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Combination of cysteamine and lipoic acid improves the post-thawed bull sperm parameters
(2016) Güngör, Şükrü; Aksoy, Adil; Yeni, Deniz; Avdatek, Fatih; Öztürk, Caner; Ataman, Mehmet Bozkurt; Coyan, Kenan; Bucak, Mustafa Numan; Başpınar, Nuri; Peker Akalın, Pınar
The present study was conducted to examine the protective roles of cysteamine, trehalose, alpha-lipoic acid and combinations of these antioxidants on post-thawed bull sperm and oxidative stress parameters. Five healthy Holstein bull (3-4 years old) were used. Eight ejaculates for each bull were collected and pooled. Pooled ejaculate, splitted into seven equal aliquots and diluted at 37 °C with base extenders containing cysteamine 2 mM, trehalose 50 mM, alpha-lipoic acid (ALA) 1 mM, cysteamine 2 mM + trehalose 50 mM, ALA 1 mM + trehalose 50 mM, cysteamine 2 mM + ALA 1 mM and no antioxidant (control), was cooled to 5 °C and then frozen. Frozen straws were thawed in a water bath for evaluation. The combination of cysteamine 2 mM and ALA 1 mM of the semen extender improved the percentages of post-thawed subjective motility (68 ± 2.7%), and progressive motility (42.9 ± 4.7%), compared with the controls (61 ± 4.2% and 37.5 ± 8%, respectively, non- significantly, P>0.05). The supplementation of the semen extender with combination of cysteamine 2 mM and ALA 1 mM produced a higher acrosome integrity and mitochondrial activity (52.02 ± 6.4% and 32 ± 4.1%, respectively), compared with the controls (30.5 ± 1.7 and 14.02 ± 3.5% respectively, P < 0.05). Combination of cysteamine and ALA antioxidants in semen extenders provided the benefit in terms of sperm motilities, acrosome integrity and mitochondrial activity on frozen-thawed bull sperm.
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.
Combination of trehalose and low boron in presence of decreased glycerol improves post-thawed ram sperm parameters: A model study in boron research
(Wiley, 2022) Bucak, Mustafa Numan; Keskin, Nazan; Bodu, Mustafa; Bulbul, Bulent; Kirbas, Mesut; Ozturk, Ali Erdem; Frootan, Fateme
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.
Cryoprotective Effect of Vitamin E Supplementation of Different Extenders on Quality and Fertilizing Ability of Frozen-Thawed Brown Trout Sperm
(Mary Ann Liebert, Inc, 2021) Bozkurt, Yusuf; Yavas, Ilker; Bucak, Mustafa Numan; Kiran, Tugba Raika; Gul, Aziz
Vitamin E is one of the most powerful antioxidants for prevention of cell damage resulting from cryopreservation, but its efficacy for cryopreserving brown trout sperm is still unclear. In this work, the protective effect of vitamin E on quality, fertilizing capacity, and DNA damage of brown trout (Salmo trutta macrostigma) sperm after cryopreservation was evaluated. Sperm samples were diluted at the ratio of 1:10 with three different extenders (E): (E-I): 300 mM glucose, 10% egg yolk; (E-II): 33.3 mM glucose, 5.1 mM NaCl, 0.5 mM NaHCO3,, 15% DMA; and (E-III): 61.6 mM NaCl, 134.2 mM KCl, 1.9 mM CaCl2, 0.8 mM MgCl2, 2.3 mM NaHCO3 in distilled water. Each extender was supplemented with 10% DMSO and different concentrations of vitamin E at 0.1, 0.5, and 1.0 mM. Spermatozoa frozen without vitamin E (0 mM, control) and fresh sperm were also used. After dilution, the sperm was aspirated into 0.25 mL straws, frozen 3 cm above the liquid nitrogen (LN2) surface, and plunged into the LN2. Cell motility, viability, fertilization, and eyeing were determined in post-thawed samples. DNA damage was determined by the comet assay after cryopreservation. Supplementation of 1 mM vitamin E to all extenders exhibited the best cryoprotective effect in terms of sperm motility, duration of motility, viability, fertility, and DNA integrity against cryopreservation damage, compared with 0.1, 0.5, and control group (0 mM) (p < 0.05). The highest post-thaw motility (62.4% +/- 0.36%), fertilization (48.2 +/- 0.84), and the lowest DNA damage (7.245%) were obtained with the extender-II including 1.0 mM vitamin E (p < 0.05). Consequently, vitamin E positively affected the motility parameters, fertility, and DNA integrity, and the results suggest the addition of extenders with vitamin E as an antioxidant for the cryopreservation of brown trout sperm.
