Kumar Abhishek, Prasad J K, Srivastava N, Ghosh S K
Germ-Plasm Centre, Division of Animal Reproduction, ICAR-Indian Veterinary Research Institute, Izatnagar, India.
Biopreserv Biobank. 2019 Dec;17(6):603-612. doi: 10.1089/bio.2019.0037. Epub 2019 Aug 20.
The aim of the article is to report a review on different sperm cryopreservation techniques, various stress-related freeze-thaw damages altering sperm structure and function during conventional cryopreservation, and strategies to minimize these stresses. Sperm cryopreservation has allowed indefinite storage and successful transportation of valuable germplasm from proven sites at distant locations, for genetic upgradation through implementation of reproductive techniques, such as artificial insemination. Different techniques for sperm cryopreservation have been proposed such as conventional freezing techniques, directional freezing, and sperm vitrification. Drawbacks related to conventional freezing methods, such as heterogeneous ice nucleation and repeated freeze-thaw cycles at the ice front that disrupts and kill sperm cells, led to the emergence of the directional freezing technique. Sperm vitrification is advantageous as there is no ice crystal-induced physical damages to sperm. However, sperm vitrification has less applicability as encouraging results are only reported in human, dog, and cat. In spite of several drawbacks, conventional freezing techniques are still most widely used for sperm cryopreservation. Spermatozoa experience stresses in the form of cold shock, osmotic stress, and mainly oxidative stress during conventional cryopreservation ultimately reduces the sperm viability and fertility. Several attempts have been made in the past to minimize all these stresses individually or in combination. Membrane fluidity was increased to prevent the cold shock and cryocapacitation-like changes by the addition of cholesterol to the membrane. Antifreeze proteins were added in semen extender to minimize freeze-thaw damages due to heterogeneous ice nucleation and ice recrystallization. Oxidative stress was reduced either by neutralizing reactive oxygen species (ROS) through enzymatic, nonenzymatic, plant-based antioxidants or reductants; or by minimizing the level of sources like the semen radiation exposure, leucocytes, and dead and defective spermatozoa, which lead to ROS production during the semen cryopreservation process. A novel approach of minimizing oxidative stress was to reduce the oxygen tension in sperm microenvironment that is, extender by partial deoxygenation process, as a number of literatures pointed out direct link of O with ROS production. When compared with other strategies, partial deoxygenation of semen extender with N gassing is found as a cost-effective, comparatively easy and a potential approach to large-scale frozen semen production.
本文旨在报道一篇关于不同精子冷冻保存技术的综述，常规冷冻保存过程中各种与应激相关的冻融损伤如何改变精子结构和功能，以及将这些应激降至最低的策略。精子冷冻保存使得宝贵的种质能够从远处的可靠地点进行无限期储存和成功运输，以便通过人工授精等生殖技术实现遗传升级。已经提出了不同的精子冷冻保存技术，如常规冷冻技术、定向冷冻和精子玻璃化。与常规冷冻方法相关的缺点，如异质冰核形成以及在冰前沿的反复冻融循环会破坏和杀死精子细胞，导致了定向冷冻技术的出现。精子玻璃化具有优势，因为没有冰晶对精子造成物理损伤。然而，精子玻璃化的适用性较低，因为仅在人类、狗和猫身上报道了令人鼓舞的结果。尽管存在一些缺点，常规冷冻技术仍然是精子冷冻保存中使用最广泛的方法。在常规冷冻保存过程中，精子会经历冷休克、渗透压应激，主要是氧化应激等应激，最终降低精子活力和生育能力。过去已经进行了多次尝试，单独或组合地将所有这些应激降至最低。通过向膜中添加胆固醇来增加膜流动性，以防止冷休克和类似冷冻获能的变化。在精液稀释剂中添加抗冻蛋白，以将由于异质冰核形成和冰重结晶导致的冻融损伤降至最低。通过酶促、非酶促、植物性抗氧化剂或还原剂中和活性氧（ROS）来降低氧化应激；或者通过尽量减少精液辐射暴露、白细胞以及死精和有缺陷精子等导致精液冷冻保存过程中产生ROS的来源水平。一种降低氧化应激的新方法是通过部分脱氧过程降低精子微环境即稀释剂中的氧张力，因为许多文献指出氧与ROS产生之间存在直接联系。与其他策略相比，用氮气充气对精液稀释剂进行部分脱氧被认为是一种经济高效、相对容易且有潜力用于大规模冷冻精液生产的方法。
