Protective effects of hesperetin on the quality of sperm, apoptosis, lipid peroxidation, and oxidative stress during the process of cryopreservation: An experimental study

Background: Hesperetin is a bioflavonoid compound, largely used in Chinese traditional medicine and found plenty in citrus fruits. Hesperetin has beneficial effects against different diseases. The sperm cryopreservation process is a common method that is used in infertility laboratories. It has been reported that during the cryopreservation process, the quality of sperm is significantly reduced. Objective: To investigate the effect of hesperetin on the quality of human spermatozoa during the cryopreservation process. Materials and Methods: In this experimental study, 22 sperm sample of normozoospermia men who referred to the infertility department of the Shariati Hospital (Tehran, Iran) Between October and November 2019 were collect and divided into three groups as: 1) fresh, 2) control (frozen-thawed group without treatment), and 3) treatment group as frozen-thawed samples supplemented with 20 μM hesperetin. Motility, Viability, morphology, Apoptotic-like changes, intracellular H2O2, intracellular O2−, and lipid peroxidation (LPO) was measured. Results: Hesperetin treatment during the cryopreservation process of human sperm significantly improved the viability, motility, and morphology rates of the spermatozoa after frozen-thawed process in control group (p < 0.01). In addition, it significantly reduced the reactive oxygen species (ROS) level, LPO level and increased the percentage of viable sperm cells with intact plasma membrane (p < 0.01) after frozen-thawed process. Conclusion: Hesperetin can improve the quality of human sperm and also protect human sperm against reactive oxygen species, LPO, and apoptosis during the cryopreservation-thawing process. Key words: Cryopreservation, Hesperetin, Spermatozoa, Reactive oxygen species

Bovine oocyte developmental competence and gene expression following co-culturing with ampullary cells: An experimental study

Background: There is no sufficient information on the impact of bovine ampullary oviductal epithelial cells (BAOECs) on in vitro oocyte maturation competence and gene expression. Objective: This study aimed to examine the oocyte developmental competence following co-culturing with a monolayer of fresh and frozen-thawed ampullary cells. Materials and Methods: Bovine cumulus-oocyte complexes (COCs) were distributed into three groups: control group; where in COCs were cultured in cell-free media for 24 hr and FML and FTML groups in which the COCs were cultured in maturation media for 18 hr and then transferred into a media containing fresh and frozen-thawed BAOECs monolayer, respectively (BAOECs were extracted from the oviducts of slaughtered cattle and were then cultured freshly or frozen-thawed) for a further 6 hr. After 24 hr, the expanded COCs were evaluated for nuclear maturation, fertilization rate, and gene expression (GDF9, StAR, CASP3, and FSHr). Results: Nuclear maturation rate in the FTML group was significantly higher than the control group (p = 0.02). The fertilization rate of FTML group was significantly higher than the control and FML groups (p = 0.05 and p = 0.03, respectively). In terms of gene expression, GDF9 were upregulated in the presence of the BAOECs during the last 6 hr of the in vitro maturation (p < 0.001). Furthermore, the expression of the StAR gene in the FTML group was higher than the other groups (p = 0.02). Conclusion: Ampullary cells co-culturing (especially frozen-thawed cells) for in vitro maturation of bovine oocytes yields encourages the results and demonstrates the beneficial effect of co-culture on gene expression and developmental competence. Key words: Ampulla, Bovine, Fertilization, Gene expression, IVM

The association between dietary inflammatory index and C-reactive protein in plasma and semen with semen quality: A cross-sectional study

Background: Infertility affects couples worldwide, with male factors being responsible for half of all cases. Objective: This study aimed to investigate the relationship between dietary inflammatory index (DII) and levels of C-reactive protein (CRP) in plasma and semen with the quality of semen in infertile males. Materials and Methods: In this cross-sectional study, 88 infertile men referring to Besat hospital, Tehran, Iran from December 2021-November 2022 were enrolled. A detailed questionnaire requesting information, and a 168-item semiquantitative food frequency questionnaire, were completed by participants. A food frequency questionnaire was used to calculate the DII. Additionally, semen and blood samples were collected from each participant for semen analysis and CRP-level assessment. Statistical analyses were performed to explore the association between DII and CRP levels with sperm quality. The correlation between DII and serum/semen CRP, besides assessing nutrients in each DII quartile group, was also explored. Results: A significant difference was observed between different DII quartiles considering sperm motility (p = 0.006) and morphology (p = 0.014). Post hoc study revealed a significant difference between the 1st and 2nd quartiles and the 1st and 4th quartiles of DII regarding sperm motility (p = 0.011, and 0.017 respectively) and a significant difference between the 1st and 2nd quartiles of DII considering sperm morphology (p = 0.009). A statistically significant inverse correlation was also observed between DII and sperm motility (p = 0.017). Carbohydrates and β-carotenes were significantly different between the 4 DII quartiles (p = 0.043 and p = 0.026, respectively). Finally, no significant correlation was observed between DII and CRP levels in blood and semen (p > 0.05). Conclusion: The findings suggested a notable correlation between DII and semen quality; however, no significant association were observed between DII and CRP levels in blood and semen. Key words: Infertility, C-reactive protein, CRP, Inflammation, Sperm

Niacin improves maturation and cryo-tolerance of bovine in vitro matured oocytes: An experimental study

