Reduction of cryopreservation-induced structural, functional and molecular damages in ram sperm by hydrated C60 fullerene.

This study aimed to investigate the morphological, functional and molecular changes in frozen-thawed ram sperm using an extender containing different concentrations of hydrated carbon 60 fullerene (C60 HyFn), a nanotechnological product. Semen taken from each of the seven Akkaraman rams were pooled. Semen collection was done twice a week and it continued for 3 weeks. Each pooled semen sample was divided into six equal groups and diluted with tris + egg yolk extender including 0 (control), 200, 400, 800 nM, 1 and 5 µM concentrations of C60 HyFn at 37°C. They were then frozen in liquid nitrogen vapour at -140°C, stored in liquid nitrogen container (-196°C) and thawed at 37°C for 25 s before analysis. In comparison with control, C60 HyFn addition prior to freezing procedure provided significant increases in total and progressive motility rates, glutathione peroxidase, catalase activities and percentage of highly active mitochondria, and significant decreases in dead and abnormal sperm rates, lipid peroxidation, caspase-3 and DNA fragmentation levels in frozen-thawed ram semen. When compared to control, C60 HyFn supplementation significantly down-regulated the expression levels of miR-200a and KCNJ11, and significantly up-regulated the expression levels of miR-3958-3p (at the concentrations of 200, 400, 800 nM and 1 µM), CatSper1 (at the concentrations of 200, 400 nM and 5 µM), CatSper2 (at the concentrations of 1 and 5 µM), CatSper3 (at the concentrations of 200, 400 nM, 1 and 5 µM), CatSper4 (at all concentrations), ANO1 (at the concentrations of 800 nM, 1 and 5 µM) and TRPV5 (at the concentrations of 200, 400 and 800 nM). The addition of C60 HyFn had no effect on global DNA methylation rates. As a result, C60 HyFn supplementation to ram semen extenders may be beneficial in reducing some of the functional, structural and molecular damages in sperm induced by the freeze-thawing procedure.

Effect of sulforaphane on long-term storage of rabbit semen.

In this study, it was aimed to determine the effect of sulforaphane (SFN) on rabbit semen cryopreservation. Semen collected from animals was divided into 5 equal volumes as Control, SFN 5 µM, SFN 10 µM, SFN 25 µM and SFN 50 µM groups. Afterwards, semen analyzes were performed. According to our results, there was no statistical difference between the groups at 4°C. However after freezing thawing, the highest total motility, progressive motility and rapid spermatozoa rate was seen in the 10 µM SFN group, while the lowest was observed in the 50 µM SFN group (P<0.05). Static sperm ratio was highest in the 50 µM group, while the lowest was observed in the 10 µM SFN group. When flow cytometry results examined the rate of acrosomal damaged and dead sperm was the lowest in the 10 µM SFN group, a statistical difference was observed between the control group (P<0.05). The highest rate of sperm with high mitochondrial membrane potential was seen in the 5 µM SFN and 10 µM SFN groups. Apoptosis and ROS rates were found to be lower in the experimental groups compared to the control groups (P<0.05). As a result, SFN supplementation at a dose of 10 µM increased the quality of sperm in the freezing and thawing processes of rabbit semen. In conclusion, 10 µM SFN improved the quality of cryopreservation of rabbit semen.

Effect of hydrated C60 fullerene on lipid, vitamin and amino acid composition in frozen-thawed ram semen.

This study was conducted to investigate the effect of different doses of hydrated C60 fullerene (C60HyFn) on freeze-thawing process-induced changes in lipid, vitamin and amino acid composition and also in motility, kinematic, sperm quality and oxidative stress parameters in ram semen. Semen was collected from seven rams twice a week for 3 weeks, so six repetitions were performed. The semen collected in each repetition was pooled. Each pooled sample was diluted with tris + egg yolk extender with (200 nM, 400 nM, 800 nM, 1 µM and 5 µM) and without (control) C60HyFn and they were frozen in mini straws. The doses of 800 nM, 1 µM and 5 µM had higher total, progressive motility, sperm membrane functionality rates, glutathione-peroxidase and catalase activities. All doses of C60HyFn significantly reduced dead and total abnormal sperm rates and malondialdehyde levels. Significant increases in vitamin A (400 and 800 nM doses), vitamin K1 (400 nM, 800 nM and 1 µM doses), total amino acid (all doses) levels, but significant decreases in vitamin D2 (800 nM, 1 and 5 µM doses), vitamin D3 (1 and 5 µM doses) and vitamin E (200 nM, 1 and 5 µM) levels were observed compared to control. In conclusion, the addition of C60HyFn to ram semen at 200 nM - 5 µM range, especially at a dose of 800 nM, provides a positive contribution to the protection of motility, vitamins A, K and total amino acid levels, and oxidant/antioxidant balance after freeze-thawing.

