INDUCTION OF XX PSEUDO-MALES, AND LARGE-SCALE REPRODUCTION OF ALL-FEMALE POPULATION IN YELLOW CATFISH
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摘要: 研究利用3种雄性化因素, 包括17α-甲基睾丸酮(MT, 5 mg/kg)、来曲唑(LZ, 300 mg/kg)和高温(33.5℃) 联合处理12—65日龄黄颡鱼幼鱼, 并将性成熟的XX伪雄鱼与正常XX雌鱼进行人工繁殖, 开展了全雌黄颡鱼(Tachysurus fulvidraco)规模化繁殖与苗种培育工作。研究发现, MT、LZ和高温共同作用可诱导XX黄颡鱼逆转为生理型雄性, 完全性逆转个体运动型精子比例与XY雄鱼无显著性差异, 组织学切片也显示其精巢中存在大量精子细胞, 推测XX伪雄鱼具有正常的繁殖功能。随后, 以XX伪雄鱼为父本, 正常XX雌鱼为母本开展了规模化人工繁殖, 获得了57万尾基因型全部为XX的黄颡鱼苗种, 并将其成功培育成大规格鱼种。在幼鱼60日龄和120日龄时取样发现, 分别有2.8%和12.0%的个体发生了不同程度的雄性化, 推测其可能受到池塘自然高温的影响而发生了性逆转。其余XX雌鱼卵巢发育良好, 来年繁殖季节可作为规模化人工繁殖的雌性亲本。研究成功开展了全雌黄颡鱼规模化繁育工作, 为全雌黄颡鱼规模化繁育体系的建立提供了基础, 也为黄颡鱼新品种选育中雌性选育提供了保障。Abstract: The shortage of female has impeded the development of aquaculture industry of yellow catfish (Tachysurus fulvidraco) in China in recent years. Therefore, large-scale production system of all-female is urgently needed. To produce an all-female population in large-scale of yellow catfish, 12—65 days post-hatching larvae were treated with the combination of three male-promoting factors, 17 α-Methyltestosterone (MT, 5 mg/kg), Letrozole (LZ, 300 mg/kg), and high temperature (33.5℃). XX pseudo-males were mated with normal XX females and large-scale production of all-female population was conducted. Results showed that the combination of MT, LZ, and high temperature-induced masculinization of XX individuals, and the completely sex-reversed XX males displayed a similar percentage of motile sperm with XY males. Histological observation found that there are a large number of spermatids in the testis of XX males, indicating normal reproductive ability. Subsequently, we mate XX males and XX females, produced 570000 genotypic XX larvae, and successfully reared them to large fingerlings in a pond. The results indicated that 2.8% and 12.0% XX individuals reversed to XX males which is probably due to the high temperature during the critical time of sex differentiation in the rearing pond. The ovary developed very well for the rest of XX females and can be used as female parents in the coming spawning season. The current work successfully conducted large-scale production of the all-female population in yellow catfish, and the results will provide an important foundation for the large-scale production system of the all-female population, and important safeguard for female selection in genetic breeding of new varieties.
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图 1 黄颡鱼XX伪雄鱼与XY雄鱼性腺形态及组织学比较
A. 对照组XY雄鱼精巢; B. XX伪雄鱼完全逆转型精巢; C. XX多精少卵型精巢; D. XX少精多卵型精巢; zr. 放射带; yg. 卵黄颗粒; st. 精子细胞; sg. 精原细胞; sc. 精母细胞; gonad. 性腺; testis. 精巢; ovary. 卵巢
Figure 1. Gonadal morphology and histology of XX male and XY male in yellow catfish
A. XY♂ gonad; B. completely sex-reversed XX male; C. mostly sex-reversed XX male; D. minorly sex-reversed XX male; zr. zona radiate; yg. yolk granules; st. spermatozoa; sg. spermatogonia; sc. spermatocytes
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图 2 黄颡鱼XX伪雄鱼与XY雄鱼精子活力比较分析
A. 对照组XY雄鱼与处理组XX伪雄鱼不同精子运动类型比较; B、C、D. 不同类型精巢前进型精子VAP、VSL、VCL随时间变化曲线; VAP. 平均路径速度; VSL. 直线运动速度; VCL. 曲线运动速度。* 表示显著性差异(P＜0.05)
Figure 2. Comparative analysis of sperm motility between XX pseudo-male and XY male in yellow catfish
A. Different sperm movement types between XY of control group and XX pseudo-male of treatment group; B, C, D. VAP, VSL and VCL of progressive sperm; VAP. Average Path Velocity; VSL. Velocity Straight Line; VCL. Curvilinear Velocity. Asterisk (*) indicates significant differences (P＜0.05)
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图 3 黄颡鱼XX伪雄鱼和XY雄鱼作为父本的人工繁殖受精率与孵化率比较
