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.

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.

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.

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 (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.

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.

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.

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.

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.

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.