Effect of Hydrated Carbon 60 Fullerene on Frozen Ram Semen Quality.

The aim of this study was to evaluate the effect of hydrated carbon 60 fullerene (C60HyFn) on ram semen quality during cryopreservation. Three ejaculates from each of seven Akkaraman rams were collected using an artificial vagina during the nonbreeding season and pooled. Pooled semen samples were divided into 10 equal parts and diluted with tris + egg yolk extender not containing (control) and containing 100, 200, 400, and 800 nM and 1, 5, 10, 20, and 40 µM C60HyFn at 37°C. After addition of 5% glycerol and an equilibration process for 3 hours, the samples were frozen in 0.25-mL straws in an automatic freezing device at -140°C and stored in a liquid nitrogen container. Straws were thawed 24 hours after freezing and analyzed immediately with no incubation period. Motility, kinematic parameters, abnormality, vitality, hypo-osmotic swelling test (HOST), and oxidative stress levels were analyzed in thawed semen. Compared with the control, 200, 400, and 800 nM and 1 and 5 µM C60HyFn doses increased motility and HOST values and decreased the dead sperm rate. When compared with the control, addition of C60HyFn significantly decreased malondialdehyde levels (between 200 nM and 40 µM doses) and significantly increased glutathione peroxidase (between 800 nM and 40 µM doses) and catalase (between 1 and 40 µM doses) activities. In conclusion, results of this study show that the C60HyFn nanoparticles are nontoxic to ram semen and their supplementation in the extender is beneficial to sperm motility and membrane integrity after freeze-thawing.

Chrysin and flunixin meglumine mitigate overloaded copper-induced testicular and spermatological damages via modulation of oxidative stress and apoptosis in rats.

This study aimed to evaluate the possible protective actions of chrysin and flunixine meglumine on testicular and spermatological injuries experimentally stimulated by copper. We separated 36 male Sprague-Dawley rats into six equal groups: control, chrysin, flunixine meglumine, copper, copper +chrysin and copper +flunixine meglumine. Chrysin (50 mg/kg/bw/po), flunixine meglumine (2.2 mg/kg/bw/ip) and copper (500 mg/kg/bw/po) were administered day to day for 21 days. Copper administration caused significant morphological, physiological and biochemical alterations compared to the control group, which are as follows: production of oxidative stress, thanks to rise in testis lipid peroxidation and fall in antioxidant enzyme concentrations, decrease in sperm quality and increase in morphologic sperm abnormalities, suppression of spermatogenesis and prominent alterations in the testis histomorphology and induction of apoptosis in the testis tissues. On the other hand, compared to the copper group, treatment with chrysin or flunixine meglumine significantly attenuated these alterations. In conclusion, chrysin and flunixine meglumine have benefits such as antioxidant, antiapoptotic and anti-inflammatory against copper-induced testicular and spermatological damages in rats via the modulation of oxidative stress and apoptosis. Consequently, chrysin is a natural product which has comparable therapeutic actions to flunixine meglumine on the male reproductive system.

Effect of freeze-thawing process on lipid peroxidation, miRNAs, ion channels, apoptosis and global DNA methylation in ram spermatozoa.

