MicroRNA profiling of royal jelly extracellular vesicles and their potential role in cell viability and reversing cell apoptosis.

MiRNAs are small non-coding RNA molecules that play important regulatory roles in diverse biological processes. Royal jelly, a milky-white substance produced by nurse honeybees (Apis mellifera), is the primary food of queen bees and plays a crucial role in their development. However, little is known about the microRNA (miRNAs) content of royal jelly and their potential functions. In this study, we isolated extracellular vesicles from the royal jelly of 36 samples through sequential centrifugation and targeted nanofiltration and performed high-throughput sequencing to identify and quantify the miRNA content of honeybee royal jelly extracellular vesicles (RJEVs). We found a total of 29 known mature miRNAs and 17 novel miRNAs. Through bioinformatic analysis, we identified several potential target genes of the miRNAs present in royal jelly, including those involved in developmental processes and cell differentiation. To investigate the potential roles of RJEVs in cell viability, RJEVs were supplemented to apoptotic porcine kidney fibroblasts induced by ethanol 6% exposure for 30 min. TUNEL assay showed a significant reduction in the apoptosis percentage after RJEV supplementation when compared with the non-supplemented control group. Moreover, the wound healing assay performed on the apoptotic cells showed a rapid healing capacity of RJEV-supplemented cells compared to the control group. We observed a significant reduction in the expression of the miRNA target genes such as FAM131B, ZEB1, COL5A1, TRIB2, YBX3, MAP2, CTNNA1, and ADAMTS9 suggesting that RJEVs may regulate the target gene expression associated with cellular motility and cell viability. Moreover, RJEVs reduced the expression of apoptotic genes (CASP3, TP53, BAX, and BAK), while significantly increasing the expression of anti-apoptotic genes (BCL2 and BCL-XL). Our findings provide the first comprehensive analysis of the miRNA content of RJEVs and suggest a potential role for these vesicles in the regulation of gene expression and cell survival as well as augmenting cell resurrection or anastasis.

Oviduct epithelial cells-derived extracellular vesicles improve preimplantation developmental competence of in vitro produced porcine parthenogenetic and cloned embryos.

Extracellular vesicles (EVs) carry bioactive cargoes involved in the early preimplantation development. This study investigated the effects of EVs obtained from an oviductal epithelial cell (OEC) conditioned medium on the developmental competence of in parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) porcine embryos. The OEC-EV-treated group showed significant increases in blastocyst formation and hatching rates compared to the control group (40.8% ± 2.2% and 20.1% ± 2.1% vs. 24.9% ± 2.0% and 5.3% ± 1.1%; p < 0.05), respectively. The 7 day OEC-EVs treatment group significantly increased blastocyst formation rate than the 3 day and 0 day-groups (45.0 ± 0.8 vs. 33.0 ± 0.7 and 26.7 ± 0.5; p < 0.05), respectively. SCNT revealed that the OEC-EV increased blastocyst formation rate compared to that of oviductal fluid EVs (OF-EVs) (35.4% ± 1.4% vs. 29.3% ± 1.3%; p < 0.05). Reactive oxygen species levels, apoptosis, and blastocyst lipid content were significantly decreased in the OEC-EVs group compared with the control group. OEC-EV group showed a significantly decreased BAX and increased BCL2, SOD1, POU5F1, SOX2, NANOG, GATA6, PNPLA2, LIPE, and MGLL gene expression than the control group (p < 0.05). In conclusion, OEC-EVs supplementation in embryo culture media improved the quality of porcine embryos, potentially helping porcine-cloned embryonic development possibly through transfer of messenger RNA and proteins to the early embryos.

Improved efficiencies in the generation of multigene-modified pigs by recloning and using sows as the recipient.

