Hamed Karami Shabankareh

Objective: This study looked at how different concentrations of curcumin (Curc), zinc chloride (ZnCl₂), zinc oxide nanoparticles (ZnO‐NPs) and Curc loaded on ZnO‐NPs (Curc‐co‐ZnO‐NPs) in cryopreservation dilution affected the quality of ram sperm after thawing.Methods: ZnO‐NPs were synthesised using Berberis vulgaris leaf aqueous extract. Then, Curc was loaded on the ZnO‐NPs that had been synthesised. We used analytical methods to look at the composition, morphology and size of green synthesised ZnO‐NPs and Curc‐co‐ZnO‐NPs, including UV‐Vis, zeta potential, EDX, DLS, FE‐SEM and FT‐IR. Using a Tris‐base extender containing various concentrations of Curc, ZnCl₂, ZnO‐NPs and Curc‐co‐ZnO‐NPs (0, 1, 10 and 100 µg/mL), semen samples from four rams were combined. Sperm motility, viability, DNA and plasma membrane integrity, total abnormalities and malondialdehyde (MDA) generation were all evaluated in treatment groups after thawing.Results: The results showed that adding 1 µg/mL of ZnO‐NPs and Curc‐co‐ZnO‐NPs significantly reduced the level of MDA and total abnormalities (p < 0.05). Additionally, following the freeze‐thawing procedure, the presence of 1 µg/mL of Curc‐co‐ZnO‐NPs in the diluent of ram sperm significantly increased the percentage of sperm viability and motility in comparison to the control and other treatment groups (p < 0.05). Furthermore, as compared to the control group and other treatments, treatments containing 1 µg/mL of Curc‐co‐ZnO‐NPs significantly improved membrane and DNA integrity (p < 0.05).Conclusions: It appears that following freeze‐thawing, the Curc‐co‐ZnO‐NPs (1 µg/mL) enhanced sperm parameters.

Background: Extensive use of different nanoparticles caused significant concerns about their biological safety.Objective: This study aimed to evaluate the effects of cryopreservation on ram semen after adding magnetic nanoparticles (MNPs) to separate X and Y chromosome‐bearing spermatozoa.Methods: The experimental ram sperms in this research included treated spermatozoa (50 μg/ml MNPs) and non‐treated spermatozoa. DNA damage of spermatozoa was examined using an acridine orange (AO) assay. Sperm viability, membrane functionality, abnormality and malondialdehyde (MDA) level were also measured.Results: Results indicated that the pre‐treatment of ram semen extender with MNPs did not significantly affect the semen parameters such as viability, membrane functionality, abnormality, as well as lipid peroxidation (LPO) levels and DNA integrity in comparison with the control group (p < 0.05).Conclusions: These observations suggest that pre‐treatment of ram semen extender with MNPs after semen sexing did not have adverse effects on different semen parameters after cryopreservation.

The present study was conducted to investigate the effects of the activator factor of the WNT pathway, chir98014, leading to the in vitro sheep oocyte maturation medium, on the cumulus cell development, different nuclear maturation stages and the following process of embryonic development. Experiments included (a) addition of different concentrations (0, 0.1, 0.5, 1 µm) of chir98014 to the maturation medium and evaluation of the cumulus cell expansion, (b) addition of different concentrations of chir98014 to the maturation medium and investigation of different nuclear maturation stages, (c) addition of different concentrations of chir98014 to the maturation medium and examination of the subsequent embryonic maturation process and (d) addition of different concentrations of chir98014 to the embryonic development culture medium (the first 48 hr) and investigation of the subsequent embryonic development process. The extracted data were analysed using the SPSS software, considering the significance level of p < .05 and making the mean comparisons. The results showed that the addition of the 0.1 µM concentration of chir98014 to the maturation medium had no significant effects on the oocyte maturation and embryo development post‐fertilization but it enhanced the Cumulus‐oocyte complexes (COCs) expansion. In the fourth experiment, the low concentration of chir98014 in the embryo culture media improved the embryo development process, whereas the high one had a detrimental effect on it, as compared to the control group. Thus, the presence of the lower concentrations of this compound in the embryonic culture medium had favourable effects on the development of embryos.

