Kaneko K, Uematsu E, Takahashi Y, Tong B, Takino S, Wajiki Y, Kimura T, Yamashiro H, Kaneko Y, Iwaisaki H, Sugiyama T, Yamada T, Yamagishi S
Faculty of Agriculture, Niigata University, Nishiku, Niigata, Japan.
Reprod Domest Anim. 2013 Dec;48(6):1001-5. doi: 10.1111/rda.12200. Epub 2013 Jun 28.
This study aimed to develop a polymerase chain reaction (PCR)-based sexing and effective semen collection methods for black-headed and straw-necked ibis species. However, most birds are not sexually dimorphic, that is, the sexes appear similar. Therefore, the gender should be determined before semen collection. DNA was extracted from the blood samples of 11 black-headed and 4 straw-necked ibis. The sex was determined after PCR amplification of the EE0.6 region of W-chromosome. The PCR products were separated using gel electrophoresis. A single band indicated the presence of the EE0.6 region and that the individual was a female, while no band indicated that the individual was a male. Further, the single bands from seven specimens were amplified. Semen collection was performed by massage or a combination of massage with electro-ejaculation and was attempted during all four seasons. The semen was successfully collected in March from male straw-necked ibis using the massage method. Limited motility, viability and concentration of straw-necked ibis sperm were observed. The sperm length was 180 μm and that of the nucleus was 30 μm with acrosome located at the tip of the nucleus. Thus, the PCR-based sexing proved to be an accurate molecular sexing method for black-headed and straw-necked ibis. Furthermore, we successfully collected semen and observed the stained sperm nucleus and acrosome of the straw-necked ibis sperm. We propose that the use of this PCR methodology can be applied as a routine method for sex determination and semen collection in ibis species for future ecological research. However, considering our limited success, further studies on semen collection method are required.
本研究旨在开发基于聚合酶链反应（PCR）的黑头白鹮和彩鹮性别鉴定及有效精液采集方法。然而，大多数鸟类并无明显的性别二态性，即雌雄外观相似。因此，在采集精液前应先确定性别。从11只黑头白鹮和4只彩鹮的血样中提取DNA。通过对W染色体的EE0.6区域进行PCR扩增后确定性别。PCR产物通过凝胶电泳进行分离。单一条带表明存在EE0.6区域，个体为雌性，而无条带则表明个体为雄性。此外，对7个样本的单一条带进行了扩增。通过按摩或按摩与电射精相结合的方式进行精液采集，并在四季均进行了尝试。3月，采用按摩法成功从雄性彩鹮采集到精液。观察到彩鹮精子的活力、存活率和浓度有限。精子长度为180μm，细胞核长度为30μm，顶体位于细胞核顶端。因此，基于PCR的性别鉴定被证明是一种准确的黑头白鹮和彩鹮分子性别鉴定方法。此外，我们成功采集到精液，并观察到彩鹮精子经染色后的细胞核和顶体。我们建议，这种PCR方法可作为一种常规方法应用于未来白鹮物种生态研究中的性别鉴定和精液采集。然而，鉴于我们的成功有限，还需要进一步研究精液采集方法。