This study was performed to improve production efficiency at the level of recipient pig and donor nuclei of transgenic cloned pigs used for xenotransplantation. To generate transgenic pigs, human endothelial protein C receptor (hEPCR) and human thrombomodulin (hTM) genes were introduced using the F2A expression vector into GalT-/-/hCD55+ porcine neonatal ear fibroblasts used as donor cells and cloned embryos were transferred to the sows and gilts. Cloned fetal kidney cells were also used as donor cells for recloning to increase production efficiency. Pregnancy and parturition rates after embryo transfer and preimplantation developmental competence were compared between cloned embryos derived from adult and fetal cells. Significantly higher parturition rates were shown in the group of sows (50.0 vs. 4.1%), natural oestrus (20.8 vs. 0%), and ovulated ovary (16.7 vs. 5.6%) compared with gilt, induced and non-ovulated, respectively (P < 0.05). When using gilts as recipients, final parturitions occurred in only the fetal cell groups and significantly higher blastocyst rates (15.1% vs. 21.3%) were seen (P < 0.05). Additionally, gene expression levels related to pluripotency were significantly higher in the fetal cell group (P < 0.05). In conclusion, sows can be recommended as recipients due to their higher efficiency in the generation of transgenic cloned pigs and cloned fetal cells also can be recommended as donor cells through correct nuclear reprogramming.

Oviduct epithelial cell­derived extracellular vesicles promote the developmental competence of IVF porcine embryos.

Assisted reproductive technology has increased the efficiency of animal reproduction. However, polyspermy is a significant limitation of porcine in vitro fertilization (IVF). Therefore, reducing the polyspermy rate and improving monospermic embryos is crucial. Recent studies have reported that oviductal fluid, along with its contents of extracellular vesicles (EVs), enhanced the fertilization process and supported embryo development. Consequently, the present study investigated the effects of porcine oviduct epithelial cells (OEC­EVs) on sperm­oocyte interactions during porcine IVF and evaluated in vitro embryo developmental competence outcomes. During IVF embryo development, the cleavage rate was significantly higher in the group treated with 50 ng/ml OEC­EVs compared with the control group (67.6±2.5 vs. 57.3±1.9; P<0.05). Furthermore, the OEC­EV group had significantly more embryos (16.4±1.2 vs. 10.2±0.8; P<0.05), and the polyspermy rate significantly decreased (32.9±2.5 vs. 43.8±3.1; P<0.05) compared with that of the control group. Additionally, the fluorescence intensities of cortical granules (3.56±0.47 vs. 2.15±0.24; P<0.05) and active mitochondria (8.14±0.34 vs. 5.96±0.38; P<0.05) were significantly higher in the OEC­EV group compared with those in the control group. In conclusion, OEC­EV adsorption and penetration crosstalk between sperm and oocytes was observed. OEC­EV treatment was demonstrated to significantly improve the concentration and distribution of cortical granules in oocytes. Furthermore, OEC­EVs also increased oocyte mitochondrial activity, reduced polyspermy and increased the IVF success rate.

Role of antioxidants in fertility preservation of sperm - A narrative review.

Male fertility is affected by multiple endogenous stressors, including reactive oxygen species (ROS), which greatly deteriorate the fertility. However, physiological levels of ROS are required by sperm for the proper accomplishment of different cellular functions including proliferation, maturation, capacitation, acrosomal reaction, and fertilization. Excessive ROS production creates an imbalance between ROS production and neutralization resulting in oxidative stress (OS). OS causes male infertility by impairing sperm functions including reduced motility, deoxyribonucleic acid damage, morphological defects, and enhanced apoptosis. Several in-vivo and in-vitro studies have reported improvement in quality-related parameters of sperm following the use of different natural and synthetic antioxidants. In this review, we focus on the causes of OS, ROS production sources, mechanisms responsible for sperm damage, and the role of antioxidants in preserving sperm fertility.

The combination of rolipram and cilostamide improved the developmental competence of cloned porcine embryos.

