Mitochondria-targeted antioxidant MitoTEMPO improves the post-thaw sperm quality

Human spermatozoa cryopreservation is an important means of assisted reproductive technology and male fertility preservation. Although this technique is particularly useful, sperm cryopreservation significantly reduces the quality of spermatozoa after freezing and thawing. The objective of the study is to examine the efficacy of mitochondria-targeted antioxidant MitoTEMPO in improving sperm quality during semen cryopreservation processes. Semen samples were collected and cryopreserved in extenders containing different concentrations (0.0, 0.5, 5, 50, and 500 μM) of MitoTEMPO. Sperm motility, viability, membrane integrity, mitochondrial membrane potential and antioxidant activities were measured and analyzed. The results showed that the addition of MitoTEMPO (5–50 μM) significantly improved post-thaw sperm motility, viability, membrane integrity and mitochondrial membrane potential (P < .05). Meanwhile, antioxidant enzymes activities were enhanced and MDA content were decreased in the group supplemented with MitoTEMPO. In conclusion, mitochondria-targeted antioxidant MitoTEMPO improves the post-thaw sperm quality and antioxidant enzymes profile.     

Antioxidant effects of clove bud (Syzygium aromaticum) extract used with different extenders on ram spermatozoa during cryopreservation

Clove bud (Syzygium aromaticum) extract was added at concentrations of 0, 35, 75, and 115 μg/ml to ovine semen extenders in order to investigate the antioxidant activities of clove bud extract and its effects on semen quality parameters after cryopreservation of ram spermatozoa. The basic extender was composed of Tris, egg yolk, and glycerol. Two other extenders were prepared by substitution of egg yolk with either LDL or egg yolk + SDS. The DPPH inhibition test was employed to assess the antioxidant activity of clove bud extract. Results showed that, compared to vitamin E, clove bud extract had a higher antioxidant activity. Better sperm motility and movement characteristics (P < 0.05) were observed in the semen diluted with medium containing egg yolk + SDS than in that containing egg yolk and LDL. Progressive motility and movement characteristics of the sperm were significantly improved (P < 0.05) by adding 35 and/or 75 μg/ml of clove bud extract to semen extenders. Sperm viability and plasma membrane integrity were also higher (P < 0.05) in the semen exposed to medium containing egg yolk + SDS and 75 μg of clove buds extract after cryopreservation processes. Higher levels of clove bud extract, however, had adverse effects on all the sperm quality parameters and significantly reduced (P < 0.05) the motility, movement parameters, viability, and plasma membrane integrity of ovine sperm. It was concluded that the clove bud extract had an antioxidant potential that makes it useful for addition to semen extenders and that the best results are obtained with a maximum clove bud extract of 75 μg/ml. Moreover, the combination of egg yolk and a detergent was found to improve sperm quality after the cooling and freeze–thawing processes.     

Elamipretide as a potential candidate for relieving cryodamage to human spermatozoa during cryopreservation

As a technique widely used in assisted reproduction, human spermatozoa cryopreservation makes it possible to conserve functional sperm for a long time, but the impact of cryodamage on sperm during the process could not be ignored. The objective of the present study was to investigate the efficacy of Elamipretide, a novel small mitochondrial targeting short cytoprotective peptide, in attenuating cryodamage during spermatozoa cryopreservation. Semen samples were collected and cryopreserved in freeze solution containing different concentrations (0.0, 0.1, 1, and 10 μM) of Elamipretide. Sperm motility, viability, membrane integrity, mitochondrial membrane potential, DNA fragmentation, antioxidant profiles, and acrosome reaction were measured and analyzed. The results showed that supplementation of the freeze media with Elamipretide (1 and 10 μM) significantly improved post-thaw sperm parameters including motility and viability, stability of the plasma membrane, and mitochondria and chromosomes. In addition, by adding Elamipretide, excessive oxidation and acrosome dysfunction in sperm cells undergoing freeze-thaw were also significantly attenuated. Therefore, Elamipretide may be a potential candidate for relieving cryodamage to human spermatozoa during cryopreservation.     