This study was carried out to investigate the effect of the semen freeze-thawing process on the functionality and molecular structure of ram spermatozoa. The temperature of pooled and diluted semen at 38°C (group 1, control) was lowered to 5°C (group 2), and it was subjected to glycerolisation-equilibration (group 3), frozen and thawed (group 4). Compared to the control, deterioration in spermatological parameters and significant increases in lipid peroxidation and global DNA methylation levels were observed in groups 3 and 4. When compared with the control, significant downregulation in the levels of miR-485 of group 2, miR-29a of group 3 and let-7a, miR-485 and miR-29a of group 4, and significant upregulation in the levels of miR-107 of group 3 and miR-127 of groups 3 and 4 were detected. In comparison to the control, significant upregulation in the levels of CatSper1, CatSper2, CatSper3, CatSper4, ANO1 and TRPM3 of group 2, CatSper4, ANO1 and TRPM3 of group 3 and KCNJ11 of group 4, and significant downregulation in the CatSper 3 level of group 4 were determined. As a result, the semen freeze-thawing process causes motility and morphological disorders in rams. This may be due to molecular changes associated with lipid peroxidation in spermatozoa.

Influence of vitamin E and vitamin E-selenium combination on arginase activity, nitric oxide level and some spermatological properties in ram semen.

The effects of vitamin E and vitamin E-selenium combination on seminal plasma arginase activity and nitric oxide level and some spermatological properties in rams were investigated in this study. For control group, animals were injected intramuscularly with physiological saline. For vitamin E group, rams were injected intramuscularly with 300 mg/ram vitamin E. For vitamin E + selenium group, animals were injected intramuscularly with 5 ml/ram vitamin E + selenium. The semen was collected by artificial vagina at 1st, 4th, 24th, 48th and 72nd hr after administration in each group. Significant decreases in seminal plasma arginase activity (at 1st, 24th and 48th hr), nitric oxide level (at 72nd hr) and abnormal sperm rate (at 1st, 24th and 72nd hr), and significant increases in semen volume (at 24th hr), semen mass activity (at 24th and 48th hr), sperm motility (at 24th, 48th and 72nd hr) and concentration (at 1st hr) were observed in vitamin E group compared with control group. Similarly, significant increase in semen volume (at 1st, 24th and 48th hr), mass activity, (at 48th hr), motility (at 48th and 72nd hr) and concentration (at 4th, 24th and 48th hr), and significant decrements in abnormal sperm rate (at 1st, 24th, 48th and 72nd hr), seminal plasma nitric oxide level (at 1st, 4th, 24th and 48th hr) and semen pH (at 24th and 48th hr) were detected in vitamin E + selenium group in comparison to the control group. As a result, it is suggested that vitamin E and/or vitamin E + selenium applications may improve reproductive performance.

The changes in semen quality, arginase activity and nitric oxide level in dexamethasone-treated rams.

This study was made to investigate the effects of intramuscular administrations of dexamethasone on seminal plasma nitric oxide levels and arginase activity, and some spermatological parameters in rams. Ten Akkaraman rams weighing 50-60 kg and 2 years old were used as material in this study. The study was performed during the breeding season (September-November) for rams. The semen was collected by artificial vagina at 1st, 4th, 24th, 48th, 72nd and 96th hours for control group before dexamethasone administration. For treatment group, 0.25 mg/kg dexamethasone was administered and semen was collected at the time points described for control group. Spermatological characteristics of semen samples (semen volume, pH, sperm motility, density and abnormal sperm rate), seminal plasma arginase enzyme activities and nitric oxide levels were determined. It was determined that the administration of dexamethasone was detected to decrease seminal plasma arginase activity (p < .05 and .01) and nitric oxide level (p < .05), semen volume (p < .05 and .01), mass activity (p < .05 and .01), sperm density (p < .05) and sperm motility (p < .05 and .01), and to increase abnormal sperm rate (p < .05 and .01). In conclusion, dexamethasone is not recommended to be used during the breeding season as it damages the sperm quality of the rams.

Evaluation of the role of L-arginine on spermatological parameters, seminal plasma nitric oxide levels and arginase enzyme activities in rams.

