Filiz Kutluyer

Cryopreservation of rainbow trout Oncorhynchus mykiss spermatozoa: Effects of extender supplemented with different antioxidants on sperm motility, velocity and fertility

In present study, it was examined whether addition of different antioxidants to the cryopreservation extenders had an effect on semen post-thaw fertility and motility in rainbow trout (Oncorhynchus mykiss) and also it was investigated the sperm characteristics post-thaw sperm characteristics and fertility. The collected semen was pooled to minimize individual variation. Each pooled ejaculate was split into 12 equal aliquots and diluted with base extenders supplemented with the antioxidants, and a base extender with no additives (control). The pooled semen samples diluted at the ratio of 1:10 by the extenders were subjected to cryopreservation. Antioxidants were separately added to the extenders (one per experimental group): catalase (250 U/l), superoxide dismutase (250 U/l), peroxidase (250 U/l), oxidized glutathione (1.5 mmol/l), reduced glutathione (1.5 mmol/l), L-methionine (1.5 mmol/l), uric acid (0.25 mmol/l), L-ascorbic acid (0.5 mmol/l), α-tocopherol (2.0 mmol/l), β-carotene (0.5 mmol/l) and carnitine (0.5 mmol/l). After dilution the semen was aspirated into 0.25 ml straws, the straws were placed on the tray, frozen for 10 min, and plunged into liquid nitrogen. Our results indicated that the post-thaw motility rate increased in extenders supplemented with uric acid, L-methionine, SOD, L-carnitine, α-tocopherol and L-reduced glutathione (p<0.05). The motility duration of frozen thawed semen increased in extenders supplemented with uric acid, L-methionine, SOD, α-tocopherol and L-reduced glutathione (p<0.05). Fertilization rate and hatching rate of frozen-thawed semen was not affected by the tested antioxidants. Consequently, the tested antioxidants affected the motility parameters and cryopreservation extenders could be supplement with antioxidants. This study suggested usage of antioxidants in the cryopreservation of rainbow trout.

The in vitro effect of cypermethrin on quality and oxidative stress indices of rainbow trout Oncorhynchus mykiss spermatozoa

There is limited information on the scientific literature about the effect of in vitro exposure of fish sperm to pesticides. In vitro effect of cypermethrin on sperm quality and oxidative stress has not yet been fully investigated. Therefore, the effects of cypermethrin, a type II pyrethroid insecticide, on quality and oxidative stress of spermatozoa were examined in vitro. To explore the potential in vitro toxicity of cypermethrin, fish spermatozoa were incubated with different concentrations of cypermethrin (1.025, 2.05 and 4.1 μg/l) for 2 h. The motility rate and duration of sperm were determined after exposure to cypermethrin. Reduced glutathione (GSH), glutathione peroxidase (GSH-Px), catalase (CAT) and malondialdehyde (MDA) in spermatozoa were analyzed for determination of oxidant and antioxidant balance. Our results indicated that spermatozoa motility and duration significantly decreased with exposure to cypermethrin. Additionally, activity of GSH-Px (P<0.05) and MDA and GSH levels increased in a concentration-dependent manner while CAT activity decreased (P<0.05). Consequently, the oxidant and antioxidant status and sperm quality were affected by quantitative changes and different concentrations of cypermethrin.

The in vitro effect of Lambda-cyhalothrin on quality and antioxidant responses of rainbow trout Oncorhynchus mykiss spermatozoa.

There is little information in the scientific literature about effect of in vitro exposure of fish spermatozoa to pesticides. In vitro effect of Lambda-cyhalothrin (LCT) on sperm quality and oxidative stress has not been fully explored yet. The effects of LCT, which is a synthetic pyrethroid insecticide, on quality and oxidative stress of spermatozoa were investigated in vitro due to extensively use to control a wide range of insect pests in agriculture, public health, and homes and gardens. To explore the potential in vitro toxicity of LCT, fish spermatozoa were incubated with different concentrations of LCT (0.6, 1.2 and 2.4 μg/L) for 2h. Reduced glutathione (GSH), glutathione peroxidase (GSH-Px), catalase (CAT) and malondialdehyde (MDA) in spermatozoa were analyzed for determination of oxidant and antioxidant balance. Our results indicated that the percentage and duration of sperm motility significantly decreased with exposure to LCT. Activity of GSH-Px and MDA (P<0.05) and GSH levels (P<0.05) increased in a concentration-dependent manner while CAT activity decreased (P<0.05). In conclusion, the oxidant and antioxidant status and sperm quality were affected by increasing concentrations of LCT.