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.
Effect of different cryoprotectants (Glycerol, methanol and dimethyl sulfoxide) on post-thaw quality, viability, fertilization ability and dna damage of cryopreserved nile tilapia (oreochromis niloticus) spermatozoa
(Cryo-Letters, 2019) Bozkurt, Yusuf; Yavaş, İlker; Bucak, Mustafa Numan; Yeni, Deniz
BACKGROUND: Cryopreservation of sperm from different fish species requires different protocols. Therefore, it is necessary to perform studies to establish reliable procedures for each species. OBJECTIVE: Experiments were designed to analyse the effect of different types of cryoprotectants on post-thaw motility, viability and fertility as well as cryoresistance of cryopreserved Nile tilapia (Oreochromis niloticus) sperm. MATERIALS AND METHODS: Sperm samples were diluted with an ionic extender containing glycerol (Gly), methanol (MeOH) and dimethyl sulfoxide (DMSO) at ratios of 5, 10 and 15 % respectively. Diluted samples were aspirated into 0.25 ml French straws and frozen 3 cm above the surface of liquid nitrogen (LN) in a styrofoam box and stored in a LN tank. DNA damage was evaluated with the comet assay technique following cryopreservation. RESULTS: Supplementation of extender with 10% glycerol gave the highest motility rate compared with the other cryoprotectant groups (P<0.05). Differences in terms of post-thaw motility duration, cell viability and fertilization rates were not significant among treatments (P>0.05). Although Gly gave the best score (5.0 ± 0.1%, P>0.05) at the concentration of 10%, 5% Me2SO caused significant DNA damage (15.0 ± 1.0%, P<0.05) with the comet test. CONCLUSION: Gly or MeOH are more suitable cryoprotectants than DMSO for the cryopreservation of Nile tilapia sperm. © CryoLetters.
EFFECT OF DIFFERENT CRYOPROTECTANTS (GLYCEROL, METHANOL AND DIMETHYL SULFOXIDE) ON POST-THAW QUALITY, VIABILITY, FERTILIZATION ABILITY AND DNA DAMAGE OF CRYOPRESERVED NILE TILAPIA (Oreochromis niloticus) SPERMATOZOA
(Cryo Letters, 2019) Bozkurt, Yusuf; Yavas, Ilker; Bucak, Mustafa Numan; Yeni, Deniz
BACKGROUND: Cryopreservation of sperm from different fish species requires different protocols. Therefore, it is necessary to perform studies to establish reliable procedures for each species. OBJECTIVE: Experiments were designed to analyse the effect of different types of cryoprotectants on post-thaw motility, viability and fertility as well as cryoresistance of cryopreserved Nile tilapia (Oreochromis niloticus) sperm. MATERIALS AND METHODS: Sperm samples were diluted with an ionic extender containing glycerol (Gly), methanol (MeOH) and dimethyl sulfoxide (DMSO) at ratios of 5, 10 and 15 % respectively. Diluted samples were aspirated into 0.25 ml French straws and frozen 3 cm above the surface of liquid nitrogen (LN) in a styrofoam box and stored in a LN tank. DNA damage was evaluated with the comet assay technique following cryopreservation. RESULTS: Supplementation of extender with 10% glycerol gave the highest motility rate compared with the other cryoprotectant groups (P<0.05). Differences in terms of post-thaw motility duration, cell viability and fertilization rates were not significant among treatments (P>0.05). Although Gly gave the best score (5.0 +/- 0.1%, P>0.05) at the concentration of 10%, 5% Me2SO caused significant DNA damage (15.0 +/- 1.0%, P<0.05) with the comet test. CONCLUSION: Gly or MeOH are more suitable cryoprotectants than DMSO for the cryopreservation of Nile tilapia sperm.
