Bio-based aromatic diamines from vanillin substrate were successfully synthesized and characterized. These amines, i.e., methylated divanillylamine (MDVA) and 3,4-dimethoxydianiline (DMAN), were then tested as curing agents for the design of bio-based epoxy thermosets. The epoxy thermosets obtained from these novel vanillin-based amines exhibited promising thermomechanical properties in terms of glass transition temperature and char residue. Epoxy thermosets are the products of the reaction between epoxy-based monomers or prepolymers with curing agents. The cross-linking agents are significant in terms of mass fraction as the latter can represent up to 50% of the formulations. Many efforts are currently undertaken to develop bio-based alternatives to traditional epoxy monomers derived from fossil resources and, in particular, bisphenol-A . 1H NMR spectrum of this orange solid demonstrated the disappearance of oxime signals at 8.10 and 11.13 ppm and the appearance of a new signal at 3.63 ppm, corresponding to the benzylic-protons of the amine . Thereafter, the hydrolysis of the methylated diester with sodium hydroxide yielded the diacid (>90%). The disappearance of the methyl ester protons at 3.38 ppm and the appearance of acid proton signals of carboxylic acid at 9.20 ppm were attributed to the formation of the methylated divanillic acid. Methylated divanillic acid was then converted into acyl azide in a two-step reaction. Ethyl chloroformate was first reacted with the acid to form in situ an acyl chloride and sodium azide was then added to the mixture, yielding the corresponding acyl azide (>60%). Finally, the di-isocyanate was obtained in good yield (>80%) by simply heating the azide compound in dry toluene. The synthesis of these latter compounds was confirmed by 1H NMR spectroscopy  and 2,500–3,300 cm−1 (O-H), acyl azide at 2,140 cm−1 (-N3), and isocyanate at 2,278 cm−1 (-N = C = O).In addition, acyl azide structure was confirmed by the disappearance of carboxylic signal at 9.20 ppm and finally the shift of the aromatic protons from 7.61 and 7.55 to 6.65 and 6.58 ppm was attributed to the so-formed di-isocyanate. These compounds were also characterized by FTIR spectroscopy . Infrare1H NMR spectroscopy of the reaction mixture revealed the presence of a by-product  was recovered by hydrolysis in basic conditions of the corresponding di-isocyanate. After extraction with ethyl acetate and washing with water a mixture of brown and white solids was obtained. However, -product . These atroscopy . 1H NMR troscopy . The folThe previously synthesized di-amines, i.e., methylated divanillylamine (MDVA) and 3,4-dimethoxydianiline (DMAN) were used as curing agents for the synthesis of epoxy thermosets.The DGEBA epoxy monomer was stirred vigorously with a stoichiometric amount of MDVA. The thermomechanical properties of the so-formed network were characterized by DSC and TGA. Bio-based thermoset was then compared with DGEBA/IPDA epoxy system. Results are summarized in the DGEBA epoxy monomer was also cured with bio-based DMAN. However, in view of the small amount recovered, crude DMAN was used for the curing reaction. Thermomechanical properties of the network formed were then determined and compared with DGEBA networks cured with 4,4′-Diaminodiphenyl sulfone (DDS). Results are summarized in At last, a bio-based polyglycidylether, tetraglycidylether of divanillyl alcohol (TetraGEDVA),  and the thermomechanical properties of thermosets obtained from epoxy precursors cured with MDVA were investigated. DGEBA epoxy prepolymers were thus cured with MDVA and the networks obtained were compared with the conventional DGEBA/IPDA system. Unfortunately, the characterization by DSC showed a weak exothermic peak, corresponding to the reaction of amine with epoxy, but no clear glass transition temperature could be observed with this system.Then, the 3,4-dimethoxyaniline (DMAN) was synthesized using the oxidative rearrangement of Curtius. From methyl divanillate and through the synthesis of acyl azide and isocyanate intermediates, the hydrolysis of this latter yielded 3,4-dimethoxyaniline (DMAN). However, the hydrolysis step was tedious and the corresponding amine failed to be isolated efficiently. Nevertheless, crude DMAN was used as curing agent in the polyaddition reaction with DGEBA. Interestingly, the so-formed semi-biobased thermoset exhibited a glass transition temperature of 176°C against 204°C for the conventional DGEBA/DDS system. Moreover, the DMAN permitted to double the char content of the network comparing to DGEBA/DDS network cured in the same conditions. In addition, a fully bio-based epoxy thermoset was obtained by curing tetraglycidylether of divanillyl alcohol (TetraGEDVA) with DMAN. The thermomechanical properties obtained were promising as the epoxy thermoset exhibited a glass transition temperature of 212°C and a char residue of 48%.Finally, new amine-type curing agents from vanillin-based starting material was successfully attempted and characterized. However, some further optimizations in the synthetic pathways described are still necessary to have a better appreciation of the potential of these new bio-based aromatic diamines in the course of thermoset designs.All datasets generated for this study are included in the manuscript/ES performed the synthesis of the chemicals and performed the polymerization experiments. CL performed the thermo-mechanical analysis. EG, SG, BD, and HC contributed equally to the supervision of the project.BD was employed by ArianeGroup company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Efficient enhancement of the toughness of epoxy resins has been a bottleneck for expanding their suitability for advanced applications. Here, polysulfone (PSF) was adopted to toughen and modify the epoxy. The influences of PSF on the mechanical and thermal properties of the epoxy resin were systematically studied by optical microscopy, Fourier transform infrared spectrometer (FT-IR), differential scanning calorimetry (DSC), thermogravimetric analyzer (TG), dynamic mechanical thermal analyzer (DMA), mechanical tests and scanning electron microscope (SEM). The dissolution experimental results showed that PSF presents a good compatibility with the epoxy resin and could be well dissolved under controlled conditions. The introduction of PSF was found to promote the curing reaction of the epoxy resin without participating in the curing reaction and changing the curing mechanism as revealed by the FT-IR and DSC studies. The mechanical properties of PSF/epoxy resin blends showed that the fracture toughness and impact strength were significantly improved, which could be attributed to the bicontinuous phase structure of PSF/epoxy blends. Representative phase structures resulted from the reaction induced phase separation process were clearly observed in the PSF/epoxy blends during the curing process of epoxy resin, which presented dispersed particles, bicontinuous and phase inverted structures with the increase of the PSF content. Our work further confirmed that the thermal stability of the PSF/epoxy blends was slightly increased compared to that of the pure epoxy resin, mainly due to the good heat resistance of the PSF component. Epoxy resins (EP) have been widely used in construction, machinery, aerospace and other related fields due to their low cost, excellent bonding performance, outstanding mechanical properties, easy processability, dimensional stability, superior thermal and chemical resistance ,2,3. UnfToughness modification of epoxy resins by rubber elastomer ,7 is ver1/2, which was an improvement