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    The comparing of homogenization methods for oxidative and antioxidative analyses of sperm
    (Scientific Technical Research Council Turkey-Tubitak, 2019) Varisli, Omer; Akyol, Numan
    Pretreatment of sperm for oxidative and antioxidative analysis has been shown to be different in studies. These differences can affect the result of analyses. The aim of this study was to compare the analyses of sperm homogenization methods. The semen of four rams were used in the study. Pretreatment procedures included the rotary, bead, and ultrasonic homogenization methods. Oxidative and antioxidative status of sperm samples was assessed by measuring serum lipid hydroperoxide, total oxidant status, free sulfhydryl groups (SH total thiol), ceruloplasmin, and total antioxidant capacity levels. As a result, the homogenization methods before oxidative and antioxidative status analyses significantly affected the result (P < 0.05), and the ultrasonic homogenization method was the most effective. It is recommended to use the ultrasonic homogenization as a pretreatment method to determine the oxidative and antioxidative status analyses in sperm.
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    Effects of different extenders and additives on liquid storage of Awassi ram semen
    (Scientific Technical Research Council Turkey-Tubitak, 2018) Varisli, Omer; Taskin, Abdullah; Akyol, Numan
    The aim of this study was to evaluate the effects of different extenders and additives on ram semen during liquid storage and to demonstrate the role of oxidative stress parameters on this process. In the present study, ejaculates taken by artificial vagina twice a week from 4 rams during the breeding season were used. They were mixed and used if motility and viability were above 70% and there was a 95% intact acrosome. The semen specimens were diluted by Tris-citrate-glucose (TRIS), Tris-TES (TEST), HEPES-buffered Tyrode lactate (TL-HEPES), and Dulbecco's phosphate-buffered saline (PBS) extenders supplemented with different additives [centrifuged egg yolk, Equex-STM, bovine serum albumin (BSA), and ethylenediaminetetraacetic acid (EDTA)]. The specimens were stored at 4 degrees C for up to 96 h and evaluated for motility, membrane integrity, acrosome integrity, mitochondrial membrane potential (MMP), and oxidative stress parameters. At the end of the 96-h storage, the highest sperm motility was 64.2 +/- 3.7% (P < 0.05) and significant loss of sperm motility and membrane integrity were not detected in extender TEST-3, but the MMP rate significantly declined. Acrosome integrity was not affected by storage time or extender types. BSA and EDTA decreased lipid peroxidation (LPO) and total oxidant status (TOS), but did not positively affect motility or membrane integrity. As a result, TRIS, TEST, and TE-FIEPES-3 were observed to provide better protection for ram semen during liquid storage at 4 degrees C than other extenders. The role of oxidative stress and MMP are considerable in liquid storage.
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    Effects Of Long-Term Storage On Some Spermatological Parameters In Cryopreserved Bull Semen
    (Cryo Letters, 2018) Akyol, Numan; Varisli, Omer; Kizil, Sedat Hamdi
    BACKGROUND: The effect of the long-term storage in liquid nitrogen on semen quality has not be reported. OBJECTIVE: The study measured the spermatological parameters of bull sperm after the long-term and short-term storage. MATERIALS AND METHODS: Vintage semen (obtained from 5 Brown Swiss bulls and frozen 30 years ago) and newly frozen semen collected from 5 bulls of the same breed and prepared at the International Center for Livestock Research and Training were used. For each bull, 10 straws (0.25 ml) were thawed and pooled. Sperm samples were analyzed by flow cytometry, computer assisted sperm analysis, and total oxidant-antioxidant levels were also tested. RESULTS: The ratios of necrotic (P < 0.001) and apoptotic (P = 0.006) spermatozoa, and the ratios of total antioxidant status (TAS) and total oxidant status (TOS) were significantly higher (P < 0.001) in long-term frozen spermatozoa. However, the early necrotic ratios (P < 0.001), velocity average pathway (VAP) (P = 0.008) and velocity curvi linear (VCL) (P = 0.01) values of long-term frozen semen were lower compared with short-term frozen semen. While necrotic and apoptotic spermatozoa ratios and total oxidant level were higher, VAPNCL ratios were lower in long-term frozen sperms compared to short-term frozen semen. CONCLUSION: Long-term storage of sperm may adversely affect the spermatological and oxidative parameters of Brown Swiss bull spermatozoa.
