Melih Aksoy

Cholesterol-loaded cyclodextrin enhances osmotic tolerance and inhibits the acrosome reaction in rabbit spermatozoa

The effects of cholesterol-loaded cyclodextrin (CLC) treatment on the osmotic tolerance and ability to undergo the acrosome reaction of rabbit spermatozoa, with an unusually high cholesterol/phospholipid ratio in plasma membranes, were examined in two successive experiments. In the first experiment, CLC-pretreated and untreated sperm cells were exposed for 15min to one of five fructose solutions, adjusted to 20, 80, 290, 500 or 1500mOsm/L. After the anisoosmotic challenge, the integrity of sperm membranes in the CLC-supplemented (at a dose level of 3mg/120x10(6)spermatozoa) and control groups was estimated by a modified hypoosmotic swelling test (HOST) associated with a supravital eosin staining test (HE-test). In the second part of the study, the influence of cholesterol supplementation on the acrosome reaction of sperm cells stimulated by either calcium ionophore A23187 (CI) or lysophosphatidylcholine (LPC) was evaluated. CLC pretreatment increased viable and live-HOST-responsive sperm rates (P<0.01) after incubation in anisoosmotic solutions varying from 80 to 1500mOsm/L. However, CLC supplementation did not influence the percentage of HOST-responsive sperm cells (P>0.05). A significant interaction was determined between CLC pretreatment and the level of osmotic pressure in maintaining the functional and physical integrities of sperm membranes undergoing osmotic challenges. Both CI and LPC successfully induced the acrosome reaction in rabbit spermatozoa (P<0.001). Compared with CI, LPC was more effective (P<0.0001). CLC pretreatment resulted in a significant reduction (P<0.01) in the percentage of acrosome reacted sperm cells irrespective of the inducing agent, either CI or LPC. In conclusion, CLC treatment enhanced the anisoosmotic tolerance of rabbit spermatozoa and reduced their ability to undergo the acrosome reaction after stimulation by CI or LPC.

Cholesterol-loaded cyclodextrin inhibits premature acrosomal reactions in liquid-stored rabbit spermatozoa.

The effect of pretreatment of rabbit sperm cells with different concentrations of cholesterol-loaded cyclodextrin (CLC) on the occurrence of premature acrosome reactions during 72 h of liquid storage was investigated in three successive experiments. The aim of the first experiment was to establish a liquid storage model to facilitate premature acrosome reactions in rabbit sperm cells and, therefore, examined the relative effects of different dilution rates (1:5, 1:25 or 1:50) and storage temperatures (4°C or 35°C) on the occurrence of premature acrosome reactions. Increasing both dilution rate (from 1:5 to 1:25; P<0.05) and storage temperature (from 4°C to 35°C; P<0.0001) significantly enhanced the percentage of sperm cells that underwent premature acrosome reactions during storage. Therefore, a constant dilution rate of 1:25 and storage temperature of 35°C was employed for the rest of the study. The second experiment examined the effect of different CLC concentrations (0, 0.5, 1.0 and 3.0mg per 120×10(6) spermatozoa) on the occurrence of premature acrosome reactions in sperm cells. CLC supplementation of the extender inhibited (P<0.001) premature acrosome reactions in sperm cells in a dose-dependent manner during 72 h of storage. In the third experiment, the ability of CLC-pretreated sperm cells to undergo acrosome reactions induced by lysophosphatidylcholine (LPC) was evaluated following 72 h of storage. A considerable proportion of sperm cells pretreated with CLC (between 68.7 and 91.8%) underwent the acrosome reaction in response to LPC following 72 h of liquid storage. However, the ability of the sperm cells to undergo the acrosome reaction varied with regards to the dose levels of CLC pretreatment (P<0.001). In conclusion, CLC supplementation prevents premature acrosome reactions in liquid-stored rabbit spermatozoa.

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.

Protective effect of cholesterol-loaded cyclodextrin pretreatment against hydrogen peroxide induced oxidative damage in ram sperm

Three experiments were conducted to determine the protective effect of cholesterol-loaded cyclodextrin (CLC) against hydrogen peroxide (H2O2) or cryo-induced damage in ram sperm. In Experiment 1, the fresh ejaculates were either treated with CLC or remained untreated. Both CLC treated and untreated samples were then incubated with 0, 250 or 500 μM H2O2 at 35°C for 12 h. After incubation period of 12 h, the motility, viability and membrane integrity remained higher in CLC treated sperm even in the presence of 250 or 500 μM H2O2. The H2O2 treatment affected all the sperm parameters adversely (P<0.05). However, compared to CLC untreated counterpart, the motility, viability and membrane integrity remained higher (P<0.05) in treated sperm, even in the presence of 250 or 500 μM H2O2 during 12 h of incubation. In Experiment 2, semen was cryopreserved in the presence or absence of CLC. The post-thaw results revealed that CLC treated sperm has higher (P<0.05) motility, viability and membrane integrity compared to the control. In Experiment 3, lipid peroxidation levels were assessed by determining malondialdehyde (MDA) concentrations during the H2O2-induced oxidative stress in CLC treated and untreated sperm. However, no difference (P>0.05) in MDA level was observed among the groups at any stage of incubation. In conclusion, the CLC incorporation in ram sperm membrane may protects it against H2O2 or cryo-induced oxidative damage. The cryoprotective influence of CLC on ram sperm might be resulted from, at least partly, its antioxidative property.

Comparison of two different cryopreservation protocols for freezing goat semen.

