Hypo-osmotic Swelling Response Research Articles

Correlation Between Dissolved Oxygen Level, Antioxidants and Oxidants in Semen Diluted with Partially Deoxygenated Extender at Various Stages of Cryopreservation

BACKGROUND: Dissolved oxygen (DO) in semen dilutor may lead to the production of reactive oxygen species (ROS) and buffalo sperm may become more prone to deleterious effects of ROS due to the presence of high amounts of polyunsaturated fatty acids (PUFAs) in their membranes. OBJECTIVE: To study the correlation between dissolved oxygen level, antioxidants and oxidants in semen diluted with partially deoxygenated extender at various stages of cryopreservation. MATERIALS AND METHODS: Each semen sample was split into two aliquots viz., Aliquot I [diluted with Extender I (control: without deoxygenation)] and Aliquot II [diluted with Extender II: partially deoxygenated by liquid nitrogen (LN) flushing], which were diluted, filled in straws, cryopreserved and evaluated post-thaw. RESULTS: The DO levels ( P < 0.05) decreased significantly after LN flushing of the extender and they increased significantly ( P < 0.05) in post-thaw semen. The progressive motility, viability, hypo-osmotic swelling response, acrosomal integrity, glutathione peroxidase (GPx), total antioxidant capacity (TAC) and superoxide dismutase (SOD) decreased significantly ( P < 0.05) in both control and treated semen after thawing. SOD and TAC were positively correlated in semen treated with normal extender at the pre-freeze stage; however, in semen treated with partially deoxygenated extender, no correlation was found between SOD and TAC at the pre-freeze stage. ROS and total TAC were negatively correlated in semen treated with partially deoxygenated extender at the pre-freeze stage; however, no correlation was found between ROS and TAC in control semen. CONCLUSION: The partial deoxygenation of extender affects the correlation between sperm quality parameters, antioxidants, and oxidants during different stages of semen cryopreservation.

Comparative evidence support better antioxidant efficacy of mitochondrial-targeted (Mitoquinone) than cytosolic (Resveratrol) antioxidant in improving in-vitro sperm functions of cryopreserved buffalo (Bubalus bubalis) semen

The present study compared the effect of mitochondria-targeted (Mitoquinone, MitoQ) and untargeted cytosolic antioxidant (Resveratrol, RESV) supplementation on lipid peroxidation (LPO) and in-vitro sperm functions of cryopreserved buffalo bull semen. To optimize additive's concentration, sperm pellet obtained from twenty-four ejaculates was supplemented with different concentrations of MitoQ (20 nM, 100 nM, 200 nM); and RESV (10 μM, 25 μM, 50 μM) against control in the extender. The post-thaw sperm motility, livability, and membrane integrity were higher (P < 0.05) in 200 nM MitoQ and 50 μM RESV than other concentrations used. In another experiment, sperm pellet from thirty-two ejaculates was supplemented with 200 nM MitoQ and 50 μM RESV in the extender. Pre-freeze and post-thaw progressive motility and livability were higher (P < 0.05) in MitoQ (200 nM) than RESV (50 μM) treatment. MitoQ supplementation improved post-thaw membrane integrity (CFDA-PI) higher (P < 0.05) than RESV, however, hypo-osmotic swelling response observed no improvement with RESV treatment. Post-thaw LPO rate was lower (P < 0.05) and Bovine cervical mucus penetration was higher (P < 0.05) in MitoQ than RESV treatment. In post-thaw semen, MitoQ showed higher (P < 0.05) proportion of acrosome intact (FITC-PNA), live non-apoptotic (P < 0.01) sperm with a higher reduction (P < 0.05) in membrane scrambling. MitoQ improved (P < 0.01) proportion of sperm with high Mitochondrial Membrane Potential and low LPO (P < 0.01) than RESV treatment. In conclusion, improvement in post-thaw in-vitro sperm functions and cryo-tolerance was more evident in MitoQ than RESV supplemented buffalo bull semen. Our study provides a better strategy to mitigate oxidative stress by enhancing mitochondrial antioxidant system with targeted antioxidants than cytosolic antioxidant supplementation.

