Combination of trehalose and low boron in presence of decreased glycerol improves post-thawed ram sperm parameters: A model study in boron research.

BACKGROUND: Sperm cryopreservation has been widely used in the field of reproductive biotechnology. It applies to certain males of economic and scientific values, including livestock breeds or endangered animal species. The development of a semen extender with a low cryoprotectant concentration and an appropriate amount of trehalose and boron can prevent the deterioration of sperm parameters. OBJECTIVE: The main goal of this study is to establish a suitable ram extender model, by examining different combinations of high (5%) and low (3%) glycerol concentrations (to reduce its toxic effects on sperm freezing), a fixed amount of trehalose and an increased dose of boron to prevent the deterioration of sperm parameters, and investigate the levels of gene expressions. MATERIALS AND METHODS: The Merino ram ejaculates were collected. The collected ejaculates providing the defined criteria were pooled. The pooled ejaculates were divided into eight aliquots and diluted with the Tris extender including different combinations of glycerol (5% and 3%) and boron (0.25, 0.5, and 1 mm) concentrations and a fixed amount of trehalose, then frozen. After freeze-thawing process, sperm motility, mitochondrial membrane activity, plasma membrane integrity, acrosomal membrane integrity, DNA damage (single cell gel electrophoresis (COMET) and TUNEL assays) as well as NAD(P)H quinone oxyreductase (NQO1), glutamate-cycteine ligase (GCLC), and glutathione S-transferase (GSTP1) for molecular mechanisms of sperm cell response to oxidative stress were assessed for different extender groups following freeze-thawing process: 5% glycerol + 0 mm boron (G5B0.00), 5% glycerol + 0.25 mm boron (G5B0.25), 5% glycerol + 0.5 mm boron (G5B0.50), 5% glycerol + 1 mm boron (G5B1.00), 3% glycerol + 0 mm boron (G3B.00), 3% glycerol + 0.25 mm boron (G3B0.25), 3% glycerol + 0.5 mm boron (G3B0.50), and 3% glycerol + 1 mm boron (G3B1.00). RESULTS: G3B0.25 presented higher percentages of subjective motility, mitochondrial activity, and viability of spermatozoa comparing with G5B0.00 and groups with boron. Supplementation of 0.25 mm boron with and without trehalose (G3B0.25 and G5B0.25) showed higher acrosome integrity, compared with G5B0.00, G5B1.00, G3B0.50, and G3B1.00. For TUNEL analysis, G3B1.00 showed the highest DNA integrity among the experimental groups which was statistically significant only with G5B0.50 (p < 0.05). The mRNA levels of NQO1 were significantly decreased in G5B1.00, G3B0.50, and G3B1.00, when compared to G5B0.00. In comparison with G5B0.00, supplementation of 1 mm boron with and without trehalose had significantly lower expression of GCLC. The level of GSTP1 gene was significantly lower (approximately threefold) in G3B1.00, compared to G5B0.00 (p < 0.05). DISCUSSION AND CONCLUSION: It can be assumed that the increase of the boron concentration in the extender may have important adverse effects on sperm parameters and antioxidant gene expression after thawing. The results obtained from this study will help to understand the toxicity limits of boron and eliminate the toxicity of glycerol in studies of gametes and tissue freezing. Therefore, it can be concluded that the use of sufficient boron can decrease cryodamages of cryopreservation of mammalian spermatozoa as well tissue engineering.

Combination of fetuin and trehalose in presence of low glycerol has beneficial effects on freeze-thawed ram spermatozoa.

