Effect of Cryoprotective Additives-Reduced Glutathione, Acetyl-L-Carnitine on Sperm Membrane Lipid Peroxidation, DNA Integrity and Recovery of Motile Human Sperm (original) (raw)
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Effects of antioxidants on motility and DNA integrity in frozen-thawed sperm
2020
This study aims to investigate whether the addition of the antioxidant complex including taurine, ascorbic acid, and glutathione into the cryopreservation medium affects the damage to sperm during the freezing process. Material and Methods: Ejaculate samples of patients who applied for semen analysis to the Assisted Reproduction Unit of Private Adatip Hospital were used. Fresh samples were analyzed for standard semen quality parameters according to the World Health Organization guidelines. Samples within the normal range were evaluated for sperm DNA fragmentation using the Halosperm technique. Remaining ejaculates were washed with the gradient method before cryopreservation. Sperm samples of each patient were divided equally for freezing in a cryopreservation medium with or without the antioxidant supplementation. One month later, the samples were thawed. Post-thaw total motility and DNA fragmentation were determined for each sample. Results: Semen samples of 40 patients were analyzed. We observed decreased total motility (34.8 ±5.32 % vs. 65.5 ±6.42 %, P= 0.002) and increased sperm DNA fragmentation (52.3±5.42 % vs. 26.4±3.12 %, P= 0.002) in post-thaw semen samples following cryopreservation in comparison to fresh samples. The addition of antioxidants to the freezing medium did not have a statistically significant effect on sperm motility (38.3± 6.22 % vs. 34.8 ±5.32 %, P =0.07) and DNA damage (47.5±4.7 % vs. 52.3±5.42, P =0.08) when compared to control samples following the freezing process. Conclusions: We observed increased sperm DNA fragmentation and decreased total motility following cryopreservation. No significant improvement in sperm motility or DNA integrity was obtained after the addition of 5 µM of the antioxidants taurine, ascorbic acid, and glutathione to the freezing media.
Reproductive BioMedicine Online, 2009
This study examined whether the addition of an antioxidant to cryopreservation medium could improve the post-thaw integrity of cryopreserved human spermatozoa, particularly from men with abnormal semen parameters. Semen samples were collected by masturbation and assessed following WHO standards. Normal (n = 23) and abnormal (n = 20) samples were divided into three aliquots prior to cryopreservation. The first aliquot remained untreated and was mixed with cryopreservation medium (in-house 1:1). The second and third aliquots were mixed with cryopreservation medium containing either 100 mol or 200 mol vitamin E analogue. Samples were frozen at -10 C per minute to -80 C, then plunged into liquid nitrogen. Thawed samples were assessed for motility, vitality and DNA integrity. Split-plot repeated-measures ANOVA was used to assess within-subject (dose) and between-group (normal/abnormal) differences in post-thaw motility index, vitality staining and DNA fragmentation. Vitamin E dose was significantly associated with post-thaw motility (P = 0.041) and the pattern of response across doses was similar for normal and abnormal groups. Post-thaw motility was significantly improved by the addition of 200 mol vitamin E (P = 0.006), but neither vitality nor sperm DNA fragmentation were altered. These results suggest that the addition of vitamin E to cryopreservation medium improves post-thaw motility.
Journal of andrology
Sperm membrane damage during cryopreservation reduces the recovery of motile sperm. The present study investigates changes in sperm motility and membrane lipid peroxidation (LPO) in response to two changes in the standard sperm cryopreservation/thawing methodology: 1) the addition of platelet-activating factor (PAF) and pentoxifylline (PTX) as cryoprotective additives, and 2) the alteration of sample thawing time. PAF (1 microM) and PTX (3 mM) were added to fresh sperm samples prior to cryopreservation. After 2 weeks the samples were thawed either quickly (5 minutes at 37 degrees C) or slowly (30 minutes at 4 degrees C) and evaluated for sperm motility and LPO. Thawing time influenced both post-thaw motility and LPO. Samples thawed quickly exhibited a 31% increase in motility recovery (35.2 +/- 4.3% in quick-thaw samples; 24.3 +/- 3.9% in slow-thaw samples) and a 23% lower LPO level (23.3 +/- 3.4% in quick-thaw samples; 30.09 +/- 4.4% in slow-thaw samples) compared to samples thawed...