Effect of partial deoxygenated extender on different post thaw incubation periods of physico-morphological and functional attributes during cryopreservation of buffalo spermatozoa

The study was designed to explore the effect of partial deoxygenated extender of different post thaw incubation periods on physico-morphological attributes during cryopreservation of buffalo semen. Semen samples with mass motility of ≥4+ and individual progressive motility ≥ 80 collected from murrah buffalo bulls were utilized. Immediately after collection, a portion of semen was evaluated for progressive motility, hypo-osmotic swelling response (HOS response) and acrosomal intactness and other portion was divided into two equal groups. Group I was kept as control and diluted with Egg yolk-Tris-Glycerol (EYTG) with a maximum of 60×106 sperm/mL. Group II was diluted with partially deoxygenated EYTG extender. Both the groups were subjected to equilibration and freezing. Both the group of semen samples were evaluated at pre-freeze and post-thaw stage of progressive motility, viability, HOS response and acrosomal intactness. Post-thaw motility and viability was studied at 0, 15, 30, 45 and 90 minutes of incubation. At pre-freeze and post thaw stage, significantly (p

Effect of cholesterol loaded cyclodextrin on activity of antioxidants during cryopreservation of buffalo (Bubalus bubalis) semen

The present study was carried out to investigate the effect of cholesterol loaded cyclodextrin on activity of antioxidants during cryopreservation of buffalo semen. Ejaculates (48) with mass motility ≥3+ from 3 bulls (16 from each bull) were collected as per standard procedure. Each ejaculate was divided into group I (control): diluted with Tris-egg yolk-glycerol dilutor up to 60×106 sperm/ml and group II: treated with cholesterol loaded cyclodextrin @ 3 mg/120×106 sperm and incubated at 37ºC for 10 min and then finally diluted with Tris-egg yolk-glycerol dilutor up to 60×106 sperm/ml. French medium straws (0.5 ml) were filled with the extended semen samples, sealed with polyvinyl alcohol powder and kept for 3 h at 5ºC for equilibration and then kept in automatic programmable freezer till temperature of straws reached –145°C followed by plunging into liquid nitrogen (– 196°C). Ejaculates were evaluated freshly, before freezing and after thawing for seminal attributes (progressive motility, viability, acrosomal integrity and hypo-osmotic swelling response) and antioxidants (superoxide dismutase, catalase, glutathione peroxidase and total antioxidants). After thawing, activity of superoxide dismutase, catalase and total antioxidants were significantly higher in group II as compared to group I. It is concluded that cholesterol loaded cyclodextrin enhances seminal antioxidant activity.

Effect of different levels of egg-yolk on freezability of Jakhrana buck semen

The present study was designed to determine the effect of different levels of egg-yolk on freezability of Jakhrana buck semen. Six healthy Jakhrana bucks (BW=30 ± 2kg, age=12 ± 0.5 month) were used for semen collection. These bucks were maintained under semi-intensive system at Jakhrana Unit of C.I.R.G. Makhdoom, Mathura. A total of 48 ejaculates (6 bucks × 8 replicates) were collected twice a week using artificial vagina. Each ejaculate was divided into 4 groups (G1, G2, G3 and G4). The G1, G2, G3 and G4 were extended with Tris-egg yolk-citric acid- fructose-glycerol (TEYCFG) extenders containing 5, 10, 15 and 20% egg yolk level, respectively. Each ejaculate was evaluated for sperm motility, viability, abnormality, and hypo-osmotic swelling (HOS) response and acrosome integrity before and after freezing. At pre-freeze stage no significant (P&gt;0.05) difference in sperm motility and viability was found among all groups. Sperm abnormality was significantly (P&lt;0.05) higher in G4 as compared to other groups (G1, G2, G3). The HOS response and acrosomal integrity was significantly (P&lt;0.05) higher in G1, G2 and G3 as compared to G4. However, no significant (P&gt;0.05) difference was observed in HOS response and acrosomal integrity among G1, G2 and G3. At post thaw stage, sperm motility, viability and HOS response was significantly (P&lt;0.05) higher in G1 and G2 as compared to G3 and G4. Sperm abnormality was significantly (P&lt;0.05) lower in G2 as compared to other groups. The acrosomal integrity was significantly (P&lt;0.01) higher in G1 and G2 as compared to G3 and G4. It is concluded that 10% egg yolk in Tris based extender may be the best for successful cryopreservation of Jakhrana buck semen.

