Mehdi Hajian

The developmental competence of oocytes is crucial for successful fertilization and embryonic development; however, optimizing this capacity remains challenging. Hydrogen sulfide (H ₂ S), a gasotransmitter recognized for its cytoprotective and antioxidant properties, has the potential to enhance oocyte quality. This study investigated the effects of sodium hydrosulfide (NaHS), an exogenous H ₂ S donor, on the developmental competence of ovine oocytes in both conventional (C‐IVM) and biphasic in vitro maturation (Pre‐IVM) systems. Immature ovine oocytes were cultured in C‐IVM medium supplemented with increasing NaHS concentrations (0–10 mM) to determine the optimal concentration based on redox state, mitochondrial status, and developmental outcomes. Subsequently, the effects of NaHS during the Pre‐IVM phase of the biphasic system were evaluated, focusing on meiotic arrest, cytoplasmic maturation, and developmental competence. The results revealed that 2.5 mM NaHS during C‐IVM significantly improved oocyte quality by reducing reactive oxygen species (ROS) levels, increasing glutathione (GSH) content, and enhancing blastocyst rates. Furthermore, a 6‐h exposure to 2.5 mM NaHS during Pre‐IVM did not alter germinal vesicle (GV) rates and cyclic AMP levels but significantly decreased ROS levels and increased mitochondrial mass index at both 6 and 24 h. In the biphasic system, 2.5 mM NaHS notably elevated blastocyst rates. These findings suggest that NaHS could serve as a promising supplement to improve IVM efficiency and embryonic development, providing valuable insights for assisted reproductive technologies.

Folic acid (FA) is widely used to support one‐carbon metabolism; however, at high doses, it can circulate predominantly as unmodified FA (UMFA), potentially leading to metabolic imbalances and adverse effects on reproductive performance. This study aimed to compare the effects of high doses (20X) of FA and 5‐methyltetrahydrofolate (5mTHF) on the developmental competence of mouse oocytes, both in vitro and in vivo, using standard dose FA as a control. For the in vitro experiment, female mice were hormonally stimulated and the collected cumulus‐oocyte‐complexes (COCs) were matured in vitro with the add‐on of variable concentrations of FA or 5mTHF, and the reproductive outcomes were recorded. In vivo, 3‐week female mice received high‐dose FA or 5mTHF supplementation during the weaning period (5 weeks), after which they were either mated with fertile males or hormonally stimulated for COCs collection. Results indicated that high FA concentrations impaired oocyte maturation, spindle assembly, fertilization, and blastocyst development, whereas similar high concentrations of 5mTHF did not induce such deterioration and even improved some outcomes. Post‐mating, high‐dose FA increased embryo reabsorption rates, while 5mTHF reduced them. Blood analyses showed comparable folate levels across groups, but high‐dose FA significantly raised formaldehyde levels—a genotoxic metabolite—whereas 5mTHF markedly decreased formaldehyde. In vitro, 5mTHF supported better oocyte maturation compared to FA, and in vivo, it provided reproductive benefits without inducing formaldehyde toxicity. These findings suggest 5mTHF as a safer, more effective folate form for reproductive health, emphasizing concerns over high‐dose FA's genotoxic potential and advocating for cautious clinical use.

Vitamin D deficiency (VDD) is a widespread situation, linked to patients’ dietary habits and/or geographical origins. On the other hand, hypervitaminosis D (VDO) is also a worldwide problem, mainly associated with uncontrolled self-administration. In this study, we investigated the effects of VDD and VDO on sex steroid production and ovarian histology in mice. In addition to addressing the rarely explored situation of VDO, the originality of our approach is to disconnect VDD/VDO situations from the well-known calciotrophic effect of vitamin D (VitD). Our data indicate that VDD led to a significant decrease in serum LH and FSH levels, independently of serum calcium levels. VDD was also associated with increased testosterone and reduced oestradiol levels. VDO animals showed increased LH and reduced testosterone levels. Hormonal changes in the VDO animal groups were correlated with a lower accumulation of transcripts of steroidogenic genes such as CYP11A1 and 3ß-HSD, whereas these transcripts were higher in the VDD groups. CYP19A1 transcripts were lower in VDD animals than in controls. This study highlights the complex interaction between vitamin D status, the regulation of reproductive hormones and, consequently, reproductive performance. It underlines the need for caution when oral vitamin D supplementation is chosen as a therapeutic action to boost female reproductive performance, as VDO can be as detrimental as VDD.

