Cryopreservation with Trehalose Reduced Sperm Chromatin Damage in Miniature Pig

Abstract

Miniature pig sperm cryopreservation is continually researched in biotechnology for breed conservation and reproduction. It is important to control the temperature at each stage of cryopreservation and cryoprotectant. It is also necessary to find the optimal cryoprotectant concentration and chemical elements of the extender. Recently, many studies have used various cryoprotectant materials, such as dimethyl sulphoxide (DMSO), ethylene glycol (EG), antifreeze protein (AFP), amides, and glycerol. Glycerol is a commonly used cryoprotectant. However, glycerol has critical cytotoxic properties, including osmotic pressure and it can cause irreversible damage to live cells. Therefore, We focused on membrane fluidity modifications can reduce cell damage from freezing and thawing procedures and evaluated on the positive effects of trehalose to the viability, chromatin integrity, and motility of boar sperm. Miniature pig sperm was separated from semen by washing with modified- Modena B (mMB) extender. After centrifugation, the pellet was diluted with the prepared first extender. This experiment was designed to compare the effects that sperm cryopreservation using two different extenders has on sperm chromatin. The control group used the glycerol only and it was compared with the glycerol and glycerol plus trehalose extender. Sperm viability and motility were evaluated using WST1assays and computer-assisted semen assays (CASA). Chromatin structure was examined using acridine orange staining. For the motility descriptors, trehalose caused a significant (p<0.01) increase in total motility (57.80 ± 4.60% in glycerol vs. 75.50 ± 6.14% in glycerol + trehalose) and progressive (51.20 ± 5.45% in glycerol vs. 70.74 ± 8.06% in glycerol +trehalose). A significant (p<0.05) increase in VAP (42.70 ± 5.73 μm/s vs. 59.65 ± 9.47 μm/s), VSL (23.06 ± 3.27μm/s vs. 34.60 ± 6.58μm/s), VCL (75.36 ± 11.36 μm/s vs. 99.55 ± 12.91μm/s), STR (54.4 ± 2.19% vs. 58.0 ± 1.63%), and LIN (32.2 ± 2.05% vs. 36.0 ± 2.45%) were also detected, respectively. The sperm DNA fragmentation index was 48.8%to glycerol only and 30.6% to glycerol plus trehalose. Trehalose added group showed higher percentages of sperm motility,stability of chromatin structure than glycerol only. In this study, we suggest that trehalose is effective in reducing freezing damage to miniature pig sperm and can reduce chromatin damage during cryopreservation.