of nearly 69.23% when the phase structure of the PSF/DGEBA composites with PSF content of 10 wt% was bi-continuous. Tanaka et al. [The frequently-used thermoplastics consist of poly(ether-sulfone) (PES) ,15,16,17a et al.  synthesiIn this work, PSF resin was used as a toughening agent in the modification of bisphenol A epoxy resin. The effect of PSF on the curing behavior of the epoxy resin at different heating rates was systematically studied and the curing mechanism of the epoxy resin in the presence of PSF was analyzed. The results indicated that the addition of PSF promoted the curing reaction of epoxy resin but does not change its curing mechanism. The toughening of the resultant resin by PSF and the effective improvement in the mechanical properties can be ascribed to the absorption of external energy by different phase separation structures formed by curing induction of the epoxy resin. Relatedly, our studies also revealed that the addition of PSF also improved the heat resistance of the epoxy resin.Bisphenol A epoxy resin (E51) with epoxy equivalent 182–192 g/mol, was obtained from Baling Petrochemical Co. Ltd. . Polysulfone (PSF) (Udel P1700), with a particle size of ca. 80 μm, from the Acomo Co., Ltd. . The curing agent was Dietheyltoluene diamine(DETDA) (Ethacure 100), supplied by the Albemarle Corporation . 1, 4-epoxy-butane (THF) was purchased from Sinopharm Chemical Reagent Co., Ltd.  and used as solvent.The PSF powder was first dissolved in the epoxy resin at 130 °C until the mixed system was completely clarified. Then an appropriate amount of curing agent was added into the mixed system, after which the solution was cooled to 80 °C, followed by mechanical stirring until a uniform mix was obtained. Finally, the mixed system was vacuum defoamed in an oven at 80 °C until there were no more visible bubbles. The mixed system was transferred to a mold coated with release agent and placed in an oven at 120 °C for 4 h. After cooling and demolding, cured product of PSF/epoxy composite were obtained. According to the different weight ratios of PSF to epoxy resin, the samples were recorded as 0, 5, 10, 15 and 20 phr, respectively.Compared to other methods, involving thermoplastic resins would improve the toughness of the EP without reducing its modulus and heat resistance . MeanwhiThe tensile strength and flexural strength/modulus tests were performed following ASTM D638-10 and ASTM D5045-14 Standards. For a typical Flexural strength and modulus test, specimen dimensions were 127 mm × 12.7 mm × 3.2 mm. The fracture toughness of the PSF/epoxy blends was determined by a single-edge notched 3-point flexural test (SENB) according to ASTM D5045-14. Rectangular specimens of 44 mm × 10 mm × 5 mm with a 5 mm length notch were prepared for measurement. A WANCE 203 B-TS micro computer controlled electromechanical universal testing system was applied to perform these measurements. Izod pendulum impact tests were performed based on ASTM D256-10 standard under impact speed and impact energy of 3.5 m/s and 5.5 J, respectively. The sample sizes was 63.5 × 12.7 × 6.35 mm and each sample possessed a constant notch. The fractured surfaces of the cured PSF/epoxy blends with and without etching were analyzed in detail with a Hitachi SU8010 Scanning Electron Microscope  system. The surfaces were etched with 1,4-epoxybutane (THF) for 24 h at room temperature to remove the thermoplastic phase. Following that the specimens were dried in vacuum overnight at 100 °C.−1) from 20 to 350 °C at a heating rate of 5, 10, 15 and 20 °C/min, respectively. The glass transition temperatures (Tg) of the PSF/epoxy blends were explored by a TA Q20 differential scanning calorimetry (DSC) instruments from 40 to 250 °C at a heating rate of 10 °C/min under a nitrogen flow (50 mL/min−1). Thermogravimetric analyzer  was used to determine the thermal stability of PSF/epoxy. The samples were carried out at 20 °C/min from room temperature up to 900 °C in nitrogen atmosphere.The curing kinetics of epoxy resin and epoxy/PSF blends were measured using a NETZSCH 204F1 Differential Scanning Calorimetry . The specimens were heated under a flow of nitrogen , the final curing temperature(Tf) and the highest curing temperature rate (Tp) gradually increased. Moreover, the shape of the exothermic peak gradually became sharp. A similar trend was observed for PSF/epoxy systems with different PSF content, which can be ascribed to the steady improvement of thermal inertia, with an increase in the heating rate. In addition, the values of Ti, Tf and Tp reduced with the increase in PSF content, which suggests that adding PSF into epoxy resin promotes the curing reaction by reducing the activation energy. This result can be traced to the gradual enhancement of the exothermic lag of the curing reaction on the addition of PSF.It is well known that the curing behaviors of epoxy resin are essentially related to the properties of the cured materials. Thus, the effect of PSF on the curing behaviors of epoxy resin was analyzed by DSC as shown in E) and reaction order (n) are crucial kinetic parameters in the curing process and thus the theoretical basis to control the curing process of epoxy resin. ΔE directly reflects the degree of difficulty of the curing reaction, which can be calculated according to the Kissinger method [n) corresponding to the complexity of the reaction can be used to estimate the curing reaction mechanism. It is quantified according to the Crane equation [β is the heating rate(°C/min), Tp is the peak temperature, R is the ideal gas constant which is 8.314 J/(mol·K), E is the activation energy (kJ/mol) and n is the reaction order.Apparently, the activation energy  of the PSF/epoxy system with different PSF content at different heating rates β was substituted into the Equations (2) and (3). After that, the ln(β/Tp2) and lnβ were plotted as a function of 1000/Tp, shown in 2 of the two linear fittings all reached 0.99 or higher, indicating an excellent fitting.According to the Kissinger and the Crane equations, the peak temperature  [n) of each system was stable between 0.87 and 0.88, demonstrating that the addition of PSF does not change the curing reaction mechanism of epoxy resin.Based on the Equations (2) and (3), the calculated values of Δ groups) , which m−1 is the characteristic absorption of epoxy group while the 1623 cm−1 is the N–H characteristic peak in the curing agent E100 [−1 vanished totally in the PSF/epoxy blends after curing indicating the completion pf the cure reaction. The peak at 1154 cm−1 is the stretching vibration peak of the sulfone group [Various outstanding properties of epoxy resin were obtained by their fully crosslinked structures. Here, in order to elucidate the influence of PSF on the chemical structures of the cured epoxy resin, FT-IR analysis was conducted on the PSF, the neat epoxy resin before curing and the cured PSF/epoxy resin blends as given in ent E100 . Both 91ne group  and it cTg of the blends. We also notice that the Tg of PSF/epoxy blends is smaller than that of pure epoxy resin and it increases with the increase in PSF content. This can be explained in terms of the added PSF reducing the curing degree of the epoxy resin, which results in a decrease in the storage modulus and Tg of PSF/epoxy blends [Tg of PSF/epoxy blends increasing with the increase in the PSF content may have been caused by the higher storage modulus Tg of PSF.The dynamic mechanical thermal analysis gave more insight into their viscoelastic properties as well as the morphologies of PSF/epoxy blends. The variations of the storage modulus of the PSF/epoxy