After cryopreservation, reactive oxygen species (ROS) can damage sperm. Antioxidants are the primary defense against oxidative damage. Berberine is a bioactive alkaloid found in Berberis vulgaris, Curcuma longa, and Ergon grape, and is a potent antioxidant. Due to the negative effects of free radicals in oxidative stress processes, antioxidant chemicals are required to protect sperm. However, berberine has low bioavailability, making it less effective. Loading techniques on nanoparticles and nanotechnology can help overcome this limitation. Selenium nanoparticles were synthesized with barberry extract, and berberine was loaded on them. Berberine nanoparticles were then synthesized using anti-solvent precipitation with a syringe pump technique. The synthesis of nanoparticles was confirmed by EDX, UV-visible, FE-SEM, Zeta-Potential, and FTIR tests. In this experiment, we aim to investigate the impact of nano-berberine and berberine loaded on Se-NPs on goat sperm parameters after freeze-thawing. We assessed the generation of reactive oxygen species (ROS), in vitro fertility, and the subsequent embryo development of zygote with treated sperm after determining the optimal concentration of various chemicals on sperm parameters. The study found that all treatments had significant differences from the control group in terms of motility, viability, DNA and membrane integrity, ROS level, lipid peroxidation, in vitro fertility ability, and the capacity to develop inseminated oocytes (p < 0.05). The most significant outcomes were observed with berberine loaded on Se-NPs and the combination of selenium nanoparticles with berberine nanoparticles.

Cryopreservation of sperm can cause oxidative stress and damage, leading to decreased different functional parameters and fertilization potential. In this study, we evaluated two types of H₂S donors: NaHS, a fast-releasing donor, and GYY4137, a slow-releasing donor during cryopreservation of goat sperm. Initially, we determined that 1.5 and 3 μM NaHS, and 15 and 30 μM GYY4137 are optimal concentrations that improved different sperm functional parameters including motility, viability, membrane integrity, lipid peroxidation, and ROS production during incubation at 38.5 °C for 90 min. We subsequently evaluated the impact of the optimal concentration of NaHS and GYY4137 supplementation on various functional parameters following thawing during cryopreservation. Our data revealed that supplementation of extender improved different parameters including post-thaw sperm motility, viability, membrane integrity, and reduced DNA damage compared to the frozen-thawed control group. The supplementation also restored the redox state, decreased lipid peroxidation, and improved mitochondrial membrane potential in the thawed sperm. Finally, we found that supplementation of the extender with NaHS and GYY4137 enhanced IVF outcomes in terms of blastocyst rate and quality of blastocysts. Our results suggest that both donors can be applied for cryopreservation as antioxidants to improve sperm quality and IVF outcomes of frozen-thawed goat sperm.

This study aims to evaluate the genomic effects of vitamin D (VitD) on steroidogenesis, sperm structure, and function in mice models with artificially induced vitamin D deficiency (VDD), while maintaining calcium and phosphorus homeostasis. qRT‐PCR was utilized to measure the steady‐state RNA levels of key steroidogenic genes, including CYP11A1, CYP19A1, 17βHSDH1, 3βHSDV1, and CYP24A1, involved in VitD metabolism, in testicular extracts from mouse models with varying degrees of VDD severity. Estradiol, testosterone, luteinizing hormone (LH), and follicle‐stimulating hormone (FSH) levels were also assessed. Furthermore, testicular histomorphometric analysis and evaluation of sperm structural and functional parameters were conducted. The study reveals a linear decrease in the expression of monitored steroidogenic genes with increasing severity of VDD. Moreover, in cases of mild and moderate VDD, testicular extracts exhibited significantly reduced levels of LH, estradiol, and testosterone. VDD has substantial effects on testicular steroidogenesis, which directly impacts spermatogenesis, independent of its established role in calcium regulation. Consequently, the reduction in steroidogenesis due to VDD contributes to the diminished efficiency of testicular spermatogenesis in individuals experiencing VDD, a prevalent condition worldwide.

