White Tea as a Promising Antioxidant Medium ... - ACS Publications

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White Tea as a Promising Antioxidant Medium Additive for Sperm Storage at Room Temperature: A Comparative Study with Green Tea Tânia R. Dias, Marco G. Alves, Gonçalo D. Tomás, Sílvia Socorro, Branca M. Silva,* and Pedro F. Oliveira* CICS − UBI − Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilhã, Portugal ABSTRACT: Storage of sperm under refrigeration reduces its viability, due to oxidative unbalance. Unfermented teas present high levels of catechin derivatives, known to reduce oxidative stress. This study investigated the effect of white tea (WTEA) on epididymal spermatozoa survival at room temperature (RT), using green tea (GTEA) for comparative purposes. The chemical profiles of WTEA and GTEA aqueous extracts were evaluated by 1H NMR. (−)-Epigallocatechin-3-gallate was the most abundant catechin, being twice as abundant in WTEA extract. The antioxidant power of storage media was evaluated. Spermatozoa antioxidant potential, lipid peroxidation, and viability were assessed. The media antioxidant potential increased the most with WTEA supplementation, which was concomitant with the highest increase in sperm antioxidant potential and lipid peroxidation decrease. WTEA supplementation restored spermatozoa viability to values similar to those obtained at collection time. These findings provide evidence that WTEA extract is an excellent media additive for RT sperm storage, to facilitate transport and avoid the deleterious effects of refrigeration. KEYWORDS: sperm, Camellia sinensis, white tea, green tea, epigallocatechin-3-gallate, reactive oxygen species, antioxidants

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INTRODUCTION Tea (Camellia sinensis (L.)) is one of the world’s most widely consumed beverages, and its medicinal properties have been widely explored.1 It can be classified in three types: unfermented (green and white teas), partially fermented (oolong tea), and completely fermented (black tea).2 To produce green tea (GTEA), freshly harvested leaves are steamed to inactivate polyphenol oxidase enzyme and then rolled and dried. Its chemical composition is very similar to that of the fresh tea leaf.1 White tea (WTEA) is exclusively prepared from young tea leaves or buds, harvested before being fully opened. The tea materials are picked and immediately sent to be steamed and dried to prevent oxidation, frequently followed by polymerization.3 Unfermented teas are known to have high polyphenolic content, mainly catechin derivatives, (−)-epigallocatechin 3-gallate (EGCG) being the most abundant and powerful antioxidant.4 With respect to processing, there are very little differences between green and white teas, although several papers suggest that WTEA presents higher levels of antioxidants than GTEA.5 Recently, antioxidant components have aroused great interest due to their ability to minimize the deleterious effects of reactive oxygen species (ROS) on a number of biological and pathological processes.6 ROS are necessary for the normal physiological function of sperm,7 although their concentration must be kept under strict control to avoid deleterious effects, such as damage to cell structures: lipids and membranes, proteins, and DNA.8 It has been reported that ROS overproduction results in oxidative stress (OS), which is related to several problems that may end up in male subfertility or infertility.9 In fact, spermatozoa are particularly vulnerable to such stress because ROS readily attack the polyunsaturated fatty acids (PUFA) of the cells membrane, initiating a self-propagating chain reaction. Endproducts of these lipid peroxidation reactions, such as © 2013 American Chemical Society

malondialdehyde (MDA), are especially dangerous for cell viability.10 Therefore, there is a growing interest in enlightening the role of ROS formation in sperm as they are responsible for lower sperm quality in freshly collected semen and poor quality of sperm after processing for usage in reproductive technologies, such as artificial insemination (AI), in vitro fertilization (IVF), or cryopreservation.11 The maintenance of mammalian sperm at room temperature (RT) for short-term periods is advantageous as the storage of sperm in a refrigerated environment induces a rapid decline in its viability.12 Establishment of optimal composition for sperm storage is of extreme relevance, as these cells are highly dependent on the supply of exogenous substrates and, due to their high metabolic rates, produce elevated amounts of ROS.12 The addition of GTEA polyphenols has proven to be of great significance on frozen−thawed spermatozoa motility.13 Here, we aimed to investigate the possible protective effect of WTEA extract on epididymal spermatozoa survival at RT, using GTEA as a comparison reference.14 For that purpose, the chemical profiles of WTEA and GTEA aqueous extracts were determined by using 1H NMR as well as the antioxidant potential of storage media containing these extracts. Furthermore, the effect of both extracts on epididymal spermatozoa maintenance at RT during 24, 48, and 72 h was evaluated by determining the spermatozoa antioxidant potential, lipid peroxidation, and viability during that time frame. Received: Revised: Accepted: Published: 608

