Kaempferol as an Alternative Cryosupplement for Bovine Spermatozoa: Cytoprotective and Membrane-Stabilizing Effects.

Kaempferol (KAE) is a natural flavonoid with powerful reactive oxygen species (ROS) scavenging properties and beneficial effects on ex vivo sperm functionality. In this paper, we studied the ability of KAE to prevent or ameliorate structural, functional or oxidative damage to frozen-thawed bovine spermatozoa. The analysis focused on conventional sperm quality characteristics prior to or following thermoresistance tests, namely the oxidative profile of semen alongside sperm capacitation patterns, and the levels of key proteins involved in capacitation signaling. Semen samples obtained from 30 stud bulls were frozen in the presence of 12.5, 25 or 50 µM KAE and compared to native ejaculates (negative control-CtrlN) as well as semen samples cryopreserved in the absence of KAE (positive control-CtrlC). A significant post-thermoresistance test maintenance of the sperm motility (p < 0.001), membrane (p < 0.001) and acrosome integrity (p < 0.001), mitochondrial activity (p < 0.001) and DNA integrity (p < 0.001) was observed following supplementation with all KAE doses in comparison to CtrlC. Experimental groups supplemented with all KAE doses presented a significantly lower proportion of prematurely capacitated spermatozoa (p < 0.001) when compared with CtrlC. A significant decrease in the levels of the superoxide radical was recorded following administration of 12.5 (p < 0.05) and 25 µM KAE (p < 0.01). At the same time, supplementation with 25 µM KAE in the cryopreservation medium led to a significant stabilization of the activity of Mg2+-ATPase (p < 0.05) and Na+/K+-ATPase (p < 0.0001) in comparison to CtrlC. Western blot analysis revealed that supplementation with 25 µM KAE in the cryopreservation medium prevented the loss of the protein kinase A (PKA) and protein kinase C (PKC), which are intricately involved in the process of sperm activation. In conclusion, we may speculate that KAE is particularly efficient in the protection of sperm metabolism during the cryopreservation process through its ability to promote energy synthesis while quenching excessive ROS and to protect enzymes involved in the process of sperm capacitation and hyperactivation. These properties may provide supplementary protection to spermatozoa undergoing the freeze-thaw process.

Cold-induced damage in boar sperm cooled to 5°C in Lactose Egg Yolk (LEY) medium with different Glycerol concentrations.

Boar sperm is highly susceptible to cold damage. When temperature drops to 5°C, the plasmatic membrane is destabilized. The freezing process causes a reduction of the fertility window because frozen/thawed boar sperm has less survivability. The aim of this work was to analyze the effect on sperm characteristics and response to capacitation stimuli of cooling to 5°C using a controlled protocol. Also, we evaluated if the addition of Glycerol 2% or 3% at 5°C was able to modify these parameters. For this purpose, we assessed motility, plasmatic membrane integrity and acrosomal membrane status. Capacitation was induced using Tyrode´s capacitating medium (TCM) and assessed by chlortetracycline stain and induction of acrosomal reaction with Progesterone. Motility patterns were analyzed using a CASA system. These tests were performed at three different points of the freezing curve: 37°C; 17°C and 5°C. Response to TCM vs TBM was only significant at 37°C. While at 37°C and 17°C capacitated sperm was below 20%, at 5°C reached 50% both in the TBM and TCM. CASA analysis showed that spermatozoa exposed to TCM had higher LIN and WOB than those in TBM. All parameters were similar in the Glycerol concentrations studied. These results suggest that the chilling process may be causing an effect similar to cryocapacitation along the cooling curve, starting subtle at 17°C and reaching 50% of the sperm population at 5°C, being independent of Glycerol concentration.

ROS-induced oxidative stress is a major contributor to sperm cryoinjury.