In vitro maturation of porcine oocytes is characterized by asynchronous cytoplasmic and nuclear maturation, leading to less competent oocytes supporting embryo development. The purpose of this study was to evaluate the combined effect of rolipram and cilostamide as cyclic Adenine monophosphate (cAMP) modulators to find the maximum cAMP levels that temporarily arrest meiosis. We determined the optimal time to maintain functional gap junction communication during pre-in vitro maturation to be four hours. Oocyte competence was evaluated by the level of glutathione, reactive oxygen species, meiotic progression, and gene expression. We evaluated embryonic developmental competence after parthenogenetic activation and somatic cell nuclear transfer. The combined treatment group showed significantly higher glutathione and lower reactive oxygen species levels and a higher maturation rate than the control and single treatment groups. Cleavage and blastocyst formation rates in parthenogenetic activation and somatic cell nuclear transfer embryos were higher in two-phase in vitro maturation than in the other groups. The relative levels of BMP15and GDF9 expression were increased in two-phase in vitro maturation. Somatic cell nuclear transfer blastocysts from two-phase in vitro maturation oocytes showed a lower level of expression of apoptotic genes than the control, indicating better pre-implantation developmental competence. The combination of rolipram and cilostamide resulted in optimal synchrony of cytoplasmic and nuclear maturation in porcine in vitro matured oocytes and there by enhanced the developmental competence of pre-implantation embryos.

Isolation, characterization, proteome, miRNAome, and the embryotrophic effects of chicken egg yolk nanovesicles (vitellovesicles).

Egg yolk constitutes about a third of the structure of the chicken egg however, the molecular structure and physiological effects of egg yolk-derived lipid membranous vesicles are not clearly understood. In this study, for the first record, the egg yolk nanovesicles (vitellovesicles, VVs) were isolated, characterized, and used as a supplement for porcine embryo culture. Yolks of ten freshly oviposited eggs were filtered and ultracentrifuged at 100,000 × g for 3 h to obtain a pellet. Cryogenic transmission electron microscopy and nanoparticle tracking analysis of the pellet revealed bilipid membranous vesicles. Protein contents of the pellet were analyzed using tandem mass spectrometry and the miRNA content was also profiled through BGISEQ-500 sequencer. VVs were supplemented with the in vitro culture medium of day-7 hatched parthenogenetic blastocysts. After 2 days of blastocyst culture, the embryonic cell count was increased in VVs supplemented embryos in comparison to the non-supplemented embryos. TUNEL assay showed that apoptotic cells were increased in control groups when compared with the VVs supplemented group. Reduced glutathione was increased by 2.5 folds in the VVs supplemented group while reactive oxygen species were increased by 5.3 folds in control groups. Quantitative PCR analysis showed that VVs significantly increased the expression of lipid metabolism-associated genes (monoglyceride lipase and lipase E), anti-apoptotic gene (BCL2), and superoxide dismutase, while significantly reducing apoptotic gene (BAX). Culturing embryos on Matrigel basement membrane matrix indicated that VVs significantly enhanced embryo attachment and embryonic stem cell outgrowths compared to the non-supplemented group. This considers the first report to characterize the molecular bioactive cargo contents of egg yolk nanovesicles to show their embryotrophic effect on mammalian embryos. This effect might be attributed to the protein and miRNA cargo contents of VVs. VVs can be used for the formulation of in vitro culture medium for mammalian embryos including humans.

Retraction notice to "Centella asiatica extract in Carboxymethyl Cellulose at its optimal concentration improved wound healing in mice model" [Heliyon 8, (2022) Article e12031].

[This retracts the article DOI: 10.1016/j.heliyon.2022.e12031.].

Epidemiological investigations of Salmonella and Escherichia coli associated morbidity and mortality in layer chickens in Hawassa city, Southern Ethiopia.