June 5, 2013 December 28, 2013 December 28, 2013 December 28, 2013 dx.doi.org/10.1021/jf4049462 | J. Agric. Food Chem. 2014, 62, 608−617

Journal of Agricultural and Food Chemistry

Article

Figure 1. Representative 1H NMR spectrum of WTEA extract showing the phytocomponent peak assignments: (EC) (−)-epicatechin; (EGC) (−)-epigallocatechin; (EGCG) (−)-epigallocatechin 3-gallate; (Ala) alanine; (The) theanine; (Lac) lactate.

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Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication 85-23, revised 1996) and the European directives for the care and handling of laboratory animals (Directive 86/609/EEC), after approbation by the National Ethics Committee of Animal Welfare. Animals were anesthetized, by intraperitoneal injection of a mixture of 90 mg/kg of ketamine and 10 mg/kg of xylazine, and euthanized by cervical displacement. Cauda epididymis were isolated and immediately placed separately in 3 mL of a prewarmed (37 °C) Krebs−Ringer bicarbonate (TYH) medium (in mM: NaCl, 118.8; KCl, 4.78; CaCl2, 1.71; KH2PO4, 1.19; MgSO4, 1.19; NaHCO3, 25; glucose, 5.56; sodium pyruvate, 1.01; sodium lactate, 29.2; plus 4.00 mg/mL BSA, 0.06 mg/mL potassium penicillin G and 0.05 mg/mL streptomycin sulfate), prepared on the day of the experiment, as described by Toyoda.24 The two cauda epididymis of each animal were minced together with a scalpel blade, to allow sperm to disperse into the medium, and the suspension was then incubated for 1.5 h at 37 °C. The number of spermatozoa was determined using a hemocytometer, and 8 × 105 spermatozoa were placed in 300 μL of a control medium (in mM: NaCl, 96.66; KCl, 4.78; CaCl2, 1.71; KH2PO4, 1.19; MgSO4, 1.19; NaHCO3, 4.15; D-glucose, 5.56; sodium pyruvate, 0.33; sodium lactate, 23.28; HEPES, 20.85; plus 4.00 mg/mL BSA, 0.06 mg/mL potassium penicillin G, 0.05 mg/mL streptomycin sulfate) as previously described.25 Moreover, the same amount of spermatozoa was placed in four other media, with the same composition of the control group but containing additional concentrations of freeze-dried WTEA or GTEA aqueous extracts to a final concentration of 0.5 or 1 mg/mL. Subsequently, sperm suspensions were kept at acclimatized RT (22−23 °C) during 24, 48, and 72 h. These sperm suspensions were used for the FRAP, TBARS, and sperm viability assays, after being processed as described in each section. Protein Quantification. After the 72 h of incubation, spermatozoa were separated from the storage media through centrifugation at 5000g for 15 min at 4 °C. Then, sperm pellets were washed with 100 μL of phosphate-buffered saline (PBS) solution (in mM: NaCl, 137; KCl, 2.7; Na2HPO4, 4.3; KH2PO4, 1.47; pH 7.4) and centrifuged again at 5000g for 15 min at 4 °C. Total proteins were isolated from the spermatozoa by the addition of an adequate amount of PBS, followed by a sonication for 15 min at 4 °C. Protein concentration was determined by Bio-Rad (Hemel Hempstead, UK) Bradford microassay according to the manufacturer’s instructions. Sperm suspensions were used in FRAP and TBARS assays. Ferric Reducing Antioxidant Power (FRAP) assay. The ferric reducing antioxidant power (FRAP) of the media samples and