blends after curing with temperatures is shown in y blends . Both thIn this work, PSF was also used to improve the mechanical and themal properties of epxoy resin. Therefore, the mechanical propeties including the tensile properties, flexural properties, fracture toughness and impact strength of the cured cured PSF/epoxy blends were illustrated against the varation of PSF content carefully. The flexural strength and flexural modulus of PSF/epoxy blends is shown in ICK) was adopted to inquire into the mechanical interfacial properties or the fracture toughness of the epoxy/PSF blends. The value of ICK was determined based on the ASTM D5045-14 standard as follows:P is the loading weight, B is the thickness of the specimen, W is the depth(width) of the specimen, a is the crack length, x is the ratio of the crack length to the depth of specimen, a/W and f(x) is the calibration factor which is represented as follows:The critical stress intensity factor (ICK) of the PSF/epoxy blends was explored carefully as displayed in ICK of the blend containing 15 phr PSF indicates a maximum fracture toughness. Still increasing the PSF content to 20 phr leads to a slightly smaller ICK than that of 15 phr PES, nevertheless, still displayed a distinguished enhancement in fracture toughness in comparison with the pure epoxy resin (0 phr).The impact of the PSF content on the fracture toughness in terms of critical stress intensity factor . Thus, the PSF/epoxy blends were etched with THF to remove the PSF phase giving more insights into the phase structures of the two components. The resulting morphologies were observed by SEM and displayed in The representative phase structures of PSF/epoxy blends could be ascribed to the reaction induced phase separation during curing process of epoxy resin which is related to the content of PSF within the resin system . Before Considering the variations of the mechanical properties of the PSF/epoxy blends, it can be contemplated that the dispersion of the thermoplastic structures within the epoxy resin formed by phase separation plays an important role in the performance of the epoxy resin. The decrease of the tensile strength and modulus with the introduction of the PSF content, arrayed in In contrast, the uniformly dispersed PSF particles in the epoxy resin matrix as shown in As illustrated by Tg) of the PSF/epoxy cured products with different PSF contents. As shown in Tg of the epoxy resin. This might be due to the macromolecular chain of PSF hindering the contact between the epoxy resin and the curing agent, thus lowering the curing degree of the epoxy resin. Meanwhile, the addition of PSF raises the viscosity of the system, which results in an incomplete curing reaction [Tg of the PSF/epoxy composites was lower than that of the pure epoxy resin. However, when the PSF content increased, the Tg of the PSF/epoxy composites increased as a whole. We attribute this to the fact that polysulfone inherently possesses a higher Tg, which could have offset its influence on the curing degree of the epoxy resin. The results of the DSC curve are in good agreement with those from the DMA results.In order to study the impact of the PSF on the thermal properties of the epoxy resin, DSC was used to analyze the glass transition temperature  and integrated program decomposition temperature (IPDT) are calculated on the basis of TG curve, as shown in xy resin .In summary, PSF was employed in this work to improve the mechanical and thermal properties of the epoxy resin. Specifically, the solubility of PSF in epoxy resin and the influence of PSF on the curing process, the chemical structures, the mechanical properties, the phase structures, viscoelastic properties and thermal performance of PSF/epoxy blends were investigated carefully. The dissolution experiment indicated that PSF had good compatibility with the epoxy resin and could be well dissolved into it to prepare the PSF/epoxy blends. The DSC results revealed that the addition of PSF does not upset the curing mechanism but promoted the curing of the epoxy resin. The addition of PSF lowered the reaction enthalpy thus promoted the curing process of the epoxy resin. In fact, PSF did not actively participate in the curing reaction as illustrated by the FT-IR measurements.The systematic studies of the mechanical properties of the PSF/epoxy blends illuminated that the addition of PSF could significantly enhance the flexural properties, the fracture toughness and impact strength of epoxy resin although it slightly reduced the tensile properties. The improvement of the mechanical properties could be attributed to the representative phase structures resulting from reaction induced phase separation, presenting dispersed particles, bicontinuous and phase inverted structures mostly depending on the PSF content within the blends. The analysis of the results further revealed that the bicontinuous phase morphology obtained during the curing process of the epoxy resin most optimized the mechanical properties. Meanwhile, due to the high heat resistance of PSF components, the thermal stability of the PSF/epoxy blends was also slightly increased compared to that of the pure epoxy resin.These findings present an interesting benchmark for further understanding the effects, mechanisms and phenomena that surround the phase structure and curing behavior of PSF and epoxy resins. Also, as an example of the modification strategies of epoxy resins, this work reveals that treatment with PSF not only enhances the overall toughness but also maximizes other important properties and tunes the resin for more sophisticated applications.


Athletes with sport-related concussions (SRC) often demonstrate deficits in postural stability. Lower cerebral blood flow in frontal cortices has been documented in athletes with symptoms after SRC, however, it is unclear if functional brain oxygenation during postural control tasks is reduced in symptomatic athletes after SRC in the same manner. We therefore compared brain oxygenation patterns in frontal cortices of symptomatic and asymptomatic athletes with SRC during postural control tasks with the hypothesis that symptomatic athletes are characterized by reduced functional brain oxygenation during postural control.n = 31 symptomatic, n = 31 asymptomatic) were investigated during four postural control tasks with eyes closed versus eyes opened conditions and stable vs. unstable surface conditions. Brain oxygenation was assessed using functional NearInfraRed Spectroscopy (fNIRS) on frontopolar cortices of each hemisphere. Postural sway was measured by the analysis of ground reaction forces.62 concussed athletes (2) within the left hemispheric frontopolar cortex were significantly reduced in symptomatic athletes when compared to asymptomatic athletes during the eyes closed condition. A stepwise linear regression analysis revealed that self-reported post-concussion symptoms such as headaches and sadness predict decreased brain oxygenation during postural control with closed eyes.Symptomatic athletes showed greater postural sway when compared to asymptomatic athletes during postural control, particularly during closed eyes and/or unstable surface conditions. Changes of oxygenated hemoglobin (∆HbOSymptomatic athletes with increased postural sway are characterized by decreased frontopolar brain oxygenation during postural control tasks, particularly during conditions with closed eyes. Because the frontopolar cortex showed to be involved in redistributing executive functions to novel task situations, we conclude that athletes with post-concussion symptoms suffer from