The purpose of this study is to investigate the effects of L-arginine on spermatological parameters, seminal plasma nitric oxide levels and arginase enzyme activities. Fertile rams that are 2-3 years old and weighing 50-60 kg were used as material. The semen was collected by artificial vagina at 1st, 4th, 24th, 48th, 72nd, 96th and 120th hours for the control group before L-arginine administration. For treatment groups, L-arginine was administered intraperitoneally at a dose of 5 mg kg-1  bw-1 and semen was collected at the time point described for the control group. Spermatological characteristics of semen samples (semen volume, pH, sperm motility, concentration and abnormal sperm rate), seminal plasma nitric oxide levels and arginase enzyme activities were determined. Increased seminal plasma nitric oxide level (p < .01), seminal plasma arginase activity (p < .01), semen volume (p < .05), semen mass activity (p < .05), sperm motility (p < .05) and concentration (p < .01) and decreased abnormal sperm rate (p < .05 and p < .01) were observed by L-arginine administration. In conclusion, it may be concluded that L-arginine application in rams during the breeding season may have positive effects on rams' reproductive performance.

Optimization of the incubation time and temperature for spermatozoa extraction in freshwater crayfish Pontastacus leptodactylus (Eschscholtz, 1823).

Determination and control of spermatozoa quality in crustacean aquaculture is an important issue for successful and controlled reproduction. Investigation of spermatozoa number in spermatophores is a basic and common parameter for determining the reproductive quality in farmed decapods. In the present study, spermatozoa extraction from spermatophores located in the ductus deferens was conducted in Pontastacus leptodactylus using different incubation times and temperatures. The results indicate that the duration of incubation and temperature affected (P < 0.05) spermatozoa extraction. Greater temperatures (40 and 75 °C) resulted in a reduction (P < 0.05) in number of extracted spermatozoa. In contrast, more spermatozoa were extracted when the 4 and 23 °C temperatures were imposed. After 4 h of incubation, the number of extracted spermatozoa were greatest in number at 23 °C. In conclusion, the greater numbers of crayfish spermatozoa can be obtained when the ductus deferens containing spermatophores is incubated at 23 °C for 4 h as compared with other temperatures and incubation durations. The results of present study are useful for assessing spermatozoa quality in aquaculture as well as the extraction of spermatozoa for research purposes.

Assessment of imidacloprid toxicity on reproductive organ system of adult male rats.

In the current study it was aimed to investigate the toxicity of low doses of imidacloprid (IMI) on the reproductive organ systems of adult male rats. The treatment groups received 0.5 (IMI-0.5), 2 (IMI-2) or 8 mg IMI/kg body weight by oral gavage (IMI-8) for three months. The deterioration in sperm motility in IMI-8 group and epidydimal sperm concentration in IMI-2 and IMI-8 groups and abnormality in sperm morphology in IMI-8 were significant. The levels of testosterone (T) and GSH decreased significantly in group IMI-8 compared to the control group. Upon treatment with IMI, apoptotic index increased significantly only in germ cells of the seminiferous tubules of IMI-8 group when compared to control. Fragmentation was striking in the seminal DNA from the IMI-8 group, but it was much less obvious in the IMI-2 one. IMI exposure resulted in elevation of all fatty acids analyzed, but the increases were significant only in stearic, oleic, linoleic and arachidonic acids. The ratios of 20:4/20:3 and 20:4/18:2 were decreased and 16:1n-9/16:0 ratio was increased. In conclusion, the present animal experiments revealed that the treatment with IMI at NOAEL dose-levels caused deterioration in sperm parameters, decreased T level, increased apoptosis of germ cells, seminal DNA fragmentation, the depletion of antioxidants and change in disturbance of fatty acid composition. All these changes indicate the suppression of testicular function.

Protective effects of nanostructures of hydrated C(60) fullerene on reproductive function in streptozotocin-diabetic male rats.