Background: This study was conducted with the objectives of estimating the morbidity and mortality rates in layer chickens, identifying the risk factors associated with morbidity and mortality, and identifying the major bacterial pathogens affecting small-scale commercial layers in Hawassa. A longitudinal observational study design was employed from November 2019 to March 2020. The chickens on selected farms were checked for morbidity and mortality twice a week. During each visit, clinical examination of sick birds and pathological investigation of dead birds were conducted. Cloaca samples were collected for isolation and identification of Salmonella spp. and E. coli. A Cox proportional hazard model was used to quantify the effects of various risk factors on the morbidity and mortality rates observed. Results: Of the 8976 chickens followed, 106 developed clinical disease, giving a morbidity of 1.18% (95% CI: 0.97, 1.43). The overall morbidity rate was 2.37 (95% CI: 1.94, 2.87) per 1000 chicken months. A total of 101 of the chickens under study were found dead, yielding a mortality of 1.13% (95% CI: 0.92, 1.37) and a mortality rate of 2.26 (95% CI: 1.84, 2.75). Multivariable Cox regression analysis showed that farm hygiene, the experience of farm manager, housing condition, housing systems, the availability of veterinary services and age of chicken were important risk factors for morbidity and mortality. Out of 58 cloacal samples collected from sick chickens, 7 (12.07%; 95% CI: 4.99, 23.29) yielded positive results for Salmonella spp., while 25 (43.10%; 95% CI: 30.16, 56.77) yielded positive results for E. coli. Out of swabs collected from 8 randomly selected sick chickens after necropsy, 3 (37.5%) were found to be positive for Salmonella spp. Four (50%) of them were positive for E. coli. Swabs were collected and cultured from 15 dead chickens, and of these, 2 (13.33%) and 7 (46.67%) were found to be positive for Salmonella spp. and E. coli, respectively. Farm hygiene, age of chickens, housing conditions and frequency antibiotics use were important risk factors for colibacillosis and salmonellosis. Conclusions: Although the incidence of chicken morbidity and mortality was relatively low in the present study, important risk factors have been identified in the poultry farms of Hawassa City, southern Ethiopia. Therefore, comprehensive poultry farm management practices are needed to mitigate risk factors for morbidity and mortality as well as colibacillosis and salmonellosis. Identification of the serotypes of Salmonella spp. and E. coli should be carried out.

MiRNA-155 inhibition enhances porcine embryo preimplantation developmental competence by upregulating ZEB2 and downregulating ATF4.

The in vitro embryo's competence is lower than in vivo counterparts and miRNAs found to affect the developmental competence, affecting pluripotency, stress level and apoptosis in embryos. We aimed to investigate the effect of miRNA-155 on parthenogenically activated early development porcine embryos at the preimplantation blastocyst stage. We designed miRNA-155 mimics and inhibitors and microinjected them into post-activated oocytes. The embryonic cleavage rate, blastocyst rate, and embryo development quality in terms of stress and apoptosis levels were investigated by confocal microscopy. Furthermore, we selected target genes, analyzed gene interaction and prediction networks, and compared the gene expression level in treatments and controls. miRNA-155 inhibition improved in vitro developmental competence by increasing cell numbers and reducing stress and apoptosis levels. The cleavage rate in the miRNA-155 inhibitor group was significantly higher (P < 0.05) than that in the miRNA-155 mimic group, but not in the control, whereas the blastocyst rate of the miRNA-155 inhibitor group was statistically significantly higher (P < 0.05) than in both control and miRNA-155 mimic groups. The relative gene expression level analysis showed downregulation of mRNAs related to stress and apoptosis, BAX, and the stress-induced autophagy gene ATF4, and TNF-ɑ in the miRNA-155 inhibitor group. Moreover, miRNA-155 inhibition showed upregulation of the relative expression of OCT4, ZEB2, BCL2, and IL-1 mRNA compared to control and mimic groups. However, the miRNA-155 mimic-injected group showed lower cleavage rates and blastocyst development rates than the other two groups. In conclusion, miRNA-155 inhibition in porcine in vitro embryos improved their preimplantation developmental competence and in vitro embryo production.

Vitamin C enhances porcine cloned embryo development and improves the derivation of embryonic stem-like cells.