EFFECT OF EXTENDER SUPPLEMENTED WITH BORON ON POST-THAW MOTILITY, VIABILITY, DNA DAMAGE AND FERTILIZATION ABILITY OF CRYOPRESERVED BROWN TROUT (Salmo trutta macrostigma) SPERMATOZOA
(Cryo Letters, 2019) Bozkurt, Yusuf; Yavas, Ilker; Gul, Aziz; Bucak, Mustafa Numan; Yeni, Deniz; Avdatek, Fatih
BACKGROUND: Boron has been considered as an essential nutrient for decreasing lipid peroxidation and improving antioxidant mechanism in different animal species. On the other hand, its effect on quality or DNA damage following cryopreservation process in fish sperm is still unclear. OBJECTIVE: Experiments were designed to analyse the effect of an ionic based extender supplemented with boron on post-thawed motility, viability, fertility and DNA integrity of cryopreserved brown trout (Salmo trutta macrostigma) sperm. MATERIALS AND METHODS: Sperm samples were cryopreserved with the ionic extender containing different boron concentrations (0.1, 0.2, 0.3 and 0.4 mM) using a controlled freezer at two different freezing rates (FR-I: 10 degrees C min(-1) from +4 degrees C to -40 degrees C and FR-II: 15 degrees C min(-1) from +4 degrees C to -40 degrees C). Sperm motility, viability, fertilization, eyeing and DNA fragmentations were determined in post-thawed samples. RESULTS: Freezing rate-I provided significantly higher fertilization and eyeing rates compared to freezing rate-II (p<0.05). Higher post-thaw motility (62.8 +/- 1.4%) and fertilization (75.2 +/- 0.9%) rates were obtained with the 0.4 mM boron concentration at freezing rate-I. CONCLUSION: Supplementation of the extender with boron increased fertilization and eyeing rates and also decreased DNA damages at both freezing rates.
The effect of raffinose and methionine on frozen/thawed Angora buck (Capra hircus ancryrensis) semen quality, lipid peroxidation and antioxidant enzyme activities
(Academic Press Inc Elsevier Science, 2010) Tuncer, Puerhan Barbaros; Bucak, Mustafa Numan; Sariozkan, Serpil; Sakin, Fatih; Yeni, Deniz; Cigerci, Ibrahim Hakki; Atessahin, Ahmet
The aim of the present study was to determine the effects of different doses of raffinose and methionine on post-thawed semen quality, lipid peroxidation and antioxidant enzyme activities of Angora buck (Capra hircus ancryrensis) sperm following cryopreservation. Ejaculates collected from three Angora bucks were evaluated and pooled at 37 degrees C. Semen samples, which were diluted with a Tris-based extender containing the additives raffinose (2.5, 5, 10 mM) and methionine (2.5, 5, 10 mM) and an extender containing no antioxidants (control), were cooled to 5 degrees C and frozen in 0.25 ml French straws. Frozen straws were thawed individually at 37 degrees C for 20 s in a water bath for evaluation. The freezing extender supplemented with 2.5 and 5 mM methionine led to higher percentages of CASA motility (63.6 +/- 7.0; 63.4 +/- 3.1%, respectively), in comparison to the controls (P < 0.01) following the freeze-thawing process. The addition of antioxidants did not provide any significant effect on the percentages of post-thaw subjective and CASA progressive motilities as well as sperm motion characteristics (VSL and VCL), compared to the control groups (P > 0.05). The freezing extender with raffinose (5 and 10 mM) and methionine at three different doses (2.5, 5 and 10 mM) led to lower percentages of acrosome abnormalities, in comparison to the controls (P < 0.001). In the comet test, raffinose (5 and 10 mM) and methionine (10 mM) gave scores lower than those of the controls, and thereby reduced DNA damage (P < 0.05). Malondialdehyde formation was found to be lower (1.8 +/- 0.1 nmol/L) in the group of 5 mM raffinose, compared to the controls following the freeze-thawing process (P < 0.01). The additives did not show any effectiveness on the maintenance of SOD, GSH-PX and GSH activities, when compared to the controls (P > 0.05). In conclusion, methionine and raffinose play a cryoprotective role against sperm CASA motility, acrosome abnormality and DNA damage. Raffinose 5 mM exhibited antioxidative properties, decreasing MDA levels. Further studies are required to obtain more concrete results on the characterization of microscopic parameters and antioxidant activities in cryopreserved goat sperm with different additives. Crown Copyright (C) 2010 Published by Elsevier Inc. All rights reserved.