BACKGROUND: Freeze-thawing process negatively affects ram spermatozoa in terms of sperm quality, DNA integrity and antioxidant defence system. Thus, antioxidant supplementation of spermatozoa during freeze-thawing is suggested to improve sperm parameters. OBJECTIVES: The aim of this study was to determine the effects of fetuin and trehalose added into ram semen extender on sperm parameters, antioxidant parameters, antioxidant-related gene expressions and DNA integrity during the freeze-thawing process, in low glycerol concentration. METHODS: Semen samples collected from six mature rams were pooled and splitted into equal aliquots and diluted with a tris-based extender containing different concentrations of glycerol (G5; %5 and G3; %3), fetuin (F; 2.5, 5 and 15 mg/mL) and trehalose (60 mm) as eight groups (G5F0, G5F2.5, G5F5, G5F15, G3F0, G3F2.5, G3F5 and G3F15). RESULTS: G3F5 group resulted in the highest motility, mitochondrial activity and viability and the lowest DNA fragmentation and DNA damage (p < 0.05). Also, G3F0 displayed considerably more cryoprotective effect compared with G5F0 group (p < 0.05) in terms of motility, mitochondrial activity and viability rates. Lipid peroxidation levels decreased in G5F5 group compared with G5F0 group (p < 0.05). The levels of total glutathione increased in G3F2.5 group (p < 0.05) in comparison with the G5F0 group. NQO1 gene levels were upregulated approximately twofold in G5F5, G5F15, G3F2.5, G3F5 and G3F15 groups compared with G5F0 group (p < 0.05). The levels of GCLC gene were approximately twofold higher in G3F0, G3F2.5, G3F5 and G3F15 groups compared with G5F0 group (p < 0.05). GSTP1 gene levels were significantly higher with different levels in all treatment groups except for G5F2.5 and G3F0 groups in comparison with G5F0 group (p < 0.05). CONCLUSIONS: Co-supplementation of tris-based extender having low glycerol (3%) with trehalose and fetuin to enhance the quality of ram spermatozoa after freeze-thawing process is recommended.

Decreasing glycerol content by co-supplementation of trehalose and taxifolin hydrate in ram semen extender: Microscopic, oxidative stress, and gene expression analyses.

This study aimed to evaluate the comparative effects of taxifolin hydrate and trehalose on the quality of frozen-thawed ram spermatozoa for the first time. Ejaculates collected from six mature rams were pooled, and divided to eight equal aliquots to extend them with different concentrations of glycerol (%5 and %3), taxifolin hydrate (10, 100, and 500 µM), and trehalose (60 mM) as eight groups (G5T0, G5T10, G5T100, G5T500, G3T0, G3T10, G3T100, and G3T500). After freeze-thawing process of cryopreservation, microscopic and oxidative stress parameters, and gene expression levels were investigated for understanding of possible impacts of taxifolin hydrate and trehalose. The study showed that G3T10 resulted in the highest post-thawed viability and mitochondrial activity. Moreover, all extenders with taxifolin hydrate reduced DNA fragmentation in comparison to G5T0, but DNA damage was prevented at the highest rate in presence of G5T10. The level of LPO significantly decreased in the groups G5T500 and G3T100, and the expression levels of NQO1, GCLC, and GSTP1 genes significantly increased in the groups G5T100, G5T500, G3T10, and G3T100 compared to the group G5T0. Finally, co-supplementation of tris-based extender having 3% glycerol with 60 mM trehalose and 10 µM taxifolin hydrate in cryopreservation extender may be recommended to improve the quality of post-thawed ram spermatozoa. However, further in vivo and in vitro studies are suggested to evaluate fertility rates of frozen-thawed ram spermatozoa co-supplemented with trehalose and taxifolin hydrate.

Cryopreservation Effects on Ram Sperm Ultrastructure.