Vitamin D (VD) deficiency reduces the chances of successful fertilization; however, it remains to be validated whether this effect is dependent or not on calcium. To address this question, we generated several situation using a mouse model in which VD content was either increased or decreased in a normo or hypocalcemia context. After the measurement of serum 25-hydroxyvitamin D₂, calcium and phosphorus levels, an analysis was carried out in terms of oocytes maturation as well as reproductive performance. VD overdose, despite the fact that it resulted in an increased number of mature oocytes, reduced developmental competence and offspring survival. VD deficiency (VDD), on the contrary, reduced the number and percentage of mature oocytes, blastocyst rate, as well as fertility rate and offspring survival. Hypo-calcemia when VD levels were normal, had a similar effect than VDD. The effects of VDD were reversed by a diet that corrected calcium level. Therefore, both VD overdose (in a context of normal calcium level) VD deficiency as well as hypo-calcemia have an effect on female reproductive function. In conclusion, although closely related, VD and calcium act in part independently of each other in defining the “optimum” for female reproductive performance.

Background: Somatic cell nuclear transfer (SCNT) is an approach for the propagation of elite animals. In vitro condition, especially the composition of culture media has a profound effect on the developmental competency of in vitro derived e mbryos. There are limited studies evaluating the effect of culture media on SCNT outcomes. To address this gap, we compare the effect of two culture media synthetic oviduct fluid (SOF) vs. commercial bracket-oliphant (BO) on developmental comptenecy. Materials and Methods: In this experimental study, embryos derived from in vitro fertilized (IVF) and SCNT were cultured in both BO and SOF media for 7 days. In addition to the assessment of cleavage and blastocyst on day 3, and 7, the quantitative expression of 16 genes in theresultant blastocysts were assessed. The resultant SCNT blastocysts from SOF and BO groups were also transferred to the synchronized recipient for developmental competency to term. Results: The blastocyst rate in the BO medium was significantly higher than that of the SOF medium in the SCNT group (P<0.05). All of the examined genes showed increased expression levels in SCNT blastocyst in both media compared to IW Blastocyst. In the IW group, Oct4, Bmpr 1, and Gcn5 showed significantly higher expression in the SOF medium compared to the BO medium while Akt, Fgfr4, Sox2 showed significantly lower expression in the SOF medium compared to the BO medium. In the SCNT group, Fgfr4. Gcn5, Fzd, Ctnnb. Bmpr 1, and Fgfr4 showed significantly higher expression in SOF compared to BO derived blastocyst. Conclusion: It appears that in SCNT blastocysts, gene regulation is less controlled compared to IVF ones, irrespective of the type of medium. In addition, there are differences regarding certain genes expressions between IVF and SCNT derived blastocysts between SOF and BO, reiterating that culture composition affects developmental competency and gene expression.

Although different strategies have been developed to generate transgenic poultry, low efficiency of germline transgene transmission has remained a challenge in poultry transgenesis. Herein, we developed an efficient germline transgenesis method using a lentiviral vector system in chickens through multiple injections of transgenes into embryos at different stages of development. The embryo chorioallantoic membrane (CAM) vasculature was successfully used as a novel route of gene transfer into germline tissues. Compared to the other routes of viral vector administration, the embryo’s bloodstream at Hamburger-Hamilton (HH) stages 14–15 achieved the highest rate of germline transmission (GT), 7.7%. Single injection of viral vectors into the CAM vasculature resulted in a GT efficiency of 2.7%, which was significantly higher than the 0.4% obtained by injection into embryos at the blastoderm stage. Double injection of viral vectors into the bloodstream at HH stages 14–15 and through CAM was the most efficient method for producing germline chimeras, giving a GT rate of 13.6%. The authors suggest that the new method described in this study could be efficiently used to produce transgenic poultry in virus-mediated gene transfer systems.

Although different strategies have been developed to generate transgenic poultry, low efficiency of germline transgene transmission has remained a challenge in poultry transgenesis. Herein, we developed an efficient germline transgenesis method using a lentiviral vector system in chickens through multiple injections of transgenes into embryos at different stages of development. The embryo chorioallantoic membrane (CAM) vasculature was successfully used as a novel route of gene transfer into germline tissues. We established a new method of eggshell windowing for embryo manipulation at different developmental stages. Compared to the other routes of viral vector administration, the embryo’s bloodstream at Hamburger-Hamilton (HH) stages 14-15 achieved the highest rate of germline transmission (GT), 7.7%. Single injection of viral vectors into the CAM vasculature resulted in a GT efficiency of 2.7%, which was significantly higher than the 0.4% obtained by injection into embryos at the blastoderm stage. Double injection of viral vectors into the bloodstream at HH stages 14-15 and through CAM was the most efficient method for producing germline chimeras, giving a GT rate of 13.6%. The authors suggest that the new method described in this study could be efficiently used to produce transgenic poultry in virus-mediated gene transfer systems.