Impact of Eurycoma longifolia extract on DNA integrity,lipid peroxidation,and functional parameters in chilled and cryopreserved bull sperm

This study aims to assess the effect of Eurycoma longifolia aqueous extract on chilled and cryopreserved quality of bull sperm. Semen samples were obtained from four Simmental–Brangus. Each sample was divided into two fractions: the first fraction was used for chilling the semen, and the second fraction was used for the freezing process. Both fractions were extended with Tris–egg yolk extender supplemented with 0.0, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 mg/ml Eurycoma longifolia aqueous extract. The diluted chilled fraction was chilled at 5 °C for 6 days, whereas the frozen–thawed fraction was frozen in liquid nitrogen. Data revealed that 1 mg/ml E. longifolia aqueous extract yielded significantly (p < .05) higher sperm motility, morphology, viability, and sperm membrane integrity compared with the control group and other treated groups in chilled semen evaluation. For cryopreserved sperm, a significant difference (p < .05) in sperm motility, viability, sperm membrane integrity, DNA integrity, and lipid peroxidation was observed between 5 mg/ml E. longifolia aqueous extract and other treated and control groups. However, no significant difference in the percentage of sperm exhibiting normal sperm morphology was observed among the groups. In conclusion, the addition of 0.25 and 1 mg/ml E. langifolia extract to chilled semen and 5 mg/ml E. longifolia aqueous extract to cryopreserved sperm into Tris–egg yolk extender helps in maintaining superior quality of bull spermatozoa during chilling and freezing.     

Cryopreservation of ram semen with antioxidant supplemented soybean lecithin-based extenders and impacts on incubation resilience

The scope of this study was investigation the affects of various antioxidants on 1% soybean lecithin-based semen extenders for ram semen cryopreservation. Ejaculates, collected via electrically stimulated ejaculation, that have a thick consistency, rapid wave motion (3–5 on a 0–5 scale) and >75% initial motility were pooled. The pooled samples were split into four equal aliquots as 5 mM Methionine, 5 mM Cysteamine, 1 mM Cysteine and a sample of antioxidant-free control group. Each sample group was diluted to a ratio of 1/5 (semen/extender, v/v) as final concentration and two step dilution method was used for cryopreservation. Extender groups were assessed for sperm motility, plasma membrane functional integrity using hypoosmotic swelling test (HOST), damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Semen samples also incubated for 6 h in humidified air with 5% CO₂ at 39 °C to evaluate post-thaw incubation resilience of semen characteristics. The results showed that freezing and thawing procedures had negative effects on motility (P < 0.05), plasma membrane integrity (P < 0.05) and acrosomal integrity (P < 0.05). After 6 h of incubation time, the Cysteine supplemented extender group yielded significantly higher results than other extender groups in terms of spermatological parameters. Furthermore MDA levels in the antioxidant groups were lower than control group (P < 0.05). Nevertheless, there were no significant differences among antioxidant groups.     

Effect of lecithin nanoliposome or soybean lecithin supplemented by pomegranate extract on post-thaw flow cytometric,microscopic and oxidative parameters in ram semen

This investigation was carried out to study the effect of soybean lecithin 1.5% (wt/vol) (0, 2.5, 5 and 7.5 mg l⁻¹ pomegranate extract (PE)) or PE-loaded lecithin nanoliposome (0, 2.5, 5 and 7.5 mg l⁻¹) to Tris-based extender. Sperm motility (CASA), viability, membrane integrity (HOS test), abnormalities, mitochondrial activity, apoptosis status, lipid peroxidation, total antioxidant capacity (TAC)) and antioxidant activities (GPX, SOD) were investigated following freeze-thawing. No significant differences were detected in motility parameters, viability, membrane integrity, and mitochondria activity after thawing sperm between soybean lecithin and lecithin nanoliposomes. It was shown that PE5 significantly improved sperm total and progressive motility, membrane integrity, viability, mitochondria activity, TAC and reduced lipid peroxidation (malondialdehyde concentration). Moreover, the percentage of apoptotic sperm in PE5 extenders was significantly the lowest among other treatments. Sperm abnormalities, SOD and GPX were not affected by the antioxidant supplements. For apoptotic status, no differences were observed between soybean lecithin and lecithin nanoliposome. We showed that lecithin nanoliposome extender can be a beneficial alternative extender to protect ram sperm during cryopreservation without any adverse effects. It was also observed that regarding pomegranate concentration, PE5 can improve the quality of ram semen after thawing.     