Cryoprotectants are known to have protective effects against cryodamage to spermatozoa. In this study, the cryoprotective effects of two cryoprotectants (glycerol, ethylene glycol) and cryoprotectants/trehalose combinations on frozen-thawed ram spermatozoa were investigated at the ultrastructural level. For this purpose, ejaculates collected from Konya Merino rams were pooled and diluted with a tris-based extender containing additives, including 5% glycerol, 3% glycerol +60 mM trehalose, 1.5% glycerol +100 mM trehalose, 5% ethylene glycol, 3% ethylene glycol +60 mM trehalose, and 1.5% ethylene glycol +100 mM trehalose. They were all cooled to 5°C and then frozen in 0.25 mL French straws in liquid nitrogen. The samples were thawed at 37°C and centrifuged to remove the diluents. Then, they were processed using a scanning transmission electron microscope. In the statistical analysis, the number of ultrastructurally cryodamaged and intact spermatozoa were counted in longitudinal and transverse ultrathin sections in all groups by electron microscopic examination. The amount of intact spermatozoa in the groups containing 5% ethylene glycol and 1.5% ethylene glycol +100 mM trehalose was found to be higher than other groups (p < 0.05). As a result, it was suggested that the groups of 5% ethylene glycol and 1.5% ethylene glycol +100 mM trehalose provided the highest protection for the ultrastructural morphology of frozen-thawed Konya Merino ram spermatozoa among the groups.

The synergistic effect of trehalose and low concentrations of cryoprotectants can improve post-thaw ram sperm parameters.

The objective of this study was to compare the effects of different concentrations of two different cryoprotectants (glycerol, G and ethylene glycol, EG) and trehalose (T), added to the semen extender, on post-thaw ram sperm parameters. Ejaculates, collected from 6 Merino rams, were pooled and evaluated at 37 °C. The pooled samples were divided into six equal aliquots, and diluted in Tris-based extenders containing 5% G, 3% G + 60 Mm T, 1.5% G + 100 Mm T, 5% EG, 3% EG + 60 mM T, and 1.5% EG + 100 Mm T. Subsequently, the samples were cooled to 5 °C, frozen in 0.25-ml French straws, and stored in liquid nitrogen (LN2). Frozen samples were thawed individually, at 37 °C for 25 s in a water bath, for evaluation. Sperm motility was assessed using a phase-contrast microscope with a warm stage maintained at 37 °C. Acrosome integrity (FITC/PNA-PI), sperm viability (SYBR-14/PI), mitochondrial activity (JC-1/PI), DNA damage (COMET assay) and DNA fragmentation (TUNEL test) were determined. The group of samples diluted in an extender containing 5% of glycerol (Group 5% G) displayed higher percentages of subjective motility, viability and mitochondrial activity of sperm, compared to the other groups (P < 0.05). On the other hand, Group 3% G + 60 mM T yielded the second-best results for subjective motility, viability and mitochondrial activity of sperm, when compared to the other groups. The post-thaw sperm parameters of Group 3% G + 60 Mm T did not show any statistically significant difference from those of Group 5% G. There were no statistically significant differences between the groups for acrosome integrity (P > 0.05). The results of the COMET assay showed that the use of low concentrations of cryoprotectants in combination with trehalose decreased sperm DNA damage. Accordingly, Group 1.5% G + 100 mM T and Group 3% EG + 60 mM T benefited from a significantly stronger cryoprotective effect on DNA integrity, in comparison to Group 5% G (P < 0.05). According to the results of the TUNEL test, the combined use of low concentrations of cryoprotectants with trehalose decreased sperm DNA damage, when compared to the use of 5% glycerol, but this difference was statistically insignificant (P > 0.05). In conclusion, G and EG concentrations can be reduced by adding various amounts of T (60 mM, 100 mM) to the semen extender. The addition of 5% of glycerol and 3% G + 60 mM T to the semen extender did not yield statistically different post-thaw sperm parameters, when compared for protection against cryoinjury. Post-thaw sperm parameters can be improved by the supplementation of the semen extender with 3% G + 60 mM T. Thus, we recommend the use of freezing extenders containing low cryoprotectant concentrations (3% G) combined with trehalose to avoid the high level of toxic and osmotic damage caused by 5% G.

Tissue reaction to urogynecologic meshes: effect of steroid soaking in two different mesh models.