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    IMPACTS OF SPECIFIC CRYOPROTECTANTS ON SPERM FREEZING AND RELATIONSHIPS BETWEEN CRYODAMAGE AND OXIDATION STRESS PARAMETERS IN AWASSI RAM SPERM
    (Cryo Letters, 2021) Varisli, Omer; Erat, Serkan; Bozkaya, Faruk; Aydilek, Nurettin; Taskin, Abdullah
    BACKGROUND: The role of oxidative stress during cryoprotectant treatment has received little attention. OBJECTIVE: To assess the effects of different cryoprotectants and discover relationships between cryodamage and oxidative stress parameters on Awassi ram sperm. MATERIALS AND METHODS: The sperm samples diluted with Salamon's tris-citrate (TRIS) containing 20% centrifuged egg yolk and 0.5, 1.0 or 1.5 M Glycerol (Gly), methanol (M), 2-methoxyethanol (2-ME), dimethylacetamide (DMA) and 1.2 propanediol (PR). After 2 h of equilibration at +4 degrees C, the sperm samples were frozen in liquid nitrogen vapour and stored. RESULTS: The best post-thaw motility (43.3%, 41.7%) of sperm was achieved when protected with 0.5 and 1.0 M glycerol. Arylesterase and ceruloplasmin parameters were significantly different after equilibration, whereas sulfhydryl groups were significantly different after freezing in their respective groups (P< 0.05). CONCLUSION: The increased use of glycerol caused greater loss of motility. The role of oxidative stress in freezing was also found to be limited.
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    Investigation of the Effects of Storage Period for Frozen Bull Semen on In Vitro Embryo Production
    (Kafkas Univ, Veteriner Fakultesi Dergisi, 2019) Akyol, Numan; Ertem, Talha Burak; Varisli, Omer
    The aim of this study was to investigate some spermatological parameters of frozen Brown Swiss bull semen 32 years ago by flow cytometry and to determine how storage time in liquid nitrogen affects in vitro embryo production ratios. For this purpose, early necrotic, necrotic, viable and apoptotic spermatozoa concentrations were analyzed using the Flow Cytometry and then the in vitro fertilization abilities of these sperms were investigated. AnnexinV/PI-FITC (R) was used to determine the apoptotic changes with flow cytometric analysis. Oocytes were obtained from slaughtered cows in a local abattoir. Brown Swiss semen, frozen in the last two years, were used for the control group. Early necrotic spermatozoa levels in semen frozen 32 years ago were lower but necrotic spermatozoa levels were higher than in the control group, but the opposite result was encountered in the control group (P<0.01) according to flow cytometry findings. The cleavage ratio in vintage spermatozoa was found to be lower than in the control group (P<0.0z), the blastocyst ratio was also lower than in the control group (P<0.01). As a result, it was observed that some spermatological parameters can be changed negatively and it can be said that a long storage period may lower the fertility capabilities of Brown Swiss bull semen.
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    RELATIONSHIP BETWEEN TOXICITY OF CRYOPROTECTANTS, OSMOTIC AND OXIDATIVE STRESSES IN AWASSI RAM SPERM
    (Cryo Letters, 2022) Varisli, Omer; Bozkaya, Faruk; Aydilek, Nurettin; Taskin, Abdullah
    BACKGROUND: The relationship between the toxicity of cryoprotectants and their osmotic and/or oxidative stresses remains to be further investigated. OBJECTIVE: To investigate the toxic effects of different cryoprotectants and osmotic stress on Awassi ram sperm and to determine the relationship between oxidative and antioxidative status of the sperm. MATERIALS AND METHODS: Pooled sperm samples were exposed to sucrose solutions of different concentrations (75 to 900 mOsm) and isosmotic condition (290-325 mOsm) was re-established by adding HEPES buffered Tyrode's lactate. Sperm samples were mixed with 0.5, 1.0 and 1.5 M of glycerol, methanol, 2-methoxyethanol, dimethylacetamide or 1,2-propanediol for 5 min and returned to isosmotic condition. Sperm samples were exposed to cryoprotectants at 4 degrees C for 2 hours and isosmotic conditions were re-established. Motility, viability, acrosome integrity and oxidative or antioxidative parameters were determined. RESULTS: Treatment with hypo- or hyperosmotic sucrose solution reduced motility and viability without affecting acrosome integrity. The addition and removal of glycerol and dimethylacetamide (1.0 or 1.5 M) decreased sperm motility, while cryoprotectants had no effect on viability except for 1.5 M glycerol. Chilling significantly reduced the motility and viability of the sperm, but not the acrosome integrity. Rapid addition or removal of cryoprotectants also did not affect the acrosome integrity. Cryoprotectants changed only the ceruloplasmin level, while there were significant post-chilling differences in lipid hydroperoxide, paraoxonase and ceruloplasmin levels. CONCLUSION: Cryoprotectants without other additives have limited protection and glycerol can be toxic to spermatozoa. The oxidative stress plays a role in cryoprotectant toxicity and chilling stress.