STUDY QUESTION: What is the mechanism behind cryoinjury in human sperm, particularly concerning the interplay between reactive oxygen species (ROS) and autophagy, and how does it subsequently affect sperm fate? SUMMARY ANSWER: The freeze-thaw operation induces oxidative stress by generating abundant ROS, which impairs sperm motility and activates autophagy, ultimately guiding the sperm toward programmed cell death such as apoptosis and necrosis, as well as triggering premature capacitation. WHAT IS KNOWN ALREADY: Both ROS-induced oxidative stress and autophagy are thought to exert an influence on the quality of frozen-thawed sperm. STUDY DESIGN, SIZE, DURATION: Overall, 84 semen specimens were collected from young healthy fertile males, with careful quality evaluation. The specimens were split into three groups to investigate the ROS-induced cryoinjury: normal control without any treatment, sperm treated with 0.5 mM hydrogen peroxide (H2O2) for 1 h, and sperm thawed following cryopreservation. Samples from 48 individuals underwent computer-assisted human sperm analysis (CASA) to evaluate sperm quality in response to the treatments. Semen samples from three donors were analyzed for changes in the sperm proteome after H2O2 treatment, and another set of samples from three donors were analyzed for changes following the freeze-thaw process. The other 30 samples were used for fluorescence-staining and western blotting. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sperm motility parameters, including progressive motility (PR %) and total motility (PR + NP %), were evaluated using the CASA system on a minimum of 200 spermatozoa. The proteomic profiles were determined with label-free mass spectrometry (MS/MS) and protein identification was performed via ion search against the NCBI human database. Subsequently, comprehensive bioinformatics was applied to detect significant proteomic changes and functional enrichment. Fluorescence-staining and western blot analyses were also conducted to confirm the proteomic changes on selected key proteins. The ROS level was measured using 2',7'-dichlorodihydrofluorescein diacetate labeling and the abundance of bioactive mitochondria was determined by evaluating the inner mitochondrial membrane potential (MMP) level. Molecular behaviors of sequestosome-1 (p62 or SQSTM1) and microtubule-associated proteins 1A/1B light chain 3 (LC3) were monitored to evaluate the state of apoptosis in human sperm. Fluorescent probes oxazole yellow (YO-PRO-1) and propidium iodide (PI) were utilized to monitor programmed cell death, namely apoptosis and necrosis. Additionally, gradient concentrations of antioxidant coenzyme Q10 (CoQ10) were introduced to suppress ROS impacts on sperm. MAIN RESULTS AND THE ROLE OF CHANCE: The CASA analysis revealed a significant decrease in sperm motility for both the H2O2-treatment and freeze-thaw groups. Fluorescence staining showed that high ROS levels were produced in the treated sperm and the MMPs were largely reduced. The introduction of CoQ10 at concentrations of 20 and 30 µM resulted in a significant rescue of progressive motility (P < 0.05). The result suggested that excessive ROS could be the major cause of sperm motility impairment, likely by damaging mitochondrial energy generation. Autophagy was significantly activated in sperm when they were under oxidative stress, as evidenced by the upregulation of p62 and the increased conversion of LC3 as well as the upregulation of several autophagy-related proteins, such as charged multivesicular body protein 2a, mitochondrial import receptor subunit TOM22 homolog, and WD repeat domain phosphoinositide-interacting protein 2. Additionally, fluorescent staining indicated the occurrence of apoptosis and necrosis in both H2O2-treated sperm and post-thaw sperm. The cell death process can be suppressed when CoQ10 is introduced, which consolidates the view that ROS could be the major contributor to sperm cryoinjury. The freeze-thaw process could also initiate sperm premature capacitation, demonstrated by the prominent increase in tyrosine phosphorylated proteins, verified with anti-phosphotyrosine antibody and immunofluorescence assays. The upregulation of capacitation-related proteins, such as hyaluronidase 3 and Folate receptor alpha, supported this finding. LARGE SCALE DATA: The data underlying this article are available in the article and its online supplementary material. LIMITATIONS, REASONS FOR CAUTION: The semen samples were obtained exclusively from young, healthy, and fertile males with progressive motility exceeding 60%, which might overemphasize the positive effects while possibly neglecting the negative impacts of cryoinjury. Additionally, the H2O2 treatment conditions in this study may not precisely mimic the oxidative stress experienced by sperm after thawing from cryopreservation, potentially resulting in the omission of certain molecular alterations. WIDER IMPLICATIONS OF THE FINDINGS: This study provides substantial proteomic data for a comprehensive and deeper understanding of the impact of cryopreservation on sperm quality. It will facilitate the design of optimal protocols for utilizing cryopreserved sperm to improve applications, such as ART, and help resolve various adverse situations caused by chemotherapy, radiotherapy, and surgery. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from the Major Innovation Project of Research Institute of National Health Commission (#2022GJZD01-3) and the National Key R&D Program of China (#2018YFC1003600). All authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Iodixanol supplementation in freezing extender improves the antioxidant capacity of semen.

The aim of this study was to evaluate the effect of supplementing bovine semen freezing extender with different concentrations of iodixanol on post-thaw sperm characteristics. Six ejaculates of three Nellore bulls were pooled and diluted in commercial extender (BotuBov®) and then divided into 4 groups: control group (without adding iodixanol); groups G1.5, G3, or G6 according to the concentration of iodixanol solution (RedCushion®). After dilution, the samples were cooled and frozen. Post-thaw semen evaluation included sperm motility by CASA immediately after thawing and after 60 min of incubation at 37°C, flow cytometry analysis for integrity of plasma and acrosomal membranes, membrane destabilization and translocation of phosphatidylserine, mitochondrial membrane potential, and formation of intracellular anion superoxide ( O 2 - ), hydrogen peroxide (H2 O2 ), and membrane lipid peroxidation. The group G6 presented significantly higher (p < .05) total and progressive motility, percentage of plasma and acrosomal membrane integrity, and H2 O2 than control and group G1.5. Furthermore, group G6 showed lower (p < .05) lipid peroxidation than control. In addition, regardless of the concentration used, the percentage of spermatozoa without phosphatidylserine translocation was higher (p < .05) in all iodixanol supplemented groups. In conclusion, iodixanol supplementation preserved the motility and integrity of sperm membranes during cryopreservation and protected against lipid peroxidation.

Antioxidant additive melatonin in tris-based egg yolk extender improves post-thaw sperm attributes in Hariana bull.

In the study, melatonin, a known antioxidant pineal peptide was used as an additive in the tris-egg yolk glycerol-based semen extender in Hariana bull semen and post-thaw sperm characters were evaluated. In the study, Group I was a control without melatonin; and Group II, III, and IV were having 0.5 mM, 1 mM, and 2 mM melatonin/80 × 106 spermatozoa, respectively were treatment groups. Thirty-two semen ejaculates from 4 Hariana bulls were processed for freezing and post-thaw sperm characteristics were evaluated. Sperm motility, velocity, the viability with intact membrane, and total antioxidant capacity were markedly (P < 0.05) improved in Group IV compared to all other groups. The lipid peroxidation and total protein carbonylation were substantially (P < 0.05) decreased in Group IV compared to all other groups. The population of cryocapacitated, acrosome-reacted, and apoptotic-like spermatozoa were significantly (P < 0.05) decreased in Group IV. Further, the relative band intensity of 74 kDa protein and percent of spermatozoa showing positive immune reactivity to tyrosine-phosphorylated proteins was decreased in Group IV. The progesterone-receptor ligand binding, in vitro capacitation response, and Vanguard distance were markedly (P < 0.05) improved in Group IV. In summary- Group IV having 2 mM melatonin was found to be optimal in providing cryoprotective effects to Hariana bull spermatozoa after freezing-thawing and can be suitably used as a semen additive during semen cryopreservation.

