İlker Yavaş

Effect of cholesterol-loaded cyclodextrin on cryosurvival and fertility of cryopreserved carp (Cyprinus carpio) sperm.

Addition of cholesterol-loaded cyclodextrin (CLC) to the diluents of mammalian semen increased stability and rigidity of phospholipid hydrocarbon chains of plasma membrane during sperm cryopreservation process. CLC has been tested successfully as cryoprotectant in various livestock sperm cryopreservation protocols but its efficacy for cryopreserving of fish sperm has not previously been tested. In the present study, different cholesterol loaded cyclodextrin concentrations were evaluated for the cryopreservation of carp (Cyprinus carpio) sperm. Sexually mature fish were induced to spermiation and ovulation with Ovopel. The extenders were prepared by using 300 mM glucose and 10% DMSO supplemented with different concentrations of CLC (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0mg per 120×10(6) spermatozoa) and without CLC (control). The pooled semen was diluted separately at a ratio of 1:3 (v/v) by using CLC extenders. Diluted semen placed into 0.25 ml straws were equilibrated at 4°C for 15 min and frozen in liquid nitrogen vapor. Fertilization was conducted using a ratio of 1×10(5) spermatozoa/egg. Fresh sperm with no treatment showed the greatest sperm motility, duration of motility, viability, and fertilization results compared to the other tested cryopreserved and control groups (p<0.05). Supplementation of 1.5 mg CLC to the extender showed the best cryoprotective effect for sperm motility, duration of motility, and viability against freezing damage in comparison to extenders containing 2.5 mg, 3.0 mg CLC, and control group (p<0.05). Cryopreserved sperm containing 1.5 mg CLC provided greater result in term of fertilization success when compared to other extenders containing 0.5, 2.5, and 3.0 mg CLC or control (p<0.05). The amount of CLC effected post-thaw sperm quality and fertility as a dose-dependent manner. It is concluded that treatment of cholesterol-loaded cyclodextrin for carp sperm cryopreservation significantly improves cell cryosurvival and fertilization.

An evaluation of soybean lecithin as an alternative to avian egg yolk in the cryopreservation of fish sperm.

Plant-derived lecithin has been used as a more sanitary alternative to avian egg yolk in livestock sperm cryopreservation protocols but its efficacy for cryopreserving fish sperm has not previously been tested comparatively. Here various concentrations of soybean lecithin were evaluated for the cryopreservation of carp (Cyprinus carpio) sperm. Sexually mature fish were induced to spermiation and ovulation with ovopel. The extenders were prepared by using 300 mM glucose, 10% DMSO, supplemented with different ratios of lecithin (5%, 10%, 15%, and 20%) and 10% egg yolk (control I). Negative control was made without egg yolk and soybean lecithin (control II). The pooled semen was diluted separately at ratio of 1:3 (v/v) by using egg yolk and soybean-based extenders. Diluted semen placed into 0.25 ml straws were equilibrated at 4 °C for 15 min and frozen in liquid nitrogen vapor. Fertilization was conducted using a ratio of 1 × 10(5)spermatozoa/egg. Supplementation of 10% lecithin to extender showed the best cryoprotective effect for sperm motility and duration of motility against freezing damage compared to 15%, 20% and control II groups (p<0.05). Cryopreserved sperm with extender containing 10% lecithin provided a greater result in terms of fertilization success when compared to extenders containing 20% lecithin or control II (p<0.05). It is concluded that the animal protein-free extender containing 10% soybean lecithin has a similar cryoprotective actions with conventional egg yolk-based extender against freezing damages and fertilization. Therefore, soybean lecithin is a suitable alternative to avian egg yolk for the cryopreservation of fish sperm.

Effect of Different Thawing Rates on Motility and Fertilizing Capacity of Cryopreserved Grass Carp (Ctenopharyngodon Idella) Sperm

ABSTRACT Cryopreservation of spermatozoa has been well developed in many fish species for resource conservation and aquaculture practices. There are limited data on the effect of cryopreservation on grass carp (Ctenopharyngodon idella) spermatozoa. This research was carried out to investigate the effect of different thawing procedures on motility and fertilizing capacity of frozen/thawed grass carp semen as preliminary data to design future cryopreservation experiments. Semen was collected by abdominal stripping from adult male grass carp. Collected semen was diluted with extender containing 350 mM glucose, 30 mM Tris and 5% glycerole (pH 8.0). Diluted samples were packaged in 0.25 ml straws and left to equilibrate for 30 min at4°C. Following equilibration, the straws were exposed to liquid nitrogen vapour for 10 min and plunged into the liquid nitrogen (-196°C). For thawing, the straws were removed from liquid nitrogen and immersed in 30, 35 and 40°C water for 10, 20 and 30 seconds. In fertilization experiments spermatozoa:egg ratio was 1x105:1. The highest mean motility (83.4±2.1) (p<0.05) and fertilization rate (85.6±2.8) (P<0.05) was determined from semen thawed at 35°C for 30 sec. The results indicate that thawing rates significantly influenced motility and fertilizing ability of cryopreserved grass carp semen.