Introduction: Cryopreservation of semen is routinely used in a variety of circumstances including before assisted reproduction treatments, pre- radiation or chemotherapy treatment and etc. The aim of this study was to compare the effect of Butylated hydroxytoluene (BHT) and Glutathione supplemented cryopreservation medium on sperm parameters and amount of DNA fragmentation during the freeze-thaw process. Methods: Semen samples were obtained from 60 donors. After the determination of basic parameters, groups of three sample with similar parameters were pooled and processed by Pure Sperm gradient centrifugation. The semen samples were then diluted with normal freezing medium (control) or a medium containing 5mM glutathione (test) and 0.5 mM BHT (test) stored in liquid nitrogen. Frozen cryovials were thawed individually for 20 seconds in a water bath (37 ˚C) for evaluation. Results: Significant differences were observed in motility, viability and DNA fragmentation. Motility and viability were significantly higher in treated groups with 0.5 Mm in 5 min BHT than the control group and Glutathione 5mM (P<0.001). Conclusion: Significant differences were observed in motility, viability and DNA fragmentation. Motility and viability were significantly higher in treated groups with 0.5 Mm in 5 min BHT than the control group and Glutathione 5mM (P<0.001).
Biomolecules
Cryopreservation-thawing of human semen was found to reduce the level of antioxidant activity surrounding the sperm, which may negatively affect post-cryopreservation (post-thaw) recovery of sperm motility. Therefore, the current manufactured cryoprotectant media have been supplemented with certain antioxidants to preserve the loss in seminal antioxidant activity. In this study, we aimed to explore the correlation between total antioxidant capacity (TAC) of human semen samples before cryopreservation and the post-thaw recovery of sperm motility. Normal semen specimens (n = 77) were recruited in this study. Sperm motility was measured for each semen sample before and after cryopreservation and the post-thaw recovery of sperm motility was calculated. Seminal TAC was measured spectrophotometrically before cryopreservation for each semen sample using the sensitive cupric ion-reducing antioxidant capacity (CUPRAC) method. The results from this study showed that the post-thaw recovery of ...
Cryobiology, 2010
This study was conducted to determine the effects of methionine, inositol and carnitine on sperm (motility, abnormality, DNA integrity and in vivo fertility) and oxidative stress parameters (lipid peroxidation, total glutathione and antioxidant potential levels) of bovine semen after the freeze-thawing process. Nine ejaculates, collected with the aid of an artificial vagina twice a week from each Simmental bovine, were included in the study. Each ejaculate, splitted into seven equal groups and diluted in Tris-based extender containing methionine (2.5 and 7.5 mM), carnitine (2.5 and 7.5 mM), inositol (2.5 and 7.5 mM) and no additive (control), was cooled to 5°C and then frozen in 0.25 ml straws. Frozen straws were then thawed individually at 37°C for 20 s in a water bath for the evaluation.
Freeze and Thaw Creates Oxidative Stress and Dna Damage in Frozen Human Spermatozoa
Oxidative stress plays a major role in induction of cell structural damage, cellular dysfunction, and loss of cell survival. Semen cryopreservation is an important part of the work of many clinical laboratories, particularly those associated with infertility clinics. Most of the studies conducted on semen storage have demonstrated a beneficial effect of in vitro antioxidant supplements in protecting spermatozoa from exogenous oxidants and cryopreservation. In contrast, the effect of antioxidants in protecting spermatozoa from endogenous ROS and sperm processing have not been established conclusively. The aim of the present investigation was to determine the oxidative stress induced cryopreserved sperm quality, DNA intactness and lipid peroxidation of human semen that was stored at -196 0 C. The semen sample stored at -196 0 C has showed significant increase of lipid peroxidation (LPO), and super oxide dismutase (SOD), and slightly, compared with fresh samples, the decrease in catalase and glutathione peroxidase (P≤0.004) activities. In addition to these the DNA damage was also found on using electrophoresis and comet assay. In conclusion ejaculates collected from donors were subjected to cryopreservation and thawing. These samples on experimental analysis conclude that the cryopreservation induces oxidative stress in the spermatozoa due to increasing rate of lipid peroxidation and suppression of the antioxidant enzyme defense mechanism. These effects further may provide an evidence for cryopreservation induced spermatozoa damage including nucleic acids.