Effect of α-tocopherol supplementation during boar semen cryopreservation on sperm characteristics and expression of apoptosis related genes

Boar semen is extremely vulnerable to cold shock and sensitive to peroxidative damage due to high content of unsaturated fatty acids in the phospholipids of the plasma membrane and the relatively low antioxidant capacity of seminal plasma. The present study evaluated the influence of α-tocopherol supplementation at various concentrations in the boar semen extender during cryopreservation on post-thawed sperm motility characteristics (total sperm motility, MOT; local motility, LCM; curvilinear velocity, VCL; straight linear velocity, VSL; and average path velocity, VAP), sperm qualities (viability, acrosomal integrity and apoptosis), expression of stress protein (HSP70), and the expression of pro-apoptotic (Bax and Bak) and anti-apoptotic (Bcl-2l and Bcl-xl) genes. Semen collected from 10 Duroc boars was cryopreserved in lactose-egg yolk buffer supplemented with various concentrations of α-tocopherol (0, 100, 200, 400, 600 and 800 μM) using the straw-freezing procedure and stored at −196 °C for a minimum period of one month. In frozen-thawed groups, sperm motility was significantly (P < 0.05) lower than that of fresh sperm. In fresh sperm, HSP70 immunoreactivity expression was observed in the equatorial region, but in frozen-thawed groups, expressions were mostly observed in the sperm head. Higher apoptosis rates were observed in 600 and 800 μM α-tocopherol supplemented frozen-thawed groups. In α-tocopherol supplemented frozen-thawed groups immediately after thawing, the expression was similar to that of fresh group. But after incubation at 37 °C for 3 h, the expression in 200 and 800 μM α-tocopherol supplemented groups was higher than that of others. Expression of pro-apoptotic genes was significantly higher and anti-apoptotic genes was significantly (P < 0.01) lower in α-tocopherol supplemented frozen-thawed groups compared to fresh sperm group. In conclusion, α-tocopherol, supplemented at 200 μM concentration in boar semen extender during cryopreservation had a positive effect on post-thawed sperm survivability.     

Improving viability of cryopreserved honey bee (Apis mellifera L.) sperm with selected diluents, cryoprotectants, and semen dilution ratios

This is the first study where the systematic application of theories and techniques used in mammalian sperm cryopreservation have been applied to honey bee (Apis mellifera L.) semen as a means to improve postthaw viability of cryopreserved sperm. Six newly designed diluents, three cryoprotectants (dimethyl sulfoxide, DMA, glycerol), and five diluent:semen ratios (1:1, 3:1, 6:1, 9:1, and 12:1) were tested. In addition, the sperm freezing tolerance of three honey bee strains was evaluated. Specific protocols were designed to control semen freezing and thawing rates. Sperm motility was assessed visually, whereas sperm viability was assessed using SYBR-14 and propidium iodide fluorescent stains. Diluent treatments did not affect fresh (nonfrozen) sperm viability yet affected fresh sperm motility (P < 0.05). Based on these assessments, two diluents were chosen and used in all successive cryopreservation experiments. Using the selected diluents, semen was collected at various diluent:semen ratios, along with one of the three cryoprotectants. Semen collected at high dilution ratios, using a hypotonic antioxidant diluent containing catalase, in combination with dimethyl sulfoxide, provided higher postthaw sperm viability than that of all other combinations tested (68.3 ± 5.4%; P < 0.05). Using this combination of dilution ratio, diluent, and cryoprotectant, there were no differences among honey bee strains for postthaw sperm viability (P = 0.805). Nevertheless, these new semen dilution and freezing methods improved postthaw viability of sperm to levels that could theoretically sustain worker populations in colonies, thus providing potential for further optimization of cryopreservation techniques for the genetic preservation and improvement of honey bee genotypes.     

Novel and traditional traits of frozen-thawed porcine sperm related to in vitro fertilization success

