漠斑牙鲆性分化及性别控制技术的初步研究
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摘要
漠斑牙鲆(Paralichthys Lethostigma),是一种具有较高营养价值和商业捕捞价值的优良鱼种,其饵料转化率高,养殖成本低,是进行人工增养殖的名优品种。本论文以工厂化养殖漠斑牙鲆为研究对象,以组织切片法为监测手段,对养殖漠斑牙鲆性腺分化、温度和激素诱导性逆转、人工雌核发育等方面进行了初步研究和探讨,所得结果如下:
1.漠斑牙鲆性腺分化的组织学观察
本试验采用组织学和形态学观察手段,对0日龄初孵仔鱼~270日龄漠斑牙鲆成鱼的性腺分化和发育情况进行跟踪观察,以期补充和完善漠斑牙鲆性腺发育的有关资料,为单性苗种的制种和培育提供技术参考。结果表明:在水温20±0.5℃条件下,漠斑牙鲆孵化后10日龄,全长4.0±0.36mm时,PGCs呈椭圆形,体积大,核大,核膜明显,刚出现生殖嵴;35日龄,全长16.9±0.84mm时,PGCs细胞聚集,从体壁游离出来,进入腹腔后部,此时生殖嵴的大小开始分化,大的生殖嵴迅速增大,生殖细胞开始有丝分裂,生殖细胞数目迅速增多,逐渐形成了原始性腺。85日龄,全长5.9±0.32cm,部分性腺中开始出现生殖细胞的有丝分裂,形成成簇发育的PGCs群,其中少数发育成为卵母细胞,标志着性腺形态学分化的开始。120日龄,全长8.3±0.81cm,漠斑牙鲆卵巢的上下两缘不断延伸,形成分化的两支,开始形成卵巢腔,同时形成数个生殖板。180日龄,全长13.4±1.2 cm时,卵巢分化完成。漠斑牙鲆精巢分化晚于卵巢,130日龄,全长为6.3±0.52cm时,原始性腺中精原细胞有丝分裂开始,性腺体积增大,生殖上皮细胞明显;160日龄,全长为6.8±0.56cm时,精小管原基出现,标志着精巢分化开始;200日龄,全长为8.7±0.93 cm时,精小叶和输精管等结构出现,输精管的出现标志着精巢分化的完成;240日龄,全长为10.3±1.1cm时,精巢中精小叶,精母细胞,精小管等结构明显,漠斑牙鲆雄鱼进入成熟发育期。漠斑牙鲆由原始性腺逐渐发育分化为不同结构和功能的卵巢或精巢,在无外界因素干扰下,分化后的分化类型稳定不变,此种性腺分化类型的漠斑牙鲆属于雌雄异体的分化型鱼类。
2.真鲷冷冻精子诱导漠斑牙鲆减数雌核发育研究
本文采用紫外线(UV)灭活的冷冻真鲷精子激活漠斑牙鲆卵子发育,冷休克法抑制第二极体排出,成功获得雌核发育杂合二倍体仔鱼。试验结果显示:冷冻真鲷精子适宜的UV灭活剂量为72mJ/cm2,随着处理时间(0~100s)延长,已灭活精子受精后的胚胎孵化率呈哈特维希效应,40s灭活组胚胎孵化率达到最高29±1.9%,单倍体率100%。冷休克处理温度0~2℃,起始时刻3min、持续时间45min时,胚胎的二倍体孵化率最高9.4±0.71%。雌核发育后代经流式细胞仪、形态学观察和染色体倍性鉴定,均为二倍体(2n=48),未发现单倍体和非整倍体现象。胚胎发育时序跟踪观察表明冷休克组和单倍体组发育同比正常孵化组时间(49h45min)差异显著,分别历时64h59min和55h49min。雌核发育二倍体与正常仔鱼数量形状差异不显著(P>0.05),但单倍体仔鱼明显畸形,与正常组和雌核组仔鱼在上述指标上存在显著差异(P<0.05)。
3.环境因子对漠斑牙鲆性腺分化的影响
在漠斑牙鲆孵化后40~160日龄期间,以17α-甲基睾酮(17α-MT)和17β-雌二醇(17β-Estradiol)为诱导因子,分别设置20、60、80、100μg/L 4种浓度梯度,采用浸泡法处理实验样本,160日龄后转入正常水体养殖至200日龄,每组分别取样以石蜡组织切片法鉴定表型性别,结果显示:60μg/L、80μg/L、100μg/L的MT浸浴处理都能显著提高群体中的雄性比例,分别达到78.5%(P<0.05)、83.4%(P<0.05)、87.3%(P<0.05)。17β-Estradiol各试验组雌性化比例差异不明显,雌性比例分别为58.2%、66.8%、71.3%、71.8%。同批漠斑牙鲆孵化后每组随机选取120尾进行温度诱导研究,分别精确控温至14℃,18℃,22℃(正常对照组),26℃,32℃温度培养至160日龄,转到21-23℃正常水温养殖至200d,石蜡组织切片鉴定性别,结果表明:26℃和32℃高温能明显提高群体中的雄性比例,使其分别达到66.3%(0.010.05)和58.8%(P>0.05);对照组群体中雌雄个体比例接近1:1。温度能促使漠斑牙鲆原始性腺向雄性方向分化,因此该鱼属于温度依赖型性别决定类型。
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 flounder
1.1 Development of primordial gonad
The 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 differentiation
After 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 differentiation
Similar 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 mojor
In 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 flounder
The 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℃, 0.010.05;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.
