The effect of quercetin on fertility of frozen-thawed ram epididymal spermatozoa

The aim of the present study was to evaluate the effects of quercetin as an antioxidant supplement on frozen-thawed ram epididymal sperm quality. Quercetin is a type of flavonoid antioxidant that is found in plants, with the ability to scavenge free radicals. Twenty testicles from mature rams were collected from a nearby slaughterhouse immediately after slaughter. Epididymal spermatozoa were recovered from the caudal of epididymides by injecting Bracket and Oliphant's (BO) medium retrogradely through the ductus deferens and extended with a tris egg-yolk-based extender and supplemented with 0, 5, 10, 15, 20, and 50 μg/mL quercetin. Following equilibration, the straws were frozen, and then plunged into liquid nitrogen. After thawing, optimized concentrations of quercetin were defined based on their viabilities and used to assess fertilization and developmental potential. The results showed that the viability of frozen-thawed spermatozoa significantly increased by using 5 and 10 μg/mL quercetin in the freezing extender. However, total and progressive motility of frozen-thawed spermatozoa were not affected by 5 and 10 μg/mL quercetin in comparison with control (0 μg/mL). The mean number of zygote, morula, and blastocyst stage embryos increased significantly by using 5 and 10 μg/mL quercetin compared with other frozen-thawed treatments(P <0.05). However, the blastocyst rate of fresh sperm was significantly higher (P <0.05). In conclusion, to improve the quality of frozen-thawed ram epididymal spermatozoa, 5 and 10 μg/mL quercetin appears to be an attractive option. Further studies are suggested to understand the synergistic effect of quercetin with other antioxidants to improve the ram freezing–thawing process.Keywords: antioxidant, cryopreservation, fertility, freezing extende

Optimization of in vitro culture and transfection condition of bovine primary spermatogonial stem cells

The present study aimed to optimize the in vitro culture and transfection efficiency of bovine primary spermatogonial stem cells (SSCs). To this end, SSCs were obtained from newborn Holstein bull calves by two-step enzymatic digestion. After enrichment and culture, SSCs were characterized by using alkaline phosphatase (AP) staining and expression of vasa and thy1 genes as specific bovine SSC markers. To evaluate the effect of antioxidants on vitality, colony formation, and the expression of pro- and anti-apoptotic genes of bovine SSCs, various concentrations of vitamin C (5, 10, 25 and 50 μg/mL) and Trolox (a water soluble α-tocopherol analogue) (12.5, 25, 50 and 100 μg/mL) were added to the SSC culture medium. The results showed that SSCs treated with 50 μg/mL of vitamin C or 25 μg/mL of Trolox individually could increase cell viability and colony formation significantly in comparison with other concentrations and the control group. Additionally, the expressions of bax (as a pro-apoptotic gene) and bcl2 (as an anti-apoptotic gene) were significantly lower and higher than the control group, respectively. To optimize the transfection condition, the effective dosages of vitamin C or Trolox, with various concentrations of two transfection reagents (X-tremeGENE HP and Turbofect) and DNA, at day 8 of culture, were studied. Results showed that 1 μl X-tremeGENE HP or 0.5 μl Turbofect and 2 μg of DNA are the best concentrations for transfecting SSCs. However, X-tremeGENE HP expressed more potential for transfecting SSCs in comparison with Turbofect. Besides, no difference was observed between the use of defined doses of vitamin C or Trolox.Keywords: Apoptosis, gene transfer, primary cells, viability, vitamin C, Trolo

Production of Buffalo Embryonic Stem Cell from HMC Embryos

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of blastocyst and differentiate into all three embryonic germ layers: ectoderm, endoderm, and mesoderm. In this study, ESCs are derived from Hand Made Cloning (HMG) blastocysts and their efficiencies compared to ESCs derived from In Vitro Fertilization (IVF) embryos. Feeder layer was used for ESCs culture, and culture medium consisting of Knockout- Dulbecco’s Modified Eagle’s Medium (Ko-DMEM) supplemented with Knockout Serum Replacement (KSR), Leukemia Inhibitory Factor (LIF), Basic Fibroblast Growth Factor-2 (FGF-2), L-glutamine, nonessential amino acids and gentamicin. The cell surface antigens used for characterization were the SSEA-1, SSEA-4, TRA-1-60 and TRA-1-81 and the pluripotency markers were NANOG, OCT3/4 and SOX2. Results showed that, the growth rate of ESCs colonies in ESCs from IVF embryos was significantly higher than ESCs from HMG embryos (120% compared with 65%, respectively). Not only real-time PCR results revealed the same expression level of SOX2, OCT3/4 and cMYC between them, but also ESCs from HMG embryos resulted to higher expression of NANOG. Both of ESCs groups maintain in pluripotency state for more than two years and differentiated to the different types of cells like neuron, epithelial, lipid and muscle cells