Porcine cloning through somatic cell nuclear transfer (SCNT) has been widely used in biotechnology for generating animal disease models and genetically modified animals for xenotransplantation. Vitamin C is a multifunctional factor that reacts with several enzymes. In this study, we used porcine oocytes to investigate the effects of different concentrations of vitamin C on in vitro maturation (IVM), in vitro culture (IVC), and the derivation of nuclear transfer embryonic stem-like cells (NT-ESCs). We demonstrated that vitamin C promoted the cleavage and blastocyst rate of genetically modified cloned porcine embryos and improved the derivation of NT-ESCs. Vitamin C integrated into IVM and IVC enhanced cleavage and blastocyst formation (P < 0.05) in SCNT embryos. Glutathione level was increased, and reactive oxygen species levels were decreased (P < 0.05) due to vitamin C treatment. Vitamin C decreased the gene expression of apoptosis (BAX) and increased the expression of genes associated with nuclear reprogramming (NANOG, POU5F1, SOX2, c-Myc, Klf4, and TEAD4), antioxidation (SOD1), anti-apoptotic (Bcl2), and trophectoderm (CDX2). Moreover, vitamin C improved the attachment, derivation, and passaging of NT-ESCs, while the control group showed no outgrowths beyond the primary culture. In conclusion, supplementation of vitamin C at a dose of 50 µg/ml to the IVM and IVC culture media was appropriate to improve the outcomes of porcine IVM and IVC and for the derivation of NT-ESCs as a model to study the pre- and post-implantation embryonic development in cloned transgenic embryos. Therefore, we recommend the inclusion of vitamin C as a supplementary factor to IVM and IVC to improve porcine in vitro embryonic development.

Oviduct Epithelial Cell-Derived Extracellular Vesicles Improve Porcine Trophoblast Outgrowth.

Porcine species have a great impact on studies on biomaterial production, organ transplantation and the development of biomedical models. The low efficiency of in vitro-produced embryos to derive embryonic stem cells has made achieving this goal a challenge. The fallopian tube plays an important role in the development of embryos. Extracellular vesicles (EVs) secreted by oviductal epithelial cells play an important role in the epigenetic regulation of embryo development. We used artificially isolated oviductal epithelial cells and EVs. In this study, oviductal epithelial cell (OEC) EVs were isolated and characterized through transmission electron microscopy, nanoparticles tracking analysis, western blotting and proteomics. We found that embryo development and blastocyst formation rate was significantly increased (14.3% ± 0.6% vs. 6.0% ± 0.6%) after OEC EVs treatment. According to our data, the inner cell mass (ICM)/trophectoderm (TE) ratio of the embryonic cell number increased significantly after OEC EVs treatment (43.7% ± 2.3% vs. 28.4% ± 2.1%). Meanwhile, the attachment ability of embryos treated with OEV EVs was significantly improved (43.5% ± 2.1% vs. 29.2% ± 2.5%, respectively). Using quantitative polymerase chain reaction (qPCR), we found that the expression of reprogramming genes (POU5F1, SOX2, NANOG, KLF4 and c-Myc) and implantation-related genes (VIM, KRT8, TEAD4 and CDX2) significantly increased in OEC EV-treated embryos. We report that OEC EV treatment can improve the development and implantation abilities of embryos.

ROCK Inhibitor (Y-27632) Abolishes the Negative Impacts of miR-155 in the Endometrium-Derived Extracellular Vesicles and Supports Embryo Attachment.

Extracellular vesicles (EVs) are nanosized vesicles that act as snapshots of cellular components and mediate cellular communications, but they may contain cargo contents with undesired effects. We developed a model to improve the effects of endometrium-derived EVs (Endo-EVs) on the porcine embryo attachment in feeder-free culture conditions. Endo-EVs cargo contents were analyzed using conventional and real-time PCR for micro-RNAs, messenger RNAs, and proteomics. Porcine embryos were generated by parthenogenetic electric activation in feeder-free culture conditions supplemented with or without Endo-EVs. The cellular uptake of Endo-EVs was confirmed using the lipophilic dye PKH26. Endo-EVs cargo contained miR-100, miR-132, and miR-155, together with the mRNAs of porcine endogenous retrovirus (PERV) and ß-catenin. Targeting PERV with CRISPR/Cas9 resulted in reduced expression of PERV mRNA transcripts and increased miR-155 in the Endo-EVs, and supplementing these in embryos reduced embryo attachment. Supplementing the medium containing Endo-EVs with miR-155 inhibitor significantly improved the embryo attachment with a few outgrowths, while supplementing with Rho-kinase inhibitor (RI, Y-27632) dramatically improved both embryo attachment and outgrowths. Moreover, the expression of miR-100, miR-132, and the mRNA transcripts of BCL2, zinc finger E-box-binding homeobox 1, ß-catenin, interferon-γ, protein tyrosine phosphatase non-receptor type 1, PERV, and cyclin-dependent kinase 2 were all increased in embryos supplemented with Endo-EVs + RI compared to those in the control group. Endo-EVs + RI reduced apoptosis and increased the expression of OCT4 and CDX2 and the cell number of embryonic outgrowths. We examined the individual and combined effects of RI compared to those of the miR-155 mimic and found that RI can alleviate the negative effects of the miR-155 mimic on embryo attachment and outgrowths. EVs can improve embryo attachment and the unwanted effects of the de trop cargo contents (miR-155) can be alleviated through anti-apoptotic molecules such as the ROCK inhibitor.