MATERIALS AND METHODS

Chemicals. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless specifically stated otherwise. Tea Extracts. WTEA and GTEA were purchased on the Portuguese market (Diese, Bestlife - Comércio e Distribuiçaõ Lda., Portugal). Samples (n = 5) were subjected to infusion (1 g/100 mL distilled water; pH 5.5) at 100 °C during 3 min, according to the manufacturer’s instructions. The resulting infusions were filtered with qualitative filter papers (catalog no. 516-0819, VWR, Leuven, France) in a vacuum system and freeze-dried overnight in a ScanVacCoolSafe Freeze-Dryer (Labogene, Lynge, Denmark). The mean extraction yields (g of lyophilized extract per 100 g of dried teas leaves) were 25% (for WTEA) and 20% (for GTEA). The lyophilized extracts were kept in a desiccator, protected from light, until analysis. Proton Nuclear Magnetic Resonance (1H NMR). 1H NMR spectra were acquired as previously described.15 Briefly, 1H NMR spectra of WTEA and GTEA aqueous extracts dissolved in D2O were acquired at 14.1 T, 25 °C, using a Bruker Avance 600 MHz spectrometer equipped with a 5 mm QXI probe and a z-gradient. 1H NMR spectra were acquired with solvent suppression and a sweep width of 6 kHz, using a delay of 14 s, a water presaturation of 3 s, a pulse angle of 45°, an acquisition time of 3.5 s, and at least 128 scans. Sodium fumarate was added to the samples for quantification purposes (in a final concentration of 1 mM), because it does not exist in our tea extracts and was used as a reference (6.50 ppm), as routinely done in our laboratory,16,17 to quantify the extract compounds whenever present in solution. The following coupling patterns, available in the literature,18−23 were used to quantify the identifiable extract compounds (multiplet, ppm): L-theanine (triplet, 1.08); lactate (doublet, 1.33); alanine (doublet, 1.45); EGCG (doublet, 2.7); caffeine (singlet, 3.29); H1-α-glucose (doublet, 5.22); sucrose (doublet, 5.4); (−)-epigallocatechin (EGC) (singlet, 6.6); (−)-epicatechin (EC) (singlet, 7.0) (for a representative image see Figure 1). The relative areas of 1H NMR resonances were quantified using the curve-fitting routine supplied with the NUTSproTM NMR spectral analysis program (Acorn, NMR Inc., Fremont, CA, USA). Isolation of Epididymal Spermatozoa. The present study used six male 3-month-old Wistar rats, obtained from our accredited animal colony (Health Sciences Research Centre, University of Beira Interior) and maintained on ad libitum food and water in a constant room temperature (20 ± 2 °C) on a 12 h cycle of artificial lighting. Rats were fed a standard chow diet (4RF21 certificate, Mucedola, Italy). All animal experiments were performed according to the Guide for the 609

