Denizli Sağlık Hizmetleri Meslek Yüksekokulu Koleksiyonu
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Article Citation - WoS: 7Citation - Scopus: 6Lipid mixtures (from a liposome kit) and melatonin improve post-thawed Angora goat sperm parameters(Academic Press Inc., 2024-06) Bucak, Mustafa Numan; Karasor, Omer Faruk; Sari, Aysa; Bodu, Mustafa; Ili, Pinar; Narlicay, Salih; Ataman, Mehmet BozkurtSemen freezing and storing has been widely used in reproductive biotechnology, being applied to certain males of livestock breeds or animal species with economic value such as the Angora goat. The development of a semen extender with the cryoprotective agents can prevent the deterioration of sperm parameters after thawing. This study aimed to investigate lipid mixtures (from a liposome kit, Lps) and melatonin (Mel) at different doses to prevent the deterioration of sperm parameters and to provide the cryoprotective effects on sperm DNA. The Angora goat ejaculates were collected and pooled. They were divided into seven equal volumes, and each of them was diluted with the extenders of the experimental groups with additives (Lps 321.99 μg/mL, Lps 841.33 μg/mL, Mel 0.25 mM, Mel 1 mM, Lps 321.99 μg/mL + Mel 1 mM, Lps 841.33 μg/mL + Mel 0.25 mM) and no additives (control group). After the freeze-thawing process, motility, viability, acrosome integrity, DNA double-strand breaks, and abnormal DNA integrity were assessed for different extender groups. It was determined that the use of Lps alone at low dose or the combination of Lps and Mel had significant cryoprotective effects on motility, viability, acrosome integrity, and DNA damage in Angora goat sperm. This study will help us to understand the effects of Lps and Mel used alone or in combination at different doses and which doses give the optimum spermatological parameter rates following the freeze-thawing process, and hence it will shed light on further studies. © 2024 Society for CryobiologyArticle Citation - WoS: 13Citation - Scopus: 13Combination of trehalose and low boron in presence of decreased glycerol improves post-thawed ram sperm parameters: A model study in boron research(Wiley, 2021-11-30) Bucak, Mustafa Numan; Keskin, Nazan; Bodu, Mustafa; Bulbul, Bulent; Kirbas, Mesut; Ozturk, Ali Erdem; Frootan, FatemeBackground 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.Article Citation - WoS: 36Citation - Scopus: 43The synergistic effect of trehalose and low concentrations of cryoprotectants can improve post-thaw ram sperm parameters(Academic Press Inc., 2020-08) Ozturk, Ali Erdem; Bodu, Mustafa; Bucak, Mustafa Numan; Agir, Vahit; Ozcan, Ayse; Keskin, Nazan; Ili, PinarThe 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. © 2020 Elsevier Inc.Article Citation - WoS: 26Citation - Scopus: 25Decreasing glycerol content by co-supplementation of trehalose and taxifolin hydrate in ram semen extender: Microscopic, oxidative stress, and gene expression analyses(Academic Press Inc., 2020-10) Bucak, Mustafa Numan; Keskin, Nazan; Ili, Pinar; Bodu, Mustafa; Akalin, Pinar Peker; Ozturk, Ali Erdem; Ozkan, HuseyinThis 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. © 2020 Elsevier Inc.Article Citation - WoS: 24Citation - Scopus: 26Cryopreservation Effects on Ram Sperm Ultrastructure(Mary Ann Liebert Inc., 2020-09-29) Keskin, Nazan; Erdogan, Cennet; Bucak, Mustafa Numan; Ozturk, Ali Erdem; Bodu, Mustafa; Ili, Pinar; Baspinar, NuriCryoprotectants 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. © Copyright 2020, Mary Ann Liebert, Inc., publishers 2020.Article Citation - WoS: 31Citation - Scopus: 37Influence of lycopene and cysteamine on sperm and oxidative stress parameters during liquid storage of ram semen at 5 °C(Elsevier B.V., 2016-04) Peker Akalin, Pinar; Bucak, Mustafa