Effect of cholesterol‐loaded cyclodextrins on cryosurvival of dog spermatozoa

Fang

et al. 2019

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.

Section: Methodsmentioning

confidence: 99%

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

Fang

et al. 2019

“…Although Tris-egg yolk buffer is the most commonly used diluent for mammalian sperm, many studies show that the supplementation of anti-apoptotic factors, anti-oxidants, post-thaw enhancing chemicals, or novel cryo-protective agents is required to get good post-thaw results [ 1 , 5 , 17 , 18 ]. Thus, various molecules such as metformin [ 19 ], cholesterol [ 20 ], or α-tocopherol [ 21 ] have been successfully used in canine sperm cryopreservation to reduce oxidative stress, DNA damage [ 19 , 20 ], improve motility [ 21 ], and protect plasma membrane and acrosome integrity [ 20 ].…”

Section: Introductionmentioning

confidence: 99%

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

Mahiddine

et al. 2020

This study investigated the effects of conditioned medium (CM) from canine amniotic membrane-derived MSCs (cAMSCs) on dog sperm cryopreservation. For this purpose, flow cytometry analysis was performed to characterize cAMSCs. The CM prepared from cAMSCs was subjected to proteomic analysis for the identification of proteins present in the medium. Sperm samples were treated with freezing medium supplemented with 0%, 5%, 10%, and 15% of the CM, and kinetic parameters were evaluated after 4–6 h of chilling at 4 °C to select the best concentration before proceeding to cryopreservation. Quality-related parameters of frozen–thawed sperm were investigated, including motility; kinetic parameters; viability; integrity of the plasma membrane, chromatin, and acrosome; and mitochondrial activity. The results showed that 10% of the CM significantly enhanced motility, viability, mitochondrial activity, and membrane integrity (p < 0.05); however, the analysis of chromatin and acrosome integrity showed no significant differences between the treatment and control groups. Therefore, we concluded that the addition of 10% CM derived from cAMSC in the freezing medium protected dog sperm during the cryopreservation process.

“…Since its domestication, the dog (Canis lupus familiaris) has emerged as one of the major companion animals, playing an imperative role in human society [1]. The increasing socio-economic value of dogs has resulted in an increasing demand for advances in assisted reproductive technologies (ART) [2], particularly artificial insemination using cryopreserved semen [3]. Semen cryopreservation permits the exchange of genetic material among populations and preserves the genes of valuable individuals over extended periods [4,5].…”

Section: Introductionmentioning

confidence: 99%

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

Fang

et al. 2019

Oxidative stress during freeze–thaw procedures results in reduced semen fertility. A decrease in free radical levels can improve the post-thaw sperm quality. We examined the effects of myoinositol supplementation in freezing medium on the structure and function of cryopreserved dog sperm. Pooled ejaculates were diluted with buffer without or with myoinositol (1 or 2 mg/mL). Analysis of fresh semen revealed that the optimal concentration of myoinositol was 1 mg/mL, and this concentration was used in further experiments. Post-thaw semen quality in the myoinositol-supplemented group was superior (p < 0.05) compared with that in the control group in terms of motility (57.9 ± 0.4% vs. 47.8 ± 0.2%), sperm viability (57.5 ± 0.5% vs. 44.6 ± 0.6%), intact plasma membrane (56.6 ± 0.4% vs. 46.2 ± 0.6%), and acrosome membrane (59.3 ± 0.5% vs. 51.8 ± 0.5%). In addition, sperm in the myoinositol-supplemented group showed a significantly lower expression of pro-apoptotic (BAX) and mitochondrial reactive oxygen species (ROS) modulator (ROMO1) genes but higher expression of anti-apoptotic (BCL2), and protamine-related (PRM2 and PRM3) genes compared with that in the control group. Therefore, myoinositol supplementation before freezing can protect against oxidative stress and improve post-thaw dog sperm quality.