An investigation on the sperm number and reproductive parameters of males in wild caught freshwater crayfish (Astacus leptodactylus, Eschscholtz)

In the present study, the sperm number of wild caught Astacus leptodactylus was counted in the beginning of reproduction season. In addition, the reproductive system, testes, and vasa deferentia were weighed, and gonado-somatic index and testicular index were calculated. Twenty-five adult males of A. leptodactylus were used for the study. Significant linear regressions were found between body length (and weight) and sperm count, reproductive system weight, testes weight and vasa deferentia weight ( P < 0 . 05 ). The mean sperm number of vas deferens for the size range of 41-56 mm carapace length crayfish was 5.72 ± 4.54 × 109 sperm/distal vas deferens (DVD) section. Sperm number ranged from 4 × 108 to 8.5 × 109 sperm/DVD section. The results revealed that an increase in body weight causes an increase in sperm number. For example, mean sperm number was 3.87 ± 2.87 × 109 for the weight range of 15-24 g crayfish, 5.01 ± 2.62 × 109 for the weight range of 25-35 g crayfish and 7.37 ± 2.87 × 109 for the weight range of 36-42 g crayfish. In conclusion, this study provides some data on the male reproduction characters and needs to be improved with details about the reproductive performance of males (i.e., sperm number and quality) in order to reach a maximum reproductive efficiency in captivity.

Combined effects of physicochemical variables (pH and salinity) on sperm motility: characterization of sperm motility in European sea bass Dicentrarchus labrax

Abstract Gamete activation in fish is an important step in terms of artificial fertilization of oocytes, cryopreservation studies and other experimental manipulations. Salinity and pH differences in activation media affect to sperm motility and fertilizing ability. These experiments were therefore designed to investigate the combined effects of pH (range 5.0–9.0) and salinity (20, 30, 37, and 45‰) of activation media on sperm motility of European sea bass Dicentrarchus labrax. The best results were obtained at salinity 37‰ and a pH of 9.0. Our results also demonstrated that non-progressive motility at salinity 45‰ was observed in the range of 5.0–9.0 pH. In conclusion, spermatozoa can be motile at a wide range of pH and salinity values although the percent of motile spermatozoa and motility duration are negatively affected by low pH values.

Effect of semen extender supplementation with cysteine on postthaw sperm quality, DNA damage, and fertilizing ability in the common carp (Cyprinus carpio)

Amino acids have an important biological role for prevention of cell damage during cryopreservation. The objective of this study is to determine the effects of cysteine on postthaw sperm motility, duration of sperm motility, DNA damage, and fertility in the common carp (Cyprinus carpio). Sperm collected from 10 individuals was cryopreserved in extenders containing different cysteine concentrations (2.5, 5, 10, and 20 mM). Semen samples diluted at the ratio of 1:9 by the extenders were subjected to cryopreservation. After dilution, the semen was aspirated into 0.25-mL straws; the straws were placed on the tray, frozen in nitrogen vapor, and plunged into liquid nitrogen. DNA damage was evaluated by comet assay after cryopreservation. Our results indicated that an increase in the concentration of cysteine caused a significant increase in the motility rate and duration of sperm in the common carp (C carpio; P < 0.05). Comparing all concentrations of cysteine, the best concentration of cysteine was 20 mM. Higher postthaw motility (76.00 ± 1.00%) and fertilization (97.00 ± 1.73%) rates were obtained with the extender at the concentration of 20 mM. Supplementation of the extender with cysteine was increased the fertilization and hatching rate and decreased DNA damage. Consequently, cysteine affected the motility, fertilization, and DNA damage positively, and extenders could be supplemented with cysteine.

Effect of the organophosphate insecticide chlorpyrifos exposure on oxidative stress and quality of Salmo coruhensis spermatozoa

Abstract The use of pesticides has been increasing along with increasing farming activities and has caused deleterious environmental impacts. Non-target organisms in particular, including fish, are affected by pesticides. In this work, the impacts of Chlorpyrifos (CPF) on sperm oxidative stress markers and sperm motility were investigated in vitro. CPF concentrations were 0 μg/L (control), 5 μg/L, 10 μg/L and 15 μg/L. Lipid peroxidation [malondialdehyde (MDA)], nonenzymatic antioxidants [glutathione (GSH)] and enzymatic [superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT)] activities in sperm cells were examined for the determination of oxidative stress status. Our findings showed that motility and survival of sperm cells significantly decreased with exposure to Chlorpyrifos. Biochemical assays revealed that CAT activity and levels of MDA and, GSH increased in spermatozoa based on CPF concentration while activity of GSH-Px and SOD decreased. Consequently, spermatozoa were highly sensitive to CPF exposure. It can be deduced that CPF has the potential to disrupt sperm quality and to cause to oxidative stress in sperm cells of S. coruhensis.

