Improved viability and fertility of frozen-thawed dog sperm using adipose-derived mesenchymal stem cells

Abstract

Cryopreservation procedures negatively affect the quality traits of sperm, causing certain changes at structural and molecular levels due to thermal, mechanical, osmotic, and oxidative damage. The objective of this study was to examine the potential of canine adipose-derived mesenchymal stem cells (Ad-MSCs) for providing protection to the dog sperm against cryo-damage. Canine Ad-MSCs were selected on the basis of the significantly higher gene expression for different proteins actively involved in the cell repair including annexin 1 (ANX1), histone H3 (H3) and high mobility group B (HMGB) protein compared to skin fibroblasts. Semen was collected from four healthy dogs by digital manipulation. The washed pooled ejaculates were diluted with buffer 2 (extender) supplemented without Ad-MSCs (Control), with 2.5 × 10 6 Ad-MSCs/mL (Group 1) or with 5 × 10 6 Ad-MSCs/mL (Group 2). Group 1 exhibited significantly higher post-thaw motility, live sperm, intact plasma membrane and normal acrosomes than the other groups. Additionally, Group 1 showed significantly higher expression levels of genes related to the repair of membranes (ANX1, dysferlin; DYSF, and fibronectin; FN1) and chromatin material (H3 and HMGB). Protein expression of ANX1, H 3, and FN1 was also statistically more in Group 1 than in Control. The results confirm that canine Ad-MSCs can effectively preserve the quality of frozenthawed sperm by a reduction in cryoinjury. At an appropriate concentration, Ad-MSCs significantly improve the quality of post-thaw dog sperm. Semen cryopreservation is regarded as the most important step of artificial insemination which is the most widely adopted assisted reproductive technology (ART) in canine practice 1. It can facilitate the storage of the genetic material for an extended period, conserve the elite individual's fertility and serve as a useful tool for preserving the endangered species. In addition, it has greatly benefited the animal-based industry by reducing the cost and the stress associated with transportation, overcoming the quarantine restrictions and breeding issues (e.g., aggressive behavior and size issues) 2. Despite many attempts to improve freezing agents and techniques, reduction in semen quality still remains the major issue associated with freezing procedures. During freezing the quality-related traits of sperm are highly compromised 3. Sperm undergoes certain detrimental changes at the structural and molecular levels as a result of thermal, mechanical, osmotic, and oxidative damage 4,5. The sperm that survives during freezing procedure suffers a reduction in fertility 6 and this has been linked with damage that adversely affects viability, motility, plasma membrane, acrosome and chromatin material 7. In addition, the activation of apoptotic pathways results in the fragmentation of sperm cell DNA 8. These changes ultimately contribute to an overall reduction in the fertility of sperm. Current protective modifications employed to minimize the above mentioned damaging effects involve the use of different ty...

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

“…In addition, fibronectin, an ECM component that acts as a growth factor [ 99 ], was found in the cAMSC-CM, which is also involved in protecting membrane integrity ( Table S2 and Table 1 ). Qamar et al [ 100 ] showed that fibronectin was expressed in adipose-derived MSCs and that plasma membrane integrity was protected when adipose-derived MSCs were added to the freezing media. This suggests that cAMSC-CM protected plasma membrane integrity in the present study ( Table 6 ).…”

Resveratrol supplementation into extender protects against cryodamage in dog post-thaw sperm

“…The sperm cryopreservation process involves freezing and thawing, leading to significant physiological and chemical changes in the sperm, which in turn causes the production of reactive oxygen species (ROS) [ 40 , 44 ]. This is mainly due to the removal of seminal plasma during the cryopreservation process, which reduces antioxidant defenses in sperm; thus, sperm become vulnerable to oxidative stress [ 31 ].…”

Semen evaluation: methodological advancements in sperm quality-specific fertility assessment

“…Methods used for analyzing sperm plasma membrane are normally based on the enhanced permeability of damaged membrane [ 107 ]. These methods include the hypo-osmotic swelling (HOS) test [ 108 , 109 ], eosin–nigrosin staining [ 110 , 111 ], and use of fluorescent probes [ 112 , 113 ]. Fluorescent probes such as Hoechst or propidium-iodide (PI) are either used alone or in combination with other permeable fluorochromes such as carboxyfluorescein diacetate or SYBR [ 114 – 116 ].…”