Cryopreserved semen allows the use of single ejaculates for repeated analyses, potentially improving IVF consistency by eliminating interejaculate variability observed with fresh semen. However, the freezing and thawing processes result in compromised sperm function and IVF success. Semen samples are often screened for motility before use for IVF. Samples that are below a designated motility threshold may be discarded. Our objectives were to determine if post-thaw sperm motility, other traits that may be indicative of sperm function, or a novel assay of oviduct binding were related to IVF success. Semen from 16 boars was cooled to 15 °C for overnight shipment before cryopreservation. Semen was thawed and motility was recorded microscopically and confirmed using computer-automated sperm assessment. Each sample was tested by IVF in two to three independent replicates. Regression and correlation analyses were employed to determine the interrelationships between sperm traits and the relationships between post-thaw motility, sperm-oviduct binding and IVF outcomes. Among the sperm traits examined, sperm acrosome integrity was negatively correlated with post-thaw motility (r² = 0.64) but not with IVF results. The number of sperm bound to oviduct aggregates was correlated with IVF polyspermy rates (r² = 0.62, P < 0.05) but less with overall IVF rates (r² = 0.31, P > 0.10). There was some relationship of post-thaw motility with IVF monospermic fertilization (P = 0.06, r² = 0.08) but not to other IVF outcomes. Our results indicate that post-thaw motility of frozen-thawed boar sperm is strongly related to acrosome integrity but has limited use for predicting IVF success. The number of sperm bound to oviduct cells was related to IVF polyspermy rates and may be more indicative of in vitro sperm function than traditional sperm motility and acrosome status evaluation.     

Protein degradation and phosphorylation after freeze thawing result in spermatozoon dysfunction

Cryopreservation is widely used in many assisted conception units. Semen cryopreservation is the only proven method that offers many couples the chance to have children. However, spermatozoa are exposed to physical and chemical stressors during freezing and thawing that result in adverse changes in membrane lipid composition, sperm motility, viability, and acrosome status. Wang et al. (Proteomics 2014, 14, 298–310) evaluate the protein content of freeze‐thawed sperm samples relative to that of fresh sperm samples from the same normozoospermic donors. Four proteins are verified via Western blot and immunofluorescent staining, which are putatively involved in spermatozoon dysfunction. These marked differences demonstrated by Wang et al. suggest that dysfunctional spermatozoon after cryopreservation may be due to protein degradation and protein phosphorylation.     

Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation

Little is known about the effects of Cholesterol-Loaded Cyclodextrin (CLC) on post-thaw semen quality in chicken. The aim of the present study is to investigate the efficacy of CLC levels (0, 1, 2 and 3 mg/mL Schramm diluent) on post-thawed semen quality and fertility in two breeds of chicken Pradu Hang Dum (native chicken) and Rhode Island Red. Semen samples of each breed were pooled, divided into 4 aliquots and diluted with Schramm diluents, cooled to 5 °C when DMF was added (6% of final volume). Semen straws were subjected to cryopreservation using the liquid nitrogen vapor method. Post-thawed sperm motility, viability, acrosome integrity, mitochondrial function, and the Malondialdehyde (MDA) level were determined. The fertility of frozen semen was tested by inseminating laying hens. Post-thaw motility between Pradu Hang Dum and Rhode Island Red was no different; but Rhode Island Red had a higher semen viability and live cell intact acrosomes than Pradu Hang Dum (P < 0.05). The percentage of high functioning mitochondria in the Pradu Hang Dum was higher than the Rhode Island Red. CLC at 2 and 3 mg/mL supplementation was associated with improved viability of frozen semen; that is, acrosome integrity and mitochondrial function (P < 0.01), albeit having no effect on MDA levels. The sperm with 1 mg/mL CLC yielded a significantly better fertility (P < 0.01). CLC (1 mg/mL) improved the quality of frozen rooster semen. There was no interaction among breeds and CLC on post-thaw semen quality and fertility.     

Caffeic acid improves microscopic sperm parameters and antioxidant status of buffalo (Bubalus bubalis) bull semen following freeze-thawing process

This study investigates the effects of cryopreservation and supplementation of buffalo's semen with Caffeic acid. It studies the effects of different Caffeic acid concentrations on cryopreservation capacity of the buffalo and evaluates their influence on various sperm parameters like motility, viability, progressive motility, sperm plasma membrane integrity, and antioxidant status. Twenty-four semen samples were collected with an artificial vaginal from three adult water buffalos. The semen samples were evaluated and the qualified ejaculates were separated and were diluted in a Tris-based extender. The resulting samples were classified into 5 groups: No antioxidant (control), Control sham (NaOH), Caffeic acid 50 μM, Caffeic acid 100 μM, and Caffeic acid 200 μM. The semen samples encountered cryodamage and the quality was deteriorating during the cryopreservation (P < 0.05). The semen evaluation after thawing showed that the groups of samples receiving 100 μM Caffeic acid had higher viability, total motility, and lower abnormal sperm and better linearity (LIN), curvilinear velocity (VCL), straight-line velocity (VSL) and path velocity and higher intact plasma membrane (P < 0.05) compared to other groups. It is notable that adding 100 μM Caffeic acid to freezing extenders enhances the CAT, GPx, SOD, and GSH and also ameliorates total antioxidant capacity of spermatozoa after thawing. It is notable that the addition of 100 μM Caffeic acid decreases the amount of Malondialdehyde. These reactions lead us to conclude that 100 μM Caffeic acid enhances the semen quality of water buffalo after thawing.     

