Strategies to Minimize Various Stress-Related Freeze-Thaw Damages During Conventional Cryopreservation of Mammalian Spermatozoa

Abstract

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 O₂ with ROS production. When compared with other strategies, partial deoxygenation of semen extender with N₂ gassing is found as a cost-effective, comparatively easy and a potential approach to large-scale frozen semen production.

Keywords: cold shock; conventional freezing; directional freezing; osmotic stress; oxidative stress; sperm cryopreservation; sperm vitrification.