Effect of incubation on freezability of cholesterol-loaded cyclodextrin treated buffalo (Bubalus bubalis) spermatozoa.

Aim:The aim of this study was to investigate the effect of incubation on freezability of cholesterol loaded cyclodextrin (CLC) treated buffalo spermatozoa.Materials and Methods:Semen samples with mass motility of 3+ and greater, collected from Murrah buffalo bulls were utilized. Immediately after collection, four equal groups of semen sample were made. Group I was kept as control and diluted with Tris upto concentration of 60×106 sperm/ml, where as Groups II, III, and IV were treated with CLC at 3 mg/120× 106 spermatozoa, incubated at 37°C for action of CLC for 10, 15 and 20 min, respectively, and diluted with tris upto concentration of 60×106 sperm/ml. All groups were subjected to equilibration and freezing. The evaluation of semen samples from all groups was carried out at fresh, pre-freeze and post-thaw stage for progressive motility, viability and hypo-osmotic swelling response (HOS response).Results:At the pre-freeze stage, significantly (p<0.05) higher percentage of progressive motility and viability was observed in treatment groups as compared to control with no significant difference among treatment groups. HOS response was significantly (p<0.05) higher in treatment groups as compared to control at pre-freeze stage. At post-thaw stage, significantly (p<0.05) higher percentage of progressive motility, viability and HOS response was recorded in Group II as compared to control and other treatment groups (III and IV). Group II retained significant post-thaw motility and viability at various post-thaw incubation periods.Conclusion:Incubation period of 10 min for CLC treated buffalo spermatozoa yielded significantly higher results in terms of freezability as compared to incubation for 15 and 20 min.

Activity of Enzymatic Antioxidants and Total Antioxidant Capacity in Seminal Plasma of Murrah Bulls during Cryopreservation

Semen samples with mass motility of 4+ or more and an initial progressive motility ≥ 80% collected from murrah buffalo bulls were utilized for study. Semen samples were evaluated for various seminal attributes, enzymatic antioxidants (superoxide dismutase, catalase and glutathione peroxidase) and total antioxidant capacity at fresh, pre-freeze and post-thaw stage. Seminal attributes (progressive motility, viability, acrosomal integrity and hypo-osmotic swelling response) were significantly (p<0.05) higher in bull II as compared to bulls (I & III) at pre-freeze and post-thaw stage. Superoxide dismutase was significantly higher in bulls (II & III) than bull I at fresh stage (p<0.05) and significantly (p<0.01) higher in bull II as compared to bulls (I & III) at pre-freeze and post-thaw stage. Catalase was significantly higher in bull II than bulls (I & III) at fresh (p<0.05) and pre-freeze (p<0.01) stage. Significantly, higher total antioxidant capacity was noticed at pre-freeze (p<0.05) and post-thaw (p<0.01) stage in bull II and bulls (II & III), than bulls (I & III) and bull I, respectively. On the basis of our findings, it could be concluded that seminal enzymatic antioxidants (superoxide dismutase & catalase) and total antioxidant capacity varied among bulls at various stages of cryopreservation. There was progressive reduction in activity of enzymatic antioxidants and total antioxidant capacity from fresh to post-thaw stage.

Effect of cholesterol supplementation on cryosurvival of goat spermatozoa.

Aim:Sperm membrane cholesterol influences cryodamage during cryopreservation. The present study was carried out to evaluate the effect of varying cholesterol levels in Tris based extenders on the freezability of sexually healthy Malabari buck semen.Materials and Methods:A total of 48 ejaculates from two adults healthy sexually healthy Malabari bucks were utilized for the study. The collected and pooled ejaculates were divided into four groups with Group I serving as Control - I, Group II and III were treated with 1 mg and 2 mg of cholesterol-loaded-cyclodextrin (CLC)/120 × 106 spermatozoa, respectively, and Group IV, treated with 1 mg methyl-β-cyclodextrin (MβCD) served as Control - II. Manual freezing was carried out to cryopreserve the treated and control spermatozoa.Results:Treatment of semen samples with CLC resulted in improved maintenance of sperm motility at pre-freeze and post-thaw stages of cryopreservation without affecting hypo-osmotic swelling response. Treatment of semen with 1 mg of CLC/120 × 106 spermatozoa was observed to be better than treatment with 2 mg of CLC/120 × 106 spermatozoa. In general, MβCD treatment was found to result in significantly lower sperm characteristics than those of Control - I and CLC treatment at pre-feeze and post-thaw stages and when incubated up to 4 h.Conclusion:Cholesterol treatment of sexually healthy Malabari buck semen was found to hold promise for improving cryopreservability of spermatozoa.