Diabetes mellitus is a well-recognized cause of male sexual dysfunction and impairments of male fertility. Streptozotocin (STZ) is used for medical treatment of neoplastic islet ß-cells of pancreas and producing of animal model of diabetes mellitus type 1 that is characterized by suppression of reproductive activity due to the hyperglycaemia-induced oxidative stress and histopathological alterations in testes. Seeking for the agents that could alleviate diabetes-induced damage to reproductive system is yet the important area of inquiry. The present study was designed to evaluate whether hydrated C(60) fullerene (C(60)HyFn), which is known to be powerful bioantioxidant, eliminate testicular dysfunction induced by STZ-diabetes in rats. Wistar strain male albino rats were divided into four groups of six animals each: (1) control group, (2) C(60)HyFn-treated nondiabetic group, (3) STZ-diabetic group and (4) C(60)HyFn-treated diabetic group. Once hyperglycaemia was induced by STZ, rats in the second and fourth groups were treated with C(60)HyFn (in the form of drinking water) at the dose of 4µg/kg daily for 5 weeks. In diabetic rats, relative weights of right cauda epididymis, seminal vesicles, prostate, sperm motility and epididymal sperm concentration were significantly less than those of control group, but which were restored in the fourth group treated with C(60)HyFn (p<0.001). In hematoxylin and eosin staining, marked histopathological changes including degeneration, desquamation, disorganisation and reduction in germinal cells, interstitial oedema and congestion were evident in the testis of diabetic rats, but C(60)HyFn treatment resulted in recovery of histopathological changes and an increase in Johnsen's testicular score significantly (p<0.001). C(60)HyFn treatment restores the increased apoptosis induced by STZ-diabetes. In diabetic rats, levels of serum testosterone, testicular reduced glutathione (GSH) and alpha-tocopherol were significantly reduced and testicular lipid peroxidation level was increased (p<0.001). Nevertheless, treatment of diabetic rats with C(60)HyFn resulted in significant corrective effects on these parameters towards the control levels. C(60)HyFn, applied alone, did not exert any toxic effects in testicular tissues. Furthermore, C(60)HyFn treatment in diabetic and nondiabetic rats resulted in considerable elevations of some important polyunsaturated fatty acids. In conclusion, we have presented for the first time substantial evidence that administration of C(60)HyFn significantly reduces diabetes-induced oxidative stress and associated complications such as testicular dysfunction and spermatogenic disruption.

The effect of vitamin E treatment during preovulatory period on reproductive performance of goats following estrous synchronization using intravaginal sponges.

The objectives of this study were to investigate whether the use of intravaginal sponge for estrous synchronization of goats causes oxidative stress, and to examine the effect of administering vitamin E during preovulatory period on reproductive performance of estrous synchronized goats. Estrus was synchronized in 36 non-lactating adult does using intravaginal sponges containing 30 mg of fluorogestane acetate (FGA) for 14 days. All females received 500 IU of eCG at the sponge withdrawal. The goats were allocated at random to two groups balanced for breed, age and body weight. Treatment group (n=18) received 200mg of vitamin E i.m. at the time of sponge removal and again at the time of second artificial insemination. The other 18 goats (control) were administered 1 ml of physiological saline instead of vitamin E on each of these two occasions. All does in estrus was intracervically inseminated at 12 and 24h after the onset of estrus. Blood samples were collected every 72h during the experimental period for evaluation of malondialdehyde (MDA) and vitamin E concentrations. Serum MDA level increased and vitamin E concentration decreased during the period of vaginal sponge application. Following the sponge removal, MDA level declined rapidly to below basal level in the treatment group but remained high in the control group. Conversely, vitamin E concentration increased in the treatment group after the sponge withdrawal and remained at a low level in the control group. No statistically significant differences (P>0.05) were observed between groups in terms of estrous response, conception rate, gestation length or kidding rate. However, the number of multiple births (70.0% versus 50.0%) and prolificacy rate (2.40+/-0.37 versus 1.63+/-0.26 kids per kidding) were significantly higher (P<0.05) for the treatment group than those of the control group. The results indicate that the use of intravaginal sponges for estrous synchronization of goats causes an increase in level of oxidative stress. However, the vitamin E treatment during preovulatory period can prevent the overproduction of reactive oxygen species (ROS), and it may improve the multiple birth rates and the number of kids born in estrous synchronized goats.

Effects of pomegranate juice consumption on sperm quality, spermatogenic cell density, antioxidant activity and testosterone level in male rats.