Supplementation of freezing media with alpha lipoic acid preserves the structural and functional characteristics of sperm against cryodamage in infertile men with asthenoteratozoospermia

The aim of this study was to examine the effects of alpha lipoic acid (ALA) supplementation during semen cryopreservation on the sperm quality, chromatin integrity, oxidative stress, and expression level of BAX, BCL2, HSP70 and iNOS genes in semen samples obtained from infertile men with asthenoteratozoospermia.MethodsTwenty freshly ejaculated semen samples were cryopreserved with sperm freezing medium supplemented with 0.00, 0.02, 0.05, 0.1, 0.5, and 1 mmol/mL of ALA. The samples were analyzed according to the WHO guidelines before and after freezing. Sperm ROS production level, DNA fragmentation and cryo-capacitation were assessed using flow cytometry, TUNEL assay and chlortetracycline (CTC) test, respectively. Expression level of stress protein (HSP70), pro-apoptotic Bax, anti-apoptotic Bcl-2, and iNOS genes was assessed by real-time PCR assay.ResultsThe effective concentrations of ALA (0.02 and 0.5 mM) significantly improved the motility, viability and morphology of the frozen-thawed sperms compared to the control group treated with 0.00 mM of ALA. During cryopreservation, treatment of semen with 0.02 mM of ALA, as the optimal concentration, significantly decreased DNA fragmentation and oxidative stress level (P < 0.05), protected the acrosome integrity, and led to insignificant reduction in BAX gene expression level and significant increase in expression level of BCL2, HSP70, and iNOS genes compared with control group.ConclusionOur findings revealed that the adding ALA to semen samples obtained from infertile men with asthenoteratozoospermia plays a significant protective role against cryodamage by preserving the sperm functional parameters.     

Lipid profiles of sperm and seminal plasma from boars having normal or low sperm motility

Sperm plasma membrane lipids have an important role to play in determining membrane fluidity and sperm motility. The objective of the present study was to determine whether there are differences in the lipid and fatty acid (FA) composition of boar sperm and seminal plasma in the ejaculates of boars having different sperm motilities. Semen was collected from two groups of boars having normal (> 60%; n = 53) or low (< 60%; n = 53) motility sperm and the semen was evaluated for motility, morphology and vitality. The semen was then centrifuged to separate the sperm from the seminal plasma and both were kept at −20 °C until analyzed for lipid content and FA profile by gas chromatography. Total antioxidant status (TAS) of seminal plasma was determined using a commercial kit. There were differences (P ≤ 0.05) in sperm total lipids, cholesterol, saturated fatty acids (SFA), phospholipids, n-3 polyunsaturated fatty acids (PUFA), docosahexaenoic acid (DHA) and the ratio of n-6:n-3 PUFA between boars with normal and low motility sperm. Total lipids, cholesterol, phospholipids, PUFA, DHA and n-3 PUFA were positively correlated with sperm motility, viability, normal morphology and normal plasma membrane. In contrast, SFA and the ratio of n-6: n-3 PUFA were negatively correlated (P ≤ 0.05) with sperm motility, viability, normal morphology and normal plasma membranes. The TAS of seminal plasma from boars having normal motility sperm was higher (P ≤ 0.05) than that of boars having low motility sperm and TAS was positively correlated (P = 0.0001) with sperm motility, viability, normal morphology and normal plasma membranes. In summary, differences in sperm motility were related to n-3 PUFA content in the sperm plasma membrane and extracellular antioxidants in seminal plasma which protect sperm plasma membranes from lipid peroxidation during periods of oxidative stress.     