INTRODUCTION AND HYPOTHESIS: Steroid soaking may decrease mesh-triggered inflammatory reaction in tissue. We aimed to investigate the tissue reaction to a steroid-soaked mesh material and an unsoaked mesh material in the rat model. METHODS: Neutral and steroid-soaked type I macroporous polypropylene (PP) monofilament and polyvinylidene fluoride (PVF) mesh materials were implanted on the rectus abdominis muscle of 20 mature Wistar albino rats. Animals were divided into four groups: PP mesh with steroid (PP-S), PP mesh without steroid, PVF mesh with steroid (PVF-S), and PVF mesh without steroid. The rats were killed after 12 weeks, and histologic, immunohistochemical and electron microscopic examinations were performed. For immunohistochemical analysis, polyclonal rabbit anti-mouse CD3, rabbit anti-mouse CD68, rabbit anti-mouse CD15, and rabbit anti-mouse CD34 antibodies were used for the detection of lymphocytes, macrophages, polymorphonuclear leukocyte foreign body giant cells, and fibromyocyte stem cells, respectively. Samples were stained with hematoxylin and eosin for the histologic evaluation of inflammation and with Masson's trichrome stain for the evaluation of collagen deposition. Pore size and mesh ultrastructure were evaluated by electron microscopy. RESULTS: Expression of CD3 was lower in the PVF, PVF-S and PP-S groups, and expression of CD34 was higher in the PVF-S and PP-S groups than in the PP groups (p < 0.05). Collagen deposition was lower in the PVF, PVF-S and PP-S groups (p < 0.05). Histologically, the intensity of inflammation was lower in the PVF-S and PP-S groups than in the PP mesh group (p < 0.05). There were no significant differences among the groups in terms of pore size and mesh ultrastructure on electron microscopic examination (p > 0.05). CONCLUSIONS: PVF mesh induces less inflammation than PP mesh, and in both mesh types steroid soaking further decreases inflammation without changing the pore size.

Chronic Exposure to Static Magnetic Fields from Magnetic Resonance Imaging Devices Deserves Screening for Osteoporosis and Vitamin D Levels: A Rat Model.

Technicians often receive chronic magnetic exposures from magnetic resonance imaging (MRI) devices, mainly due to static magnetic fields (SMFs). Here, we ascertain the biological effects of chronic exposure to SMFs from MRI devices on the bone quality using rats exposed to SMFs in MRI examining rooms. Eighteen Wistar albino male rats were randomly assigned to SMF exposure (A), sham (B), and control (C) groups. Group A rats were positioned within 50 centimeters of the bore of the magnet of 1.5 T MRI machine during the nighttime for 8 weeks. We collected blood samples for biochemical analysis, and bone tissue samples for electron microscopic and histological analysis. The mean vitamin D level in Group A was lower than in the other groups (p = 0.002). The mean cortical thickness, the mean trabecular wall thickness, and number of trabeculae per 1 mm2 were significantly lower in Group A (p = 0.003). TUNEL assay revealed that apoptosis of osteocytes were significantly greater in Group A than the other groups (p = 0.005). The effect of SMFs in chronic exposure is related to movement within the magnetic field that induces low-frequency fields within the tissues. These fields can exceed the exposure limits necessary to deteriorate bone microstructure and vitamin D metabolism.

Immunohistochemical investigation of galectin-3 in the skin of mice applied with Origanum hypericifolium essential oil and irradiated with ultraviolet B.