a deficit in coordinating postural adjustments to balance control tasks with reduced sensory input. Potential post-concussion health deficits concern symptoms such as headaches, dizziness, memory problems, etc. that usually last for about a week . HoweverIn concussed athletes who were already cleared for sport participation, the application of electroencephalography (EEG) revealed a decrease in EEG power in all bandwidths especially during standing postures . The com22.162 active athletes with a history of SRC  from various sports  from local sports-clubs participated in the study as part of a concussion assessment protocol of the German Sports University. Written informed consent was obtained from each participant. The local Ethics Committee of the GSU approved the study.2.2Participants were clinically questioned using a standardized questionnaire to obtain the athletes’ sports participation, age, education, and the occurrence of a mild traumatic brain injury as defined by the recent consensus statement on concussion in sport , time po2.3Participants performed a working memory task, which had proven effective in the investigation of functional abnormalities of concussed athletes . During 2.4eyes opened (c1); condition 2: eyes closed (c2). The two conditions (c1 and c2) were performed either on a firm (/stable) surface or on an unstable surface: condition 3 (c3): eyes opened and unstable surface; condition 4 (c4): eyes closed and unstable surface. The unstable surface was created using a piece of six cm thick foam pad . Each balance condition comprised two blocks, each of which included three trials , resulting in a total of six trials per condition. The subjects were instructed to stand still on both feet (distance between feet: 2 cm) without losing balance in a standardized position and posture  the third parameter velocity (V) [mm/second] represents the mean velocity during the postural control trials per second. Mean parameters of the values of PL, SuA, and V were exported for each subject and condition for statistical analyses.During balance tasks, a force plate system  was used to register center of mass displacement  by measuring ground reaction forces. This system provides three parameters of information about the ability to keep postural control, i.e., (i) it registers the deviations from the Center of Pressure (CoP) by the mean length of the movement path per time [millimeters/second] ; PL is defined as the absolute length of the CoP path movements throughout the testing period; (ii) the second parameter surface area (SuA) [mm2.5Cerebral oxygenation changes were recorded during postural control tasks using a near-infrared optical tomographic imaging device . Methodology and underlying physiology are explained in detail elsewhere . A totalData were analyzed using the “nirslab” software package . 8 channels (ch) were converted to hemoglobin concentration changes according to 2.62), because these appear to reflect task-related cortical activation more directly than changes of deoxygenated hemoglobin, as evidenced by the stronger correlation between the former and the blood-oxygenation level- dependent signal measured by fMRI  concussed athletes with a PCS score > 10 (symptomatic), and (ii) concussed athletes with a PCS score < 10 (asymptomatic). Repeated within-subjects factors constitute visibility  with either opened eyes or (ii) closed eyes) and stability  on a stable or (ii) on an unstable surface). For fNIRS analyses, the additional within-subjects factor channels (8) was statistically calculated by uniANOVA. Significant results are reported from p < 0.05. Multiple post hoc pairwise comparisons were corrected with Bonferroni corrections. To determine a relationship of postural control, brain oxygenation, the PCS score, and post-concussion symptoms, we calculated a correlation  and (stepwise) regression analyses. Because the aim of the present paper is to better understand differences between symptomatic and asymptomatic athletes, we focus onto the effects between groups in the results section.Comparisons of the mean(s) (repeated (rmANOVA) and univariate (uniANOVA) analyses of variance) were performed using IBM SPSS statistics (Version 25). The parameters path length (PL), parameter surface area (SuA), and velocity (V) were used for statistical analyses of postural control. Statistical analyses of brain oxygenation data focused on the changes of oxygenated hemoglobin  and the response times during working memory performances (t(60) = −2.289, p < 0.05; M = 0.9 ± 0.9). Furthermore, symptomatic athletes (M = 1071.7 ± 162.8) showed significantly increased response times during the working memory task when compared to asymptomatic athletes (M = 981.8 ± 145.9).Significant differences between groups were found for the PCS score (t(60) = −9.703, 2.1.2groups  = 7.874, p < 0.01, η2 = 0.116), an interaction effect of group x visibility  = 7.093, p < 0.05, η2 = 0.106), group x stability  = 6.646, p < 0.05, η2 = 0.100), and of group x visibility x stability  = 5.096, p < 0.05, η2 = 0.078; The rmANOVA revealed for the parameter surface area (SuA) (but neither for path length nor velocity) significant differences between group effect revealed a significantly greater SuA for symptomatic athletes when compared to asymptomatic (p < 0.01). Post-hoc comparisons of the interaction effect of group x visibility revealed significantly greater SuA for symptomatic athletes when compared to asymptomatic during the sighted (p < 0.01) as well as during the condition with closed eyes  as well as during the unstable conditions (p < 0.01). Post-hoc comparisons of the interaction of group x stability x visibility revealed significantly greater SuA for symptomatic athletes when compared to asymptomatic during the stable condition with closed eyes (p < 0.01), and during the unstable condition with opened (p < 0.05) and closed eyes (p < 0.01).Post-hoc comparisons of the 2.1.3eyes opened and stable surface condition (rs(62) = 0.306, p < 0.05), during the eyes closed and stable surface condition (rs(62) = 0.376, p < 0.01), during the eyes opened and unstable surface condition (rs(62) = 0.282, p < 0.05), and during the eyes closed and unstable surface condition (rs(62) = 0.275, p < 0.05). A following stepwise linear regression analysis with the PCS score as the dependent variable and the significantly correlated SuA parameters as independent variables revealed significance  = 9.893, p < 0.01, R2 = 0.142), i.e., the SuA during the eyes closed and stable surface condition significantly predicted the PCS score  PCS score and worse postural control by increased mean surface areas during the 2.1.4eyes closed condition and increased) response times during the working memory task (rs(62) = 0.279, p < 0.05).It exists a significant positive correlation between (increased) mean surface area during the 2.1.5group x visibility  = 3.071, p < 0.05, η2 = 0.317). The uniANOVA showed significant effects above the right and left hemispheric (LH) frontopolar cortex (FPC) for the interaction of group x visibility for channel 1  = 4.799, p < 0.05, η2 = 0.074), ch4  = 7.215, p < 0.05, η2 = 0.107; p = 0.07, η2 = 0.05; 2 in symptomatic when compared to asymptomatic athletes during the condition with closed eyes in all three channels, however, only in channel 4 post-hoc comparisons reached significance (p < 0.05).The rmANOVA revealed a significant effect for the interaction of 2.1.6eyes closed condition in channel 4 (rs(62) = −0.345, p < 0.01; p < 0.01, R2 = 0.125), i.e., left hemispheric FPC (channel 4) during the eyes closed and stable surface condition significantly