Antimicrobial host defence peptide, LL-37, as a potential vaginal contraceptive

Does antimicrobial peptide, LL-37, inhibit sperm fertilizing ability? Our results indicate that LL-37 inhibits mouse and human sperm fertilizing ability. LL-37, a cationic antimicrobial peptide, exerts its microbicidal effects through the disruption of microbial cytoplasmic membranes following its interaction with microbial surface anionic phospholipids. ALL-38 (an LL-37 close analogue: LL-37 + Ala at the N-terminus) is produced in the vagina 2-6 h post-intercourse from its precursor hCAP-18, a seminal plasma component. At this time, motile sperm have already swum into the uterine cavity, thus unexposed to ALL-38. Since sperm contain a substantial amount of acidic sulfogalactosylglycerolipid (SGG) on their surface, treatment of sperm with LL-37 may cause their membrane disruption in an analogous manner to that occurring on microbial membranes. Mouse/human sperm treated (2-30 min) with LL-37 in a physiological concentration range (up to 10.8 µM) were assessed for SGG-dependent LL-37 binding, and parameters relevant to fertilizing ability, namely motility and intactness of the sperm acrosome and plasma membrane. Ability of mouse sperm to fertilize eggs in vitro was also evaluated. Each study was performed with greater than or equal to three different sperm samples. The efficacy of LL-37 to inhibit sperm fertilizing ability in vivo was determined in female mice (n = 26 each for LL-37 treatment and no treatment), using sperm retrieved from 26 males. Human sperm samples were donated by fertile men. LL-37 was chemically synthesized and was biotinylated for sperm binding studies. Sperm motility was assessed by videomicroscopy and the acrosomal status by Coomassie blue staining of acrosome-intact mouse sperm or the exposure of CD46, an inner acrosomal membrane protein, of acrosome reacted human sperm. Sperm membrane permeabilization/disruption was assessed by the loss of hypo-osmotic swelling response, an incorporation of Sytox Green (a membrane impermeable fluorescent DNA dye), and electron microscopy. Mouse IVF was scored by the presence of two pronuclei in eggs 6 h post-insemination. Ability of mouse sperm to fertilize eggs in vivo was determined by the pregnancy outcome of female mice injected transcervically with sperm with or without LL-37. Biotinylated LL-37 bound to both mouse and human sperm and the binding was partially dependent on sperm surface SGG. Mouse and human sperm became immotile and underwent a premature acrosome reaction upon treatment with LL-37 at 3.6 and 10.8 µM, respectively. The initial action of LL-37 on both mouse and human sperm appeared to be through permeabilization/disruption of sperm surface membranes evidenced by the loss of hypo-osmotic swelling response, Sytox Green staining and electron microscopy revealing ultrastructural damage. Mouse sperm treated with 3.6 µM LL-37 lost the ability to fertilize eggs both in vitro and in vivo. All 26 female mice inseminated with sperm and LL-37 did not become pregnant. No apparent damage to the reproductive tract was observed as revealed by histological characterization in LL-37-inseminated mice and these females resumed fecundity following mating with fertile males. Direct demonstration that LL-37 treated human sperm fail to fertilize eggs was limited by legal restrictions on obtaining human eggs for such use. Our results reveal selective inhibitory effects of LL-37 on sperm fertilizing ability in mice without apparent impairment to the female reproductive tract. LL-37 is therefore a promising candidate to be developed into a vaginal contraceptive with microbicidal activity. This work was supported by Grand Challenges Explorations grant from the Bill & Melinda Gates Foundation (OPP1024509), Canadian Institutes of Health Research (MOP119438 & CCI82413) and International Collaboration and Exchanges NSFC of China (No.30611120525). There are no competing interests to declare.