Oksijen Radikal Absorbans Kapasitesi (ORAK) Seviyeleri Farklı Bitki Masere Yağlarının Yoğun Stoklanmış Gökkuşağı Alabalıklarının (Oncorhynchus mykiss) Bazı Kan Parametrelerine Etkileri

Bu calismada, oksijen radikal absorbans kapasiteleri (ORAK) farkli bitkilerin [sogan (Allium cepa), sarimsak (Allium sativum), reyhan (Ocimum basilicum), hashas (Papaver somniferum), zencefil (Zingiber officinale), zerdecal (Curcuma longa)], masere yaglari elde edilmistir. Calisma, kontrol grubu (aycicek yagi) dahil olmak uzere toplam 7 grupta, bir hafta boyunca 30 litrelik havlandirmali tanklarda, su sirkulasyonu olmadan gerceklestirilmistir. Her tanka 20 adet (14,6±1,0 gr) gokkusagi alabaligi yuksek yogunlukta stoklanmistir. Masere yaglar, ilk gun baligin lateral cizgisi boyunca derisine surulmustur. Masere yaglarin su ve balik uzerine etkilerini belirlemek icin suyun Oksidasyon-reduksiyon potansiyeli (ORP) ve gokkusagi alabaliklarinin hematolojik parametreleri [granulosit (GRAN), beyaz kan hucresi (WBC), kirmizi kan hucresi (RBC), hemoglobin (HGB), kirmizi kan hucresindeki ortalama hemoglobin miktari (MCH), eritrositlerin hacmindeki degisikligin standart sapmasi (RDW-SD), trombositlerin ortalama buyuklugu (MPV), kan hucrelerindeki trombositlerin dagilim araligi (PDW), buyuk hucreli trombosit orani (P-LCR)] baliklarda incelenmistir. Sonuclar suyun ORP seviyesinde azalmalar oldugunu gostermistir. En yuksek yasama orani hashas yagi uygulamasinda oldugu belirlenmistir. 7 gunluk uygulama sonunda, en yuksek WBC, LYM, HGB, MCV, MCH, MPV degerleri zencefil yagi uygulamasinda (ORAK: 28.811) belirlenmistir. En yuksek RBC degeri zerdecal yagi (ORAK: 159.277) uygulamasinda, en yuksek GRAN degeri hashas yagi (ORAK: 481) uygulamasinda, en yuksek HCT ve PLT degerleri reyhan yagi (ORAK: 67.553) uygulamasinda oldugu tespit edilmistir. Calisma sonunda masere yaglarin deriye surulmesinin kan parametrelerini uyardigi tespit edilmistir.

Influence of stock density on digestive enzyme activity (trypsin), heat shock protein 70 (HSP70), and oxidative stress biomarkers of narrow clawed crayfish, Astacus leptodactylus Eschscholtz, 1823 (Decapoda, Astacidae)

Stocking density causes changes in behaviour and physiology of aquatic animals and high density can cause oxidative damage in living cells. Therefore, experiments were designed to investigate the effects of stocking density on the oxidant-antioxidant response (glutathione (GSH), glutathione peroxidase (GSH-Px), catalase (CAT), and malondialdehyde (MDA)), digestive enzyme activity (trypsin), and heat shock protein 70 (HSP70) of narrow clawed crayfish ( Astacus leptodactylus ). The experiment was conducted to assess effects by stocking 3, 6, 9 and 12 crayfish/m 2 . Crayfish were stocked into 12 fiberglass tanks, three replicates per treatment and fed for 45 days. Our results indicated that activity of GSH-Px, MDA, and GSH levels increased in hepatopancreas (p<0.05) and muscle tissues (p<0.05) with increasing stock concentration while CAT activity decreased (p<0.05). Trypsin and HSP70 insignificantly increased in haemolymph. In conclusion, oxidative stress biomarkers were negatively influenced by increased quantitative changes in stocking density.