Investigation of the effects of thymoquinone (TQ) and polyvinylpyrrolidone (PVP) on the motility, viability, and chromatin status of sperm in normozoospermic semen samples.

Objective: Adverse effects of polyvinylpyrrolidone (PVP) on sperm membrane and chromatin have been proven in many studies. Among the natural products proposed as an alternative for PVP, thymoquinone (TQ)-a major constituent of Nigella sativa plant- has been suggested as a potential natural spermostasis. Therefore, this study aimed to compare the effects of TQ with PVP for sperms motility, survival, DNA denaturation, and DNA fragmentation in normozoospermic men (men with a normal or healthy sperm profile). Methods: An experimental trial was carried out on 30 normozoospermic men of the Andrology Unit of (Shahid Beheshti Hospital, Isfahan, Iran). Each washed semen samples were divided into four fractions and was randomly treated with TQ (50 µg/ml), %5 PVP, and %10 PVP (M/V) which was compared to untreated fraction (control). Results: There was a significant difference between the four groups in terms of motility, viability, DNA denaturation, and fragmentation (P <0.05). TQ caused sperm immobility, while 5% PVP and 10% PVP decreased (98 and 99%, respectively) sperm motility compared to control. TQ did not affect sperm viability compared to the control group, but PVP decreased it. Besides, TQ did not affect DNA denaturation and fragmentation, but PVP increased it. Conclusion: TQ could be used as an alternative natural spermostasis with less adverse effects rather than PVP which causes more efficient immobilization and isolation of individual sperm cells.

Effects of temperature and storage time on the motility, viability, DNA integrity and apoptosis of processed human spermatozoa.

The aim of this study was to evaluate motility, viability, DNA integrity and apoptosis of spermatozoa when washed semen samples were kept for up to 12 days at 4-6°C and 25°C. In this experimental study, 26 normozoospermic semen samples were washed twice in Modified Ham's F10 and resuspended in IVF fertilisation medium. Half of the specimens were stored at 4-6°C, and the other half was kept at 25°C for 12 days. The proportions of viable, motile, spermatozoa with double-stranded DNA and apoptotic spermatozoa were examined during storage time. Apoptosis was measured using annexin V-PI staining followed by flow cytometry. Results showed that sperm motility and viability decreased during 12 days of sample storage (p < .001). There was no significant difference between the two temperatures in terms of motility and viability for up to 2 days (p < .05). The percentage of spermatozoa with double-stranded DNA remained unchanged during the 12 days of storage at both temperatures (p > .05). Although there was no difference between the two temperatures in terms of motility, viability and apoptosis during the first two days of storage, storage of spermatozoa at 4-6°C is better than storage for a longer period than storage at 25°C. Sperm DNA resisted against denaturation during storage.

Correlation between sperm motility and sperm chromatin/DNA damage before and after cryopreservation and the effect of folic acid and nicotinic acid on post-thaw sperm quality in normozoospermic men.

Cryopreservation exposes sperm to physical and chemical stresses causing cell damages and impairs sperm functions. The aim of this study was to evaluate the association between motility and sperm chromatin/DNA damage before and after cryopreservation and investigate the effects of folic acid and nicotinic acid on post-thaw sperm quality. Thirty semen samples were obtained from 30 normozoospermic men, aged between 25 and 45 years old. Each sample were divided into five aliquots to form the following groups: fresh, cryopreserved with sperm-freeze only (control), with nicotinic acid (10 mM), with folic acid (50 nM), and with a combination of folic acid (50 nM) + nicotinic acid (10 mM). Sperm viability and motility in each group were assessed by eosin-nigrosine staining and computer-aided sperm analysis respectively. Sperm chromatin quality was studied by aniline blue, toluidine blue, acridine orange staining methods and sperm chromatin dispersion test. Cryopreservation led to a significant reduction in sperm quality in comparison to fresh sample groups (p < 0.05). Sperm chromatin damage was negatively correlated with the percentage of progressively motile cells. Supplementation of the cryopreservation medium with folic acid or nicotinic acid induced a significant improvement in sperm parameters and chromatin quality, compared to control groups (p < 0.05). Meanwhile, the combination of folic acid + nicotinic acid showed a significant protective effect in post thaw sperm. In conclusion, cryopreservation generated oxidative stress, inducingsperm cryodamage, reducing progressive motility and sperm quality, as an indicator of significant chromatin/DNA damage. Folic acid and nicotinic acid exhibited a potential cryoprotective effect by enhancing sperm quality.

Effect of preservation of human semen sample at 4-6 and 25 °C on sperm motility.