Epicatechin Prevents Cryocapacitation of Bovine Spermatozoa through Antioxidant Activity and Stabilization of Transmembrane Ion Channels.

Epicatechin (EPC) is a flavonoid belonging to the family of catechins; it has been described as a powerful scavenger of a wide spectrum of reactive oxygen species (ROS) and a modulator of ex vivo sperm vitality. In this study, we assessed the potential protective abilities of EPC on cryopreserved bovine spermatozoa. We focused on conventional quality parameters, as well as the oxidative profile of spermatozoa alongside capacitation patterns, and expression profiles of proteins involved in the process of capacitation. Semen samples were cryopreserved in the presence of 25, 50 or 100 µmol/L EPC and compared to native semen (negative control) as well as ejaculates frozen in the absence of EPC (positive control). A dose-dependent improvement of conventional sperm quality parameters was observed following EPC administration, particularly in case of the sperm motility, membrane, acrosome and DNA integrity in comparison to the positive control. Experimental groups exposed to all EPC doses presented with a significantly lower proportion of capacitated spermatozoa as opposed to the positive control. While no significant effects of EPC were observed in cases of superoxide production, a significant decrease in the levels of hydrogen peroxide and hydroxyl radical were recorded particularly in the experimental groups supplemented with 50 and 100 µmol/L EPC. Western blot analysis revealed that supplementation of particularly 100 µmol/L EPC to the semen extender prevented the loss of the cation channel of sperm (CatSper) isoforms 1 and 2, sodium bicarbonate cotransporter (NBC) and protein kinase A (PKA), which play important roles in the process of sperm capacitation. In summary, we may hypothesize that EPC is particularly effective in the stabilization of the sperm membrane during the freeze-thaw process through its ability to quench ROS involved in damage to the membrane lipids and to prevent the loss of membrane channels crucial to initiate the process of sperm capacitation. These attributes of EPC provide an additional layer of protection to spermatozoa exposed to low temperatures, which may be translated into a higher post-thaw structural integrity and functional activity of male gametes.

Curcumin in a tris-based semen extender improves cryosurvival of Hariana bull spermatozoa.

In this study, the cryoprotective potential of natural antioxidant curcumin in Hariana bull semen was evaluated as an additive in a tris-based extender with the assessment of motility and motion parameters of spermatozoa, membrane intactness, progesterone-receptor binding, protein carbonyl content, cervical mucus penetration, cryocapacitation-associated and apoptotic-like changes. The collected ejaculates were divided into five groups in the tris-based extender (control without curcumin-I, 10 µM-II, 25 µM-III, 50 µM-IV and 75µM-V) and were cryopreserved. Groups II and III containing 10 and 25 µM curcumin substantially (p < .05) improved the post-thaw sperm parameters like viability, motility, and velocity parameters; intact acrosome and membrane; lowered protein carbonyl content; DNA fragmentation and cryocapacitation-associated changes in comparison to control. It was interesting to note that early apoptotic-like changes in sperm cells were significantly (p < .05) decreased in Group II along with an increase in a higher population of sperm cells having high mitochondrial transmembrane potential. Higher progesterone-receptor binding, Vanguard distance and in vitro capacitation response were observed only in Group II (10µM) compared to other groups. In conclusion, curcumin in a semen extender manifests cryoprotective effects and may be incorporated at 10 µM concentration in a Hariana bull semen extender for better post-thaw sperm quality.

Beneficial effects of hypotaurine supplementation in preparation and freezing media on human sperm cryo-capacitation and DNA quality.

BACKGROUND: Although widely used, slow freezing considerably modifies the functions of human spermatozoa. Cryopreservation induces nuclear sperm alterations and cryo-capacitation, reducing the chances of pregnancy. Hypotaurine is naturally present in the male and female genital tracts and has capacitating, osmolytic and anti-oxidant properties. The analysis were performed on surplus semen of men with normal (n = 19) or abnormal (n = 14) sperm parameters. Spermatozoa were selected by density gradient centrifugation before slow freezing. For each sample, these steps were performed in parallel with ("H+" arm) or without ("H-" arm) hypotaurine supplementation. After thawing, we measured total and progressive mobility, vitality, acrosome integrity, markers of capacitation signaling pathway and nuclear quality. For the latter, we focused on sperm chromatin packaging, DNA fragmentation and the presence of vacuoles in the sperm nucleus. RESULTS: Post-thaw spermatozoa selected and frozen in the presence of hypotaurine had a higher vitality (+ 16.7%, p < 0.001), progressive and total motility (+ 39.9% and +  21.6% respectively, p < 0.005) than spermatozoa from the control "H-" arm. Hypotaurine also reduced the non-specific phosphorylation of the capacitation protein markers P110 and P80 (p < 0.01), indicating a decrease in cryo-capacitation. Hypotaurine supplementation reduced chromatin decondensation, measured by chromomycin A3 (- 16.1%, p < 0.05), DNA fragmentation (- 18.7%, p < 0.05) and nuclear vacuolization (- 20.8%, p < 0.05). CONCLUSION: Our study is the first to demonstrate beneficial effects of hypotaurine supplementation in preparation and freezing procedures on human spermatozoa sperm fertilization capacity and nucleus quality. Hypotaurine supplementation limited cryo-capacitation, increased the proportion of live and progressively motile spermatozoa and reduces the percentage of spermatozoa showing chromatin decondensation, DNA fragmentation and nuclear vacuolation. TRIAL REGISTRATION: Clinical Trial, NCT04011813 . Registered 19 May 2019 - Retrospectively registered.