dx.doi.org/10.1021/jf4049462 | J. Agric. Food Chem. 2014, 62, 608−617


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spermatozoa pellets was performed according to the colorimetric method described by Benzie and Strain.26 Briefly, working FRAP reagent was prepared by mixing acetate buffer (300 mM, pH 3.6), 2,4,6-tripyridyl-s-triazine (TPTZ) (10 mM in 40 mM HCl), and FeCl3 (20 mM) in a 10:1:1 ratio (v/v/v); 180 μL of this reagent was mixed with 6 μL of each sample. The reduction of the Fe3+−TPTZ complex to a colored Fe2+−TPTZ complex by the samples was monitored immediately after addition of the sample and 40 min later, by measuring the absorbance at 595 nm using an Anthos 2010 microplate reader (Biochrom, Berlin, Germany). The antioxidant potential of the samples was determined against standards of ascorbic acid, which were processed in the same manner as the samples. Absorbance results were corrected by using a blank, with water instead of sample. The changes in absorbance values of test reaction mixtures were used to calculate FRAP values as described elsewhere.26 Thiobarbituric Acid Reactive Substances (TBARS) Assay. TBARS are formed as a byproduct of lipid peroxidation, which can be detected by the TBARS assay using thiobarbituric acid (TBA) as a reagent. This peroxidation reaction produces MDA, which reacts with TBA in conditions of high temperature and low pH, generating a pink complex that absorbs at 532 nm.27 The TBARS assay was carried out according to the method described by Iqbal et al.,28 with slight adaptations. Briefly, the reaction mixture in a total volume of 0.1 mL contained 0.01 mL of the sample, 0.01 mL of Tris-HCl buffer (150 mM, pH 7.1), 0.01 mL of ferrous sulfate (1.0 mM), 0.01 mL of ascorbic acid (1.5 mM), and 0.06 mL of H2O. This mixture was incubated at 37 °C for 15 min. The reaction was stopped by the addition of 0.1 mL of trichloroacetic acid (10% w/v). Subsequently, 0.2 mL of thiobarbituric acid (0.375% w/v) was added, and all samples were incubated for 15 min at 100 °C. Finally, samples were subjected to centrifugation at 3000g for 10 min at 4 °C. The amount of MDA formed in each of the samples was estimated by measuring the optical density at 532 nm using a UV−vis spectrophotometer (Shimadzu, Kyoto, Japan) against a blank. The results were expressed as nanomoles of TBARS per milligram of protein. Sperm Viability Evaluation. To assess sperm viability, an eosin/ nigrosin staining was used because it is effective, simple, and, in addition to allowing sperm to be readily visualized, permits assessment of sperm membrane integrity. This method was performed with slight modifications of a method previously described.29 A total of 5 μL of the sperm suspensions obtained as described above (see Isolation of Epididymal Spermatozoa) was mixed with 10 μL of 0.5% eosin/ nigrosin stain and placed on a prewarmed glass microscope slide. Samples were analyzed at 0, 24, 48, and 72 h of the experiment. The number of viable and nonviable spermatozoa was determined by counting a total of 333 spermatozoa per slide in continuous random fields under a light microscope, with oil immersion (×1000 magnification), to determine the percentage of viable sperm, as previously described.16 Live sperm remained white, whereas dead sperm stained pink, because the integrity of their plasma membrane was compromised, causing an increase in membrane permeability, which led to the uptake of the dye (Figure 5A). Statistical Analysis. Statistical significance was assessed by twoway ANOVA, followed by Bonferroni post-test using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA). All data are presented as the mean ± SEM. Differences with p < 0.05 were considered statistically significant. Further analysis of the statistical power (SP) of differences of experimental data was evaluated with a one-tailed test assuming an α value of 0.05, which corresponds to a 0.95 confidence interval, as described by Levin,30 using the software provided by http://www.dssresearch.com/KnowledgeCenter/toolkitcalculators/ statisticalpowercalculators.aspx.

biological and pharmaceutical properties that have been linked to beneficial effects on human health.33 The 1H NMR data obtained from the aqueous extracts allowed the assignment of the following phytochemicals: three catechin derivatives, EC, EGC, and EGCG; one methylxanthine, caffeine; two free amino acids, L-theanine and alanine; two carbohydrates, glucose and sucrose; and one organic acid, lactate (for a representative image see Figure 1). As expected, the most abundant class of phytochemicals was the catechin family, representing 133 ± 14 and 91 ± 7 g/kg of WTEA and GTEA extracts, respectively. Among catechins, EGCG was the one present in the highest amount in both tea extracts, although in WTEA extract the quantity of this compound (82 ± 7 g/kg of WTEA extract) was nearly twice that verified in GTEA extract (42 ± 2 g/kg of GTEA extract). Concerning caffeine and sucrose, WTEA extract also demonstrated a larger amount (71 ± 8 and 60 ± 4 g/kg of WTEA extract, respectively) in comparison with GTEA (21 ± 3 and 26 ± 2 g/kg of GTEA extract, correspondingly). L-Theanine, alanine, and glucose were also present in considerable amounts, accounting for 19 ± 2, 0.7 ± 0.1, and 6 ± 1 g/kg of WTEA extract and 22 ± 1, 0.20 ± 0.04, and 11 ± 2 g/kg of GTEA extract, respectively (Table 1). Table 1. White (WTEA) and Green Tea (GTEA) Extract Phytochemical Profiles As Determined by 1H NMR Spectroscopy contenta (g of compd/kg of tea extract) compound glucose sucrose lactate alanine caffeine L-theanine ECb EGCc EGCGd