Oxidative and nitrosative stress in frozen-thawed pig spermatozoa. II: Effect of the addition of saccharides to freezing medium on sperm function

Saccharides have bioprotective properties, with a high capacity to preserve biological proteins and membranes during sperm cryopreservation. The aim of this study was to evaluate how replacing the lactose of cryopreservation media by sucrose (SUC) or trehalose (TRE) at concentrations of 0.2 M (SUC-1 and TRE-1) and 0.25 M (SUC-2 and TRE-2) affects frozen/thawed pig spermatozoa. The media used were composed of medium A (saccharide/egg yolk) and B (saccharide/egg yolk/glycerol), their osmolality being determined prior to freezing. Cell viability, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), lipid peroxidation, thiol group oxidation, total reactive oxygen species (ROS), peroxynitrite and superoxide anion (O₂^●-) were determined through flow cytometry; total motility (TM), progressive motility (PM) and kinetic parameters motility were determined immediately after thawing (T0) and again 30 (T30) and 60 (T60) minutes later. The SUC-2 and TRE-2 groups maintained viability significantly and presented fewer lipid membrane disorders, respectively, both with a significant increase in MMP. The production of O₂^●- and peroxynitrite was lower in the TRE-2 groups compared to the control (P < 0.05). Total motility at T0 was greater in the TRE-2 group (P < 0.05). Sperm kinetics was not affected by the treatment. The use of saccharides SUC and TRE at a concentration of 0.25 M improves sperm quality, so that both non-penetrating cryoprotectants can be utilized in pig sperm freezing media.     

Cryopreservation of epididymal sperm from ibexes (Capra pyrenaica) using short equilibration time with glycerol

Two experiments were conducted to study the effect of shortening the equilibration time with the cryoprotectant glycerol before freezing epididymal sperm recovered postmortem from Iberian ibex. In the first experiment, the standard equilibration time of 3 hours was compared with 2 hours, and subjective sperm motility and quality of movement were greater (P < 0.05) in the latter group. In the second experiment, reducing the equilibration time from 2 hours to 15 minutes did not affect sperm motility (evaluated subjectively and objectively), viability, acrosomal integrity, or membrane functional integrity. In conclusion, shortening the equilibration time can be used as a technique to simplify the cryopreservation process and this provides practical advantages under field conditions.     

Protective effects of propolis on cryopreservation of common carp (Cyprinus carpio) sperm

Cryopreservation of sperm is common procedures in aquaculture, particularly used for routine in artificial insemination. However, these application cause damages and adversely affected sperm motility, viability and consequently lower hatching rates. The objective of this study is to determine whether propolis has an effect on cryopreservation and fertilization ability and to investigate the potential protective effect of propolis on spermatozoa of Cyprinus carpio. Many studies have been done in cryopreservation offish spermatozoa, but none of them contain propolis in extender composition. The extenders were prepared by using modified Kurokura Solution to which 10% Me₂SO added with different levels of propolis (0.2, 0.4, 0.6, 0.8 and 1 mg ml⁻¹) and 10% egg yolk (as a control without propolis). The pooled semen samples diluted at the ratio of 1:9 by the extenders were subjected to cryopreservation. The percentage and duration of motility and fertilization tests of cryopreserved sperm samples have been done immediately after thawing and compared with control and fresh semen. The extenders containing propolis exhibited higher percentage motility and motility duration than control group (P < 0.05). Especially the group IV (0.8 mg ml⁻¹ propolis) and the group V (1 mg ml⁻¹ propolis) showed significant positive effects on both post thaw motility and hatching ability. The propolis maintained the integrity of the spermatozoa during the cryopreservation process. Evaluating with its contents, it has been shown that propolis is an appropriate cryoprotective agent in fish semen.     

Addition of procyanidine to semen preserves progressive sperm motility up to three hours of incubation

Several studies on semen physiology and sperm fertilizing capacity have shown a beneficial effect of antioxidants. Procyanidine is a natural antioxidant, more efficient compared with vitamin C and E, with many applications in the food, agriculture, pharmaceutical and cosmetic industry. Thus, we tested whether the addition of procyanidine to the semen of infertile men has a beneficial effect on spermatozoa during their in vitro incubation and during the cryopreservation process. Semen samples of 25 infertile men were divided in to two aliquots, in which procyanidine was added or not. Semen analysis, measurement of sperm DNA fragmentation index (DFI) and measurement of reactive oxygen species (ROS) were performed 3 h after incubation at 37 °C and after sperm cryopreservation and thawing. In-vitro addition of procyanidine to semen of infertile men resulted in a lesser decrease in progressive motility [−4 (−31:+6) vs. −6 (−31:+5), p < 0.001] and total motility [−5 (−29:+3) vs. −9 (−32:+2), p < 0.001] after 3 h of incubation compared with no addition of procyanidine. Sperm morphology was decreased only in the control group after 3 h of incubation [2 (0:+6) vs. 1 (0:+4), p = 0.009]. Furthermore, a larger increase in sperm DFI was observed in the control compared with the procyanidine group [9 (−7:+27) vs. 3 (−3:+18), p = 0.005] after thawing of cryopreserved semen samples. In conclusion, in-vitro addition of procyanidine to the semen of infertile men exerts a protective effect on progressive motility during handling and after 3 h of incubation as well as on sperm DFI during the process of cryopreservation.     

