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.

Protective effects of L-arginine against aluminium chloride-induced testicular damage in rats.

Aluminium (Al) is a toxic metal with frequent exposure because it is a common element in nature and is found in many products used in daily life. L-arginine is a semi-essential amino acid that is involved in many biochemical pathways in the body and has antioxidant effects. The current research evaluated the possible protective effects of L-arginine against aluminium chloride (AlCl3 ) induced testicular damage. In this animal-based experimental study, 28 male Wistar Albino rats were separated into four groups: control, Al (20 mg/kg/day Al), Al + L-arginine (20 mg/kg/day Al + 50 mg/kg/day L-arginine), and L-arginine (50 mg/kg/day L-arginine). All applications were carried out intraperitoneally (i.p.) for 4 weeks. The histopathological changes caused by exposure to Al in the testicular tissue and the protective effects of L-arginine were investigated by using biochemical, histochemical, immunohistochemical [4-hydroxynonenal (4-HNE) and 8-hydroxy-2'-deoxyguanosine (8-OHdG)] and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay techniques. The testicles in the AlCl3 group showed increased 4-HNE, 8-OHdG expressions, apoptotic index, and abnormal sperm content, while serum testosterone levels, sperm motility, and sperm number were decreased (p < 0.05). Furthermore, histomorphometric examinations of testicular tissue indicated significant structural impairments such as vacuolization in the seminiferous tubule epithelium, edema, and vascular congestion in the interstitial area (p < 0.05). However, the structural alterations were largely ameliorated in the Al + L-arginine group (p < 0.05). Thus, L-arginine, which is an antioxidant, may protect against the harmful effects of Al and may help improve male fertility.

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.

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.

The in-vivo assessment of Turkish propolis and its nano form on testicular damage induced by cisplatin.

OBJECTIVE: Chemotherapeutic drugs, such as cisplatin (CP), which are associated with oxidative stress and apoptosis, may adversely affect the reproductive system. This study tests whether administration of propolis and nano-propolis (NP) can alleviate oxidative stress and apoptosis in rats with testicular damage induced by CP. METHODS: In this study, polymeric nanoparticles including propolis were synthesized with a green sonication method and characterized using Fourier transform-infrared spectroscopy, Brunauer-Emmett-Teller, and wet scanning transmission electron microscopy techniques. In total, 56 rats were divided into the following seven groups: control, CP, propolis, NP-10, CP + propolis, CP + NP-10, and CP + NP-30. Propolis (100 mg/kg), NP-10 (10 mg/kg), and NP-30 (30 mg/kg) treatments were administered by gavage daily for 21 d, and CP (3 mg/kg) was administered intraperitoneally in a single dose. After the experiment, oxidative stress parameters, namely, malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GPx), and catalase (CAT), and apoptotic pathways including B cell leukemia/lymphoma-2 protein (Bcl-2) and Bcl-2-associated X protein (Bax) were measured in testicular tissues. Furthermore, sperm quality and weights of the testis, epididymis, right cauda epididymis, seminal vesicles and prostate were evaluated. RESULTS: Propolis and NP (especially NP-30) were able to preserve oxidative balance (decreased MDA levels and increased GSH, CAT, and GPx activities) and activate apoptotic pathways (decreased Bax and increased Bcl-2) in the testes of CP-treated rats. Sperm motility in the control, CP, and CP + NP-30 groups were 60%, 48.75%, and 78%, respectively (P < 0.001). Especially, NP-30 application completely corrected the deterioration in sperm features induced by CP. CONCLUSION: The results show that propolis and NP treatments mitigated the side effects of CP on spermatogenic activity, antioxidant situation, and apoptosis in rats.

Protective role of yeast beta-glucan on lead acetate-induced hepatic and reproductive toxicity in rats.

Lead (Pb) is one of the most common environmental pollutants and causes adverse effects on human and animal health. This study aimed to evaluate the protective role of beta-glucan against hepatic and reproductive toxicity induced by lead acetate. A total of 28 Sprague Dawley male rats were distributed into four groups (n = 7). The control group was intraperitoneally injected saline (1 ml/kg b.w.) daily for 21 days, the Pb group was intraperitoneally injected lead acetate (15 mg/kg b.w.) daily for 21 days, the beta-glucan group was orally administrated beta-glucan (50 mg/kg b.w.) daily for 21 days, and the Pb + beta-glucan group was intraperitoneally injected lead acetate (15 mg/kg b.w.) daily following the oral administration of beta-glucan (50 mg/kg b.w.) daily for 21 days. Results showed that feed intake in the Pb + beta-glucan group was significantly increased in comparison with that of the Pb group (p < 0.001). We also found that liver malondialdehyde (MDA) level was increased significantly in the Pb group (p < 0.01), while glutathione (GSH) level (p < 0.05), glutathione peroxidase (GSH-Px) (p < 0.05), and catalase (CAT) (p < 0.01) activities were reduced when they were compared with control. Moreover, Pb administration increased expression of pro-apoptotic protein Bax, the ratio of Bax/Bcl-2, and decreased the expression of the antiapoptotic protein Bcl-2 (p < 0.01). Also, Pb was found to cause a significant decrease in sperm motility (p < 0.01) and sperm concentration (p < 0.05) but increase in sperm tails and total sperm anomalies (p < 0.05). These findings were partially preserved by the administration of beta-glucan. Taken together, these results indicated that beta-glucan has the potential to alleviate the Pb-induced toxicity.

