,and the levels of ACTH,FSH,E₂,FT₃and T₄in O.mykiss larvae were significantly increased at 35d after hatching.3)the absorption rate of yolk sac of O.mykiss larvae was promoted by 10⁶pg/m L cortisol treatment,and the fertilization rate,hatching rate and survival rate of O.mykiss larvae were improved.Therefore,exogenous cortisol significantly affects the expression of growth-related genes and hormone levels of O.mykiss larvae through maternal transmission,and then promotes the growth and development of O.mykiss larvae,and the effect of 10⁶pg/m L cortisol treatment on the growth and development of O.mykiss larvae is more obvious.In conclusion,cortisol can be used as an important inducing factor to regulate the rapid development of oocytes in the yolk accumulation stage of O.mykiss by regulating the expression of serum hormones and proteins,as well as HPG axis related genes,so as to improve the quality of eggs.At the same time,cortisol can also promote the growth and development of offspring through maternal transmission.Therefore,in the process of artificial reproduction,the key to obtain high quality fish fry is to increase the serum cortisol level of O.mykiss by inducing exogenous cortisol,promote the development and maturation of O.mykiss oocytes and obtain better quality eggs in advance.At the same time,this study provides a reference for solving the breeding problem of domestic O.mykiss and improving the breeding rate in the future.