WTEA 6 60 0.40 0.7 71 19 5 46 82

± ± ± ± ± ± ± ± ±

1.00 4.00 0.01 0.10 8.00 2.00 1.00 6.00 7.00

GTEA 11 26 0.46 0.20 21 22 28 21 42

± ± ± ± ± ± ± ± ±

2.00 2.00 0.02 0.04 3.00 1.00 2.00 3.00 2.00

Results are presented as the mean ± SEM (n = 5). bEC, (−)-epicatechin. cEGC, (−)-epigallocatechin. dEGCG, (−)-epigallocatechin 3-gallate. a

In fact, (−)-epicatechin 3-gallate (ECG), (−)-catechin (C), and (−)-gallocatechin-3-gallate (GCG) were absent in our extracts. Generally, these polyphenols are minor tea components but sometimes are absent in tea extracts. For instance, Carvalho et al.34 and Rusak et al.35 have not found GCG and C, respectively, in tea extracts. Hence, the white and green tea qualitative phytochemical profiles obtained using 1H NMR (Table 1) are in accordance with the phenolic and methylxanthine profiles obtained by HPLC-UV, HPLC-DAD, or HPLC-MS previously reported by Carvalho et al.,34 Rusak et al.,35 and Unachukwu et al.36 The observed differences in the quantitative profile may be mainly due to the use of different extraction conditions (solvents, temperatures, times of extraction, and ratio leaves/water). Also, the natural variability of plants caused by edapho-climatic factors, harvesting techniques, or agricultural practices may contribute to these differences. Storage Media Supplemented with White Tea Extract Have the Highest Antioxidant Potential. As the aqueous extracts of WTEA and GTEA demonstrated to be rich in

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RESULTS White Tea Extract Presents the Highest Content in Catechin Derivatives, Especially EGCG. Tea has been characterized by its high content in flavonoids,31 such as catechin derivatives and other polyphenols,32 which have 610



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Figure 2. Ferric reducing antioxidant power (FRAP) of the epididymal spermatozoa storage media, control medium (CTRL), and media supplemented with freeze-dried WTEA or GTEA aqueous extracts to a final concentration of 0.5 or 1 mg/mL. The antioxidant power is expressed by the FRAP value (μmol of antioxidant potential/L). Results are presented as the mean ± SEM (n = 6). Significant results (p < 0.05) are indicated: b, versus CTRL; c, versus GTEA, 0.5 mg of GTEA extract/mL; d, versus WTEA, 0.5 mg of WTEA extract/mL; e, versus GTEA, 1 mg of GTEA extract/mL.

Figure 3. Ferric reducing antioxidant power (FRAP) of the epididymal spermatozoa stored in control medium (CTRL) and media supplemented with freeze-dried WTEA or GTEA aqueous extracts to a final concentration of 0.5 or 1 mg/mL, during the 24, 48, and 72 h of the experiment. The antioxidant power is expressed by the FRAP value in μmol of antioxidant potential/μg of protein and is presented as the mean ± SEM (n = 6). Significant results (p < 0.05) are indicated: b, versus CTRL; c, versus GTEA, 0.5 mg of GTEA extract/mL.

