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    Combination of fetuin and trehalose in presence of low glycerol has beneficial effects on freeze-thawed ram spermatozoa
    (Wiley, 2021) Bucak, Mustafa Numan; Akalin, Pinar Peker; Keskin, Nazan; Bodu, Mustafa; Ozturk, Ali Erdem; Ili, Pinar; Ozkan, Huseyin
    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.
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    Decreasing glycerol content by co-supplementation of trehalose and taxifolin hydrate in ram semen extender: Microscopic, oxidative stress, and gene expression analyses
    (Academic Press Inc Elsevier Science, 2020) Bucak, Mustafa Numan; Keskin, Nazan; Ili, Pinar; Bodu, Mustafa; Akalin, Pinar Peker; Ozturk, Ali Erdem; Ozkan, Huseyin
    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 mu M), 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 mu M 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.
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    Influence of lycopene and cysteamine on sperm and oxidative stress parameters during liquid storage of ram semen at 5°C
    (Elsevier Science Bv, 2016) Peker Akalin, Pinar; Bucak, Mustafa Numan; Gungor, Sukru; Baspinar, Nuri; Coyan, Kenan; Dursun, Sukru; Ili, Pinar
    Ejaculates 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 x 10(6) 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 degrees C) with the Tris based extender, containing 0 (control), 0.5, 1 and 2 mM lycopene, at a final concentration of approximately 400 x 10(6) 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 degrees to 5 degrees C in a cold cabinet, and maintained at 5 degrees C. Sperm and oxidative stress parameters were evaluated after 0, 24, 48 and 72 h of storage at 5 degrees 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. (C) 2016 Elsevier B.V. All rights reserved.