Santiani Alexei, Cucho Miguel, Delgado Josselyn, Juárez Javier, Ruiz Luis, Evangelista-Vargas Shirley
Laboratory of Animal Reproduction, Faculty of Veterinary Medicine, Universidad Nacional Mayor de San Marcos (UNMSM), 2800 Circunvalación Av., San Borja, Lima 15021, Peru.
Grupo de Investigación en Biotecnología Reproductiva Animal, Dirección General de Investigación, Desarrollo e Innovación, Universidad Científica del Sur (UCSUR), Panamericana Sur Km. 19, Villa el Salvador, Lima 15018, Peru.
Animals (Basel). 2025 Aug 5;15(15):2282. doi: 10.3390/ani15152282.
In remote alpaca breeding regions, access to advanced sperm analysis laboratories is limited. This study validates a practical cytometric method for evaluating sperm viability and acrosomal integrity in epididymal alpaca sperm using early fluorochrome staining, formaldehyde fixation, and intermediate storage. Thirty-two testes were transported at 5 °C, and spermatozoa were collected from the cauda epididymis. After morphometric screening, 26 samples were included. Aliquots were stained with Zombie Green (viability) and FITC-PSA (acrosomal integrity), at time zero. Each aliquot was divided for cytometric analysis at T0 (immediately), T24 (24 h after formaldehyde fixation) and T1w (1 week post-fixation). Fixed samples showed higher viability and acrosomal integrity values (T24: 70.75%, 97.24%; T1w: 71.80%, 97.21%) than T0 (67.63%, 95.89%). This may reflect fluorescence alterations associated with fixation. Strong correlations and Bland-Altman analysis confirmed consistency across time points. This method enables accurate sperm quality evaluation up to one week after collection, offering a useful tool for reproductive monitoring in field conditions without immediate analysis. Further research on ejaculated semen and field protocols is recommended.
在偏远的羊驼养殖地区，使用先进的精子分析实验室存在限制。本研究验证了一种实用的细胞计数方法，该方法通过早期荧光染色、甲醛固定和中间储存来评估附睾羊驼精子的活力和顶体完整性。32个睾丸在5°C下运输，从附睾尾部收集精子。经过形态学筛选，纳入了26个样本。在时间零点，等分试样用Zombie Green（活力）和FITC-PSA（顶体完整性）染色。每个等分试样在T0（立即）、T24（甲醛固定后24小时）和T1w（固定后1周）进行细胞计数分析。固定后的样本显示出比T0（67.63%，95.89%）更高的活力和顶体完整性值（T24：70.75%，97.24%；T1w：71.80%，97.21%）。这可能反映了与固定相关的荧光变化。强相关性和布兰德-奥特曼分析证实了各时间点之间的一致性。该方法能够在采集后长达一周的时间内准确评估精子质量，为在没有即时分析的现场条件下进行生殖监测提供了一种有用的工具。建议对射出精液和现场方案进行进一步研究。