predicted the PCS score .It exists a significant negative correlation between the (increased) PCS score and the (decreased) brain oxygenation during the 2.1.72 in channel 4 during the eyes closed and stable surface condition and each post-concussion symptom revealed significance, i.e., the data showed a significant negative correlation between (decreased) ∆HbO2 in the LF FPC (ch4) and the (increased) symptoms headaches (rs(62) = −0.461, p < 0.001), pressure in the head (rs(62) = −0.276, p < 0.05), sensitivity to light (rs(62) = −0.316, p < 0.05), sensitivity to noise (rs(62) = −0.329, p < 0.01), difficulty remembering (rs(62) = −0.309, p < 0.05), fatigue or low energy (rs(62) = −0.267, p < 0.05), confusion (rs(62) = −0.276, p < 0.05), difficulty falling asleep (rs(62) = −0.376, p < 0.01), irritability (rs(62) = −0.352, p < 0.01), and sadness (rs(62) = −0.347, p < 0.01). A following stepwise linear regression analysis with ∆HbO2 in ch4 during the eyes closed and stable surface condition as the dependent variable and the significantly correlated symptoms as independent variables revealed significance  = 12.327, p < 0.001, R2 = 0.295), i.e., the (increased) symptoms headaches  and sadness  significantly predicted decreased ∆HbO2 in LF FPC (ch4) during the eyes closed and stable surface condition.A following correlation analysis of the ∆HbO4eyes closed condition, the unstable surface condition, and the combination of closed eyes and unstable surface condition. The SuA during the eyes closed and stable surface condition showed to predict the PCS score. The analysis of the fNIRS data revealed that symptomatic athletes are characterized by a lack of activation  in frontopolar cortices when compared to asymptomatic athletes during postural control with eyes closed on a stable surface. The symptoms headaches and sadness significantly predicted reduced ∆HbO2 in frontopolar cortices when controlling posture with closed eyes.The present study compared postural control performances in concussed athletes with and without post-concussion symptoms by analyzing postural sway and functional brain oxygenation in frontopolar cortices using fNIRS. Symptomatic athletes presented increased postural sway (/surface areas (SuA)) when compared to asymptomatic athletes overall balance conditions as well as during the 4.1In line with previous findings , the preThe present analyses also revealed that symptomatic athletes respond slower (/increased response times) during a working memory task when compared to the asymptomatic group. Increased symptoms post concussion have been previously been associated to increased response times . Further2 when compared to asymptomatic athletes) in frontopolar cortices when performing postural control tasks with closed eyes. When balancing with closed eyes, the attention of an individual shifts from external reference points towards the perception of proprioceptive information from the own body (The analysis of the fNIRS data revealed that symptomatic athletes are characterized by a lack of activation (i.e., reduced changes of ∆HbOown body . Marx etown body , the redown body . FPC-lesown body . Furtherown body . Thus, t4.2headaches and sadness predicted decreased brain oxygenation patterns in frontopolar regions during postural control conditions with closed eyes. Symptoms of fatigue or low energy have been reported previously to be related to balance deficits (sadness and headaches, must be taken into account as depressive patients have been characterized by decreased brain oxygenation in the frontal cortex as well (Further analyses revealed that increased self-reported symptoms such as deficits . It has deficits . Individdeficits . Thus, wdeficits  and vertdeficits . Becausedeficits , it has deficits . In factdeficits . Thus, ddeficits , i.e., m as well . Thus, f4.3Because sport-related concussions and potential long-term effects are a major concern in sports , it is oThe authors disclose no conflicts of interest.


Ugni molinae Turcz) on boar sperm stored at 17°C in order to reduce oxidative stress and improve sperm quality in the long term. Five experiments were performed: first, characterization of the polyphenol content from five genotypes of murtilla; second, determination of the genotype with the best antioxidant effect (MT-Ex); third, the antioxidant capacity on O2− and lipid peroxidation; fourth, the influence of MT-Ex on motility, calcium movement, cAMP, and metabolic parameters; and fifth, analysis of long-term refrigeration. The average phenolic content was 344 ppm; gallic acid, catechin, quercetin, myricetin, and kaempferol were detected. All extracts evaluated presented a concentration-dependent antioxidant effect. MT-Ex reduces intracellular O2−/peroxides but low lipid peroxidation. MT-Ex in nonstimulated ROS conditions reduces sperm motility, mitochondrial membrane potential, cAMP, and ATP, but the succinate dehydrogenase activity remained normal; also, we observed a reduction in calcium movement in in vitro sperm capacitation. The long-term analyses showed that MT-Ex improved sperm motility decay and reduced membrane damage and ROS at 168 h. Based on this study, we propose MT-Ex as a supplement in semen extenders.The production of reactive oxygen species (ROS) in boar spermatozoa increases in refrigeration; this can have an impact on sperm quality and fertilization capacity. We evaluated the effect of polyphenol-rich aqueous extract of murtilla ( The world pork market grows larger every year. Pork is considered a good and cheap alternative source of animal protein and a rich source of B complex vitamins. In the swine reproduction industry, artificial insemination (AI) is the best system for increasing the number of animals and decreasing the interval between generations, a combination of factors which improves breeding programs. One of the most used systems in the swine industry is the refrigeration of semen dosage and it is recommended to maintain sperm viability during transportation/storage, and its efficiency is determined by many factors including the medium, the semen extender used, and the refrigeration time and temperature usually 17°C) [7°C [1]. Boar spermatozoa have been described as very sensitive to thermal shock because the membrane has a high content of polyunsaturated fatty acids . In addiα-tocopherol, butylated hydroxytoluene, superoxide dismutase, catalase, cysteine, and glutathione, generates an improvement in boar sperm quality 3−, and NaOH were obtained from Merck. Luminol  and ATP were obtained from Sigma-Aldrich. Phosphate-buffered saline (PBS), Hank's buffered saline solution, and all fluorescent dyes were obtained from Thermo Fisher.Folin-Ciocalteu, NaUgni molinae Turcz.)  