Effects of thymol and carvacrol on sperm quality and oxidant/antioxidant balance in rats

Abstract In this study, we have investigated the effects of different doses of thymol (T) and carvacrol (C) on sperm quality oxidative stress and antioxidant system. For this purpose, 49 rats were divided into seven groups (7 rats in each group): 1st Group (control); 2nd Group T-10 (thymol 10 mg/kg), 3rd Group T-20 (thymol 20 mg/kg), 4th Group C-10 (carvacrol 10 mg/kg), 5th Group C-20 (carvacrol 20 mg/kg), 6th Group T + C-10 (thymol 10 mg/kg + carvacrol 10 mg/kg) and 7th Group T + C-20 (thymol 20 mg/kg + carvacrol 20 mg/kg). The duration of the experiment was 10 weeks for all animals. During the study, sperm quality parameters (motility, concentration, abnormal spermatozoa and live–dead sperm ratio), biochemical parameters [malondialdehyde (MDA), reduced glutathione(GSH), glutathione peroxidase (GSH-Px), catalase (CAT), AST, ALT, GGT, urea and creatinine] were analysed, and histopathological examination was performed. The study results showed that monotherapies of thymol and carvacrol significantly decreased MDA levels in testicles, liver and kidney tissues compared to the control group (p < .001). GSH levels increased only with the thymol administration and GSH-Px and catalase activity increased only with the carvacrol administration compared to the control group (p < .05). The combined administration of these two agents did not cause any significant change in any parameter. Regarding the sperm quality parameters, only the spermatozoa concentration and motility increased significantly in the thymol and carvacrol groups compared to the control group (p < .01). However, these parameters decreased in the 7th Group (T + C-20) compared to the control group (p < .001). Considering the dead sperm ratio decreased significantly in the 2nd (T-10), 3rd (T-20), 4th (C-10), 5th (C-20) and 6th Group (T + C-10) compared to the control group (p < .001). In respect of spermatozoon anomaly, there was a significant decrease in thymol and carvacrol monotherapy groups. The histopathological analysis of the testicle, liver and kidney tissues of the animals showed no difference between the groups. In conclusion, we have determined that thymol and carvacrol administration decreased the oxidative damage and increased the antioxidant levels and improved the sperm quality parameters. However, the combined use of these two active ingredients had a limited therapeutic effect on the mentioned parameters.

Cryopreservation of Nile Tilapia (Oreochromis niloticus) Sperm

The main aim of this study is to determine the effect of the straw volume (0.25 vs. 0.5 mL) on Nile tilapia sperm quality after cryopreservation. Sperm was frozen according to conventional slow freezing procedure and diluted at ratio of 1:3 with ionic extender containing 350 mM glucose and 30 mM Tris containing 10% dimethylacetamide. Diluted semen was equilibrated at 4°C for 10 min and drawn into 0.25-mL or 0.5-mL plastic straws and sealed with polyvinyl alcohol. Samples were frozen 3 cm above of the liquid nitrogen surface and exposed to the liquid nitrogen vapor (≈−140°C) for 10 min. After this, frozen sperm cells were kept into the liquid nitrogen container (−196°C). The frozen sperm in different volume of straws were thawed in a water bath at 30°C for 20 s (0.25-mL straws) or at 30°C for 30 s (0.5-mL straws), respectively. Fertilization was conducted using 1 ×105 spermatozoa/egg ratio with each straw type. The findings of the present study indicated that cryopreservation of sperm in glucose-Tris–based extender using 0.5-mL straws improved post-thaw progressive motility, duration of progressive motility, and fertilization results (P 0.01). In conclusion, our results suggest that Nile tilapia sperm can be successfully cryopreserved in Tris-based extenders supplemented with glucose containing 10% dimethylacetamide in 0.5-mL straws.