RéSUMé: CONTEXTE: Bien que largement utilisée, la congélation lente modifie considérablement les fonctions des spermatozoïdes humains. La cryoconservation induit des altérations nucléaires du sperme et une cryocapacitation, réduisant les chances de grossesse. L'hypotaurine est. naturellement présente dans les voies génitales masculines et féminines et possède des propriétés capacitantes, osmotiques et anti-oxydantes. Les mesures ont été réalisées sur le reliquat de sperme d'hommes avec des paramètres spermatiques normaux (n = 19) ou anormaux (n = 14). Les spermatozoïdes ont été sélectionnés par centrifugation sur gradient de densité (test de migration survie) avant congélation lente. Pour chaque prélèvement, ces étapes ont été réalisées en parallèle avec des milieux supplémentés en hypotaurine (bras « H+ ¼) ou sans hypotaurine (bras « H- ¼). Après décongélation, nous avons mesuré la mobilité totale et progressive, la vitalité, l'intégrité de l'acrosome, des marqueurs de la voie de signalisation de la capacitation et la qualité nucléaire. Pour cette dernière, nous nous sommes concentrés sur la condensation de la chromatine, la fragmentation de l'ADN et la présence de vacuoles dans le noyau du sperme. RéSULTATS: Post-décongélation, les spermatozoïdes sélectionnés et congelés en présence d'hypotaurine avaient une vitalité plus élevée (+ 16,7%, p < 0,001), une motilité progressive et totale (+ 39,9% et + 21,6% respectivement, p < 0,005) que les spermatozoïdes du bras « H- ¼ sans suplémentation. L'hypotaurine a également réduit la phosphorylation non spécifique des marqueurs protéiques de capacitation P110 et P80 (p < 0,01), indiquant une diminution de la cryocapacitation. La supplémentation en hypotaurine a réduit la décondensation de la chromatine, mesurée par la chromomycine A3 (− 16,1%, p < 0,05), la fragmentation de l'ADN (− 18,7%, p < 0,05) et la vacuolisation nucléaire (− 20,8%, p < 0,05). CONCLUSION: Notre étude est. la première à démontrer les effets bénéfiques de la supplémentation en hypotaurine dans les milieux de préparation et de congélation sur la capacité de fécondation des spermatozoïdes humains et leur qualité nucléaire. La supplémentation en hypotaurine a limité la cryocapacitation, augmenté la proportion de spermatozoïdes vivants et progressivement mobiles et réduit le pourcentage de spermatozoïdes présentant une décondensation de la chromatine, une fragmentation de l'ADN et une vacuolisation nucléaire. ENREGISTREMENT DE L'ESSAI: essai clinique, NCT04011813 . Enregistré le 19 mai 2019 - Enregistré rétrospectivement.

piR-121380 Is Involved in Cryo-Capacitation and Regulates Post-Thawed Boar Sperm Quality Through Phosphorylation of ERK2 via Targeting PTPN7.

Cryopreservation induces capacitation-like (cryo-capacitation) changes, similar to natural capacitation, and affects the fertility potential of post-thawed sperm. The molecular mechanism of sperm cryo-capacitation during cryopreservation remains unknown. PIWI-interacting RNAs (piRNAs) have been reported to be involved in cryo-capacitation of post-thawed sperm and regulation of sperm motility, capacitation, and chemotaxis. In this study, protein tyrosine phosphatase nonreceptor type 7 (PTPN7) was positively targeted by piR-121380 after a dual luciferase assay. The mRNA expression of PTPN7 and piR-121380 was significantly decreased (p < 0.01); however, PTPN7 protein was significantly increased (p < 0.01) in post-thawed boar sperm. Furthermore, E1RK1/2 phosphorylation was reduced during cryopreservation. Six hours after transfection with piR-121380 mimic and inhibitor, the phosphorylation of ERK2 was significantly increased and decreased (p < 0.01), respectively. Furthermore, the highest and lowest total sperm motility, forward motility, and capacitation rate were observed after piR-121380 mimic and inhibitor treatments, respectively. The concentration of intracellular calcium ([Ca2+]i) showed no significant difference after transfection with either piR-121380 mimic or inhibitor at 1, 3, and 6 h. In conclusion, we demonstrated that piR-121380 modulates ERK2 phosphorylation by targeting PTPN7, which induces sperm cryo-capacitation, and eventually affects the motility and fertility potential of post-thawed sperm.

Freezing-thawing induces deprotamination, cryocapacitation-associated changes; DNA fragmentation; and reduced progesterone sensitivity in buck spermatozoa.