group during all of the experiments (SP = 100%). Comparing GTEA supplementation, the medium with 1 mg/mL of GTEA extract presented a significantly higher antioxidant potential compared to the medium with 0.5 mg of GTEA extract/mL (11 ± 0.2 vs 6 ± 0.3 μmol of antioxidant potential/L) (SP = 100%). In relation to WTEA, the FRAP value of the medium supplemented with 0.5 mg/mL of WTEA extract was 14 ± 0.3 μmol of antioxidant potential/L, which was significantly higher compared to the medium with the same concentration of GTEA extract (SP = 100%). Moreover, the medium containing 1 mg/mL of WTEA presented a significantly higher antioxidant potential of 24 ± 0.6 μmol of antioxidant potential/ L in comparison with the medium supplemented with 0.5 mg/

catechin derivatives, namely, EGCG, we performed a FRAP assay to evaluate the antioxidant potential of all experimental media supplemented with these extracts in comparison with the control media. The FRAP assay measures the potential of an antioxidant to reduce ferric(III) to ferrous(II) in a redox-linked colorimetric reaction that involves single electron transfer.37 The reducing power of a compound/extract serves as a significant indicator of its potential antioxidant activity (FRAP value). The control medium showed a FRAP value of 1.64 ± 0.05 μmol of antioxidant potential/L (Figure 2). On the other hand, the FRAP value of the media supplemented with WTEA and GTEA revealed a dose-dependent reducing power (Figure 2), with significantly higher FRAP values relative to the control 611



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Figure 4. Sperm TBARS values produced in epididymal spermatozoa stored in control medium (CTRL) and media supplemented with freeze-dried WTEA or GTEA aqueous extracts to a final concentration of 0.5 or 1 mg/mL, during the 24, 48, and 72 h of the experiment. Results are expressed in nmol/mg of protein and are presented as the mean ± SEM (n = 6). Significant results (p < 0.05) are indicated: b, versus CTRL; c, versus GTEA, 0.5 mg of GTEA extract/mL; e, versus GTEA, 1 mg of GTEA extract/mL.

Spermatozoa Lipid Peroxidation Is Significantly Reduced by White Tea Supplementation. During spermatozoa storage, ROS production is known to induce several deleterious effects38 such as lipid peroxidation. Because spermatozoa antioxidant potential was increased after incubation with the tea extracts, we hypothesized that it could also decrease ROS production, thus decreasing lipid peroxidation. Therefore, we performed the TBARS assay to determine the levels of lipid peroxidation that occurred in spermatozoa during storage in the different media. Because ROS have extremely short half-lives, they are difficult to measure directly. Instead, several products induced by OS, such as TBARS, can be measured and used as an accurate indicator of OS.39 Using the TBARS assay, it was possible to detect a significant decrease in lipid peroxidation in spermatozoa stored in media supplemented with WTEA and GTEA extracts. At 24 h, there was a significant decrease in lipid peroxidation from spermatozoa stored in the control medium (73 ± 4 nmol TBARS/mg protein) relative to the media containing 0.5 mg/mL of GTEA (47 ± 7 nmol TBARS/mg protein) and 0.5 and 1 mg/mL of WTEA (54.8 ± 4.7 and 46 ± 2 nmol TBARS/mg protein, respectively) (Figure 4) (SP = 100%). After 48 h, the lipid peroxidation significantly decreased from 81 ± 10 nmol TBARS/mg protein in the control group to 44 ± 6 and 41 ± 8 nmol TBARS/mg protein (SP = 100%) in the spermatozoa stored in the media supplemented with 0.5 and 1 mg of WTEA extract/mL, respectively (Figure 4). Moreover, the spermatozoa stored in the medium with 1 mg of WTEA extract/mL also presented significantly less lipid peroxidation than the spermatozoa maintained in the medium with 1 mg of GTEA extract/mL, which presented a value of 69 ± 10 nmol TBARS/ mg protein (SP = 99.9%). At the end of the experiment (72 h), the control group presented a lipid peroxidation of 79 ± 4 nmol TBARS/mg protein that significantly decreased to 58 ± 8, 42 ± 5, and 41 ± 3 nmol TBARS/mg protein (SP = 100%) after storage in the media supplemented with 0.5 mg of GTEA extract/mL and 0.5 and 1 mg of WTEA extract/mL,