AIM: To demonstrate galectin-3-immunoreactivity in the undiluted essential oil of Origanum hypericifolium when applied to the ultraviolet B (UVB) irradiated skin of mice. MATERIALS AND METHODS: Female BALB/c mice were allocated to 4 groups, each comprising 6 mice (Group 1: control; Group 2: UVB irradiated control; Group 3: undiluted O. hypericifolium essential oil applied; Group 4: undiluted O. hypericifolium essential oil applied before UVB irradiation). One week prior to UVB irradiation, the undiluted O. hypericifolium essential oil was applied to, the shaved dorsal skin of mice 3 times a week. Subsequently, the mice were irradiated 3 times per week with UVB (week 1: 50 mJ/cm2, week 2: 70 mJ/cm2, and weeks 3 and 4: 80 mJ/cm2) for 4 weeks. At the end of this period, immunohistochemical staining for galectin-3 was performed on frozen sections of skin specimens, and then they were photographed. RESULTS: Numerous galectin-3-immunoreactive cells, whichwere considered to be immune system cells, were observed in the dermis of Group 3. CONCLUSION: It is suggested that undiluted O. hypericifolium essential oil may cause an increase in the galectin-3-immunoreactive cells. However, there is a need to research these findings with further molecular analyses.

Raffinose and hypotaurine improve the post-thawed Merino ram sperm parameters.

The aim of this study was to determine the effects of raffinose and hypotaurine on sperm parameters after the freeze-thawing of Merino ram sperm. Totally 40 ejaculates of five Merino ram were used in the study. Semen samples, which were diluted with a Tris-based extender containing 10mM raffinose, 5mM hypotaurine, 5mM raffinose +2.5mM hypotaurine (H+R) and no antioxidant (control), were cooled to 5 °C and frozen in 0.25 ml French straws and stored in liquid nitrogen. Frozen straws were then thawed individually at 37 °C for 25s in a water bath for evaluation. The addition of raffinose led to higher percentages of subjective and CASA motilities (47.5 ± 12.2%, 46.3 ± 13.6%) compared to controls (38.8 ± 13.8%, 30.5 ± 11.7%, P<0.05). For the CASA progressive motility, 5mM raffinose (20.12 ± 8.82%) had increasing effect in comparison to control (10 ± 7.94%, P<0.05) following the freeze-thawing process. Raffinose and hypotaurine led to higher viability (40.8 ± 4.68%, 40.8 ± 4.7%), high sperm mitochondrial activity (29.5 ± 5.4%, 27.3 ± 4.9%) and acrosome integrity (50.8 ± 8.1, 50.7 ± 4.4) percentages, compared to control groups (31.5 ± 3.5%, 9.5 ± 8.2%, 42.8 ± 7.3%, P<0.05). H+R group only led to high sperm mitochondrial activity when compared to control group. In the comet test, raffinose and hypotaurine resulted in lower sperm with damaged DNA (6.2% and 3.9%) than that of control (9.1%), reducing the DNA damage. For TUNEL assay, The TUNEL-positive cell was distinguished by distinct nuclear staining. Raffinose and H+R groups resulted in lower sperm with TUNEL-positive cell (1.5 ± 1.2% and 2.1 ± 0.9%) than that of control (4.9 ± 2.5%) (P<0.05). In conclusion, findings of this study showed that raffinose and hypotaurine supplementation in semen extenders provided a better protection of sperm parameters against cryopreservation injury, in comparison to the control groups.

The effects of Crataegus aronia var. dentata Browicz extract on biochemical indices and apoptosis in partially hepatectomized liver in rats.

Crataegus species have been widely used in herbal medicine, especially for the hearth diseases. In the present study, the effect of Crataegus aronia var. dentata Browicz extract on partially hepatectomized rats was investigated with biochemical and TUNEL apoptosis assays. The extracts of the plant at the concentrations of 0.5 and 1 ml/100 g body weight/day were administered orally to the two experimental groups including partially hepatectomized rats for 42 days. At the end of the experimental period, animals were sacrificed, blood was collected for the assessment of serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT), and the liver tissue was used for TUNEL assay. In biochemical assay, it was found a significant decrease in the levels of serum ALT and AST in the experimental groups. On the other hand, the plant extract did not cause any significant changes in the level of GGT in these groups. In apoptosis assay, TUNEL positive hepatocytes could not be detected in both experimental groups. The present findings can suggest that Crataegus aronia var. dentata Browicz extract can decrease the levels of serum ALT and AST and play a role in apoptosis of hepatocytes in the liver of partially hepatectomized rats. However, further studies are required to confirm the effects of the plant extract on hepatoprotection and apoptosis in the regenerating liver after partial hepatectomy in animal models.