Numan; Gungor, Sukru; Baspinar, Nuri; Coyan, Kenan; Dursun, Sukru; Ili, PinarEjaculates were collected from six Merino rams with the aid of an artificial vagina twice a week. The ejaculates containing spermatozoa with >80% forward progressive motility and concentrations higher than 2 × 109 spermatozoa/ml were pooled. The present study included two experiments. In experiment 1, each pooled ejaculate was divided into four equal aliquots and diluted (37 °C) with the Tris based extender, containing 0 (control), 0.5, 1 and 2 mM lycopene, at a final concentration of approximately 400 × 106 sperms/ml (single step dilution), In experiment 2, cysteamine at concentrations of 0 (control), 0.5, 1 and 2 mM, was used as an additive in the extender, and the procedure explained above was applied for the division of aliquots and the dilution of semen. Diluted semen samples were kept in glass tubes and cooled from 37° to 5 °C in a cold cabinet, and maintained at 5 °C. Sperm and oxidative stress parameters were evaluated after 0, 24, 48 and 72 h of storage at 5 °C. The extender supplemented with 0.5 mM lycopene resulted in higher mitochondrial activity rate (p < 0.05) in comparison to the control group at 72 h of storage. Lycopene at 0.5 mM dose led to higher sperm motility rate (p < 0.05) when compared to 2 mM lycopene group at 72 h of liquid storage. As regards oxidative stress parameters, only 2 mM lycopene increased total glutathione levels (p < 0.05) at 0 h of storage. The extender supplemented with 1 mM cysteamine gave higher motility (p < 0.05) at 48 h compared to control. As regards oxidative stress parameters, 1 and 2 mM cysteamine at 48 h and 1 mM cysteamine at 72 h increased total glutathione levels (p < 0.05) compared to control groups. Cysteamine at 1 and 2 mM doses decreased lipid peroxidation (p < 0.05) at 0 h of liquid storage compared to control. Our data suggest that lycopene at 0.5 and 2 mM and cysteamine at 1 and 2 mM doses can be added to Tris based extender for improving the ram sperm motility, viability, mitochondrial activity and oxidative stress parameters during the liquid storage. © 2016 Elsevier B.V.Article Citation - Scopus: 22Effects of arginine and trehalose on postthawed bovine sperm quality(Akademia Kiado Rt, 2017) Ozturk, Caner; Gungor, Şükrü; Ataman, Mehmet Bozkurt; Bucak, Mustafa Numan; Başpınar, Nuri; İli, Pınar; Inanc, Muhammed EnesThe present study was conducted to examine the protective role of arginine and trehalose on post-thaw bull sperm and oxidative stress parameters. Five ejaculates for each bull were used in the study. Each ejaculate, split into three equal aliquots and diluted at 37 degrees C with base extenders containing 2 mM arginine, 25 mM trehalose and no antioxidant (control) was cooled to 5 degrees C and then frozen. Frozen straws were thawed in a water bath for evaluation. Supplementation of the semen extender with arginine decreased the percentages of post-thawed subjective motility (29 +/- 8.21%), CASA motility (12.2 +/- 5.69%) and progressive motility (3.52 +/- 2.13%), compared with the controls (43 +/- 2.73%, 55.4 +/- 6.78% and 33.48 +/- 4.14%, respectively, P < 0.05). Supplementation of the semen extender with trehalose produced a higher mitochondrial activity and sperm viability (36.3 +/- 3.99% and 44.1 +/- 2.18%) compared with the control (13 +/- 8.15 and 31.7 +/- 3.94%, respectively, P < 0.05). It was established that trehalose (95.1%) and arginine (92.8%) protect DNA integrity compared to the control (90.4%) (P < 0.05). Trehalose supplementation in semen extenders provided great benefit in terms of viability, mitochondrial activity, and intact sperm DNA on frozen-thawed bull sperm.Article Citation - WoS: 16Citation - Scopus: 20Influence of ellagic acid and ebselen on sperm and oxidative stress