In this study, two different semen cryopreservation protocols were compared to freeze goat semen. The ejaculates (n=12) were collected by using electro-ejaculator from six mature bucks (two ejaculates per each buck). Each ejaculate was divided into two groups as Protocol 1 (P1) and Protocol 2 (P2). In P1, semen was diluted directly in an extender containing 15% egg yolk, 300mM Tris, 28mM glucose, 95mM citric acid 5% glycerol to a concentration of 200×10(6)sperm/mL. In P2, after the removal of seminal plasma by centrifugation, the semen sample was diluted with the first portion of milk extender consist of 100mg/mL skimmed milk powder and 27.75mM glucose (without glycerol) to a concentration of 400×10(6)sperm/mL. The second portion of the milk extender containing 14% glycerol was added to semen gradually in order to achieve sperm concentration 200×10(6)sperm/mL and 7% glycerol level in the final volume. Extended semen was loaded in 0.25mL straws, held for 2h at 4°C, frozen in nitrogen vapor and stored in liquid nitrogen. Post-thaw motility and live sperm rate (mean±SEM) were significantly lower (P<0.05) in P1 as compared to P2 (47.50±1.23% vs. 55.63±1.72%; 80.04±1.29% vs. 84.04±1.08%, respectively). However, live intact, total intact, abnormal, reacted acrosome and DNA damaged sperm rates were similar (P>0.05) in both protocols. It was concluded that both protocols used in this study provided reasonable post-thaw parameters; however, P2 yielded better motility and live sperm rate compared to P1.

Trehalose enhances osmotic tolerance and suppresses lysophosphatidylcholine-induced acrosome reaction in ram spermatozoon.

This study was aimed to investigate the influence of trehalose on osmotic tolerance and the ability of ram spermatozoon to undergo acrosome reaction induced by lysophosphatidylcholine (LPC). In experiment 1, the diluted ejaculates were exposed to anisosmotic fructose solutions (70, 500, 750 and 1000 mOsm l−1) with or without 50 mm trehalose. The presence of trehalose in hyperosmotic conditions enhanced (P < 0.05) the percentage of live, live‐intact and intact spermatozoa. Similarly, trehalose enhanced (P < 0.05) the live and live‐intact spermatozoa during hypo‐osmotic conditions. In experiment 2, the centrifuged ejaculates were diluted with TCG only or TCG containing either 50 or 100 mm trehalose. The acrosome reaction was induced by LPC. The percentage of acrosome‐reacted spermatozoon was less (P < 0.05) in trehalose‐supplemented groups compared to control. In experiment 3, the ejaculates were cryopreserved in an extender containing 0 mm (control), 50 mm or 100 mm trehalose. Supplementation of extender with trehalose, either 50 mm or 100 mm, enhanced the cryosurvival rate (P < 0.05) compared to the control. In conclusion, the presence of trehalose in anisosmotic conditions enhances the osmotic tolerance, cryosurvival rate of ram spermatozoon and suppresses their ability to undergo LPC and cryo‐induced acrosome reaction.

Dietary quercetin maintains the semen quality in rabbits under summer heat stress

This study focused to determine beneficial impact of feeding quercetin supplemented diet on semen quality in summer heat imposed rabbits. Twelve heat stressed (HS) adult rabbits bucks were either fed with basal diet (HS; n = 06) or quercetin supplemented diet (QU-HS; n = 06) for a period of 56 days. Semen samples were collected and evaluated for volume, osmolality, morphology, concentration, motility, motion kinetics, viability, acrosome integrity, mitochondrial potential, and seminal plasma MDA level. Semen volume, concentration, motility and sperm kinetics parameters were affected by diet supplementation. Diet affected the sperm mitochondrial potential and day of treatment affected the viable sperm percentage. There was an effect of diet, day of treatment and diet by day interaction on acrosome reaction rate. Sperm head abnormalities were influenced by diet provision, sperm mid-piece abnormalities were affected by diet and day of treatment, whereas, the effect of diet and diet by day of treatment interaction were observed for total sperm abnormalities. There was an effect of diet and diet by day interaction for seminal plasma MDA level. In conclusions, quercetin reduces the damaging effects of HS and maintains the semen quality by lowering the oxidative stress in rabbits.

Seasonal variations in sperm acrosome reaction, osmotic tolerance and serum testosterone concentrations in rams

The study was conducted to evaluate the seasonal dynamics in the sperm acrosome reaction (AR), osmotic tolerance and serum testosterone concentration in rams. Blood and semen samples were collected from six mature rams during the winter, spring, summer and autumn seasons. The AR in sperm samples was induced by using lysophosphatidylcholine (LPC) and calcium ionophore (CI). The AR was monitored at various incubation times (15, 30, 60, 120 and 180 min) post-induction. To determine the osmotic tolerance through hypo-osmotic swelling test (HOST) the sperm were exposed to different osmotic conditions (50, 100, 290, 500 and 1000 mOsm/L). The serum testosterone concentration was assessed using a solid phase enzyme-linked immunosorbent assay (ELISA). The proportion of acrosome reacted sperm was greater (P <  0.05) during the winter season in the LPC and control groups, whereas, it was greater (P <  0.05) in two different seasons (winter and summer) at all incubation time points (15-180 min) in the CI group. The live HOST +ve, total HOST +ve and total live sperm were least (P <  0.05) during the winter season. The greatest membrane integrity (P <  0.05) was in the autumn season. The peak (P <  0.05) serum testosterone concentration was during the summer season. In conclusion, the osmotic tolerance and sensitivity of ram sperm to undergo the AR in response to LPC and CI vary with season. The increased testosterone concentration in the summer season might be the reason for a greater membrane integrity during the subsequent autumn season in Kivircik ram sperm.