were obtained from the Agricultural Research Institute, Vilcún, Chile (INIA Carillanca). The fruits were harvested in April 2012 at the INIA Carillanca experimental station located close to Puerto Saavedra, Araucanía Region, Chile . Six grams of fruit of each genotype was ground in a mortar and transferred to a bottle containing 20 mL−1 of prewarmed (30°C) distilled water. The mixture was shaken in an incubator  at 170 rpm, 30°C, for 20 min and vacuum-filtered (Whatman N° 1 filter paper). The aqueous extract was stored under refrigeration, protected from light and oxygen, until each analysis.Fresh fruits of five genotypes of murtilla (μL) was mixed with distilled water (3.16 mL) in a test tube, and then 200 μL of Folin-Ciocalteu reagent was added. After 5 min at 20°C, 600 μL of 20% Na2CO3 was added to the reaction mixture, which was maintained at 20°C for 120 min in the dark. The absorbance was measured at 765 nm using a spectrophotometer , and the results were expressed as μg of gallic acid equivalent per mL (μgGAE mL−1) of aqueous extract.The total polyphenol content was determined using the Folin-Ciocalteu method as described by Alfaro et al. . Aqueousμm C18 RP Inertsil ODS-3 column  was used with a 250 mm × 4.60 mm i.d., maintained at 25°C. The original extract was concentrated to dryness using a rotary evaporator  at 140 rpm, 30°C, and then resuspended in 5 mL−1 of methanol : formic acid . The sample extract was filtered through a 0.45 μm filter, and 20 μL was injected for polyphenol analysis. The identification of compounds was confirmed both by comparison of their retention time with pure standards and by coinjection. A linear gradient solvent system consisting of 1% formic acid (A) and acetonitrile (B) was used at a flow rate of 1 mL−1 min as follows: 0-2 min, 100% A; 2-15 min, 80% A/20% B; 15-20 min, 70% A/30% B; 20-30 min, 40% A/60% B; and 30-35 min, 100% A. Phenolic compounds were detected at 280 nm 3− in 0.1 M of NaOH, pH 8) and immediately measured with an integration of 1000 milliseconds (ms). The data were compared with a control treated with H2O2 and without antioxidant treatment. Transformation of the X-axis to log10 was performed followed by a nonlinear regression (dose response) to determine the EC50 (effective concentration to reduce stress by 50%) of the antioxidant effect of each extract evaluated. The extract which presented the lowest dispersion of data was selected as the best antioxidant; subsequent experiments were executed with the murtilla extract selected, G14-4, hereafter “MT-Ex”.For antioxidant screening, we used the luminol assay  modified6 cells in MR-A semen extender. Each sample was incubated with 122.7 μM of H2O2 for 6 h at 17°C to stimulate ROS production, then centrifuged at 700g for 10 min. The resulting pellets were resuspended in the MR-A semen extender supplemented with 0.0001–100 μgGAE mL−1 of MT-Ex and incubated for 30 min to 6 h at 17°C. The antioxidant effect was evaluated following the Luminol protocol described above.The method was performed using 25 × 106 cells in the MR-A semen extender, divided into 4 groups: “control”—sperm samples incubated in the MR-A semen extender, “oxidant”—sperm samples treated with an oxidant agent, “MT-Ex”—sperm samples treated with 0.0315 μgGAE mL−1 as antioxidant treatment (concentration obtained in experiment 2), and “MT-Ex + oxidant”—sperm samples coincubated with oxidant agent and 0.315 μgGAE mL−1 as antioxidant treatment. The oxidant agent used was 122.7 μM of H2O2 to stimulate superoxide anion/hydrogen peroxide production and 100 μM of FeSO4 to stimulate lipid peroxidation. All groups were incubated for 30 minutes at 17°C.To detect the superoxide anion and lipid peroxidation, we used 25 × 10μM of DHE and 0.04 μM of SYTOX Green for 20 minutes at 37°C in the MR-A semen extender. At the end of incubation, the samples were suspended in 300 μL of prewarmed PBS (1x) and immediately analysed in a flow cytometer  using the spectra 488/583 (DHE) and 488/523 (SYTOX Green) analysing 10,000 events per sample. The data was analysed in Flowing Software 2.0, gating the SYTOX Green negative events (viable cells); the population was then assessed with high DHE fluorescence (viable cells with high ROS production).The capacity of the selected extract to reduce superoxide anion and hydrogen peroxide was measured using dihydroethidium (DHE) as a fluorescent indicator of ROS in combination with SYTOX Green as a membrane damage indicator . At the Membrane lipid peroxidation was determined using the BODIPY C-11 fluorescent sensor, which exhibits a red basal fluorescence in the nonoxidized state, while oxidation shows a green fluorescence. The method was carried out according to Aitken et al. with some modifications . Thirty The same groups described in experiment 3 were used in this experiment. We characterised sperm motility, succinate dehydrogenase activity, mitochondrial membrane potential, and ATP content.μgGAE mL−1, then 25 × 106 cells/ml were added to the solution and incubated for 30 minutes at 17°C. The second step was carried out with the same groups described in experiment 3. In both cases, the temperature was increased to 37°C five minutes before the end of incubation to recover sperm motility. Two microliters of sample was loaded in a slide chamber (Leja), and the motile, static, and progressive populations were analysed (%) in Automatic Sperm Analyzer System according to the following parameters: 45 frames at a frame rate of 60 frames/s; minimum contrast = 46; minimum cell size = 7 pixels; motility > 45 μm/s; progressive motility > 45 μm/s; and straightness > 45%.Sperm motility evaluation was performed in two steps: first, an assay to determine the concentration effect of MT-Ex on sperm motility. The MR-A semen extender was supplemented with 0 to 10 5 cells were incubated with 76.6 μg/mL of JC-1 and 0.5 μM propidium iodide for 5 minutes at 37°C. At the end of incubation, the cells were suspended in 300 μL of prewarmed PBS (1x) and immediately analysed in a flow cytometer (FACSCanto II BD) analysing 10,000 events per sample.The mitochondrial membrane potential was evaluated by a JC-1 probe  determinμg/mL) for 1 hour at 37°C; at the end of incubation, 100 μL of DMSO was added to lysate cells, the formazan crystals were solubilized, and the reaction was stopped. The colorimetric reaction was measured in a microplate reader using an absorbance of 514 nm . The data was compared with the nontreated group .The succinate dehydrogenase activity assay was based on reduction of tetrazolium salts to insoluble formazan in the presence of succinate by the mitochondrial enzyme succinate dehydrogenase, which allowed the metabolic rate related to aerobic respiration to be determined . Each saμL of each sperm sample (5 × 105 cells) was incubated with 100 μL of the ATP probe mixture following the manufacturer's recommendations. The solution was incubated for 10 minutes in a shaker (180 rpm), and the luminescence was immediately measured in a luminometer  with integration of 1000 ms. The luminescence measured was compared with the standard curve for ATP (0.0001 to 1000 nmol), based on a linear regression, and the data were interpreted as nmol of ATP in 1 × 106 cells.The influence of MT-Ex on ATP content was measured using a luminescence-dependent indicator  . At the 2+- and Mg2+-free). cAMP was measured following the manufacturer's instruction for suspended cells. The data were compared with a standard curve for cAMP (0.0006 to 6000 pmol), analysed by sigmoidal equation, and interpreted as pmol of cAMP in 1 × 106 cells.cAMP was determined using the commercial kit cAMP-Screen Direct (Invitrogen) . At the 6 cells) were incubated with 1 μM of FLUO-4 AM (without pluronic acid F-127) for 45 minutes at 37°C in the MR-A semen extender. The samples were divided into 4 groups and washed twice  to remove the unbound stain. Each group was diluted in each prepared buffer and maintained at 17°C until analysis. Then 15 × 106 cells were placed in a microplate , and the fluorescence was recorded for 50 min at 60-second intervals using a microplate reader (BioTek Synergy HT) in the 488/523 spectrum.The transient calcium was measured to determine the effect of MT-Ex on the capacitation state of sperm samples. The measurement method used was the green fluorescent probe FLUO-4 AM (Invitrogen) to detect the calcium signal . First, 6 cells were recuperated of each treatment and incubated with 2 mmol−1 of dithiothreitol  for 45 min at room temperature in order to relax the