In the present study, there was evaluation of cryocapacitation-associated changes, apoptotic-like changes, deprotamination, total antioxidant capacity (TAC), and in vitro sperm functional attributes in Barbari bucks after freezing-thawing. The correlation between deprotamination and sperm functional characteristics was established. Using immunoblotting procedures, there was detection of the presence of a single 28-kDa protein band corresponding to protamine-1. The localization in the head region of the spermatozoa was further validated by an immunofluorescence test. Capacitated (B-) and acrosome-reacted (AR-) pattern spermatozoa, spermatozoa with the externalization of phosphatidylserine and a relatively lesser mitochondrial transmembrane potential, and deprotamination and DNA fragmentation was greater (P < 0.05) after freezing-thawing and indicated there were cryocapacitation- and apoptotic-like changes, respectively. Furthermore, the detection of phosphorylation of tyrosine-containing proteins with use of immunoblotting and immunofluorescence procedures confirmed there were cryocapacitation-like changes in the buck spermatozoa after freezing-thawing. Total antioxidant capacity (TAC), in vitro thermal resistance response, Vanguard distance, progesterone sensitivity, and in vitro capacitation response were less (P < 0.05) in the spermatozoa after freezing-thawing compared with spermatozoa after initial dilution and equilibration. Deprotamination (chromomycin A3-positive cells, CMA3+) and DNA fragmentation (TUNEL+ve) were positively correlated with B- and AR-pattern spermatozoa, while other values for other variables were negatively correlated. In conclusion, the results of this study indicated there was protamine-1 in buck spermatozoa and after freezing-thawing there was a loss of protamine-1 combined with cryocapacitation-associated changes and apoptotic-like changes in buck spermatozoa. Spermatozoa deprotamination might be attributed to increased DNA fragmentation, resulting in compromised fertilizing capacity of buck spermatozoa.

Exogenous cholesterol prevents cryocapacitation-like changes, membrane fluidity, and enhances in vitro fertility in bubaline spermatozoa.

A study was conducted to determine the optimum dosage of the exogenous cholesterol-loaded cyclodextrins (CLC) to get maximum cryoprotection for bubaline spermatozoa. In the present study, 120 × 106 spermatozoa were incubated in 2, 3 and 4 mg of CLC as grouped as Gr II, III and IV, respectively, and sperm progressive motility, intracellular Ca2+ , capacitation status by protein tyrosine phosphorylation (PTP) assay and zona binding per cent (ZBP) and cleavage rate (CR) of the cryopreserved buffalo spermatozoa by in vitro fertility assay were assessed in comparison with an untreated control group (Gr I). Results revealed that there was a significant (p < .05) linear decrease in percentage of sperm population with higher intracellular Ca2+ and percentage of sperm population with medium or high capacitated by PTP in CLC treated from 2 to 3 mg and then increased to 4 mg/120 × 106 spermatozoa whereas sperm progressive motility, percentage of sperm population with low capacitated, ZBP and CR were increased significantly (p < .05) in sperm population treated from 2 to 3 mg CLC and then decreased to 4 mg/120 × 106 spermatozoa. The study has clearly indicated that CLC at 3 mg/120 × 106 spermatozoa has maximum beneficial effects in protection of sperm progressive motility, membrane fluidity (low intracellular Ca2+ ); prevention of cryocapacitation (low capacitation pattern in immunolocalization) and enhancement of in vitro ZBP and CR. Post-thaw motility of the CLC-treated sperm has shown positively significant (p < .05) correlation with sperm population with low intracellular Ca2+ , low capacitated sperm population, ZBP and CR, whereas it was negatively (p < .05) correlated with sperm population with high intracellular Ca2+ , medium or high capacitated sperm. The present study has revealed for the first time that incubation of spermatozoa with CLC of higher dose (>3 mg/120 × 106 spermatozoa) had adverse effects on sperm cryopreservation, although incubation of sperm with 3 mg/120 million prior to processing had minimised the freezing-thawing-associated damages in bubaline species.

Membrane stability and mitochondrial activity of bovine sperm frozen with low-density lipoproteins and trehalose.

Cryopreservation results in the destabilization of the sperm plasma membrane, leading to negative side effects such as premature cryocapacitation, apoptosis and the low mitochondrial activity of bovine spermatozoa. Low-density lipoproteins (LDL) and trehalose have been used in seminal freezing to protect the integrity and stability of sperm membranes. Likewise, trehalose can increase the mitochondrial activity of sperm. The objective of this study was to evaluate the membrane stability and mitochondrial activity of bovine sperm after being frozen and treated with LDL sources and trehalose. Ten ejaculates from five bulls were cryopreserved under the treatments, CEY: chicken egg yolk (20% v/v); CCEY: centrifuged CEY (20% v/v); LDL: LDL (8% v/v); T: trehalose (100 mM); and TLDL: T (100 mM) plus LDL (8% v/v). After thawing, membrane stability and mitochondrial membrane potential (ΔΨM) were assessed by flow cytometry through the M-540/Yopro-1 and DiOC6/PI probes. The structural membrane integrity (SMI) was evaluated by fluorescence microscopy using SYBR14/PI dyes. A generalized linear model was adjusted, and the means were compared using the Tukey test. Centrifuged chicken egg yolk and LDL had a higher proportion of non-cryocapacitated non-apoptotic sperm (M-Y-), while CEY and T had the largest populations of cryocapacitated non-apoptotic sperm (M+Y-) and cryocapacitated apoptotic sperm (M+Y+). Centrifuged chicken egg yolk also showed a higher proportion of sperm with high-ΔΨM. Treatments that included egg yolk or purified LDL had a positive effect on SMI. Centrifuged chicken egg yolk has a superior cryoprotective effect on membrane stability and mitochondrial activity of bovine semen over the conventional use of CEY or the individual or simultaneous use of LDL and trehalose.

Boar sperm protein tyrosine phosphorylation in the presence of egg yolk soluble and low density lipoprotein fractions during cooling.