parameters during liquid preservation of ram semen(Royan Inst., 2019) Bucak, Mustafa Numan; Bodu, Mustafa; Başpınar, Nuri; Güngör, Şükrü; İli, Pınar; Acibaeva, Begimay; Topraggaleh, Tohid RezaeiObjective: The purpose of the present study was to assess the effects of ellagic acid and ebselen on sperm and oxidative stress parameters during liquid preservation of ram semen. Materials and Methods: In this experimental study, sixty ejaculates from six mature Merino rams were used. In experiment 1, the ejaculates were diluted in base extender contained ellagic acid at 0 (control), 0.5, 1, and 2 mM. In experiment 2, ebselen at 0 (control), 10, 20, and 40 mu M were added to the extender. Sperm motility, viability, mitochondrial membrane potential, DNA integrity, lipid peroxidation (LPO), the antioxidant potential (AOP), and total glutathione (tGSH) were evaluated at 0, 24, 48, and 72 hours of preservation. Results: Supplementation of ellagic acid at 1 and 2 mM resulted in higher sperm motility and viability at 0 hours of storage. Ellagic acid at 2 mM led to higher motility and viability compared to controls after 0, 24, and 48 hours of preservation and increased AOP after 24 and 72 hours. Higher tGSH was at 1 mM ellagic acid, compared to control after 72 hours. Addition of ebselen at a concentration of 40 mu M increased motility at 24 and 48 hours and 10 mu M produced the same effect after 48 and 72 hours of storage as well as higher viability, compared to the controls after 0 hours of storage. Sperm DNA integrity was significantly improved after 24, 48, and 72 hours with the addition of ebselen at 10 mu M, and after 72 hours at 40 mu M. Addition of 40 mM ebselen also reduced the LPO levels after 24 hours of storage compared to the controls. Conclusion: The results showed that supplementation of ellagic acid and ebselen in semen extender has a potential effect on sperm and oxidative stress parameters during liquid preservation of ram semen.Article Citation - WoS: 14Citation - Scopus: 16DNA damaging effect of paclitaxel in the epididymal sperms as a chemotherapeutic agent and possible remedies to prevent this effect: A study on reproductive potential of male cancer patients of reproductive age(Elsevier Science, 2019) İli, Pınar; Sari, Fikret; Bucak, Mustafa Numan; Öztürk, Caner; Güngör, Şükrü; Ataman, Mehmet Bozkurt; Pınar İliCancer is a major public health problem, young cancer patients therefore undergo chemotherapy, and most of them may lose their fertility. DNA damage level provides important clues about the quality and reproductive potential of spermatozoa. In this study, we evaluated the levels of both DNA fragmentation and abnormal DNA integrity in the epididymal sperms of New Zealand rabbit (Oryctolagus cuniculus) after cryopreservation using the terminal deoxyribonucleotidyl transferase-mediated dUTP nick endlabelling (TUNEL) assay and the toluidine blue (TB) staining methods and assessed the effects of paclitaxel, resveratrol, L-glutamine (LG), and basal medium eagle (BME) solution on DNA damage. Paclitaxel induced the levels of both DNA damages in the sperms, but resveratrol ameliorated this effect. LG and BME supplementation to the extender prevented the sperm samples from DNA fragmentation after cryopreservation. Chemotherapy drugs containing paclitaxel can cause the sperm DNA to be damaged, and hence adversely affect the fertility of male cancer patients of reproductive age. The administration of resveratrol together with paclitaxel may ameliorate the DNA damage inducing effect of paclitaxel. Sperm banking and cryopreservation with the appropriate cryoprotectants such as LG and BME prior to cancer treatment can also be suggested to all male cancer patients of reproductive age facing cancer treatment for fertility preservation.