BACKGROUND & AIM: Pomegranate fruit is inescapably linked with fertility, birth and eternal life because of its many seeds. The aim of this study was to investigate the effects of pomegranate juice (PJ) consumption on sperm quality, spermatogenic cell density, antioxidant activity and testosterone level of male healthy rats. METHODS: Twenty-eight healthy adult male Wistar rats were divided into four groups; each group containing seven rats. One milliliter distilled water, 0.25 mL PJ plus 0.75 mL distilled water, 0.50 mL PJ plus 0.50 mL distilled water and 1 mL PJ were given daily for seven weeks by gavage to rats in the first, second, third and fourth groups, respectively. Body and reproductive organ weights, spermatogenic cell density, sperm characteristics, levels of antioxidant vitamins, testosterone, and lipid peroxidation and, antioxidant enzyme activities were investigated. All analyses were done only once at the end of the seven week study period. Data were compared by analysis of variance (ANOVA) and the degree of significance was set at P<0.05. RESULTS: A significant decrease in malondialdehyde (MDA) level and marked increases in glutathione (GSH), glutathione peroxidase (GSH-Px) and catalase (CAT) activities, and vitamin C level were observed in rats treated with different doses of PJ. PJ consumption provided an increase in epididymal sperm concentration, sperm motility, spermatogenic cell density and diameter of seminiferous tubules and germinal cell layer thickness, and it decreased abnormal sperm rate when compared to the control group. CONCLUSION: The results suggest that PJ consumption improves sperm quality and antioxidant activity of rats.

Protective role of lycopene on cisplatin-induced changes in sperm characteristics, testicular damage and oxidative stress in rats.

The aim of this study was to investigate the possible protective role of lycopene on cisplatin (CP)-induced spermiotoxicity using quantitative, biochemical and histopathological approaches. Adult male Sprague-Dawley rats were randomly divided into four groups. The control group received physiological saline; animals in cisplatin group received only cisplatin; pre-treatment group received a 10-day of lycopene before administration of cisplatin while animals in post-treatment group received a 5-day of lycopene following administration of cisplatin. Cisplatin (7 mg kg(-1)) was intraperitoneally (i.p.) injected as a single dose and lycopene (4 mg kg(-1)) was administered by gavage in corn oil. Traits of reproductive organs; sperm characteristics, testicular histological findings, plasma testosterone levels and the testicular tissue oxidative status were determined. Administration of cisplatin to rats decreased sperm concentration (p < 0.05) and sperm motility (p < 0.001), increased total abnormal sperm rates (p < 0.05) as compared with the control group. While a marked normalization was achieved only in sperm concentration with lycopene in pre-treatment group, significant normalizations were achieved in the sperm concentration, sperm motility, total abnormal sperm rates in post-treatment group. No significant differences in levels of testosterone were observed among all groups. An increase in testes malondialdehyde concentrations (p < 0.05) and glutathione peroxidase activities (p < 0.001) were detected while significant decreases in glutathione levels (p < 0.001) in cisplatin alone group when compared to control group. While pre-treatment with lycopene restoring only malondialdehyde concentrations, its post-treatment caused normalization in both malondialdehyde and glutathione levels when compared with the cisplatin alone group. On the other hand, significant increases were determined in GSH-Px activities in all experimental groups when compared with the control group. Although the mechanism is not clear, the results from this experimental study suggest that the lycopene have a possible protective effect against cisplatin-induced spermiotoxicity, effect of giving lycopene after cisplatin being superior to the giving it before cisplatin.

Short term effects of dexamethasone on hyaluronidase activity and sperm characteristics in rams.

The aim of this study was to determine the effects of dexamethasone on sperm characteristics and hyaluronidase activity of serum and semen. In this investigation, 14 healthy Akkaraman rams, at the age of 2 years and weighing between 50-60 kg, were used. The rams were randomly divided into two groups. After the last administration of dexamethasone intramuscularly at a dose of 0.25 mg/kg, semen and blood samples were taken at different times. The results showed that the serum hyaluronidase activity was increased significantly (p<0.001) in the treatment group when compared with the control group except for the 1st hour. There was a significant difference (p<0.001, 0.01, 0.05) in the hyaluronidase activity of semen between the treatment group and the control group. Furthermore, there was a significant difference (p<0.01) in sperm concentration between both groups at all the times except the 96th hour. There were statistically significant (p<0.05) differences in semen volume between the treatment and control groups. There were also significant differences (p<0.05) in sperm motility between the treatment and control groups except for the 72 and 96th hours. These findings indicate that dexamethasone increases hyaluronidase activity of serum and semen, but it decreases sperm concentration, semen volume and sperm motility in rams. Therefore the use of these drugs in breeding rams during breeding season is not suitable.