Pre‐conceptual sex selection is still a highly debatable process whereby X and Y chromosome bearing spermatozoa are isolated before oocyte fertilization. Recently, magnetic nanoparticles (MNP) have been used to determine X and Y chromosomes bearing spermatozoa as a result of searching for a cheap, highly efficient method using non‐toxic materials. This study aimed to recover the sperm bearing X chromosomes in ram with different concentrations of MNP and then evaluate the success of this method using polymerase chain reaction (PCR). Ram sperms were divided into four groups, treated with 0 (control), 50, 100 and 200 μg/ml MNP, respectively. MNP was used to restore sperm cells bearing X chromosomes. Upon recovery, the PCR was performed to identify the X and Y sperms, Methyl ThiazoleTetrazolium (MTT), to assess MNP toxicity and sperm viability and acridine orange (AO) to evaluate sperm DNA integrity. The results of PCR revealed that the treatment of spermatozoa‐ bearing X chromosomes with 50 μg/ml MNP had the highest effects on the recovery of X sperm rather than the other concentrations of MNP. However, the concentrations of MNP did not have any toxic effects on spermatozoa, sperm viability and, DNA integrity, but the high concentration of MNP (200 μg/ml) significantly reduced DNA integrity. According to MTT and AO results, the concentrations of MNP used in this study had no toxic effects on spermatozoa and did not reduce the sperm viability and DNA integrity, except that 200 μg/ml MNP significantly reduced DNA integrity.

This study evaluated the effect of resveratrol administered at different concentrations (0, 0.1, 0.25, 0.5, 2.0, and 5.0 μM) via IVM medium on cumulus expansion, nuclear maturation and subsequent embryonic development of Sanjabi sheep oocytes, and total cell number of embryos. Meiotic progression was evaluated based on cumulus expansion, the frequency of germinal vesicles, germinal vesicle breakdown and metaphase II (MII). The effects of the resveratrol concentrations in the in vitro maturation (IVM) medium on cleavage and developmental potential to blastocyst stage were also investigated. After maturation, a sample of oocytes were fixed and stained to evaluate nuclear maturation, while the remaining oocytes were fertilized in vitro with fresh semen, then cultured for 8 days in synthetic oviductal fluid (SOF) medium for assessment of oocyte developmental capacity. Addition of 0.25 and 0.5 μM of resveratrol to the maturation medium was shown to significantly increase cumulus expansion (P < 0.05). There was no significant difference in the percentage of MII oocytes between control, and 0.1, 0.25, 0.5 and 2.0 μM resveratrol treatment groups. Furthermore, addition of 0.25 and 0.5 μM of resveratrol to IVM medium also significantly improved blastocyst formation, and addition of 0.5 µM resveratrol in maturation media increased trophectoderm cell numbers, inner cell mass and the total cell number of ovine embryos. However, addition of high resveratrol concentrations (5.0 μM) to IVM medium had a negative effect on the maturation rate and blastocyst formation. Together, these findings show that supplementation of IVM medium with 0.25 and 0.5 μM resveratrol can improve the meiotic competence and early embryonic development of Sanjabi sheep oocytes.