Increased protein tyrosine phosphorylation and the appearance of a phosphorylated protein of 32 kD (p32) are reported among the capacitation-like changes in cryopreserved boar sperm. Egg yolk freezing extenders are composed by two fractions: insoluble granules and soluble plasma, which contains the low density lipoproteins (LDL) proposed as responsible for the egg yolk cryoprotective action. The aim of this work was to analyze the effects of complete egg yolk and its insoluble, soluble and LDL fractions on boar sperm quality and protein tyrosine phosphorylation after the first stage of a standard cryopreservation protocol. Semen samples in Androstar® Plus diluent were centrifuged and resuspended in the different egg yolk extenders. Temperature was decreased from 17 °C to 5 °C and sperm quality, protein tyrosine phosphorylation and protein pattern were analyzed. Results showed that complete egg yolk as well as soluble and LDL egg yolk fractions maintained sperm quality after temperature decrease. Cooling without any lipid component or in the presence of the insoluble fraction, significantly reduced sperm motility. About sperm protein tyrosine phosphorylation analysis, the p32 band appeared before treatments or after cooling in Androstar® Plus diluent. Complete egg yolk and its insoluble fraction interfered with sperm tyrosine phosphorylation even after cells were extensively washed. Analysis of extenders revealed a high amount of tyrosine phosphorylated proteins in the insoluble fraction, which may have co-precipitate with sperm in experiments. Samples submitted to temperature decrease from 17 °C to 5 °C in the presence of soluble and LDL egg yolk fractions in Androstar® Plus diluent did not show any change in the p32 band associated with sperm capacitation. However, a tyrosine-phosphorylated protein of 33 kD present in clarified egg yolk was also observed in sperm treated with this extender. Protein transference from plasma and LDL egg yolk extenders was also observed in sperm protein profile. Results suggested that soluble and LDL fractions might have a protective action preventing sperm protein tyrosine phosphorylation during cooling from 17 °C to 5 °C. Further studies are needed to expand the knowledge of the LDL protection mechanism as well as to determine the possible benefits of clarified egg yolk in freezing protocols.

Cryopreservation of boar semen in 0. 5 mL straws at low spermatozoa concentration is better than high concentration to maintain sperm viability / A manutenção da viabilidade do sêmen suíno criopreservado em palhetas de 0, 5 mL em baixas concentrações espermáticas é melhor do que em altas concentrações

To date, no studies have been performed evaluating the effect of boar spermatozoa concentration in 0.5mL freezing straws, leading us to examine this question. Each sperm-rich fraction of the ejaculate (n=25) was diluted at five different sperm concentrations (100, 200, 300, 600 and 800 x 106 spermatozoa/mL), packaged in 0.5mL straws, and subsequently frozen. After thawing, the sperm from all of treatment groups were analyzed to determine motility characteristics using a sperm class analyzer (SCA-CASA), and their plasma and acrosomal membrane integrity, mitochondrial membrane potential, sperm membrane lipid peroxidation and fluidity were analyzed by flow cytometry. An increase in spermatozoa concentration above 300x106 spermatozoa/mL in a 0.5mL straw impaired (p<0.05) the total and progressive motility, curvilinear velocity, straight-line velocity, linearity and beat cross frequency. However, the plasma and acrosomal membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation and fluidity were not influenced (p>0.05) by high spermatozoa concentrations at freezing. Therefore, to increase spermatozoa survival and total and progressive motility after thawing, boar spermatozoa should be frozen at concentrations up to 300x106 spermatozoa/mL.(AU)

Até o momento, não foram realizados estudos que avaliassem o efeito da concentração de espermatozoides/mL em palhetas (0,5mL) para a criopreservação, levando-nos a analisar esta questão. Cada fração-rica do ejaculado (n=25) foi diluída em cinco diferentes concentrações de espermatozoides (100, 200, 300, 600 e 800x106 espermatozoides/mL), envasadas em palhetas de 0,5mL e posteriormente congeladas. Após a descongelação, os espermatozoides de todos os tratamentos foram avaliados a fim de determinar as características de motilidade usando um sistema de análise computadorizada dos espermatozoides (SCA-CASA). A integridade das membranas plasmática e acrosomal, o potencial de membrana mitocondrial, a peroxidação lipídica e a fluidez da membrana foram analisadas por citometria de fluxo. O aumento na concentração de espermatozoides acima de 300x106 espermatozoides/mL diminuiu (p<0,05) a motilidade total e progressiva, velocidade curvilínea, velocidade linear, linearidade e frequência de batimento. No entanto, a integridade da membrana plasmática e acrosomal, potencial de membrana mitocondrial, peroxidação lipídica e fluidez de membrana não foram influenciados (p>0,05) por altas concentrações de espermatozoides durante a criopreservação. Portanto, a fim de melhorar a sobrevivência dos espermatozoides suínos e a motilidade total e progressiva após a descongelação, os espermatozoides suínos devem ser congelados a concentrações não superiores a 300x106 espermatozoides/mL.(AU)

Effect of various levels of dissolved oxygen on reactive oxygen species and cryocapacitation-like changes in bull sperm.

The present investigation was carried out to study the effect of various levels of dissolved oxygen (DO) on reactive oxygen species (ROS) and cryocapacitation-like changes in bull sperm. Egg yolk-Tris-glycerol (EYTG) extender was split into four subextenders; viz., Extender I (control; no flushing with liquid nitrogen (LN2 )), Extender II, Extender III and Extender IV were flushed with LN2 for 40, 16 and 8 min, respectively. The DO levels were standardized to 11.7, 2, 4 and 8 ppm, respectively, in control (Extender I), Extender II, Extender III and Extender IV. Ejaculates with mass motility of ≥ 3+ were divided into group I (diluted with Extender I), group II (diluted with Extender II), group III (diluted with Extender III) and group IV (diluted with Extender IV) up to 80 × 106  sperm/ml. Extended semen samples were packed in French mini straws (0.25 ml), equilibrated and cryopreserved. Semen samples were evaluated at prefreeze and post-thaw stage for various parameters (DO, progressive motility (PM), viability (VIB), acrosomal integrity (AI), hypo-osmotic swelling (HOS) test, ROS, cholesterol (C) and phospholipid (P). The percentage of PM, VIB, AI, HOS test, cholesterol (C) and phospholipid (P) levels, and capacitated sperm were significantly (p < 0.05) higher in groups III and IV as compared to groups I and II. However, the acrosome-reacted sperm (%; pattern AR) were significantly (p < 0.05) decreased in group III as compared to all other groups. Besides the proportion of sperm displaying tyrosine-phosphorylated pattern, EA (fluorescence at both equatorial and anterior acrosomal regions, i.e. high capacitation level) was significantly (p < 0.05) reduced in group III compared to all other groups. In conclusion, varying DO levels in the extender significantly affect sperm quality, ROS production and capacitation-like changes in bulls.

