Effects of the cryopreservation process on dog sperm integrity

Abstract

Sperm cryopreservation has become an indispensable tool in reproductive biology. However, frozen/thawed semen has a short lifespan due to loss of sperm cell integrity. To better understand which sperm cell structures are compromised by the cryopreservation process and apoptosis markers, the sperm of five healthy mature dogs was analyzed in this study. Analysis was performed after collection, cooling, and thawing via computer assisted sperm analyzer (CASA) and evaluation of membrane fluidity and permeability, phosphatidylserine translocation (Annexin V), membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation (LPO) and activity of the apoptotic markers caspases 3 and 7 by flow cytometry. Cryopreservation decreased total and progressive motility and the percentage of rapid sperm (P < 0.01). Damage to sperm cells was confirmed by Annexin V (P < 0.01), indicating that capacitation-like changes were induced by the cryopreservation procedures. An increase in sperm membrane fluidity was also noted in frozen/thawed samples (P < 0.01). Plasma and acrosomal cell membranes were affected (P < 0.01), with decreases in the subpopulation displaying high membrane potential (P < 0.01). Membrane LPO was increased in thawed sperm compared to cooled sperm (P < 0.05) but was not different from that in fresh sperm. No differences were observed in caspase 3 and 7 activity after cooling, freezing, or thawing. In conclusion, total and progressive motility, plasma membrane integrity and mitochondrial membrane potential suffered from the deleterious effects caused by cryopreservation, unlike the activity of caspases that remained stable during the freezing process.

Conditioned Medium from Canine Amniotic Membrane-Derived Mesenchymal Stem Cells Improved Dog Sperm Post-Thaw Quality-Related Parameters

“…These events ultimately result in a weakened oxidative stress defense that exposes the sperm cells to reactive oxygen species (ROS). An increase in the ROS production during cryopreservation destroys sperm lipid matrix structures and, subsequently, causes the loss of membrane integrity, an increase in lipid peroxidation, and excessive DNA fragmentation [ 7 , 8 ].…”

A triple stain method in conjunction with an in-depth screening of cryopreservation effects on post-thaw sperm in dogs

“…Cryopreservation process is recognized to reduce mitochondrial membrane potential of the spermatozoa [34], causing pore formation and the release of proapoptotic factors into the cytoplasm, damaging plasma and acrosomal membranes [35,36]. In addition, sperm structural damage has a pivotal role in generating increased reactive oxygen species (ROS) that triggers an oxidative environment, ultimately leading to damage of sperm DNA [37].…”

First successful frozen semen of the maned wolf (Chrysocyon brachyurus)

“…(2009) and Sicherle et al. (2020). Briefly, the samples were centrifuged at 720 g/10 min, the supernatant was discarded, and the pellet was resuspended to ~50 × 10 6 spermatozoa/mL in BotuCrio ® (Botupharma, Botucatu, Brazil, adjusted to osmolarity ~280 mOsmol) for both Wolf 1 and 2.…”

“…The ejaculates were analysed to volume, motility (0%-100%) and vigour (score 0-5) subjectively, and sperm concentration. Then, the semen was frozen in according to Martins et al (2009) and Sicherle et al (2020). Briefly, the samples were centrifuged at 720 g/10 min, the supernatant was discarded, and the pellet was resuspended to ~50 × 10 6 spermatozoa/mL in BotuCrio ® (Botupharma, Botucatu, Brazil, adjusted to osmolarity ~280 mOsmol) for both Wolf 1 and 2.…”