Sperm motility is the result of transverse movements that exist along its tail. It plays an important role in male fertility. The aim of this study was to evaluate the influence of keeping washed normozoospermic semen samples at 4-6 and 25 °C on the motility of spermatozoa. 26 semen samples of normozoospermic were washed twice in modified Ham's F10 medium. Then, thirteen of the semen samples were kept in refrigerator (4-6 °C) and the remaining samples were stored in incubator (25 °C) for 12 days. On the 0 (immediately after sampling as control group), 1st, 2nd, 5th, 7th and the 12th days, the percentage of fast progressive (grade a), slow progressive (grade b), non-progressive (grade c) and immotile (grade d) sperm cells were calculated for each temperature. The data obtained from this study showed that the percentages of a, b and c grades of motile spermatozoa were significantly decreased (p < 0.001) during 12 days at the both temperatures but reduction of these percentages has a gentle slope at 4-6 °C. There was no motile sperm after 12 days of storage. This study suggests that motile spermatozoa could be retrieved up to 7 days after the storage of washed normozoospermic men semen samples at 4-6 and 25 °C. Also, there were no motile sperm cells 12 days after sampling.

Thymoquinone as a natural spermostatic substance in reproductive medicine: An experimental study.

BACKGROUND: Nonoxynol-9 a nonionic surfactant is widely used for its spermicidal effects. Finding new sperm immobilizing agents is necessary because Nonoxynol-9 damages the tissues of female reproductive system. OBJECTIVE: The aim of this study was to evaluate the effects of Thymoquinone (TQ) as a potential spermostatic compound on the motility and viability of human spermatozoa. MATERIALS AND METHODS: In this experimental study, the effects of 5, 10, 20, 50, 100 µg/ml, 1 and 10 mg/ml of TQ on normozoospermic semen samples were investigated. Sperm motility and viability were compared between untreated and TQ-treated aliquots of each semen sample. To evaluate the effects of TQ on the alteration of mitochondrial membrane potential (MMP), 32 semen samples were examined using 50 µg/ml of TQ. Flow cytometric analysis was performed after staining of spermatozoa with JC-1. RESULTS: Doses above 20 µg/ml of TQ could eventually immobilize all spermatozoa in culture medium. Adding 50 µg/ml of TQ did not significantly diminish the percentage of viable spermatozoa and flow cytometry results revealed that this amount of TQ could decrease sperm MMP. CONCLUSION: TQ could discontinue the movement of sperm cells in medium without reducing the population of live spermatozoa. It is more likely that TQ exerts its spermostatic action by mitigating the MMP of spermatozoa. Therefore, TQ could be considered as a potential new natural spermostatic chemical.

Coadministration of calcium chloride with lead acetate can improve motility of cauda epididymal spermatozoa in Swiss white mice.

BACKGROUND: Lead is an industrial heavy metal that can decrease sperm motility. OBJECTIVE: The aim was to investigate the protective effects of calcium against lead on motility of spermatozoa. MATERIALS AND METHODS: In total 40 adult male Swiss white mice were randomly divided into 5 groups (control, lead of 1(st) wk, lead of 2(nd) wk, lead/calcium of 1(st) wk and lead/calcium of 2(nd) wk). The lead groups of mice were injected by a single dose of lead acetate (200 mg/kg) intraperitoneally. Lead/calcium groups of mice were injected by a single same dose of lead acetate along with three doses of 80 mg/kg calcium chloride. The control group of mice was injected only with same volume of distilled water through the same route. Mice of 1(st) and 2(nd) wk groups were sacrificed through cervical dislocation one and two weeks after injections respectively. RESULTS: Mean of the progressive motile spermatozoa of cauda epididymis in lead/calcium group of the first week was higher than the lead group of the first week and this difference was significant. There was not any significant difference among weight of testes and epididymides of all groups. CONCLUSION: It can be concluded that calcium can decrease the effects of lead on sperm motility.

The epididymal sperm viability, motility and DNA integrity in dead mice maintained at 4-6oC.

BACKGROUND: When male animals die, spermatozoa within the body of animal will be degenerated. Because of unique chromatin structure of sperm, maybe this degeneration is different from other cells. However there is not any research which considered directly the integrity of sperm DNA by keeping the cadaver in refrigerator. OBJECTIVE: The aim of this study was to assess viability, total motility and DNA integrity of sperm cells after death. MATERIALS AND METHODS: In this experimental study, 24 male Swiss white mice were killed by cervical dislocation and then kept in refrigerator (4-6(o)C) for up to 12 days. On the 0 (immediately after death as control group), 1(st), 2(nd), 3(rd), 5(th), 7(th), 10(th) and the 12(th) days after death cauda epididymides were removed and squeezed in Ham's F10 medium. The proportion of viable, motile and double stranded DNA spermatozoa was examined. Viability and DNA integrity of sperm cells were examined consecutively by eosin nigrosin and acridine orange stainings. RESULTS: The data obtained from this study showed that viability and total motility of sperm cells were significantly decreased during 12 days after death (p<0.001). In contrast with viability and motility, DNA integrity was without significant changes (even 12 days after death). CONCLUSION: This study suggests that integrity of sperm DNA would not change even after 12 days after death if the cadaver kept in refrigerator.