Low concentrations of 3-O-methylglucose improve post thaw recovery in cryopreserved bovine spermatozoa

A number of studies have explored the use of membrane permeable (usually metabolizable) and membrane impermeable saccharides to protect cells in general, and sperm in particular during cryopreservation. Critical concentrations for protective levels of sugars frequently range between 50 mmol/L and 500 mmol/L, where efficacy is attributed to the sugar's membrane stabilizing and glass forming attributes and colligative effects that reduce intra- and extracellular salt concentrations during freezing. Many studies on bull sperm have demonstrated that both permeating and non-permeating sugars have negligible positive effects on post-thaw viability. Recently, however, a non-metabolizable sugar, 3-O-Methylglucose (3-OMG), was shown to protect hepatocytes during liver cryopreservation at 0.1–0.3 mol/L. Because glucose is readily transported into sperm, we hypothesized that 3-OMG could be a new class of cryoprotectant to explore in bull sperm. Here we present positive results demonstrating that 3-OMG improves post thaw viability in bull sperm, and that this effect is not likely due to improved glass forming capabilities. In particular, in experiment 1, 3-OMG was added to the Tris-egg yolk-glycerol base media at levels from 0 mmol/L to 200 mmol/L. Semen from four bulls was collected and diluted with one of the cryopreservation media, cooled, and frozen following industry standard practices. Motility and mitochondrial activity were negatively impacted when concentration of 3-OMG was more than 25 mmol/L. Therefore, we explored lower concentrations in experiment 2, where semen from eight bulls was used to evaluate concentrations 5 mmol/L, 15 mmol/L and 25 mmol/L of 3-OMG compared with control. Motility and progressive motility in 5 mmol/L 3-OMG and in the control were significantly higher than 15 mmol/L and 25 mmol/L 3-OMG, whereas mitochondrial activity and acrosome integrity in 5 mmol/L 3-OMG were significantly better than the control freezing medium. In experiment 3, to evaluate whether the improved effects of 3-OMG are due to its non-metabolizing property, or due to colligative effects, we compared post-thaw viability in semen from four bulls cryopreserved with 5 mmol/L glucose, sucrose, or 3-OMG. Motility and progressive motility was significantly improved in 3-OMG compared to glucose or sucrose groups which were comparable to the EY control. In conclusion, 3-OMG at a concentration of 5 mmol/L in Tris-egg yolk-glycerol medium improves the post thaw motility, progressive motility and viability of bull sperm. The mechanism of action is not understood but because the efficacy is maximal at low concentrations, it is not likely due to improved intra- or extracellular glass forming abilities and may demonstrate a different protective mechanism than was shown in hepatocytes.     

Freeze–thaw induced genotoxicity in buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) spermatozoa in relation to total antioxidant status

Use of cryopreserved semen has become an important tool in assisted reproduction but freezing and thawing cause sub-lethal damage to spermatozoa. This is detrimental to sperm because of the membrane damage including permeability and integrity. An excess generation of reactive oxygen species (ROS) creates oxidative stress due to reduced antioxidant status of the cryopreserved spermatozoa. In the present study fresh buffalo semen was collected and divided into two aliquots. One aliquot was used for fresh semen analysis and the other was cryopreserved in Tris-egg yolk-citrate extender. The semen samples were used to study different sperm quality parameters like motility, viability, membrane integrity and total antioxidant status. The DNA integrity in fresh and cryopreserved spermatozoa was also studied using comet assay. The sperm quality parameters like post-thaw sperm motility, viability, membrane integrity and total antioxidant status of cryopreserved spermatozoa were significantly lowered (P < 0.05) compared to fresh spermatozoa. The DNA fragmentation in cryopreserved spermatozoa was significantly higher (P < 0.01) as compared to fresh spermatozoa. The results show that the irreversible DNA damage occurs in spermatozoa during cryopreservation.