Contents: The objective of this study was to examine the different concentrations of antipain and trehalose combination on post‐thawed quality of ram semen cryopreserved in tris extender. Ejaculates were collected from four rams using the artificial vagina, pooled at 37°C and diluted with (A₀ Tre₀: antipain 0 μM and trehalose 0 mM(Control); A₁₀ Tre₀; A₅₀ Tre₀; A₀ Tre₃₀; A₀ Tre₆₀; A₁₀ Tre₆₀; A₁₀ Tre₃₀; A₅₀ Tre₃₀and A₅₀ Tre₆₀). Diluted semen samples were gradually cooled down from 37 to 5°C in a cold cabinet; then, they were loaded into 0.25 mlstraws, frozen and stored in liquid nitrogen. Sperm motility (CASA), viability, membrane functionality and abnormality were evaluated after thawing process. Progressive motility in extender supplemented with A₁₀ Tre₀, A₀ Tre₃₀and A₁₀ Tre₆₀significantly (p < 0.05) higher as compared to the control (A₁₀ Tre₀). A₁₀ Tre₆₀(47.50 ± 0.73) provided the best maintenance of progressive motility in comparison with the control (40.50 ± 0.73). No significant differences were observed between all treated groups in terms of total motility,VAP,VSL,VCL,ALH,BCF,STRandLIN. The percentages of sperm with viable were significantly higher in extenders supplemented with A₁₀ Tre₀, A₅₀ Tre₀, A₀ Tre₃₀and A₁₀ Tre₆₀, compared to control. Addition of A₁₀ Tre₀, A₅₀ Tre₀and A₁₀ Tre₆₀to extenders improved the percentages of sperm abnormality, compared to the controls. A₁₀ Tre₆₀(67.84 ± 1.51) treatment provided the best maintenance of normal morphology compared to the other treatments. The supplementation with A₁₀ Tre₀, A₀ Tre₆₀and A₁₀ Tre₆₀improved the percentage of sperm membrane functionality when compared to the control (p < 0.05). Comparing these results with those of control diluents, the effects of supplementation were better except for A₅₀ Tre₆₀group. In conclusion, when combination of antipain (10 μM) and trehalose (30 and 60 mM) was added, they conferred a great cryosurvival capacity with their synergic effects during freeze‐thawing process.

Previous studies reported many discrepancies about the effects of corpus luteum (CL) and ovarian follicle size on the developmental competence of oocytes. The aim of this study was to investigate the effects of CL and different size of follicle on the developmental potential of bovine oocytes. After ovarian classification based on presence or absence of CL, sample follicles were placed in three groups according to their diameter; small (S; 3–6 mm), medium (M; 6–9 mm), and large (L; 10–20 mm). Collected oocytes in each group were subjected to the in vitro embryo production processes. Results showed that, the percentages of blastocyst obtained from oocytes originating from small and medium follicles of ovaries bearing a CL (CL+S-oocytes and CL+M-oocytes, respectively) were lower (p<0.001) than those of small and medium follicles of ovaries not bearing a CL (CL-S-oocytes and CL-M-oocytes, respectively) (30.8% and 33.6% vs. 36.9% and 38.7% respectively). Although, the percentages of blastocyst obtained from CL-M-oocytes and CL-L-oocytes were greater (p< 0.001) than those of CL+S-oocytes and CL+M-oocytes. There were no significant differences in the percentages of blastocyst formation between controls (C-oocytes), CL-S-oocytes and CL+L-oocytes. According to the results of this study, the negative effect of CL on the developmental competence of bovine oocyte depends on the follicle size. Therefore, oocytes originating from large grown follicles were not influenced by negative effects of CL as much as those originating from small and medium follicles did.

Many studies reported that follicle size has an essential role in developmental potential of oocytes. Also, the brilliant cresyl blue (BCB) test is one of the most important criteria in selection of more competent oocytes. Selection of developmentally competent bovine oocytes. A total of 1730 bovine cumulus oocyte complexes (COCs) were recovered from the ovaries by follicles isolation and classified into 3 categories according to the diameters of the follicles (small, 6 mm). Oocytes were exposed to the BCB stain, diluted in Dulbecco's phosphate-buffered saline, modified with 0.4% bovine serum albumin (BSA) for 90 min. Oocytes with or without blue coloration of the cytoplasm were designated as BCB+ and BCB-, respectively. The BCB+ and control oocytes originated from large and medium follicles exhibited a higher (p<0.0001) cleavage and blastocyst rate than BCB- oocytes. Furthermore, the BCB+ oocytes from large and medium follicles had the highest (p<0.0001) proportion of blastocyst than other treatment groups. In contrast, the BCB- oocytes from small follicles had the lowest (p<0.0001) proportion of blastocyst than other treatment groups. Interestingly, the percentage of the BCB+ oocytes from the large and medium ovarian follicles was significantly higher (p<0.0001), than the BCB+ oocytes from the small follicles. Current results confirmed that each BCB+ oocyte could not lead to perfect embryo development and the BCB test is not sufficient enough for the identification of oocytes that are competent for in vitro embryo development.