Irisin may have a role in pubertal development and regulation of reproductive function in rats.

Physical exercise and body muscle/fat mass are known to affect the endocrine system, puberty onset and reproductive health. However, the potential effects of irisin, an adipo-myokine and exercise-induced hormone, have not yet been fully elucidated on reproductive maturation. Therefore, the present study aimed to determine the effects of irisin administration on pubertal maturation and reproductive system in female and male rats. Daily i.p. injection of irisin (100 ng/kg; from postnatal day 21 for about 10 weeks) delayed the ages at the vaginal opening (as an external index of puberty onset) and first estrus. Furthermore, continuous administration of irisin to female rats caused a significant decrease in serum follicle-stimulating hormone levels and an increase in serum luteinizing hormone and 17ß-estradiol levels, as well as causing histopathological changes in the ovarian tissue. On the contrary, irisin administration to male rats did not modify the timing of puberty, as estimated by age at preputial separation. However, chronic exposure to irisin produced significant increases in serum luteinizing hormone and testosterone levels and also sperm concentration and seminiferous tubule diameter in male rats. In conclusion, irisin exposure has different effects on both pubertal maturation and reproductive system in female and male rats. The present findings reveal that chronic irisin exposure may lead to disorders in the female reproductive system and may have androgenic potential on the hypothalamic-pituitary-gonadal axis in males.

Bee glue (propolis) improves reproductive organs, sperm quality and histological changes and antioxidant parameters of testis tissues in rats exposed to excess copper.

This study was designed to determine the effects of propolis on the sperm quality, antioxidant and histological parameters in the testicular tissues of male Sprague Dawley rats exposed to excessive copper (Cu). In this aim, 24 rats were randomly divided into four groups as follows: the control, Cu, Propolis and Cu+Propolis. When compared to control group, Cu administration significantly decreased sperm motility and concentration, increased total abnormal sperm rate. It caused a significant induction the malondialdehyde (MDA), and reduction the superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) in testicular tissues. Also, it caused loss, disorganisation and vacuolation of the germinal epithelium, oedema of the interstitial tissues, proliferation of the interstitial cells, spilled immature spermatogenic cells in the lumen of some seminiferous tubules. A large number of active caspase-3-positive stained apoptotic cells and a significant decrease in Johnsen's testicular score were determined. However, significant ameliorations were observed in all sperm characteristics, MDA, SOD, CAT, GSH, seminiferous tubules, number of apoptotic cells and Johnsen's testicular score in Cu+Propolis group. Our results showed that oral supplementation of propolis had curative effect on the sperm quality, antioxidant and histological parameters in the testicular tissues of male Sprague Dawley rats exposed to Cu.

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.

Evaluation of ameliorating effect of lycopene against testicular toxicity due to diethylnitrosamine using biochemical, spermatological and histopathological data.

The aim of this study was to evaluate the possible therapeutic or protective effects of lycopene on diethylnitrosamine (DEN)-induced testicular lipid peroxidation and on the associated changes in spermatological parameters and histopathological architecture of rat testis. DEN is a carcinogenic substance that can be derived from chemicals used in agriculture, such as insecticides and nitrate. The rats were assigned to control, lycopene, DEN(1), DEN(2), lycopene + DEN(1), lycopene + DEN(2), DEN(1) + lycopene and DEN(2) + lycopene groups. During the study, lycopene was administered by oral gavage at a dose of 10 mg kg-1  bw-1 every other day for 10 days and DEN was administered at a dose of 200 mg  kg-1  bw-1 as a single dose intraperitoneally. DEN was applied for 30 days in group DEN(1) and for 90 days in group DEN(2). Malondialdehyde (MDA) and reduced glutathione (GSH) levels, antioxidant enzymes activities, spermatological parameters, the weight of the reproductive organs (v. seminalis, prostate, testis and epididymis) and the histopathological structure were determined. MDA levels significantly increased, while GSH and antioxidant enzymes' activities decreased in DEN groups (p < 0.001). There was an increase in the rate of abnormal spermatozoa and a decrease in sperm density and motility, and reproductive organ weight (the weight of the right and left testis) in both DEN groups. Lycopene has normalised biochemical and spermatological parameters and reproductive organ weight. The histopathological examination of testicular tissue showed that the most significant histopathological change in DEN groups was the seminiferous tubule dilatation. These results suggest that besides the protective effects, the therapeutic effect of lycopene is possibly due to its antioxidant effects on DEN-induced testicular toxicity.