Background: Nicotinic acid (niacin) is a broad-spectrum lipid-modifying agent that has potent antioxidant properties and reduces the production of lipid peroxidation. Objective: The purpose of the present study was to investigate the maturation, embryo development and cryo-tolerance merit, and levels of malondialdehyde (MDA), total oxidant status, and total antioxidant capacity following the supplementation of bovine oocytes maturation medium with different concentrations of niacin. Materials and Methods: Immature cumulus-oocyte complexes were cultured in tissue culture medium-199 maturation media supplemented with 0, 100, 200, and 400 µM niacin under a standard in vitro culture condition. After 24 hr of culture, the nuclear maturation rate was assessed. Then, two groups of immature cumulus-oocyte complexes were cultured in TCM-199 either with or without 400 µM niacin and evaluated for embryo development. Also, matured cumulus-oocyte complexes in both groups were frozen using a standard vitrification procedure. After vitrification, oocytes were warmed in two steps and evaluated for embryo development. In addition, the level of total antioxidant capacity, total oxidant status, and MDA were measured. Results: The results indicated that although the treatment with 400µM niacin increased in vitro nuclear maturation (87.6 ± 5.3), it did not improved the embryo development to the blastocyst stage. Higher cleavage and blastocyst rates were observed in vitrified oocytes that were cultured with supplemented 400 µM niacin compared to the control group (without niacin) (53.6 ± 2.7 and 10.6 ± 1.6 vs. 46.2 ± 4.1 and 6.3 ± 2.4, respectively). Also, the addition of 400 µM niacin to the maturation media could decrease MDA levels after maturation. Conclusion: Niacin could improve the quality of in vitro embryo production (IVP) embryos and tolerance of bovine oocytes to vitrification. Key words: Bovine, Embryonic development, Niacin, Oocytes, Vitrification

DNA Methylation and Histone Acetylation Patterns in Cultured Bovine Adipose Tissue-Derived Stem Cells (BADSCs)

Objective: Many studies have focused on the epigenetic characteristics of donor cells to improve somatic cell nuclear transfer (SCNT). We hypothesized that the epigenetic status and chromatin structure of undifferentiated bovine adipose tissue-derived stem cells (BADSCs) would not remain constant during different passages. The objective of this study was to determine the mRNA expression patterns of DNA methyltransferases (DNMT1, DNMT3a, DNMT3b) and histone deacetyltransferses (HDAC1, HDAC2, HDAC3) in BADSCs. In addition, we compared the measured levels of octamer binding protein-4 expression (OCT4) and acetylation of H3K9 (H3K9ac) in BADSCs cultures and different passages in vitro. Materials and Methods: In this experimental study, subcutaneous fat was obtained from adult cows immediately post-mortem. Relative level of DNMTs and HDACs was examined using quantitative real time polymerase chain reaction (q-PCR), and the level of OCT4 and H3K9ac was analyzed by flow cytometry at passages 3 (P3), 5 (P5) and 7 (P7). Results: The OCT4 protein level was similar at P3 and P5 but a significant decrease in its level was seen at P7. The highest and lowest levels of H3K9ac were observed at P5 and P7, respectively. At P5, the expression of HDACs and DNMTs was significantly decreased. In contrast, a remarkable increase in the expression of DNMTs was observed at P7. Conclusion: Our data demonstrated that the epigenetic status of BADSCs was variable during culture. The P5 cells showed the highest level of stemness and multipotency and the lowest level of chromatin compaction. Therefore, we suggest that P5 cells may be more efficient for SCNT compared with other passages

Supplementation of L-carnitine during in vitro maturation of mouse oocytes affects expression of genes involved in oocyte and embryo competence: An experimental study

Background: Oocyte developmental competence is one of the key factors for determining the success rate of assisted reproductive technique. Objective: The aim of the current study was to investigate the effect of L-carnitine (LC) supplementation during in vitro maturation (IVM), on preimplantation embryo development and expression of genes involved in embryo competence derived from oocytes selected with brilliant cresyl blue (BCB) test. Materials and Methods: Cumulus-oocyte complexes (COCs) were obtained from NMRI mice ovaries. COCs were stained with BCB and then BCB+ (colored cytoplasm) oocytes cultured in IVM medium supplemented with 0.3 or 0.6 mg/ml LC. COCs untreated with LC were used as control. Fertilization rate and blastocyst development rate were determined after in vitro fertilization. In addition, quantitative reverse transcriptase polymerase chain reaction was used to measure relative genes expression related with development (Ccnb1, Mos, Ces5, and Dppa2) and apoptosis (Bax and Bcl-xL) in oocytes and embryos. Results: Oocytes treated with both LC concentrations showed higher blastocyst development rate compared with untreated oocytes (p<0.01). Moreover, fertilization rate was increased in oocytes treated with 0.6 mg/ml LC (p<0.01). Treatment of oocytes with both LC concentrations increased (p<0.01) the level of Ccnb1 mRNA in MII oocytes. The two-cell stage embryos and blastocysts derived from LC-treated oocytes (0.6 mg/ml) showed increased the expression levels of Dppa2 and Bcl-xl mRNA, respectively (p<0.01). Conclusion: The results of the present study show that adding of LC to the IVM medium of BCB+ oocytes can ameliorate reproductive success following in vitro fertilization