Centella asiatica extract in carboxymethyl cellulose at its optimal concentration improved wound healing in mice model.

Centella asiatica (C. asiatica) has reported to be one of the traditional herbal remedies, whereas poor water solubility leads to lower bioavailability thereby affecting it remedial efficacy. Therefore, we aimed to evaluate its efficacy through increased bioavailability by using high viscosity Carboxymethyl Cellulose (CMC) as solvent on methanol-based extract on wound healing, in vivo. The preparation was applied as 0.0% (control, CMC alone), 0.25. 0.5 and 1% concentrations of extract of C. asiatica. We evaluated the efficiency of preparations on wound healing progression as progression of wound contraction, tissue proliferation and cells deposition, and relative level of gene expression for genes associated with wound healing. The results showed that 0.5% extract in CMC had significantly higher (P < 0.05) wound contraction than control and other concentrations. The level tissue deposition and the infiltration of polymorphonuclear cells in groups treated with 0.5 % concentration preparation were higher than that other treatments and control. Similarly, the relative level of gene expression in 0.5% concentration treated group were statistically significantly higher (P < 0.05) than that of control. It is believed that the lower concentration of the extract would have lessor effect on wound healing, whereas higher concertation would be interfering the optimal inflammatory tissue deposition; and there by negatively affecting wound healing. The results indicated that C. asiatica can be optimally used at 0.5 % of extract in CMC for wound healing as indicated by speeding the progression of wound closure and by increasing the expression of collagen II and III together with reducing the expression of TGFß1. However, higher concentrations of the crude extract of C. asiatica could paradoxically resulting in undesired effects. It is recommended that further evaluation should be performed on wider scale and the economic feasibility evaluation should be performed.

Quercetin improves the apoptotic index and oxidative stress in post-thaw dog sperm.

Freeze storage of ejaculated sperms is a crucial technique for the semen preservation of valuable pet animals such as dogs. The current study was conducted to investigate if quercetin (QRN) may ameliorate apoptosis and oxidative stress in post-thaw dog sperm. Herein, we evaluated the post-thaw apoptosis and oxidative stress after treatment with QRN (control, 25, 50, and 100 µM) in the freezing of dog semen. Reactive oxygen species levels were significantly affected (p < 0.05) between the various concentrations of QRN and the control (17.56 ± 1.02, 7.54 ± 0.48, 5.66 ± 0.80, and 10.41 ± 0.69), respectively. The apoptosis index was 9.1 ± 1.34, 6.66 ± 0.58, 6.77 ± 0.66, and 5.38 ± 0.86 in the control, and 25, 50, and 100 µM QRN treatment groups, respectively (p < 0.05). The effects of ameliorated cryo-induced damage by QRN on post-thaw sperm quality were also observed through improved structural and functional tests. Sperm treated with 50 µM QRN showed significantly higher motility (51.8 ± 2.1% vs. 43.1 ± 1.4%, P < 0.05), survival rates (46.9 ± 0.7% vs. 43.9 ± 0.4%, P < 0.05), and mucus penetration than control group, respectively. Results also indicated that higher concentrations of QRN (100 µM) were not effective on sperm quality and parameters when compared with the medium levels (50 µM). In conclusion, supplementation of freezing buffer with 50 µM QRN reduced oxidative damage and improved the quality of post-thaw dog sperm.