Effect of reduced glutathione supplementation in semen extender on tyrosine phosphorylation and apoptosis like changes in frozen thawed Hariana bull spermatozoa.

To provide new insights into the mechanisms through which reduced glutathione (GSH) is able to protect spermatozoa, we tested the hypothesis that cryocapacitation and apoptosis like changes can contribute to the negative effect of freezing and thawing on bull spermatozoa, and that GSH prevent this damage. Having known protective effects of GSH in terms of a potent antioxidant, we evaluated capacitation, tyrosine phosphorylation and apoptosis like changes in bull spermatozoa after freezing and thawing in egg yolk tris glycerol extender containing (0.5m M-GSH-T1 & 1mM GSH-T2) and without GSH serving as the control (C). Forty ejaculates were collected from four Hariana bulls and were pooled due to non significant variations among the bull ejaculates for the evaluation of sperm attributes. Capacitation like changes, tyrosine phosphorylation, localization of tyrosine phosphorylated proteins, apoptosis like changes in terms of mitochondrial transmembrane potential and DNA fragmentation after final dilution, 4h of equilibration at 4°C and 24h after freezing and thawing were evaluated. GSH supplementation at 0.5mM showed significant reduction in B- and AR- pattern spermatozoa during all stages of semen freezing and thawing. Immunoblot revealed six proteins which were tyrosine phosphorylated and protein of 30 and 75kDa (p30, p75) were the major tyrosine phosphorylted proteins. On further analysis, the p30 showed differential variation in intensity in all the three groups after freezing and thawing. Positive immune reactivity for tyrosine phosphorylated proteins was found in neck, middle piece and post-acrosomal regions of spermatozoa. Addition of 0.5mM GSH decreased percentage of spermatozoa showing fragmented DNA and increased the percentage of spermatozoa having high transmembrane mitochondrial potential (P<0.05). This study demonstrates that GSH favours survival of bull spermatozoa by interfering with apoptotic and cryocapacitation pathways, and thereby protects the spermatozoa from deleterious effects of cryopreservation. The findings of the study indicated that GSH at 0.5mM can be effectively used as an additive in bull semen extender for freezing and thawing.

Effect of cholesterol loaded cyclodextrin supplementation on tyrosine phosphorylation and apoptosis like changes in frozen thawed Hariana bull spermatozoa.

The beneficial effects of cholesterol loaded cyclodextrin (CLC) addition were evaluated in cryopreserved bull semen. Forty ejaculates were collected from Hariana bulls (n = 4), pooled and divided into 4 aliquots. All the aliquots were initially diluted in to egg yolk tris citrate and supplemented with CLC @ 0.5 mg (Group-II), 1.0 mg (G-III) and 2.0 mg (G-IV) CLC/120 × 106 spermatozoa or without CLC (G-I) that served as control. Extended semen was cryopreserved at -196 °C for 24 h. Seminal attributes like motility, viability, cryocapacitation like changes, tyrosine phosphorylation, apoptosis like changes in terms of mitochondrial transmembrane potential and DNA integrity were evaluated after equilibration and thawing. Results showed a significant increase in the motility, viability and acrosome intact spermatozoa in Group II as compared to other three groups. Further, the proportion of spermatozoa showing capacitation and acrosome reaction was also decreased (P < 0.05) significantly in Group II as compared to Group I, III, and IV. Immunoblot demonstrated a 32 kDa (p32) protein showing differential variation in the band intensity in all the four groups being lower in Group II. Further, the immunolocalization study revealed positive immune reactivity for tyrosine phosphorylated proteins at middle piece and neck (high fluorescence), post-acrosomal region (medium fluorescence), and principal piece (low fluorescence) of spermatozoa. Addition of CLC significantly increased (P < 0.05) the percentage of spermatozoa showing high transmembrane mitochondrial potential, and also, CLC @ 0.5 mg/120 × 106 in semen extender significantly decreased (P < 0.05) spermatozoa showing fragmented DNA after thawing as compared to control. Results of the present study indicate beneficial effects of CLC supplementation on cryodamage of spermatozoa by reducing the cryocapacitation and apoptosis like changes.

Cholesterol-loaded cyclodextrins prevent cryocapacitation damages in buffalo (Bubalus bubalis) cryopreserved sperm.

