Gonadal Development Of Quasipaa Spinosa And Effects Of Letrozole On Gender Differentiation

Quasipaa spinosa is a unique economic frog in China.In artificial breeding,the economic benefit of males is higher than that of females,and the prospect of all-male breeding is broad.In this study,anatomical and histological methods were used to systematically study the gonadal development of Q spinosa in Shimen mountain of Hunan Province at 18°C-20°C.Different concentrations of letrozole（20 mg/Kg,100 mg/Kg,400mg/Kg,1000 mg/Kg）were added to feed to induce tadpoles masculinize.The feeding cycle was 90 days,at the same time,the control groups were set up which were recorded as CK,L₂₀,L₁₀₀,L₄₀₀,and L₁₀₀₀ groups,respectively.The effects of different concentrations of letrozole on sex ratio,growth and sex hormone（estradiol E₂,testosterone T）levels of Q spinosa were analyzed.The main results are as follows:1.The genital ridge of Q spinosa tadpoles in Shimen,Hunan appeared at 10 days after hatching（Gosner 25）at 18℃-20℃.Then,the primordial germ cells migrated into the genital ridge at 12 days after hatching（Gosner 25 stage）,and the primordial gonads began to form.No primary cavity was observed in the primary gonadal stage.Tadpoles developed to32 days after hatching（Gosner stage 26）and the ovarian cavity appears,marking the beginning of ovarian differentiation.Tadpoles develop to 46 days after hatching（Gosner stage 29）,germ cells moved to the center of the gonads,testes began to differentiate.Shimen,Hunan Q spinosa gonad differentiation belongs to differentiation type,the original gonad directly differentiated into testis or ovary.The testis of male Q spinosa belongs to tubule type,and seminiferous tubules can be observed when the metamorphosis is completed.Mature sperm is observed in the testis of 1-year-old sexual maturation after metamorphosis.Female Q spinosa belongs to the type that lays eggs repeatedly,mature eggs can be observed in the 2 years after metamorphosis to sexual maturity.2.The male rate of Q spinosa was low at 18°C-20°C,the male rate of CK group was only 33.33%.Feeding different concentrations of letrozole could significantly improve the male rate of Q spinosa,the male rates of L₂₀,L₁₀₀,L₄₀₀ and L₁₀₀₀ groups were 58.49%,55.26%,48.84%and 56.82%,respectively.Different concentrations of letrozole had no significant effect on body length,head length,head width,forearm and hand length,hind limb length and liver-to-body ratio of young frogs,indicating that letrozole had no effect on the growth of Q spinosa under low temperature.3.The levels of E₂ and T significantly changed from 20 days after hatching to 70 days after hatching（from primitive gonad to the end of gonad differentiation）.The levels of E₂reached a high level at 30 days after hatching and 50 days after hatching,while the level of T reached the maximum at 50 days after hatching.In terms of groups,there was no significant difference in E₂ and T levels among the letrozole concentration groups.Compared with the control group,the levels of E₂ and T in the concentration groups of letrozole were significantly increased on the whole,and the significant increase of T level in the concentration groups of letrozole from 40 days after hatching to 50 days after hatching（during testis differentiation）was the key to inducing male sterility.This study enriched the reproductive data of different geographical populations of Quasipaa spinosa,explored the effect of letrozole on gonadal differentiation of Quasipaa spinosa,enriched the research content of letrozole-induced gender differentiation of amphibian species,and provided theoretical reference for subsequent monosexual breeding of Quasipaa spinosa.