Cryopreservation of Pig Semen Using a Quercetin-Supplemented Freezing Extender.

Reactive oxygen species (ROS) produced during freeze−thaw procedures cause oxidative damage to the sperm, reducing fertility. We aimed to improve the post-thaw quality of pig sperm by quercetin (QRN) supplementation to reduce the cryodamage associated with the freeze−thaw procedure. Four equal aliquots of pooled boar semen were diluted with a freezing extender supplemented with different concentrations of QRN (0, 25, 50, and 100 µM) and then were subjected to cryopreservation in liquid nitrogen. Semen analysis was performed following 7 days of cryopreservation. Results demonstrated that the semen samples supplemented with 50 µM QRN significantly improved the post-thaw sperm quality than those subjected to other supplementations (p < 0.05). Semen samples supplemented with 50 µM QRN showed significantly improved plasma membrane functional integrity (47.5 ± 1.4 vs. 43.1 ± 4.1, 45.3 ± 1.7, and 44.1 ± 1.4) and acrosome integrity (73.6 ± 3.4 vs. 66.3 ± 2.4, 66.7 ± 3.6, and 68.3 ± 32.9) as compared to the control, 25 µM, and 100 µM QRN groups, respectively. The mitochondrial activity of the 50 µM QRN group was greater than control and 25 µM QRN groups (43.0 ± 1.0 vs. 39.1 ± 0.9 and 41.9 ± 1.0) but showed no difference with the 100 µM QRN group. Moreover, the 50 µM QRN group showed a higher sperm number displaced to 1 cm and 3 cm points in the artificial mucus than other groups. Therefore, supplementing the freezing extender with QRN can serve as an effective tool to reduce the magnitude of oxidative damage associated with sperm freezing.

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

Antioxidants have multiple protective roles in a variety of cells and thus can be used to protect sperm against cryo-damage during freezing, which affects fertility. The antioxidant resveratrol (3,5,4-trihydroxytrans-stilbene; RSV) has been reported to protect the animal sperm during cryopreservation, including human sperm. In this study, we assessed the protective effects of RSV supplementation on dog sperm cryopreservation. Semen was collected from four dogs and the effect of different concentrations of RSV (0, 100, 200, and 400 µM) on post-thaw sperm quality was examined. After thawing, sperm motility was assessed using computer-aided sperm analysis, and the structural integrity of the plasma membrane, acrosome, and chromatin was examined. In addition, their mitochondrial activity and gene expression were also assessed. Dog sperm cryopreserved with 200 µM RSV showed significant improvement in post-thaw sperm motility and viability compared with that of the control group (P<0.05). Moreover, RSV-supplemented samples showed significantly higher numbers of sperm with an intact plasma membrane, active mitochondria, and structural integrity of acrosomes and chromatin than that of control samples (P<0.05). Furthermore, gene expression showed that RSV supplemented samples showed lower expression of pro-apoptotic (BAX), reactive oxygen species (ROS) modulator oxidative stress-related (ROMO1) and 8-oxoguanine DNA glycosylase 1 (OGG1) whereas higher expression levels of anti-apoptotic (BCL2), protamine-2 (PRM2), protamine-3 (PRM3) and sperm acrosome-associated 3 (SPACA3) genes than control. Our results suggest that RSV, at its optimum concentration, can be efficiently used as an antioxidant in the cryopreservation of dog sperm.

The Role of Stem Cells and Their Derived Extracellular Vesicles in Restoring Female and Male Fertility.