chromatin and allow the enzyme better access to the DNA [The In Situ Cell Death Detection kit , which is based on the terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick-end labeling (TUNEL) technique, was used to analyse the sperm DNA fragmentation. At the end of the incubation, 5 × 10 the DNA . Subsequ2− production. The samples were divided into two groups: control—semen diluted in the MR-A semen extender; MT-Ex—semen diluted in the MR-A semen extender and supplemented with 0.315 μgGAE mL−1 of MT-Ex. Both groups were refrigerated at 17°C for 168 h and evaluated at 24 h intervals.In this experiment, long-term analyses were performed to evaluate sperm motility, viability, mitochondrial membrane potential, and ROS and On = 15; 5 animals × 3 collections), aged 15 to 28 months, were used in this study; they were obtained from the Swine Research Center, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga, São Paulo, Brazil. The sperm samples were collected weekly from each boar using the gloved-hand technique. Semen samples were purified, and the sperm-rich fraction was placed in the MR-A extender supplemented with 0.315 μgGAE mL−1 prewarmed to 37°C and then refrigerated at 17°C (25 × 106 cells/ml). In attempt to normalize the possible individual variations, we performed seminal analysis of fresh ejaculate from the 10 boars, which included total motility (>70%), concentration, and morphology , and according to the parameters established by the Brazilian College of Animal Reproduction, all of them were approved by these criteria.Five mature boars  system . The parameters used were 45 frames at a frame rate of 60 frames/s; minimum contrast = 46; minimum cell size = 7 pixels; motility > 45 5 cells were obtained every 24 h to evaluate mitochondrial membrane potential (JC-1), and production of superoxide anion/hydrogen peroxides (DHE) following the procedures described above. One extra protocol was performed in this experiment, to determine ROS production: determination of total ROS by CellROX Green/PI [5 cells were stained with 5 μM of CellROX Green for 30 minutes at 37°C; in the last 10 minutes, PI was added to a final concentration of 6 μM. All fluorescent reactions were performed in a Guava easyCyte flow cytometer (Merck Millipore) analysing 10,000 events per sample using a 488 laser and fluorescence channels according to the manufacturer's recommendations.Aliquots of 2 × 10Green/PI . AliquotP < 0.05 were considered significant. The differences between means were determined using Tukey's multiple comparison tests. The results were expressed as the means of the measurements and their corresponding standard deviations. For experiment 2, the concentration-response analysis was performed by polynomial regression to determine EC50. The EC50 obtained for each genotype was subjected to analysis of variance (ANOVA), and the significance of the difference between means was determined by Tukey's test (P < 0.05). The Time and Concentration analysis was evaluated by ANOVA to determine the interaction between variables. Once the absence of the interaction between variables was verified, a prediction analysis of the surface plot and equation that describes the mathematical model was carried out using the software Curve Expert (Microsoft 2.2). The equation was solved by log transformation of the concentration parameter and full cubic polynomial regression. The EC50 analysis for each time was performed by polynomial regression using GraphPad Prism. For experiment 3, evaluation of O2−/peroxide production and lipid peroxidation was subjected to analysis of variance (ANOVA) and the significance of the difference between means was determined by Tukey's test (P < 0.05). For experiment 4, sperm motility data were analysed by log transformation of the concentration variable and sigmoidal nonlinear regression. The mitochondrial membrane potential, SDH activity, ATP, and cAMP content were subjected to analysis of variance (ANOVA), and the significance of the difference between means was determined by Tukey's test (P < 0.05). Calcium analysis was performed by linear regression comparing the linear trends. For experiment 5, long-term analysis was performed by calculation of means and S.D., and statistical analysis of the results was performed using GraphPad Prism 5.0. Analysis of repeated measures of variance was done to compare semen motility, mitochondrial membrane potential, viability, ROS production, and mitochondrial O2− between the control and MT-Ex groups and over time, followed by a Bonferroni significant difference test (to locate differences). Values were considered significant when P < 0.05.For experiment 1, the data were analysed using one-way analysis of variance (ANOVA). The values obtained at μgGAE mL−1; the highest value was obtained from genotype 19-1 , 28.9 μgGAE mL−1 (G19-1), 15.3 μgGAE mL−1 (G22-1), 23.2 μgGAE mL−1 (G23-1), and 24.8 μgGAE mL−1 (G27-1). The average of the five genotypes was 21.76 μgGAE/mL−1. Genotype G14-4 was selected as the ideal extract in the characterization and antioxidant analysis because of the low dispersion of the data in the curve (R2: 0.995). The results below were performed using only G14-4, hereafter “MT-Ex”.We evaluated the antioxidant potential of fruit extract from five ncreased . We obsex1 corresponds to time (in hours) and x2 to the log of the concentration in μgGAE mL−1. Evaluation of the surface plot revealed that the effect was concentration-dependent  and partμgGAE mL−1, respectively  on O2− production was evaluated by the DHE probe. As expected, the oxidant group presented the highest percentage of population with high O2− production (93.6% ± 4.1). This was followed by the MT-Ex + oxidant group (37.67% ± 5.6) and the control (30.3% ± 3.2) with no statistical difference between them. The lowest percentage  of viable cells with high superoxide production (19.3% ± 4.9) was found in the antioxidant group (MT-Ex) (The EC50 which produced an antioxidant effect (0.315  (MT-Ex) .Lipid peroxidation was evaluated by the BODIPY C-11 probe; we observed a different response profile between treatment groups when compared to superoxide-anion reduction. In this case, the oxidant and MT-Ex + oxidant groups presented the highest fluorescence intensity (4938 ± 455.2 and 3796 ± 147.2), while the control and MT-Ex groups presented lower fluorescence intensities  .The effect of MT-Ex on sperm motility was evaluated in two steps. First, we examined the effect of increasing concentrations of MT-Ex on sperm motility during refrigerated incubation: we observed a concentration-dependent decrease in total motility and a consequent increase in static cells, without any significant change in the progressive population .In the second trial, we determined the influence of MT-ex on sperm motility in oxidant and antioxidant conditions. A reduction in sperm motility was observed in all groups treated. As expected, the oxidant group resulted in a reduction in the motile sperm population from 89.6% ± 3.1(control) to 56.3% ± 2.6 (oxidant); however, treatments with MT-Ex on cells without oxidative stimulation resulted in a reduction in sperm motility (65.3% ± 1.5), while the addition of MT-Ex to oxidized sperm did not improve the motility parameter; on the contrary, a substantial reduction was observed with respect to the oxidant group (46.3% ± 1.9) .2O2 as an oxidative stimulator reduced this parameter, independently of the presence or absence of MT-Ex, with similar results between groups . However, the addition of MT-Ex in nonoxidized sperm presented a higher percentage of basal MMP (65.7%) when compared to