Dietary rosemary oil alleviates heat stress-induced structural and functional damage through lipid peroxidation in the testes of growing Japanese quail.

Supplementation of natural antioxidants to diets of male poultry has been reported to be effective in reducing or completely eliminating heat stress (HS)-induced reproductive failures. In this study, the aim is to investigate whether rosemary oil (RO) has a protective effect on HS-induced damage in spermatozoa production, testicular histologic structures, apoptosis, and androgenic receptor (AR) through lipid peroxidation mechanisms in growing Japanese quail. Male chicks (n=90) at 15-days of age were assigned to two groups. The first group (n=45) was kept in a thermo-neutral (TN) room at 22°C for 24h/d. The second group (n=45) was kept in a room with a greater ambient temperature of 34°C for 8h/d (from 9:00 AM to 5:00 PM) and 22°C for 16h/d. Animals in each of these two groups were randomly assigned to three subgroups (RO groups: 0, 125, 250ppm), consisting of 15 chicks (six treatment groups in 2×3 factorial design). Each of subgroups was replicated three times with each replicate including five chicks. The HS treatment significantly reduced the testicular spermatogenic cell counts, amount of testicular Bcl-2 (anti-apoptotic marker) and amount of AR. In addition, it significantly increased testicular lipid peroxidation, Bax (apoptotic marker) immunopositive staining, and the Bax/Bcl-2 ratio in conjunction with some histopathologic damage. Dietary supplementation of RO to diets of quail where the HS treatment was imposed alleviated HS-induced almost all negative changes such as increased testicular lipid peroxidation, decreased numbers of spermatogenic cells, and decreased amounts of Bcl-2 and AR, increased ratio of Bax/Bcl-2 and some testicular histopathologic lesion. In conclusion, dietary supplementation of RO for growing male Japanese quail reared in HS environmental conditions alleviates the HS-induced structural and functional damage by providing a decrease in lipid peroxidation.

Effect of cinnamon (Cinnamomum zeylanicum) bark oil on heat stress-induced changes in sperm production, testicular lipid peroxidation, testicular apoptosis, and androgenic receptor density in developing Japanese quails.

The aim of this study was to investigate the effect of cinnamon bark oil (CBO) on heat stress (HS)-induced changes in sperm production, testicular lipid peroxidation, testicular apoptosis, and androgenic receptor (AR) density in developing Japanese quails. Fifteen-day-old 90 male chicks were assigned to two main groups. The first group (45 chicks) was kept in a thermoneutral room at 22 °C for 24 h/day. The second group (45 chicks) was kept in a room with high ambient temperature at 34 °C for 8 h/day (from 9 AM-5 PM) and at 22 °C for 16 h/day. Each of these two main groups was then divided into three subgroups (CBO groups 0, 250, 500 ppm) consisting of 15 chicks (six treatment groups in 2 × 3 factorial order). Each of subgroups was replicated for three times and each replicate included five chicks. Heat stress caused significant decreases in body weight, spermatid and testicular sperm numbers, the density of testicular Bcl-2 (antiapoptotic marker) and AR immunopositivity, and significant increases in testicular lipid peroxidation level, the density of testicular Bax (apoptotic marker) immunopositivity, and a Bax/Bcl-2 ratio along with some histopathologic damages. However, 250 and 500 ppm CBO supplementation provided significant improvements in HS-induced increased level of testicular lipid peroxidation, decreased number of spermatid and testicular sperm, decreased densities of Bcl-2 and AR immunopositivity, and some deteriorated testicular histopathologic lesions. In addition, although HS did not significantly affect the testicular glutathione level, addition of both 250 and 500 ppm CBO to diet of quails reared in both HS and thermoneutral conditions caused a significant increase when compared with quails without any consumption of CBO. In conclusion, HS-induced lipid peroxidation causes testicular damage in developing male Japanese quails and, consumption of CBO, which has antiperoxidative effect, protects their testes against HS.