The aim of this study was to investigate the effect of cholesterol-loaded cyclodextrins (CLC) on motility, viability, capacitation status, and in vivo fertility of buffalo frozen-thawed sperm. After the initial semen assessment, buffalo sperm were diluted in BULLXcell extender containing 0- (control), 1.5-, and 3-mg/mL CLC and cryopreserved. At thawing, sperm motility was evaluated by phase contrast microscopy, and viability-capacitation status was assessed by Hoechst 33258-chlortetracycline (CTC) assay. Capacitation status was also evaluated by an indirect immunofluorescence assay to localize phosphotyrosine-containing proteins. Moreover, buffaloes were artificial inseminated to assess the in vivo-fertilizing potential of CLC-treated semen. No differences among control, 1.5-, and 3-mg/mL CLC-treated groups were recorded in both sperm motility (66.5 ± 5.6, 68.8 ± 4.8, and 68.8 ± 4.8, respectively) and viability (86.5 ± 1.9, 87.6 ± 1.5, 88.4 ± 2.3, respectively). However, the extender supplementation with CLC significantly reduced sperm cryocapacitation. Indeed, CLC treatment decreased (P < 0.01) the proportion of sperm showing the CTC pattern B (capacitated sperm) compared with the control (69.6 ± 3.4, 37.8 ± 1.5, and 51.3 ± 4.7, respectively, with 0, 1.5-, and 3-mg/mL CLC; P < 0.01). Furthermore, the percentage of sperm displaying tyrosine-phosphorylated pattern EA (i.e. high capacitation level) was reduced (P < 0.01) in both CLC-treated groups (10.8 ± 3.3 and 5.6 ± 1.6, respectively, with 1.5- and 3-mg/mL CLC) compared with the control (37.3 ± 6.9), reaching values similar to those recorded in fresh semen (11.0 ± 3.5). In addition, treating sperm with 3-mg/mL CLC increased (P < 0.01) the percentage of nonfluorescent (pattern NF), i.e., non-capacitated sperm (41.8 ± 3.6) compared with fresh semen (11.0 ± 6.9). No differences were recorded in pregnancy rates at 60 days post-artificial insemination among control, 1.5- and 3-mg/mL CLC groups (59.7%, 65.6%, and 56.9%, respectively). In conclusion, CLC treatment of buffalo sperm strongly decreases sperm cryocapacitation damages, without affecting the in vivo fertilizing capability.

Resveratrol prevents capacitation-like changes and improves in vitro fertilizing capability of buffalo frozen-thawed sperm.

The aim of this study was to evaluate the effect of resveratrol supplementation of semen extender on fertility parameters of frozen-thawed buffalo sperm. After the initial semen assessment, buffalo semen was cryopreserved in BioXcell containing 0 (control group), 0.5, 1, 10, and 50-µM resveratrol. After thawing, viability, motility, and capacitation status (assessed by localization of phosphotyrosine-containing proteins) were evaluated. Based on the results of the dose-response trial, the concentration of 50 µM was selected for further assessments, such as membrane integrity, total antioxidant capacity, reactive oxygen species, and lipid peroxidation (LPO) levels. Moreover, in vitro fertilizing ability by heterologous IVF and in vivo fertility were assessed. No differences among groups were recorded in sperm motility and viability (on average 52.3 ± 2.1% and 76.6 ± 1.3%, respectively). However, data showed a resveratrol dose-dependent effect on sperm capacitation status, with a significant reduction of the cryopreservation-induced capacitation with the higher concentrations tested. In particular, both 10- and 50-µM resveratrol increased (P < 0.01) the percentage of sperm displaying pattern A (low capacitation level), but treatment with 50-µM resveratrol also decreased (P < 0.01) the proportion of sperm exhibiting pattern EA (high-capacitation level) compared with the control. Interestingly, supplementation of semen extender with resveratrol increased membrane integrity, indicated by the higher percentage of hypo-osmotic swelling positive sperm (55.6 ± 0.6 vs. 48.4 ± 0.7; P < 0.01), and total antioxidant capacity (1.36 ± 0.01 vs. 1.32 ± 0.02 mM/L; P < 0.05) compared with the control. Intracellular reactive oxygen species decreased in resveratrol-treated sperm compared with the control, as indicated by dihydroethidium values (0.17 ± 0.01 and 0.22 ± 0.01 µM/µL dihydroethidium, respectively; P < 0.01). Moreover, when IVF was carried out by using semen treated with 50 µM resveratrol, the normal fertilization rate considerably improved (60.8%, P < 0.05) compared with the control (51.3%). However, no differences were recorded in pregnancy rates at 60 days post-AI with resveratrol-treated semen (50 µM) compared with the control (48.7 vs. 46.5%, respectively). In conclusion, the inclusion of 50-µM resveratrol in the extender decreases capacitation-like changes and oxidative stress, improving membrane stability and in vitro fertilizing ability of buffalo semen.

Spermine reduces reactive oxygen species levels and decreases cryocapacitation in canine sperm cryopreservation.

The objective of this study was to determine the ability of spermine to act as an antioxidant in scavenging reactive oxygen species (ROS), maintaining sperm function and decreasing cryocapacitation after cryopreservation. Although motility did not increase with spermine treatment, values for membrane integrity were significantly increased (P < 0.05). Higher percentages of linearity and straightness with a lower amplitude of lateral head displacement (ALH) indicated that spermine inhibits hyperactivation. Concentrations of intracellular and extracellular ROS were decreased in the treatment group (P < 0.05). Higher expression of an anti-apoptotic gene (Bcl-2) and lower expression of a pro-apoptotic gene (Bax), together with decreased expression of the mitochondrial ROS modulator ROMO1, DNA repair due to oxidative damage (OGG1), spermine synthase (SMS), NADPH oxidase associated with motility (NOX5) and spermine amino oxidase (SMOX), all showed that 5.0 mM spermine treatment was beneficial to spermatozoa. Furthermore, the proportion of live spermatozoa with intact acrosomes after thawing in the treatment group was higher than in the control. After incubation in canine capacitating medium, numbers of live capacitated spermatozoa with reacted acrosomes were higher than in the control. Our results indicate that 5.0 mM spermine is an optimal concentration for maintaining sperm function, reducing ROS production, preventing apoptosis and adverse effects of cryocapacitation during canine sperm cryopreservation.