The effects of different extenders and myo-inositol on post-thaw quality of ram semen
(2011) Kulaksız, Recai; Bucak, Mustafa Numan; Akçay, Ergun; Sakin, Fatih; Daşkın, Ali; Ateşşahin, Ahmet
Bu çalışma koç spermasının çözüm sonu kalitesi, lipit peroksidasyonu ve antioksidan aktiviteleri üzerine farklı sulandırıcıların ve inositolün etkilerini değerlendirmek amacıyla yapıldı. Sperma 4 baş Karayaka koçundan suni vajen yardımıyla haftada üç kez alındı. Alınan sperma örneklerinden normospermi özellik gösterenler birleştirildi. Birleştirilen sperma örnekleri iki farklı dozda myo-inositol (5, 10 mM) içeren ve içermeyen (kontrol) üç farklı sulandırıcı (tris, yağsız süt tozu, sodyum sitrat) ile sulandırıldı. Örnekler dokuz ayrı çalışma grubuna ayrıldı: T-5I, T-10I, T (kontrol); M-5I, M-10I, M (kontrol); Na-5I, Na-10I, NaC (kontrol) sulandırılmış sperma içeren payetler 4°C’de 2 saat ekilibre edildi, sıvı azot buharında (-120°C’da 15 dakika) donduruldu ve sıvı azot (-196°C) içinde saklandı. Dondurulmuş spermalar su banyosunda 37°C’de 30 saniyede çözdürüldü. Sulandırıcılara eklenenen myo-inositol mikroskopik sperm ve oksidatif stres parametelerine önemli bir etkiye neden olmadı (P>0.05). T ve M sulandırıcıları, NaC sulandırıcısına göre donma-çözünme sonrası spermatozoon motilitesinde (%50.00±2.24% ve 55.00±3.42) ve HOS testte (%49.00±3.32% and 48.17±2.9) daha yüksek oranlar verdi (P<0.01). Myo-inositol ilave edilen sulandırıcılar MDA seviyeleri ile CAT, SOD, GSH ve GSH-PX aktivitelerini, 10 mM inositol içeren T sulandırıcısının MDA seviyesi hariç, kontrol gruplarına göre önemli oranda etkilemedi (P>0.05). MDA seviyesi T sulandırıcısında (1.22±0.07 nmol/ml), M ve NaC sulandırıcılarına göre daha düşük bulundu (P<0.05). GSH ve GSH-PX aktiviteleri için, T ve NaC sulandırıcıları M sulandırıcısına göre daha yüksek değerler verdi (P<0.01).
The Effects of Different Extenders and Myo-Inositol on Post-thaw Quality of Ram Semen
(Kafkas Univ, Veteriner Fakultesi Dergisi, 2011) Kulaksiz, Recai; Bucak, Mustafa Numan; Akcay, Ergun; Sakin, Fatih; Daskin, Ali; Atessahin, Ahmet
The study was conducted to evaluate the effects of different extenders and inositol additions on post-thaw semen quality, lipid peroxidation (LPO) and antioxidant activities. Semen was collected from four Karayaka rams from by artificial vagina three times a week. Semen samples showing normospermy quality were pooled. The pooled semen samples were extended in three extenders (Tris, T-, skimmed milk, M-and sodium citrate, NaC) with myo-inositol at two different doses (5 mM, 10 mM) and no antioxidant (control). Nine experimental groups were assigned as follows: T-5I, T-10I, T (control); M-5I, M-10I, M (control); Na-5I, Na-10I, NaC (control). Straws containing extended semen were equilibrated at 4 degrees C for 2 h, frozen in vapor of (15 min at -120 degrees C) liquid nitrogen and stored in liquid nitrogen. Frozen semen was thawed in a water bath at 37 degrees C for 30 seconds. The use of all the extenders supplemented with different doses of myo-inositol did not lead to any significant improvement in microscopic sperm and oxidative stress parameters (P > 0.05). Extenders of T and M resulted in higher sperm motility (50.00+/-2.24% and 55.00+/-0.42%) and HOST (49.00+/-3.32% and 48.17+/-2.97%) rates, compared to NaC (37.00+/-3.74% and 31.80+/-2.96%, P < 0.01), following the freeze/thawing process. Extenders supplementated with myo-inositol not significantly affect malondialdehyde (MDA) levels and activities of catalase (CAT), superoxide dismutase (SOD), glutathione (GSH) and glutathione peroxidase (GSH-PX) in comparison to the control groups (P > 0.05), except for MDA level of T extender containing 10 mM inositol. MDA level was found lower (1.22+/-0.07 nmol/ml) in T than those of the M and NaC (P < 0.05). For GSH and GSH-PX activities, T and NaC gave the higher values, compared to M, following the freeze/thawing process (P < 0.01).
