Domingo Paula, Olaciregui Maite, González Noelia, De Blas Ignacio, Gil Lydia
Department of Animal Pathology, Obstetric and Reproduction Area, Faculty of Veterinary Medicine, Universidad de Zaragoza, Miguel Servet 177, 50013, Zaragoza, Spain.
Department of Animal Pathology, Obstetric and Reproduction Area, Faculty of Veterinary Medicine, Universidad de Zaragoza, Miguel Servet 177, 50013, Zaragoza, Spain.
Cryobiology. 2018 Apr;81:174-177. doi: 10.1016/j.cryobiol.2018.01.004. Epub 2018 Jan 31.
Freeze-drying (FD) technique has been applied as an alternative technology to preserve gene resources to allow simple sperm preservation and shipment at 4 °C. Nevertheless, DNA sperm might be damaged by mechanical or oxidative stress throughout FD procedure. Therefore, suitable protection to maintain DNA integrity is required. The aim of this study was to determine the effect of rosmarinic acid (RA) as an antioxidant and two chelating agents (EGTA and EDTA) on the DNA integrity of freeze-dried rabbit sperm after storage of the samples at 4 °C and room temperature for 8 months. Rabbit sperm were freeze-dried in basic medium (10 mM Tris-HCl buffer and 50 mM NaCl) supplemented with 50 mM EGTA (1), 50 mM EGTA plus 105 μM RA (2), 50 mM EDTA (3) or 50 mM EDTA plus 105 μM RA (4). Semen samples were kept at 4 °C and room temperature during 8 months. After rehydration, DNA integrity was evaluated with Sperm Chromatin Dispersion test observing that DNA fragmentation was higher when semen samples were freeze-dried with EGTA (10.9%) than with EDTA (4.1%) (p < 0.01). Furthermore, RA acted better under adverse conditions and no significant differences were found in temperature storage. Summarizing, FD is a method that can allow simple gene resources preservation among 4 °C to 25 °C during 8 months and transportation without the need for liquid nitrogen or dry ice. EDTA chelating agent is the most suitable media for freeze-dried rabbit sperm and the addition of RA protects the DNA against the oxidative stress caused during FD procedure.
冷冻干燥（FD）技术已被用作一种保存基因资源的替代技术，以便在4°C下实现简单的精子保存和运输。然而，在整个冷冻干燥过程中，精子DNA可能会受到机械或氧化应激的损伤。因此，需要合适的保护措施来维持DNA的完整性。本研究的目的是确定迷迭香酸（RA）作为抗氧化剂以及两种螯合剂（乙二醇双四乙酸（EGTA）和乙二胺四乙酸（EDTA））对冷冻干燥后的兔精子DNA完整性的影响，这些样本在4°C和室温下储存8个月。兔精子在添加了50 mM EGTA（1）、50 mM EGTA加105 μM RA（2）、50 mM EDTA（3）或50 mM EDTA加105 μM RA（4）的基础培养基（10 mM Tris-HCl缓冲液和50 mM NaCl）中进行冷冻干燥。精液样本在4°C和室温下保存8个月。复水后，通过精子染色质扩散试验评估DNA完整性，结果观察到，用EGTA冷冻干燥的精液样本（10.9%）的DNA片段化程度高于用EDTA冷冻干燥的样本（4.1%）（p < 0.01）。此外，RA在不利条件下表现更好，且在温度储存方面未发现显著差异。综上所述，冷冻干燥是一种能够在4°C至25°C下实现简单的基因资源保存8个月并进行运输的方法，无需液氮或干冰。EDTA螯合剂是冷冻干燥兔精子最合适的介质，添加RA可保护DNA免受冷冻干燥过程中引起的氧化应激。