Cryobiology, 2011
In this study, total glutathione content was determined in human spermatozoa before and after cryopreservation. Total GSH in fresh semen was 4.47 ± 0.46 nmol/10 8 cells. Following semen cryopreservation, GSH decreased to 1.62 ± 0.13 nmol/10 8 cells, a 64% reduction (p < 0.01). This decrease in GSH content was associated with a decrease in sperm progressive motility (68% of reduction, p < 0.01). Addition of 1 mM GSH to the freezing extender increased the percentage of total motility and sperm viability. It also modified the motility pattern measured by CASA with changes in the straight-line and average path velocities and wobble of the curvilinear trajectory. Addition of GSH to the freezing media reduced spermatozoa ROS levels and increased the level of sulfhydryl groups on membrane proteins. Nevertheless, no effect of GSH addition on lipid membrane disorder or chromatin condensation was detected. Addition of 1 or 5 mM GSH to the thawing media increased the percentage of motile and progressively motile spermatozoa, but no effect on viability was detected. In conclusion, the antioxidant defensive capacity of the GSH is severely altered by the freeze-thawing process. The addition of GSH to the freezing and thawing extender could be of partial and limited benefit in improving the function of frozen human spermatozoa.
Reproduction in Domestic Animals, 2019
Cryopreservation of stallion semen has not reached the level of efficiency and positive results described in other species. This is mainly due to the greater sensitivity of stallion sperm to the freezing process, showing higher rates of oxidative stress and plasma membrane damage, which trigger the activation of several cell damage pathways that ultimately culminate in DNA fragmentation and cell death. Therefore, finding molecules that improve the efficiency of this technique in stallion by preventing oxidative stress and cell damage is required. Thus, the aim of the present study was to evaluate the effect of adding three antioxidants (MnTBAP, NAC and FeTPPS) to the freezing medium on the quality and functional parameters of stallion sperm. Semen samples from three stallions frozen with the antioxidants were evaluated in two conditions: i) adding the antioxidants before freezing, and ii) before and after freezing. Plasma membrane integrity, mitochondrial membrane potential, lipid peroxidation, intracellular ROS levels, membrane lipid disorder, DNA damage, sperm motility and binding to the zona pellucida were assessed. The results showed that MnTBAP was the antioxidant treatment that best controlled the oxidative stress process and post-thaw cell damage, showing higher plasma membrane integrity, mitochondrial membrane potential, sperm motility, number of spermatozoa bound to the zona pellucida of bovine oocytes and lower lipid disorder. Additionally, it was determined that a second post-thaw application of antioxidants is detrimental since induced higher cell damage and lower sperm motility, without showing any beneficial effect on the spermatozoa.
The Roles of Antioxidants and Fatty Acids in Sperm Cryopreservation
Cryopreservation in Eukaryotes, 2016
Despite research developments in the area of sperm storage, it has become inevitable to realize a marked reduction in the quality of fresh semen following cryopreservation. As a result, research has continued and will also continue in the future looking forward for a much better and improved methods of sperm cryopreservation along with better understanding of the physical and biochemical challenges that the sperm has to face to survive during freezing. Among the various attempts made to improve the cryopreservation process and subsequently result in superior quality of sperm after thawing include manipulating the composition of semen extenders by addition of exogenous products including antioxidants and fatty acids. While fatty acids are added to strengthening plasma membrane stability, Antioxidants are incorporated to compensate the reduction in the endogenous antioxidants level of seminal plasma due to dilution as well as to combat with the excess reactive oxygen species (ROS) production during freezing. In this chapter, the roles of antioxidants and fatty acids in mammalian sperm cryopreservation, both from endogenous and exogenous perspectives, will be discussed with reference to the latest research findings.