1.漠斑牙鲆性腺分化的组织学观察
本试验采用组织学和形态学观察手段,对0日龄初孵仔鱼~270日龄漠斑牙鲆成鱼的性腺分化和发育情况进行跟踪观察,以期补充和完善漠斑牙鲆性腺发育的有关资料,为单性苗种的制种和培育提供技术参考。结果表明:在水温20±0.5℃条件下,漠斑牙鲆孵化后10日龄,全长4.0±0.36mm时,PGCs呈椭圆形,体积大,核大,核膜明显,刚出现生殖嵴;35日龄,全长16.9±0.84mm时,PGCs细胞聚集,从体壁游离出来,进入腹腔后部,此时生殖嵴的大小开始分化,大的生殖嵴迅速增大,生殖细胞开始有丝分裂,生殖细胞数目迅速增多,逐渐形成了原始性腺。85日龄,全长5.9±0.32cm,部分性腺中开始出现生殖细胞的有丝分裂,形成成簇发育的PGCs群,其中少数发育成为卵母细胞,标志着性腺形态学分化的开始。120日龄,全长8.3±0.81cm,漠斑牙鲆卵巢的上下两缘不断延伸,形成分化的两支,开始形成卵巢腔,同时形成数个生殖板。180日龄,全长13.4±1.2 cm时,卵巢分化完成。漠斑牙鲆精巢分化晚于卵巢,130日龄,全长为6.3±0.52cm时,原始性腺中精原细胞有丝分裂开始,性腺体积增大,生殖上皮细胞明显;160日龄,全长为6.8±0.56cm时,精小管原基出现,标志着精巢分化开始;200日龄,全长为8.7±0.93 cm时,精小叶和输精管等结构出现,输精管的出现标志着精巢分化的完成;240日龄,全长为10.3±1.1cm时,精巢中精小叶,精母细胞,精小管等结构明显,漠斑牙鲆雄鱼进入成熟发育期。漠斑牙鲆由原始性腺逐渐发育分化为不同结构和功能的卵巢或精巢,在无外界因素干扰下,分化后的分化类型稳定不变,此种性腺分化类型的漠斑牙鲆属于雌雄异体的分化型鱼类。
2.真鲷冷冻精子诱导漠斑牙鲆减数雌核发育研究
本文采用紫外线(UV)灭活的冷冻真鲷精子激活漠斑牙鲆卵子发育,冷休克法抑制第二极体排出,成功获得雌核发育杂合二倍体仔鱼。试验结果显示:冷冻真鲷精子适宜的UV灭活剂量为72mJ/cm2,随着处理时间(0~100s)延长,已灭活精子受精后的胚胎孵化率呈哈特维希效应,40s灭活组胚胎孵化率达到最高29±1.9%,单倍体率100%。冷休克处理温度0~2℃,起始时刻3min、持续时间45min时,胚胎的二倍体孵化率最高9.4±0.71%。雌核发育后代经流式细胞仪、形态学观察和染色体倍性鉴定,均为二倍体(2n=48),未发现单倍体和非整倍体现象。胚胎发育时序跟踪观察表明冷休克组和单倍体组发育同比正常孵化组时间(49h45min)差异显著,分别历时64h59min和55h49min。雌核发育二倍体与正常仔鱼数量形状差异不显著(P>0.05),但单倍体仔鱼明显畸形,与正常组和雌核组仔鱼在上述指标上存在显著差异(P<0.05)。
3.环境因子对漠斑牙鲆性腺分化的影响
在漠斑牙鲆孵化后40~160日龄期间,以17α-甲基睾酮(17α-MT)和17β-雌二醇(17β-Estradiol)为诱导因子,分别设置20、60、80、100μg/L 4种浓度梯度,采用浸泡法处理实验样本,160日龄后转入正常水体养殖至200日龄,每组分别取样以石蜡组织切片法鉴定表型性别,结果显示:60μg/L、80μg/L、100μg/L的MT浸浴处理都能显著提高群体中的雄性比例,分别达到78.5%(P<0.05)、83.4%(P<0.05)、87.3%(P<0.05)。17β-Estradiol各试验组雌性化比例差异不明显,雌性比例分别为58.2%、66.8%、71.3%、71.8%。同批漠斑牙鲆孵化后每组随机选取120尾进行温度诱导研究,分别精确控温至14℃,18℃,22℃(正常对照组),26℃,32℃温度培养至160日龄,转到21-23℃正常水温养殖至200d,石蜡组织切片鉴定性别,结果表明:26℃和32℃高温能明显提高群体中的雄性比例,使其分别达到66.3%(0.01
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 flounder
1.1 Development of primordial gonad
The 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 differentiation
After 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 differentiation
Similar 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 mojor
In 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 flounder
The 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
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