| Female southern flounder (Paralichthys lethostigma) grow faster than males.Therefore, all-female production will maximize farming profit of this species. In the present study, the gonadal sexdifferentiation and sex control were studied through histological observation, sex reversal induced by temperature and hormone and meiotic gynogenesis,the results and conclusions are as follows:1. Gonadal sex differentiation of southern flounder1.1 Development of primordial gonadThe Southern flounder, 0 to 65 days after hatching (DAH), 2.3±0.31 to 37.5±3.4mm in total length (TL),were sampled and processed for histological observation.In the yolk sac stage, the primordial germ cells (PGCs), 7.1±1.5μm in diameter and nuclear 3.2±0.9μm in diameter, located between the gut and the mesonephric ducts.In larvae of 10 DAH, PGCs reached the germinal ridge and began to form the primordial gonad. In larvae of 10~35 DAH, the primordial gonad began to prolongate. In larvae of 45 DAH, the primordial gonadstretched from the ventral end of the kidney to the posterior end of the abdominal cavity after mitotic multiplication. There is no evidence of gonadal differentiation in primordial gonad until 65 DAH.1.2 Ovarian differentiationAfter matining at undifferentiation stage for certain time, when larvae were 85 DAH with 5.9±0.32cm in TL, the primordial gonad started the mitosis procedure, and clusters of oogonia appeared which indicated the beginning of ovarian differentiation of Southern flounder. Presumptive ovarian cavity begin to form at 7.1±0.36cm in TL (100DAH), the dorsal and ventral edges of the gonad expanded laterally and eventually fused, leaving a gap between them. At 8.3±0.81cm in TL (120 DAH), the ovarian cavity increased in size, and began to form lamellae which would ultimately develop into ovarian follicles containing clusters of oogonias. Ovarian cavity was finally formed at 180DAH. Formation of clusters of germ cells and ovarian cavity are regarded as the distinguishing cytological and anatomical features of ovarian differentiation of southern flounder.1.3 Testicular differentiationSimilar to other fishes, testicular differentiation of Southern flounder was posterior to ovarian differentiation. At 5.3±0.42cm in TL (100DAH), presumptive testis began to enlarge through mitosis quickly. Formation of efferent duct and blood vessels occurred at 130DAH (6.3±0.52cm in TL), which were the anatomical features of testicular differentiation. At 160 DAH ( 6.8±0.56cm TL), the stromal cells in the testis began to diffuse and form interstitial tissue which surrounded the future seminal lobule anlage, meantime,clusters of spermatogonia (SG) appeared. At 200 DAH (8.7±0.93 cm TL), the testis began to develop spermatogonial clusters of cysts and form seminal lobule, which was the cytological features of testicular differentiation. By Testis developed into maturation stage when total length reached 15.3±1.2cm at 310 DAH. In no instance was there any evidence of intersexuality,or dimorphic characteristics in any histological sections before,during,or after gonadal sex differentiation.Southern flounder belongs to differentiated gonad type, developing directly into a testis or an ovary.2. Gynogenetic induction in southern flounder by cryopreserved sperm of Pagrosomus mojorIn the present study, we developed an effective protocol to produce all-female southern flounder by using meiotic gynogenesis activated with the cryopreserved heterologous sperm of Pagrosomus mojor. UV was used to inactivate sperm and cold shock was applied to prevent extrusion of the second polar body.The results showed that diploid gynogenesis was successfully induced by activating egg development with UV irradiated sperm (72 mJ/cm2) at 3 min after fertilization,and then cold shocked (0~2℃) for 45 min. The fertilization rate was 37.2±5.1%, the lowest malformation rate was 8.3±2.5%, diploid hatching rate was up to 9.4±0.71%.Gynogenetic progeny were examined by flow cytometry, morphology and chromosome ploidy, these methods all verified that the progenies were diploid (2n = 48), haploid and aneuploid phenomena were not found.Embryonic development observations showed that the embryonic development of two treatment groups were significantly different from control group. Gynogenetic diploid larvae and normal larvae showed no significant difference in Morphology index (P> 0.05) except for haploid larvae (P <0.05).3. Effects of temperature and MT on sex ratio in progenies of southern flounderThe effects of temperature and MT on sex ratio of progenies of southern flounder were assessed by distinguishing the phenotypic sexthrough histological sectioning.Juvenile southern flounder were exposed to at 14℃, 18℃, 22℃, 26℃, and 32℃from 40 DAH to 160 DAH respectively. High temperatures(26℃,32℃) produced a higher proportion of males(66.3% males at 26℃, 0.01<P<0.05, 72.5% males at 32℃, 0.01<P<0.05).Low temperature (14℃,16℃) caused a slightly high proportion of males (58.8%males at 14℃,P>0.05;63.8%males at 16℃, P>0.05) in experimental fish. Raising southern flounder at temperature of 22℃held sex ratios close to 1:1. In a separate study, juvenile southern flounder were exposed to MT and E2 of 20μg/L,60μg/L, 80μg/L, 100μg/L from 40 DAH to 160DAH, respectively. High and low concentration of MT induced significantly higher proportion of males(78.5%males at 60μg/L, P<0.05; 83.4%males at 80μg/L, P<0.05;87.3%males at 100μg/L, P<0.05). The E2 treatment did not cause significant difference in sex ratio of southern flounder (58.2 % males at 20μg/L·H2O;66.8 % males at 60μg/L·H2O;71.3%males at 80μg/L·H2O; 71.8%males at 100μg/L·H2O). These findings indicate that sex ratio in southern flounder could be affected and controlled by temperature and MT. |
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