Improved Post-Thaw Quality of Canine Semen after Treatment with Exosomes from Conditioned Medium of Adipose-Derived Mesenchymal Stem Cells

Abstract

Simple SummaryIn this study, we have tried to exploit the potential role of exosomes-derived from canine adipose-derived mesenchymal stem cells in the protection and repair of damaged sperm during cryopreservation. The treatment of sperm with an optimal level of exosomes resulted in improved post-thaw semen quality. This improvement was due to the exosomal proteins that protect the sperm against oxidative damage and induce the repair of membranes and chromatin. The effect of exosomal proteins was also confirmed by higher expression of genes related to the repair of membranes and chromatin accompanied by the lower expression of a gene associated with reactive oxygen species production.AbstractFreezing decreases sperm quality, ultimately affecting fertilizing ability. The repair of freeze-damaged sperm is considered crucial for improving post-thaw viability and fertility. We investigated the effects of exosomes derived from canine adipose-derived mesenchymal stem cells on dog sperm structure and function during cryopreservation. The pooled ejaculate was diluted with buffer, without (Control), or with exosomal proteins (25, 50, or 100 µg/mL). Using fresh semen, the determined optimal exosomal protein concentration was 50 µg/mL (Group 2) which was used in further experiments. Post-thaw sperm treated with exosomes were superior to control (p < 0.05) in terms of motility (56.8 ± 0.3% vs. 47.2 ± 0.3%), live sperm percentage (55.9 ± 0.4% vs. 45.4 ± 0.4%), membrane integrity (55.6 ± 0.5% vs. 47.8 ± 0.3%), and acrosome integrity (60.4 ± 1.1% vs. 48.6 ± 0.4%). Moreover, expression of genes related to the repair of the plasma membrane (ANX 1, FN 1, and DYSF), and chromatin material (H3, and HMGB 1) was statistically higher in exosome-treated sperm than control, but the expression of the mitochondrial reactive oxygen species modulator 1 gene was significantly higher in control. Therefore, exosomal treatment may improve the quality of post-thaw dog semen through initiating damaged sperm repair and decreasing reactive oxygen species production.

Myoinositol Supplementation of Freezing Medium Improves the Quality-Related Parameters of Dog Sperm

“…All chemicals used were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Semen processing and evaluation was performed following the same protocol as previously explained [28].…”

The effect of astaxanthin supplementation on the post‐thaw quality of dog semen

“…All the ejaculates were assessed using phase-contrast microscopy (Leica DMIL), and ejaculates with ≥70% motility, ≥80% viability and ≥100 × 10 6 sperm/ml concentration were pooled together before use. Eosin-nigrosin staining procedure was used for the assessment of sperm viability (Qamar et al, 2019a(Qamar et al, , 2019b. Briefly, a 10 µl drop of fresh semen was mixed with an equal volume of stain.…”

“…The extended semen samples were filled into 0.5-ml straws (Minitube, Tiefenbach, Germany) followed by incubation at 4°C for 1 hr. Freezing was performed by placing the straws horizontally 2 cm above the surface of liquid nitrogen (LN 2 ) for 10 min as explained previously (Qamar et al, 2019a(Qamar et al, , 2019bSetyawan et al, 2016). Finally, the frozen straws were plunged into LN 2 and stored in the LN 2 container.…”

“…The eosin-nigrosin staining method was used for the assessment of sperm viability (Qamar et al, 2019a(Qamar et al, , 2019b. After staining, at least 200 sperm per sample were examined using ×1,000 objective of the phase-contrast microscope (Eclipse Ts 2, Nikon).…”

“…Analysis of gene expression was performed as previously explained (Qamar et al, 2019a(Qamar et al, , 2019b. F I G U R E 1 Assessment of sperm chromatin using aniline blue staining.…”