精子冷冻保存可以成为生殖管理和保护生物多样性的有用工具。然而，冷冻方法会导致细胞结构和功能受到一些损害，可能会损害解冻后的精子活性。事实证明，补充糖类冷冻保护剂是一种成功的策略，可以减少几种物种精子的冷冻损伤，同时可以稳定质膜成分。因此，本研究旨在了解葡萄牙牡蛎精子冷冻保存过程中糖对质膜、DNA 完整性和氧化反应的影响。使用三种冷冻保护剂溶液，初始浓度为 20% 二甲基亚砜 (DMSO) 和 20% DMSO，并在人工海水中补充 0.9 M 海藻糖或蔗糖。单独收集成熟雄性的精子样本，并在人造海水中按 1:10 (v/v) 稀释，然后添加冷冻保护剂 [1:1 (v/v)]。此后，将精子装入0.5 ml细管中，在4°C下维持10分钟，在可编程生物冷冻机中以-6°C/分钟从0°C冷冻到-70°C，并储存在液氮中。样品在 37°C 浴中解冻 10 秒。采用多种技术来评估解冻后的质量。使用计算机辅助精子分析（CASA）软件和彗星试验分析精子活力和 DNA 完整性。采用流式细胞术测定膜和顶体的完整性并检测细胞内活性氧（ROS）和细胞凋亡活性。使用分光光度法检测丙二醛（MDA）来测定脂质过氧化。通过谷胱甘肽过氧化物酶、谷胱甘肽还原酶和超氧化物歧化酶来评估精子的抗氧化能力。 运动性不受含糖补充剂的影响；这些化合物并没有减少DNA损伤。然而，海藻糖和蔗糖都通过增加细胞活力和显着降低MDA含量来保护细胞的质膜。对于 ROS 也观察到了相同的结果，其中活细胞在用糖冷冻保存的样品中记录到的 ROS 水平显着降低。在补充糖的处理中，抗氧化酶的活性较高，但并不显着。总之，糖的添加似乎在冷冻保存过程中保护角牡蛎精子膜方面发挥着重要作用，显示出提高解冻后精子质量并保护细胞免受冷冻损伤的潜力。
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Effect of Trehalose and Sucrose in Post-thaw Quality of Crassostrea angulata SpermSperm cryopreservation can be a helpful tool in reproductive management and preservation of biodiversity. However, the freezing methodologies lead to some damage in structure and function of cells that may compromise post-thaw sperm activity. Cryoprotectant supplementation with sugars proved to be a successful strategy to reduce cryodamage in sperm of several species, once allowing to stabilize the plasma membrane constituents. Therefore, this study intends to understand the effects of sugars in the plasma membrane, DNA integrity, and oxidative response during Portuguese oyster sperm cryopreservation. Three cryoprotectants solutions with an initial concentration of 20% dimethyl sulfoxide (DMSO) and 20% DMSO complemented with 0.9 M trehalose or sucrose in artificial seawater were employed. Sperm samples of mature males were individually collected and diluted 1:10 (v/v) in artificial seawater followed by addition of cryoprotectants [1:1 (v/v)]. Thereafter, sperm was loaded into 0.5 ml straws, maintained at 4°C for 10 min, frozen in a programmable biofreezer at −6°C/min from 0 to −70°C, and stored in liquid nitrogen. Samples were thawed in a 37°C bath for 10 s. Several techniques were performed to evaluate post-thaw quality. Sperm motility and DNA integrity were analyzed by using computer-assisted sperm analysis (CASA) software and comet assay. Flow cytometry was employed to determine membrane and acrosome integrity and to detect intracellular reactive oxygen species (ROS) and apoptosis activity. Lipid peroxidation was determined by malondialdehyde (MDA) detection by using spectrophotometry. Sperm antioxidant capacity was evaluated through glutathione peroxidase, glutathione reductase, and superoxide dismutase. Motility was not affected by the extenders containing sugars; these compounds did not reduce the DNA damage. However, both the trehalose and sucrose protected plasma membrane of cells by increasing cell viability and significantly reducing MDA content. The same finding was observed for the ROS, where live cells registered significantly lower levels of ROS in samples cryopreserved with sugars. The activity of antioxidant enzymes was higher in treatments supplemented with sugars, although not significant. In conclusion, the addition of sugars seems to play an important role in protecting the Crassostrea angulata sperm membrane during cryopreservation, showing potential to improve the post-thaw sperm quality and protect the cells from cryoinjuries.