Bezerra Luana Grasiele Pereira, Silva Andréia Maria, Dantas Maiko Roberto Tavares, Dos Santos Romário Parente, Moreira Samara Sandy Jeronimo, Pereira Ana Glória, de Oliveira Moacir Franco, Comizzoli Pierre, Silva Alexandre Rodrigues
Laboratory of Animal Germplasm Conservation, Department of Animal Sciences, Universidade Federal Rural do Semi-Árido, Mossoró, RN, Brasil.
Smithsonian National Zoo and Conservation Biology Institute, Washington, USA.
Anim Reprod. 2024 Jan 5;20(4):e20230113. doi: 10.1590/1984-3143-AR2023-0113. eCollection 2023.
The objectives of the study were to (1) describe the kinematic parameters of spermatozoa (2) compare methods of evaluating sperm viability (3) validate assays of functionality and integrity of the sperm membrane and (4) evaluate possible changes between spermatozoa from the epididymis and the vas deferens of the greater rhea. Semen samples were recovered from 7 adult individuals. Sperm motility was characterized by adjusting the set-up for Computer-assisted semen analysis (CASA) to that new species. For sperm viability evaluation, smears of bromophenol blue and eosin-nigrosine dyes were used. Five solutions of different osmolarities were then tested for the hypoosmotic swelling test (HOST). The combination of fluorescent probes (propidium iodide - IP and Hoechst 33342) was also used to assess plasma membrane integrity. Data were presented as mean ± SEM. Rhea spermatozoa from the vas deferens had an overall motility of 14.6 ± 2.5%. The bromophenol blue staining technique revealed that 64.6 ± 5.2% sperm were viable, while that proportion was 72.1 ± 2.5% using eosin-nigrosine. An average of 77.6 ± 4.8% of spermatozoa reacted to the HOST with distilled water at 0 mOsm/l. Fluorescent probes indicated that 65.3 ± 2.6% of spermatozoa had intact membranes. Interestingly, no statistical differences were observed between the parameters analyzed in the epididymal spermatozoa and the vas deferens. These new assays set reference values that can now be used to further exploration of sperm handling conditions and freezing protocols in rheas.
（1）描述精子的运动学参数；（2）比较评估精子活力的方法；（3）验证精子膜功能和完整性的检测方法；（4）评估大美洲鸵附睾和输精管精子之间可能存在的差异。从7只成年个体采集精液样本。通过调整计算机辅助精液分析（CASA）设置以适应该新物种来表征精子活力。对于精子活力评估，使用溴酚蓝和伊红-黑色素染料涂片。然后测试了五种不同渗透压的溶液用于低渗肿胀试验（HOST）。还使用荧光探针组合（碘化丙啶 - IP和Hoechst 33342）评估质膜完整性。数据以平均值±标准误表示。来自输精管的美洲鸵精子总体活力为14.6±2.5%。溴酚蓝染色技术显示64.6±5.2%的精子具有活力，而使用伊红-黑色素时该比例为72.1±2.5%。平均77.6±4.8%的精子对0 mOsm/l蒸馏水的HOST有反应。荧光探针表明65.3±2.6%的精子膜完整。有趣的是，附睾精子和输精管精子分析的参数之间未观察到统计学差异。这些新检测方法设定了参考值，现在可用于进一步探索美洲鸵精子处理条件和冷冻方案。
