Military Horse Breeding Center in Zaragoza, Spain.
Department of Animal Pathology, Faculty of Veterinary Medicine, Instituto Agroalimentario de Aragón IA2 (Universidad de Zaragoza-CITA), Zaragoza, Spain.
Anim Reprod Sci. 2019 Dec;211:106205. doi: 10.1016/j.anireprosci.2019.106205. Epub 2019 Oct 22.
The aim of this study was to evaluate the effect of trehalose and lactose extenders on ejaculated and epididymal stallion sperm vitrification. Ejaculated semen samples were collected from seven fertile stallions, and cauda epididymis samples were collected from ten stallion carcasses after slaughter. Both the ejaculated and the epididymis samples were diluted and vitrified using INRA 96® and bovine serum albumin as well as trehalose or lactose. As a control, ejaculated and epididymal samples were collected and frozen using the conventional method. Vitrification was performed by immersing sperm suspensions directly in LN2. After thawing or devitrification, there was assessment of samples for sperm motility using computer-assisted analysis. Viability was assessed using SYBR-14 and propidium iodide (PI) and acrosome integrity by fluorescein using isothiocyanate combined with peanut agglutinin (FITC-PNA) and PI. Epididymal sperm vitrification with trehalose (EPT) or lactose (EPL) resulted in greater progressive sperm motility than sperm of the control sample (EPC). After post-thaw/devitrification of sperm in the EPT group, sperm motility was greater (P<0.001) compared to that using EPL (50.72 ± 5.09% compared with 34.21 ± 3.02%). The results from assessment of ejaculated sperm samples after undergoing the vitrification process indicated cells were less viable (P<0.001) than the control (EJC) sample. In conclusion, vitrification of epididymal stallion sperm using trehalose might be a beneficial alternative for the long-term storage of sperm samples with great economic value. Spermatozoa from vitrified ejaculates of stallions, however, had lesser motility and viability rates than samples subjected to conventional freezing.
本研究旨在评估海藻糖和乳糖添加剂对精子玻璃化的影响。从 7 头健康公马中采集了精液样本，从 10 头屠宰后公马的附睾中采集了附睾样本。使用 INRA 96® 和牛血清白蛋白以及海藻糖或乳糖对精液和附睾样本进行稀释和玻璃化。作为对照，使用常规方法收集和冷冻精液和附睾样本。玻璃化是通过将精子悬浮液直接浸入 LN2 中进行的。解冻或去玻璃化后，使用计算机辅助分析评估精子运动能力。使用 SYBR-14 和碘化丙啶（PI）评估活力，通过异硫氰酸荧光素结合花生凝集素（FITC-PNA）和 PI 评估顶体完整性。使用海藻糖（EPT）或乳糖（EPL）对附睾精子进行玻璃化处理可使精子的运动能力显著提高（P<0.001）。在 EPT 组中，精子解冻/去玻璃化后，精子运动能力明显提高（P<0.001），与 EPL 组相比（50.72±5.09%与 34.21±3.02%）。经过玻璃化处理的精液样本的评估结果表明，与对照组（EJC）相比，活细胞较少（P<0.001）。结论：使用海藻糖对附睾精子进行玻璃化处理可能是具有重要经济价值的精子样本长期储存的有益替代方法。然而，与常规冷冻相比，玻璃化处理的精液精子的运动能力和活力较低。
