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龙群妹,唐志哲,唐家兴,张海成,罗庆华,王杰.2024.中国大鲵幼体的性别分子鉴定与性腺形态.动物学杂志,59(2):217-224.
中国大鲵幼体的性别分子鉴定与性腺形态
Molecular Sex Identification and Gonadal Morphology of Andrias davidianus Juveniles
投稿时间:2023-07-31  
DOI:10.13859/j.cjz.202423155
中文关键词:  中国大鲵  幼体  性腺  分子鉴定  解剖学实验
英文关键词:Chinese Giant Salamander, Andrias davidianus  Juvenile  Gonads  Molecular identification  Anatomical experiments
基金项目:国家自然科学基金项目(No. 32170435,32060238,E1J1040001);
作者单位
龙群妹 吉首大学生物资源与环境科学学院大鲵资源保护与综合利用湖南省工程实验室 吉首 416000
中国科学院成都生物研究所 成都 610299
长沙学院生物与化学工程学院 长沙 410022 
唐志哲 四川省成都市玉林中学 成都 610041 
唐家兴 中国科学院成都生物研究所 成都 610299
四川师范大学生命科学学院 成都 610101 
张海成 陕西汉中多成大鲵开发有限公司 略阳 724305 
罗庆华 吉首大学生物资源与环境科学学院大鲵资源保护与综合利用湖南省工程实验室 吉首 416000
长沙学院生物与化学工程学院 长沙 410022 
王杰 中国科学院成都生物研究所 成都 610299 
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中文摘要:
      为探明中国大鲵(Andrias davidianus)雌雄幼体的性腺发育特征,确定适合的性别分子鉴定方法,对15尾5月龄和17尾17月龄养殖个体进行形态测量、解剖观察、性腺组织切片及PCR扩增雌性特异DNA片段。结果发现,引物adf225和adf340的扩增效果好,判定5月龄个体8雌7雄;17月龄个体8雌9雄,与依据性腺形态结构区分的结果一致。体视显微镜下5月龄幼体中肾腹侧有两条半透明细条状的原始生殖嵴;组织切片显示生殖细胞形态分化不明显。17月龄卵巢波浪状弯曲,有颗粒感,精巢呈光滑的白条状,形态分化明显;组织切片显示,卵巢分化出体积较大的卵母细胞,同时保留原始卵泡,精巢分化出生精小叶和精原细胞、支持细胞。外形测量显示,5月龄与17月龄性二型不明显,不能根据外形判断性别。本研究确定了大鲵幼体性别分子鉴定的最佳引物,可用于养殖过程中雌雄选配,以节约资源。
英文摘要:
      [Objectives] The Chinese Giant Salamander Andrias davidianus is the largest amphibian on earth. Although its captive population has exceeded 10 million individuals, it is listed as Critically Endangered on TheIUCN Red Listof Threatened Species. Sexual identification of giant salamanders has long been challenging, although it is crucial to captive breeding and conservation. Consequently, the development of molecular markers targeting female-specific DNA fragments could offer a dependable method for distinguishing between males and females. [Methods] In this study, 15 5-month-old and 17 17-month-old individuals from an artificial breeding farm in Hanzhong, Shaanxi were randomly selected and dissected. The gonad was utilized for tissue section and Hematoxylin and Eosin staining. DNA was extracted from each individual and the female-specific DNA fragments were amplified using four pairs of primers. [Results] The primers adf225 and adf340 yielded the best amplification results, which were consistent with the morphological analysis of gonads (Fig. 1). In both male and female 5-month-old juveniles, the gonads were undifferentiated, appearing as slender, semi-transparent stripes (Fig. 2a, b). In contrast, 17-month-old juveniles displayed obvious differentiation in gonads, with granular ovaries or smooth, and white testes (Fig. 2c, d). Tissue section analysis revealed no significant morphological differences in germ cells between male and female 5-month-old individuals (Fig. 3a, b). However, in the 17-month-old individuals, ovarian cells had differentiated into larger oocytes, while still maintaining the primordial follicles (Fig. 3c). Additionally, sperm nests of testes had differentiated into spermatogonia lobules composed of spermatogonia and sertoli cells (Fig. 3d). Interestingly, there were no visible morphological differences between male and female 5-month-old or 17-month-old juveniles, indicating that external morphology alone is insufficient for sex determination (Fig. 4). [Conclusion] This study successfully identified molecular primers that are highly effective in determining the gender of juveniles. This significant finding provides a practical solution to the challenges posed by sex allocation and holds promising implications for the conservation and management of this species.
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