The frequency of factor V Leiden and concomitance of factor V Leiden with prothrombin G20210A mutation and methylene tetrahydrofolate reductase C677T gene mutation in healthy population of Denizli, Aegean region of Turkey.

Factor V Leiden causing activated protein C resistance is the most common inherited form of thrombophilia leading to thrombosis. Its frequency shows great ethnic and geographic variations. The aim of this study was to determine the frequency of FV Leiden and coinheritance of FV Leiden with two other frequent hereditary thrombophilia causes, namely, prothrombin G20210A and methylene-tetrahydrofolate reductase (MTHFR) C677T mutation in the Aegean region of Turkey. The study population consisted of 1030 (500 men and 530 women) apparently healthy subjects. Functional resistance to activated protein C (APC) was measured by using the test kit STA staclot APC-R ((Diagnostica Stago, Asnieres, France, Cat. No. 00721). In subjects with APC resistance, molecular analyses of FV Leiden and of prothrombin G20210A and MTHFR C677T mutation were performed by using FV-PTH-MTHFR StripA (Vienna Lab, Labordiagnostika GmbH, Austria) kit, which was based on hybridization of polymerase chain reaction (PCR) amplified DNA products with mutation-specific oligonucleotide probes. Functional APC resistance was present in 93 subjects (9%). FV Leiden mutation was found in 87 of 93 subjects with APC resistance by PCR method. The FV Leiden carrier frequency was found to be 8.4% (87/1030). Seventy-six individuals were heterozygous (7.3%), and 11 were homozygous (1.06%). Among the 87 subjects with FV Leiden mutation, 45 subjects had MTHFR C677T gene mutation (7 homozygous, 38 heterozygous) and 4 subjects had heterozygote prothrombin G20210A gene mutation. A combination of FV Leiden and prothrombin G20210A and MTHFR C677T gene mutation was detected in 3 subjects. The results indicate that FV Leiden prevalence is quite high and coexistence of FV Leiden with other hereditary causes of thrombosis such as prothrombin G20210A mutation and MTHFR enzyme defect is not rare in healthy population of Aegean region of Turkey.

Incidence and molecular analysis of glucose-6-phosphate dehydrogenase deficiency in the province of Denizli, Turkey.

BACKGROUND: G6PD deficiency is a widespread abnormality of glucose-6-phosphate dehydrogenase, a red cell enzyme, which gives rise to hemolysis under oxidative stress. In Turkey, G6PD deficiency has a variable frequency in different regions. The prevalence and genotypes of G6PD deficiency are not known in Denizli province of the Aegean region of Turkey. Accordingly, this study was designed to investigate the prevalence of enzyme deficiency and the distribution of the Mediterranean mutation of G6PD in this region. MATERIAL/METHODS: A total of 1950 students (918 females, 1032 males, ages between 14 and 17) were screened by the Fluorescent Spot Test, and the G6PD deficiency was confirmed by quantitative spectrophotometric assay. The G6PD deficient subjects were further analyzed by the PCR/RFLP technique to identify the presence of the 563 T Mediterranean mutation. RESULTS: 24 of the subjects were found to be deficient in this enzyme, a frequency of 1.23%. Of 24 deficient subjects, 19 (79%) had the 563 T Mediterranean mutation. CONCLUSIONS: The frequency of G6PD enzyme deficiency appears to be low compared with those found in the malaria-endemic Mediterranean region of Turkey. The molecular pathology of G6PD deficiency is related to the G6PD-563 T mutation in the Denizli region.