Infertility is a globally recognized issue caused by different reproductive disorders. To date, various therapeutic approaches to restore fertility have been attempted including etiology-specific medication, hormonal therapies, surgical excisions, and assisted reproductive technologies. Although these approaches produce results, however, fertility restoration is not achieved in all cases. Advances in using stem cell (SC) therapy hold a great promise for treating infertile patients due to their abilities to self-renew, differentiate, and produce different paracrine factors to regenerate the damaged or injured cells and replenish the affected germ cells. Furthermore, SCs secrete extracellular vesicles (EVs) containing biologically active molecules including nucleic acids, lipids, and proteins. EVs are involved in various physiological and pathological processes and show promising non-cellular therapeutic uses to combat infertility. Several studies have indicated that SCs and/or their derived EVs transplantation plays a crucial role in the regeneration of different segments of the reproductive system, oocyte production, and initiation of sperm production. However, available evidence triggers the need to testify the efficacy of SC transplantation or EVs injection in resolving the infertility issues of the human population. In this review, we highlight the recent literature covering the issues of infertility in females and males, with a special focus on the possible treatments by stem cells or their derived EVs.

Cellular Therapy via Spermatogonial Stem Cells for Treating Impaired Spermatogenesis, Non-Obstructive Azoospermia.

Male infertility is a major health problem affecting about 8-12% of couples worldwide. Spermatogenesis starts in the early fetus and completes after puberty, passing through different stages. Male infertility can result from primary or congenital, acquired, or idiopathic causes. The absence of sperm in semen, or azoospermia, results from non-obstructive causes (pretesticular and testicular), and post-testicular obstructive causes. Several medications such as antihypertensive drugs, antidepressants, chemotherapy, and radiotherapy could lead to impaired spermatogenesis and lead to a non-obstructive azoospermia. Spermatogonial stem cells (SSCs) are the basis for spermatogenesis and fertility in men. SSCs are characterized by their capacity to maintain the self-renewal process and differentiation into spermatozoa throughout the male reproductive life and transmit genetic information to the next generation. SSCs originate from gonocytes in the postnatal testis, which originate from long-lived primordial germ cells during embryonic development. The treatment of infertility in males has a poor prognosis. However, SSCs are viewed as a promising alternative for the regeneration of the impaired or damaged spermatogenesis. SSC transplantation is a promising technique for male infertility treatment and restoration of spermatogenesis in the case of degenerative diseases such as cancer, radiotherapy, and chemotherapy. The process involves isolation of SSCs and cryopreservation from a testicular biopsy before starting cancer treatment, followed by intra-testicular stem cell transplantation. In general, treatment for male infertility, even with SSC transplantation, still has several obstacles. The efficiency of cryopreservation, exclusion of malignant cells contamination in cancer patients, and socio-cultural attitudes remain major challenges to the wider application of SSCs as alternatives. Furthermore, there are limitations in experience and knowledge regarding cryopreservation of SSCs. However, the level of infrastructure or availability of regulatory approval to process and preserve testicular tissue makes them tangible and accurate therapy options for male infertility caused by non-obstructive azoospermia, though in their infancy, at least to date.

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

Assessment of male fertility is based on the evaluation of sperm. Semen evaluation measures various sperm quality parameters as fertility indicators. However, semen evaluation has limitations, and it requires the advancement and application of strict quality control methods to interpret the results. This article reviews the recent advances in evaluating various sperm-specific quality characteristics and methodologies, with the help of different assays to assess sperm-fertility status. Sperm evaluation methods that include conventional microscopic methods, computer-assisted sperm analyzers (CASA), and flow cytometric analysis, provide precise information related to sperm morphology and function. Moreover, profiling fertility-related biomarkers in sperm or seminal plasma can be helpful in predicting fertility. Identification of different sperm proteins and diagnosis of DNA damage has positively contributed to the existing pool of knowledge about sperm physiology and molecular anomalies associated with different infertility issues in males. Advances in methods and sperm-specific evaluation has subsequently resulted in a better understanding of sperm biology that has improved the diagnosis and clinical management of male factor infertility. Accurate sperm evaluation is of paramount importance in the application of artificial insemination and assisted reproductive technology. However, no single test can precisely determine fertility; the selection of an appropriate test or a set of tests and parameters is required to accurately determine the fertility of specific animal species. Therefore, a need to further calibrate the CASA and advance the gene expression tests is recommended for faster and field-level applications.