Effects of ultrasonication on damaged spermatozoa and mitochondrial activity rate
(2016) Akalın Peker, Pınar; Başpınar, Nuri; Çoyan, Kenan; Bucak, Mustafa Numan; Güngör, Şükrü; Öztürk, Caner
The aim of this study was to describe an optimal sonication procedure for sperm cells. Therefore, we used several parameters such as damaged spermatozoa rate (%), mitochondrial activity rate (%), levels of lipid peroxidation, and total antioxidant potential. Ejaculates were collected from rams (n = 3) and were divided into aliquots and 3-, 6-, and 10-s duration times; 1, 3, 5, and 8 repetitive application groups were established. In the groups with 3-, 6- and 10-s duration times, with the increasing number of repeated applications, damaged spermatozoa rates increased (P < 0.05) while mitochondrial activity rates decreased (P < 0.05). In relation with sonication duration time, total antioxidant potential levels increased (P < 0.05) in single-application groups compared to those in control groups and gradually decreased as the repetitions increased. The most effective results were obtained in the group with 8 repetitions and 10-s duration based on damaged spermatozoa rate and mitochondrial activity rate.
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.
Prevention of embryonic death using different hormonal treatments in ewes
(2013) Ataman, Mehmet Bozkurt; Aköz, Mehmet; Sarıbay, Mustafa Kemal; Erdem, Hüseyin; Bucak, Mustafa Numan
The purpose of this study was to evaluate different treatment protocols to prevent embryonic death in ewes. A total of 180 Akkaraman crossbred ewes and 10 healthy rams were used as material. The ewes were divided into 3 equal groups, with each of the 3 groups then separated into 3 subgroups. Ewes in estrus, determined with teaser rams, were exposed to mating. Three different treatment protocols of gonadotropin-releasing hormone (GnRH) analog (buserelin, intramuscularly) at a dose of 20 μg, vaginal sponges containing 30 mg fluorogestone acetate (FGA), and saline at a dose of 1 mL (control, intramuscularly) were applied on days 4, 12, and 16, respectively, for each subgroup after mating. No significant differences were observed in the pregnancy or multiple birth rates among any of the treatment groups. In the groups treated on days 4 and 12 after mating, the hormonal treatments gave lower rates of embryonic death compared to the control group (P < 0.05). In conclusion, the application of GnRH or FGA on days 4 and 12 after mating was found to be effective in preventing embryonic death in ewes.
Relationship of blood and seminal plasma ceruloplasmin, copper, iron and cadmium concentrations with sperm quality in Merino rams
(Elsevier Science Bv, 2015) Akalin, Pinar Peker; Bulbul, Bulent; Coyan, Kenan; Baspinar, Nuri; Kirbas, Mesut; Bucak, Mustafa Numan; Gungor, Sukru
The aim of the current study was to investigate the concentrations of ceruloplasmin, copper, iron, zinc and cadmium concentrations in blood serum and seminal plasma obtained from Merino rams. In addition, their relationship with sperm parameters, fertility rate and litter size were also studied. Blood and ejaculate samples (6 replicates) were taken in October from 19 Merino rams, aged between 18 and 24 months. Ceruloplasmin, copper, iron, zinc and cadmium in blood serum and seminal plasma were determined. Sperm parameters including volume, mass motility, motility, concentration, Hos-test, viability, abnormal sperm and acrosome abnormality in semen, fertility rate and litter size were also evaluated. Highly positive correlation was found between blood ceruloplasmin and blood copper concentrations (r=0.812, p<0.001), whereas negative correlation were determined between these parameters in seminal plasma (r=0.195, p<0.05). Seminal plasma copper concentration was positively correlated with seminal plasma cadmium (r=0.206, p<0.05) and seminal plasma iron (r=0,305, p<0.01) concentrations. Negative correlation was determined between blood ceruloplasmin level and acrosomal defect (r=0.443, p<0.05). Seminal plasma ceruloplasmin level was positively correlated with volume (r=0.255, p<0.01) and negatively correlated with abnormal sperm (r=0.186, p=0.058) and acrosome abnormality (r=0.213, p<0.05). Seminal plasma iron concentration was positively correlated with other abnormality (r=0.257, p<0.01). Seminal plasma cadmium concentration was positively correlated with sperm abnormality (r=0.207,p=0.052) and other abnormality (r=0.262,p <0.05) and negatively correlated with fertility rate (r=0.449,p =0.054). Blood cadmium concentration was negatively correlated with litter size (r=0.579, 9<0.01). In conclusion, blood and seminal plasma ceruloplasmin may be suggested to have positive influence regardless of copper with its antioxidant property whereas iron and cadmium have negative influence on sperm parameters and fertility in Merino rams. (C) 2015 Elsevier B.V. All rights reserved.

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