CRYOPRESERVATION OF GOLDFISH (Carassius auratus) SPERMATOZOA: EFFECTS OF EXTENDER SUPPLEMENTED WITH TAURINE ON SPERM MOTILITY AND DNA DAMAGE.

BACKGROUND: Amino acids, present in seminal plasma at high concentration, protect spermatozoa against cell damage during cryopreservation. OBJECTIVE: Experiments were designed to analyze the effect of semen extender supplemented with taurine on post-thawed sperm motility and duration, as well as DNA damage. MATERIALS AND METHODS: Extenders were supplemented with 1, 2 or 4 mM taurine. Semen samples were diluted at the ratio of 1:9 with the extenders. Diluted samples were aspirated into 0.25 ml French straws and 0.1 ml pellets. DNA damage was assessed with the comet assay after cryopreservation. RESULTS: The percentage and duration of sperm motility were significantly increased by taurine. Additionally, sperm motility and the motility period in pellets were higher than in straws. The best concentration of taurine was 4 mM, and the highest post-thaw motility rate (72.50 ± 3.54 %) and duration (17.50 ± 0.71 s) were obtained from the extender with 4 mM in pellets. DNA damage was decreased after taurine supplementation. CONCLUSION: Pellets could be used for goldfish sperm cryopreservation. The addition of 4 mM taurine increases the post-thaw motility and decreases DNA damage on goldfish semen.

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.

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.

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), ß-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.

Protective effects of propolis on cryopreservation of common carp (Cyprinus carpio) sperm.

Cryopreservation of sperm is common procedures in aquaculture, particularly used for routine in artificial insemination. However, these application cause damages and adversely affected sperm motility, viability and consequently lower hatching rates. The objective of this study is to determine whether propolis has an effect on cryopreservation and fertilization ability and to investigate the potential protective effect of propolis on spermatozoa of Cyprinus carpio. Many studies have been done in cryopreservation offish spermatozoa, but none of them contain propolis in extender composition. The extenders were prepared by using modified Kurokura Solution to which 10% Me(2)SO added with different levels of propolis (0.2, 0.4, 0.6, 0.8 and 1 mgml(-1)) and 10% egg yolk (as a control without propolis). The pooled semen samples diluted at the ratio of 1:9 by the extenders were subjected to cryopreservation. The percentage and duration of motility and fertilization tests of cryopreserved sperm samples have been done immediately after thawing and compared with control and fresh semen. The extenders containing propolis exhibited higher percentage motility and motility duration than control group (P < 0.05). Especially the group IV (0.8 mgml(-1) propolis) and the group V (1 mgml(-1) propolis) showed significant positive effects on both post thaw motility and hatching ability. The propolis maintained the integrity of the spermatozoa during the cryopreservation process. Evaluating with its contents, it has been shown that propolis is an appropriate cryoprotective agent in fish semen.