Effects of Butylated Hydroxytoluene on Freezability of Ram Spermatozoa

The present study was conducted to investigate the effects of butylated hydroxytoluene (BHT) supplementation on diluted, cooled and frozen-thawed ram spermatozoa. After primary evaluation of collected ejaculates, only semen samples with motility of more than 70% and sperm concentration higher than 3x10 3 sperm/ml were used for cryopreservation. The selected semen samples were then pooled and diluted 1:4 with Tris Citrate Fructose Yolk (TCFY) extender supplemented with different concentrations of BHT (0.5, 10, 2.0 and 3.0 mM). As the control, semen was diluted and frozen in the diluent without BHT. Motility, progressive motility, viability, membranes and acrosome integrity were evaluated after dilution (part 1), cooling (part 2) and freezing and thawing (part 3). The results of the first part of the experiment showed that there were no significant difference between treatments in the motility, progressive motility, viability, membranes and acrosome integrity of spermatozoa, but the results with 2.0 mM BHT were slightly better than obtained with other levels of BHT and control extender. Significantly better results (p<0.05) were observed in the second part of the experiment for cooled spermatozoa characteristics, when extender was supplemented with 2.0 and 3.0 mM BHT. Furthermore, the results obtained in the third part of the experiment indicated that, after freezing and thawing, all evaluated semen characteristics were improved significantly (p<0.05) by increasing BHT levels, with the best results obtained for extender containing 2 mM BHT. Comparison of these results with those of control diluent, the effects of supplementation were significantly (p<0.01) better. However, the higher concentration of BHT (3.0 mM) reduced the motility, acrosomal integrity, viability and hypo-osmotic swelling response of spermatozoa compared to extender containing 2.0 mM BHT. In conclusion, the results obtained in this study showed that the semen quality of rams was improved when BHT was added to extender used before the freezing process.

Butylated hydroxytoluene inclusion in semen extender improves the post-thawed semen quality of Nili-Ravi buffalo ( Bubalus bubalis)

The study was carried out to evaluate the potential impact of butylated hydroxytoluene (BHT) on the frozen–thawed semen quality of Nili-Ravi buffalo bulls. Ejaculated bull semen was extended in a Tris–citrate egg yolk extender containing various concentrations of BHT (0.5, 1.0, 2.0 and 3.0 mM). Semen was frozen at −196 °C using 50 × 10 6 spermatozoa per 0.5 mL straws. Five straws from each treatment were thawed to assess the semen quality in terms of sperm motility, viability, plasma membrane integrity and acrosomal integrity. Post-thawed sperm motility was determined using a phase-contrast microscope. Viability, plasma membrane integrity and acrosomal integrity were evaluated by the supravital staining, hypo-osmotic swelling test and normal acrosomal reaction, respectively. The highest ( P < 0.05) motility, acrosomal integrity and hypo-osmotic swelling response of spermatozoa was achieved by addition of 1.0 and 2.0 mM BHT to semen extender. However, highest ( P < 0.05) viability of spermatozoa was achieved by inclusion of 2.0 mM BHT. The higher concentration of BHT (3.0 mM) reduced the motility, acrosomal integrity, viability and hypo-osmotic swelling response of the spermatozoa compared to other concentration used. In conclusion, BHT when added in the semen extender can improve the semen quality of buffalo bulls.

Development of a rapid, sensitive, and reproducible laboratory test kit for the assessment of plasma membrane integrity of human sperm

To develop a rapid, sensitive, and reproducible hypoosmotic swelling test kit for the assessment of plasma membrane integrity of human sperm in vitro. Prospective comparison of results with the World Health Organization (WHO) method, performed simultaneously. Infertility center in a major city in India. Couples who presented for infertility evaluation. The sperm tail-coiling pattern representing sperm plasma membrane integrity was analyzed by using different concentrations of NaCl and also with plain double distilled water (ddH(2)O). Hypoosmotic swelling solution with 2% NaCl in ddH(2)O equivalent to 68 Osm/L was selected for further analysis because it provided the highest and the qualitatively best type (g) of tail coiling among the various other options tried in comparison. A rapid laboratory test kit for the assessment of plasma membrane integrity of sperm was developed that is equally sensitive and reproducible as that described in the WHO protocol. The test was validated by using different normal and subnormal semen samples and in comparison with the standard WHO protocol. After the screening with different hypoosmotic solutions by using semen samples from fertile volunteers, NaCl (2%) provided the highest and the best types of typical tail coiling, characteristic of sperm with good plasma membrane integrity. The study was then extended to 60 normozoospermic semen samples, which demonstrated a hypoosmotic swelling response of 69.5% +/- 5.23% and 71.5% +/- 4.89%, as per WHO or the present modified method, respectively (coefficient of correlation, r = 0.741). In 13 oligospermic and 18 teratozoospermic subjects, the hypoosmotic swelling response of sperm as per the new method were 36.4% +/- 5.75% and 34.2% +/- 7.78%, respectively, which were comparable to those obtained through the WHO method. There was a 15% drop in viability of sperm after the hypoosmotic challenge (5 min), irrespective of the hypoosmotic solution used. No other morphological alterations in sperm were observed after the hypoosmotic challenge. The new test kit can be used routinely in laboratories for assessment of plasma membrane integrity of sperm in vitro.