groups with H2O2, but still lower than in the control group . However, for the oxidant group, the percentage of cells was reduced to 41.5%, while in the MT-Ex + oxidant group, the MMP did not present substantial improvements .2O2 (oxidant) and with MT-Ex and H2O2 (MT-Ex + oxidant) were reduced by 0.20 ± 0.03 nmol and 0.23 ± 0.04 nmol, respectively  to 1.06 ± 0.23 nmol (MT-Ex group). On the other hand, the ATP contents in the groups treated with Hectively .2O2 on the cAMP content of sperm samples under refrigeration and subsequently the influence of MT-Ex on calcium movement. The results suggest a reduction in the cAMP content in all the treated groups: in oxidant conditions, the cAMP concentration was reduced, reaching 0.19 pmol ± 0.03, while in the MT-Ex group the concentration was 0.95 pmol ± 0.21. However, treatment with MT-Ex did not improve the loss of cAMP . The MT-Ex supplementation in nonoxidizing conditions did not show significant differences with respect to basal conditions, while the antioxidant treatment decreased the count of sperm cells with fragmented DNA with respect to the oxidant group (P < 0.005)  .Long-term analysis was carried out to determine the antioxidant capacity and verify the stabilizing properties of MT-Ex over time. In the study, it was observed that the addition of MT-Ex to the MR-A semen extender increased the number of motile cells at 168 h (17.5 ± 8.3%) as compared to the control (1.3 ± 1.9%). The reduction rates of sperm motility in the two groups were control 12.17% per day and MT-Ex 9.08% per day .The analysis of sperm viability was performed by a propidium iodide probe, which depends on membrane stability. We observed an increase in cell survival in samples with long exposure times, with significant differences observed between groups at 144 to 168 h. At the last time evaluated, the MT-Ex group presented an undamaged cell count of 17.1% ± 3.6, versus 5.2% ± 1.2 of cells with intact membrane in the control group. The reduction in viability over time was control -15.05% per day and MT-Ex -11.45% per day .The MMP analysis showed a higher angular coefficient of fall in the control group than in the MT-Ex group , indicating that populations with basal MMP are rapidly reduced over time in the absence of MT-Ex. Thus, the addition of MT-Ex to the MR-A semen extender delayed the loss of membrane potential in the last hours of the analysis (16.3% ± 3.5), in contrast to the control which presented a substantial decrease in populations with basal potential (2.4% ± 1.4) .2− production showed a reduction in this parameter in both groups, with no significant difference when the storage time was increased , with antioxidant activity shown by DPPH radical bleaching and FRAP test [Some of these compounds have been reported in fruit extracts from the berries of other endemic Chilean plants  in the MR-A semen extender.2O2 is one of the initiators of lipid peroxidation in sperm [2O2 as an oxidizing agent, which were subsequently washed to study the intracellular antioxidant potential of MT-Ex. We observed that the addition of MT-Ex to cells under oxidative stimulation by H2O2 significantly reduced the production of O2−. However, we observed that the addition of MT-Ex at EC50 concentrations reduced the population with high ROS production by more than 50%, while MT-Ex supplementation in cells without oxidative stimulation did not result in a reduction in the basal oxidation count. Thus, ROS reduction was intrinsically limited to the cells after induced stress, while the oxidative levels of cells with normal endogenous ROS production were not affected, suggesting that MT-Ex reduces the impact of H2O2, when the cells already initiated a process of substantial ROS increase, indicating then that the scavenging effect of MT-Ex should be limited only under conditions where the sample presents a high endogenous production of ROS. However, due to the large conformation of the MT-Ex components, it is not possible to suggest a specific target different to H2O2 and O2− in ROS reduction.The superoxide anion is known to be a highly reactive molecule, unstable over time. It is one of the main stimulators of oxidative damage  and in cin sperm . In our 4 (Fenton's pathway) or in samples in the basal condition. Studies in rat liver microsomes have shown that the application of high concentrations of flavonoids (75-100 μM) in media supplemented with Fe3+ and EDTA favour the production of OH and therefore the triggering of lipid peroxidation; the concentrations of flavonoids applied in our assays were too low to stimulate endogenous production of ROS and induce peroxidation, as postulated by Laughton et al.'s study [We did not observe a substantial reduction in lipid peroxidation by MT-Ex in sperm samples stimulated by FeSO's study .On the other hand, evaluation of the effect of MT-Ex on metabolic parameters showed a clear influence of the extract on the normal metabolism of spermatozoa under refrigeration conditions. An effect on MMP was observed, with a substantial reduction in the sperm count with basal MMP in samples without oxidant stimulation, while in spermatozoa under oxidative conditions, treatment with MT-Ex to reduce the oxidizing effect did not prove effective in stabilizing MMP.Furthermore, we did not observe any regulatory or modulating effects of MT-Ex on succinate dehydrogenase by the MTT assay in either oxidant or antioxidant conditions. This enzyme (complex II of electron transport chain) is involved in the transformation of succinate to fumarate to generate an influx of electrons during the oxidative phosphorylation process in aerobic respiration . We did 2O2 at concentrations of 122.7 μM; however, the application of MT-Ex to sperm samples without ROS stimulation by H2O2 generated a significant reduction of ATP (>1 nm of ATP).In the functional context, a study in isolated mitochondria reported important effects of the flavonoids quercetin, taxifolin, catechin, and galangin, which act as inhibitors of the respiratory chain of mitochondria or cause uncoupling . Other a2O2 production [Some polyphenols, such as epigallocatechin gallate, epicatechin, quercetin, and kaempferol, have been associated with inhibition of ATP synthase complex (T0), occluding proton movement in subunits T0 and T1 . Other soduction .μM) could improve sperm motility in bovine spermatozoa [It is not possible to generate an efficient balance when an oxidative process is triggered, and this could be directly related to sperm motility. Some studies suggest that the addition of catechin . This effect has been observed in long-term refrigeration using cysteine as an antioxidant, where motility and cell viability are substantially improved, while adverse effects were observed with tocopherol, which reduces mitochondrial potential, motility, and viability , 66. Ourrylation , 67, so 2O2 and reduces the impact of the oxidizing agent on DNA fragmentation; however, it also generates an important decrease in sperm metabolism, especially ATP production. This could directly affect the fertilization potential due to the reduction in sperm motility and the calcium pathway to activate capacitation and subsequent acrosome reaction. Nevertheless, we consider that the metabolic decrease and subsequent modulation of sperm capacitation are an advantage in extending the useful life of boar sperm samples refrigerated at 17°C.The five genotypes of murtilla tested contain different phenolic compounds in different concentrations but present the same pattern of antioxidant activity. The antioxidant activity of MT-Ex controls the harmful prooxidant effect of H