A. XX伪雄鱼与XY雄鱼作为父本人工繁殖受精率比较; B. XX伪雄鱼与XY雄鱼作为父本孵化率比较; XX♂和XY♂分别表示发生了性逆转的XX伪雄鱼和正常XY雄鱼
Figure 3. Comparison of fertilization rate and hatching rate between XX pseudo-male and XY male as male parents in yellow catfish
A. Comparison of fertilization rate between XX pseudo-male and XY male as male parents; Comparison of hatching rate between XX pseudo-male and XY male as male parents. XX♂ and XY♂ represent sex-reversed XX males and normal XY males
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图 4 池塘养殖温度记录及全雌黄颡鱼种群基因型性别鉴定
A. 当地6—9月份气温和全雌黄颡鱼种群0—60日龄池塘水温记录; B. 全雌黄颡鱼基因型鉴定电泳图(X-955bp; Y-826bp); 1—16为一条带, 基因型为XX. 17为对照XY基因型; M. DNA marker
Figure 4. Pond temperature records and genotypic sex identification of the all-female yellow catfish population
A. The local highest air temperature from June to September and pond water temperature; B. Genotypic sex identification by using sex-linked markers in all-female yellow catfish population (X-955 bp; Y-826 bp); 1—16. XX genotype; 17. control XY genotype; M. molecular ladder
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图 5 池塘高温期培育的XX雌鱼与性逆转XX雄鱼性腺与生长比较分析
A、A1、A2. 60日龄正常发育的XX雌鱼; B、B1、B2. 60日龄XX雄鱼; C、C1、C2. 120日龄正常发育的XX雌鱼; D、D1、D2. 120日龄精巢有分枝状结构的XX雄鱼; E、E1、E2. 120日龄精巢无分枝状结构的XX雄鱼; F、G. 60日龄XX雌鱼与XX雄鱼全长与体重比较; H、I. 120日龄XX雌鱼与XX雄鱼全长与体重比较; n. 细胞核; nu. 核仁; zr. 放射带; yg. 卵黄颗粒; sg. 精原细胞; sc. 精母细胞; st. 精子细胞。DPH.日龄; * 表示显著性差异(P＜0.05); 性逆转数据首次报道于Yu等[37]
Figure 5. Comparison of gonadal histology and growth performance of XX female and sex-reversed XX male of pond-raised yellow catfish population during high temperature phase
A, A1, A2. normal developed XX female at 60 DPH; B, B1, B2. sex reversed XX male at 60 DPH; C, C1, C2. normal developed XX female at 120 DPH; D, D1, D2. XX male with dendroid testis at 120 DPH; E, E1, E2. XX male without dendroid testis at 120 DPH; F, G. total length and body weight of XX female and XX male at 60 DPH; H, I. total length and body weight of XX female and XX male at 120 DPH; n. nucleus; nu: nucleolus; zr. zona radiate; yg. yolk granules; sg. spermatogonia; sc. spermatocytes; st. spermatozoa. Asterisk (*) indicates significant differences (P＜0.05). Data for sex reversal is first reported in Yu, et al.[37]
表 1 黄颡鱼性别逆转XX个体与XY雄鱼体指标比较分析
Table 1 Comparative analysis of body index of XX pseudo-male and XY male
指标Index 对照组XY♂
CR group XY♂处理组 XX
Treatment group XX genotype完全逆转型 多精少卵型 少精多卵型 全长Total length (cm) 18.23±1.06b 19.80±1.09a 21.20±0.10a 17.49±0.41b 体重Body weight (g) 63.38±13.96a 71.50±10.94ab 85.08±1.58ab 50.58±3.59b 性腺指数GSI (%) 0.88±0.12a 0.48±0.08b 0.41±0.04b 0.18±0.08b 精巢面积/性腺面积Proportion of testis area (%) 100±0.00a 100±0.00a 70.08±14.38b 13.53±2.56c 性腺特征Gonadal characteristics 只有精巢,
呈乳白色分枝状只有精巢,
少量分枝结构精卵并存,
多乳白色精巢精卵并存,
多卵巢少精巢注: 对照组与处理组样品数量均为50尾, 其中具有繁殖能力的XX遗传型个体数量分别为完全逆转型n=4, 多精少卵型n=2, 少精多卵型n=5; 每行数据不同上标字母表示显著性差异(P<0.05)Note: Sampling numbers for control group (CR) and treatment group are both 50. In XX genotype of treatment group, the number of fertile XX male, including completely sex-reversed XX male, mostly sex-reversed XX male, minorly sex-reversed XX male are n=4, n=2, and n=5, respectively. Different superscript letters in each line indicate significant difference (P<0.05) 
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表 2 全雌黄颡鱼规模化繁殖明细
Table 2 Breeding details of the all-female yellow catfish population
序号
Num-berXX个体
Number of XX精巢总重
Testis weight (g)所用卵重
Ovum weight (g)卵粒规格(粒/g)
Egg size卵黄苗(万)
Yolk-sac larvae1 6 0.878 1609 450 10 2 5 1.390 2075 416 47 注: 所用XX个体包括完全逆转型和间性XX个体Note: XX individuals include totally sex reserved and intersex XX individuals
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