Della Togna Gina, Trudeau Vance L, Gratwicke Brian, Evans Matthew, Augustine Lauren, Chia Han, Bronikowski Edward J, Murphy James B, Comizzoli Pierre
Smithsonian Conservation Biology Institute, Smithsonian National Zoological Park, 3001 Connecticut Ave., NW, Washington, DC, 20008, USA.
Department of Biology, University of Ottawa, 30 Marie Curie St., Ottawa, ON, K1N 6N5, Canada.
Theriogenology. 2017 Mar 15;91:27-35. doi: 10.1016/j.theriogenology.2016.12.033. Epub 2016 Dec 27.
Knowledge of basic gamete biology is critical to better protect and propagate endangered amphibian species and also to develop reproductive technologies combined with germplasm cryopreservation. The objectives of the study were to test different hormonal stimulations and then characterize the quantity and quality of Panamanian golden frog (Atelopus zeteki) spermatozoa. Following intraperitoneal injection of the gonadotropin-releasing hormone agonist (des-Gly, D-Ala, Pro-NHEt-GnRH 1, 2 or 4 μg/g of body weight), human chorionic gonadotropin (hCG; 5 or 10 IU/gbw), or Amphiplex™ (0.4 μg/gbw GnRH-A + 10 μg/gbw metoclopramide hydrochloride), spermic urine samples from 29 males were collected at different time points (from 0.5 to 24 h post-injection) to analyze the concentration, motility, and morphology of the spermatozoa. Peak of sperm concentration was observed at 3.5 h post injection for all hormonal treatments. Amphiplex™ led to the highest sperm concentrations (4.45 ± 0.07 × 10 cells/mL) followed by 4 μg/gbw GnRH-A (2.65 ± 0.21 × 10 cells/mL). Other stimulation protocols and doses induced sperm production, but at lower levels (ranging from 1.34 to 1.70 × 10 cells/mL). More than 60% of spermatozoa were motile following all treatments but the highest motility (>90%) was obtained from the 10 IU/gbw hCG treatment. Spermic urine samples obtained with all hormone treatments had higher pH (ranging from 7.1 to 7.8) than the urine alone (6.7-6.8). Spermatozoa were filiform and elongated with an apical acrosome, a mitochondrial sheath, a small midpiece and a long tail with an undulating membrane. More than 80% of cells were morphologically normal and 50-70% had intact DNA. These sperm characteristics were not influenced by hormonal treatments. This first comprehensive characterization of sperm samples following optimized hormonal stimulations in A. zeteki lays the foundation for more fundamental studies, reproductive technologies, and future preservation strategies.
了解基本的配子生物学对于更好地保护和繁殖濒危两栖动物物种以及开发与种质冷冻保存相结合的生殖技术至关重要。本研究的目的是测试不同的激素刺激，然后对巴拿马金蛙（泽氏斑蟾）精子的数量和质量进行表征。在腹腔注射促性腺激素释放激素激动剂（去甘氨酸、D - 丙氨酸、脯氨酸 - N - 乙基 - 促性腺激素释放激素1，2或4μg/g体重）、人绒毛膜促性腺激素（hCG；5或10IU/gbw）或Amphiplex™（0.4μg/gbw促性腺激素释放激素 - A + 10μg/gbw盐酸甲氧氯普胺）后，在不同时间点（注射后0.5至24小时）收集29只雄性的精液样本，以分析精子的浓度、活力和形态。所有激素处理在注射后3.5小时均观察到精子浓度峰值。Amphiplex™导致最高的精子浓度（4.45±0.07×10⁶细胞/mL），其次是4μg/gbw促性腺激素释放激素 - A（2.65±0.21×10⁶细胞/mL）。其他刺激方案和剂量诱导了精子产生，但水平较低（范围为1.34至1.70×10⁶细胞/mL）。所有处理后超过60%的精子具有活力，但最高活力（>90%）来自10IU/gbw hCG处理。所有激素处理获得的精液样本的pH值（范围为7.1至7.8）高于单独尿液（6.7 - 6.8）。精子呈丝状且细长，具有顶端顶体、线粒体鞘、小中段和带有波动膜的长尾巴。超过80%的细胞形态正常，50 - 70%具有完整的DNA。这些精子特征不受激素处理的影响。在泽氏斑蟾中经过优化激素刺激后对精子样本进行的首次全面表征为更基础的研究、生殖技术和未来的保护策略奠定了基础。