Assessment of human sperm function after hydrogen peroxide exposure: development of a vaginal contraceptive

The reactive oxygen species (ROS) that is most damaging to human spermatozoa is hydrogen peroxide. Using an artificial medium Ham’s F-10, we have evaluated the effect of different concentrations of hydrogen peroxide (H 2O 2) on various sperm function characteristics to develop a water-based vaginal contraceptive. H 2O 2 at 30 and 60 μM had no effect on sperm motility. At 120 μM of H 2O 2, a significant reduction (90–95%) in motility was observed after 120 min. However, at 600 μM of H 2O 2, all the sperm were found immotile within 15 min of incubation. At much higher concentrations (1200–1500 μM), the effect was immediate. After immobilization with H 2O 2 (600 μM in media), viability in sperm declined to 36% after 30 min, and was further reduced to 5% in 60 min. On the other hand, hypo-osmotic swelling response in these sperm went down drastically from 72 to 46% immediately after immobilization and there was no response at all after 60 min. In contrast, when Triton-x-100 (0.01%), a known spermicidal agent, was used in place of H 2O 2, the immediate loss of sperm motility corresponded simultaneously with the loss of the other two functional parameters. Further, H 2O 2 immobilized sperm revealed reduced (25–30%) nuclear chromatin decondensation as compared to 70% for the controls. A composition containing 2% H 2O 2 in 0.9% NaCl was tested in rats as a vaginal contraceptive, depicting 100% efficacy in mating studies after 2 h of vaginal application. The findings indicate the potential of developing a water-based vaginal contraceptive using H 2O 2 at an optimal concentration as utilized in the present study.

Effect of storage on sperm membrane integrity and other functional characteristics of canine spermatozoa: In vitro bioassay for canine semen

The effect of storage of canine semen on sperm membrane integrity, as determined by the hypoosmotic swelling test, and on other functional characteristics of the canine spermatozoa was evaluated by established procedures. The results of this study indicated that storage of canine semen at a chilling temperature of 5°C for 24 h did not significantly impair the physical and functional characteristics of the canine spermatozoa. The overall mean percentage of motility, hypo-osmotic swelling response, which assessed sperm membrane integrity, acrosome-reacted spermatozoa, acrosomal defects, and the percentage of live spermatozoa, did not significantly differ between the fresh and chilled semen samples. However, storage altered the rate of motility and acrosome reaction. The percentage of acrosome reaction in the canine capacitating medium peaked earlier in chilled than in fresh semen. It is probable that storing semen at 5°C initiated/triggered the acrosome reaction. This did not amount to impairment of functional properties. Significant correlations were observed between hypo-osmotic swelling vs motility (r=0.98, P<0.002); hypo-osmotic swelling vs acrosome reaction (r=0.83, P<0.08); and acrosome reaction vs motility (R=0.89, P<0.04) in the fresh semen, and between hypo-osmotic swelling vs motility (r=0.87, P<0.05) and hypo-osmotic swelling vs acrosome reaction (r=0.56, P<0.05) in the chilled semen. It was concluded: that 1) storage of canine semen at 5°C for 24 h did not significantly impair the physical and functional integrity of the spermatozoa; 2) the significant association between motility or acrosome reaction vs hypo-osmotic swelling indicates their value in assessing sperm viability; and 3) the hypo-osmotic swelling assay could have predictive value in screening out subfertile males with apparently normal spermiograms.