| Procambarus clarkii is one of the popular key economic freshwater aquaculture shrimp in China,due to the weak research on the reproductive biology of P.clarkii,artificial propagation technology has not been overcome,the large-scale breeding degree of seeds is low,and the self-propagation mode has led to germplasm degradation,lack of high-quality seedlings and other problems,which has become an obstacle to the development of the aquaculture industry of P.clarkii.Among them,the development of ovaries is not synchronized,and the low synchronization rate is the main reason restricting the large-scale breeding of seedlings.Therefore,exploring the development law of the reproductive system of female P.clarkii and inducing the synchronous development of the ovaries of P.clarkii by injection of exogenous hormones play an important role in the large-scale breeding of P.clarkii seeds.In this experimentation,the ovarian development cycle of P.clarkii was studied histologically by tissue sectioning technology.Through anatomical and histological observations,the growth and development related index,progesterone(Prog)hormone level,vitellinogen(VTG)content,and m RNA expression level of ovarian developmentrelated genes were determined,and the effects of 17α-hydroxyprogesterone and serotonin on the growth and ovarian development of P.clarkii were analyzed by removing the eye stem as a positive control.The main results are as follows:1.Morphological and histological observations of ovarian development in P.clarkii:The ovarian development of P.clarkii has a certain regularity.According to the size and color of the ovary and the proportion of each female germ cell in the ovary,the ovarian development can be divided into six stages: I the proliferation period,II the minor growth period,III the rapid growth period,IV the major growth period,V the approximatively mature period and VI the mature period.The GSI of female P.clarkii increased significantly with the development stage(P<0.05),while the HSI had no significant difference in each stage(P>0.05).2.Effects of 17α-hydroxyprogesterone on growth and ovarian development of P.clarkii: Different concentrations of 17α-hydroxyprogesterone and eye stalk significantly reduced the survival rate of P.clarkii.The injection of 20μg/m L dose group and the eye removal stem group significantly increased the maturity rate and synchronization rate of P.clarkii(P<0.05),compared to this negative control group,different doses of 17α-hydroxyprogesterone had no significant effect on the body length and weight gain of P.clarkii(P>0.05);injection of 17α-hydroxyprogesterone 20μg/m L in the ml dose group and the ocular stalk removal group increased significantly(P<0.05),reflecting the promotion effect on gonadal growth;in the experiment 16 days,the HSI in the eye stalk removal group was substantially greater than that in this negative control group,no significant difference was identified between HSI and this negative control group in the 17α-hydroxyprogesterone concentration group(P>0.05);On the 16 th day,the content of VTG in the ovaries of P.clarkii injected with 17α-hydroxyprogesterone at a dose of 20 μg/m L was substantially greater than that in this negative control group(P<0.05),and no significant difference was revealed in the content of VTG in the ovaries of the remaining concentration groups(P>0.05);on the 8th day of the experiment,the content of progesterone in the serum of P.clarkii in the 17α-hydroxyprogesterone injection 20μg/m L dose group was substantially increased than that in this negative control group,and on the16 th day of the experiment,the content of progesterone in the serum of the experimental group injected with 17α-hydroxyprogesterone was substantially increased than that in this negative control group(P<0.05).Further analysis showed that injection of 20 μg/m L of17α-hydroxyprogesterone significantly promotes the rapid development of the ovaries of P.clarkii,whereas it decreased the survival rate of P.clarkii.3.Effects of 5-HT on growth and ovarian development of P.clarkii: Different concentrations of 5-HT had not revealed any significant effect on the survival rate of P.clarkia(P>0.05).The maturation rate,synchronization rate and molt rate of P.clarkii were significantly increased by injection of 20ng/g and 10ng/g groups and eye stalk removal group(P<0.05).Compared to this negative control group,different doses of serotonin significantly increased the body length and body weight of P.clarkii(P<0.05);The GSI of the 5-HT group and the eye stalk group were significantly increased(P<0.05),which reflected the promoting effect on gonadal growth.However,no significant difference was observed in HSI between the hormone treatment group and this negative control group(P>0.05).The content of VTG in ovary of P.clarkii in all dose groups was substantially superior than that in negative control group(P<0.05),and the content of VTG in ovary of20ng/g group was substantially more advanced than that in 10ng/g and 1ng/g groups(P<0.05).On 16 d before the experiment,the 5-HT progesterone content in groups with different concentrations of 5-hydroxytryptamine was substantially superior than that in this negative control group(P< 0.05),and no considerable difference was found between the serum progesterone content in groups with different concentrations of 5-hydroxytryptamine and this negative control group throughout the experimentation 24d(P>0.05).These results demonstrate that injection of 5-HT at a concentration of 20 ng/g can efficiently elevate the rapid development of the ovaries of P.clarkii,meanwhile,it maintained a high survival rate.4.For P.clarkii,the relative expression of Vg,FOXL2,Pcna,Cyclin A and PGES-2 in the ovaries after injection of 17α-hydroxyprogesterone and 5-hydroxytryptamine increased greatly in the short term(P<0.05),Pcna and Cyclin A regulated the division and proliferation of ovarian cells,Vg participated in regulating the formation and development of germ cells,FOXL2 promoted follicle development,PGES-2 participated in hormone synthesis,and regulated ovarian corpus luteum formation.It was shown that 17α-hydroxyprogesterone and 5-hydroxytryptamine stimulated the division,proliferation and vitellin accumulation of germ cells in the ovary by upregulating Vg,FOXL2,Pcna,Cyclin A,PGES-2 and other genes,thereby inducing the ovarian development of P.clarkii. |
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