mL of WTEA extract and 1 mg/mL of GTEA extract (SP = 100%). White Tea Extract Considerably Increases the Antioxidant Potential of Spermatozoa. Because the media supplemented with WTEA extract proved to have the highest antioxidant potential, we expected to verify the same profile in the antioxidant potential of the spermatozoa. Therefore, we also measured the antioxidant potential of the spermatozoa pellets at 24, 48, and 72 h with the FRAP assay. Accordingly, spermatozoa kept in control medium had always a lower antioxidant potential than spermatozoa stored in the media supplemented with WTEA and GTEA extracts, although these differences were statistically significant only after 48 and 72 h of storage, respectively (Figure 3). Indeed, at 48 h the spermatozoa stored in the medium with 0.5 mg of WTEA extract/mL presented a FRAP value of 2.8 ± 0.2 μmol of antioxidant potential/μg of protein, significantly higher than that of spermatozoa maintained in the control medium (1.1 ± 0.3) and in medium supplemented with 0.5 mg of GTEA extract/mL (1.4 ± 0.1) (SP = 100%). At 72 h, the FRAP values of spermatozoa maintained in media with 0.5 and 1 mg of GTEA extract/mL were 1.5 ± 0.2 (SP = 84%) and 1.8 ± 0.1 (SP = 100%) μmol of antioxidant potential/μg of protein, respectively, being significantly higher than that verified in the control group. At this time of incubation, the FRAP value determined for spermatozoa kept in the control medium was significantly lower than that at 48 h, reaching a value of 0.1 ± 0.3 μmol of antioxidant potential/μg of protein (SP = 100%) (Figure 3). In contrast, the antioxidant potentials of spermatozoa stored in the media supplemented with WTEA extract (0.5 and 1.0 mg of WTEA extract/mL) were remarkably higher than those observed at 48 h (8.4 ± 2.7 (SP = 99.9%) and 12.2 ± 3.1 (SP = 100%) μmol of antioxidant potential/μg of protein, respectively) and also than the FRAP value of the control group. Moreover, at 72 h there were no significant differences between both concentrations of the same extract. 612



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Figure 5. (A) Spermatozoa staining using eosin/nigrosin showing a viable (left) and a nonviable (right) cell. (B) Spermatozoa viability at collection time (0 h) and during the 3 day storage in control medium (CTRL) and media supplemented with freeze-dried WTEA or GTEA aqueous extracts to a final concentration of 0.5 or 1 mg/mL. Results are presented as the mean ± SEM (n = 6). Significant results (p < 0.05) are indicated: a, versus 0 h; b, versus CTRL; c, versus GTEA, 0.5 mg of GTEA extract/mL; d, versus WTEA, 0.5 mg of WTEA extract/mL; e, versus GTEA, 1 mg of GTEA extract/mL.

respectively. Additionally, lipid peroxidation in spermatozoa kept in the media with 0.5 and 1 mg of WTEA extract/mL were significantly lower than in spermatozoa stored in the media supplemented with 0.5 (SP = 99.5%) and 1 mg of GTEA extract/mL (SP = 100%), correspondingly. White Tea Supplementation Increases Spermatozoa Viability after Room Temperature Storage. Sperm viability is one of the most important parameters to assess sperm quality.40 Once the WTEA and GTEA supplementation significantly increased the antioxidant potential of storage media and spermatozoa and decreased spermatozoa lipid peroxidation, we hypothesized that sperm viability could be improved by these extracts. Therefore, we evaluated the sperm viability at the time of epididymal collection (0 h) and at 24, 48, and 72 h. At collection, sperm viability averaged 67 ± 2%. During spermatozoa storage at RT in control medium we observed that viability was continuously decreasing, presenting values of 31 ± 2, 19 ± 2, and 14 ± 1% at 24, 48, and 72 h, respectively (Figure 5B) and being significantly lower compared to viability at collection time (SP = 100%). There was a dose-dependent significant increase in viability, during

the 3 day storage, in spermatozoa stored in media supplemented with both tea extracts when compared with the control. At 24 h, viability of spermatozoa kept in the media supplemented with any of the tea extracts was very similar to that observed at 0 h. At 48 h, viability of spermatozoa kept in the media with 0.5 mg/mL of GTEA or WTEA significantly decreased (49 ± 2 and 53 ± 5%, respectively) in comparison with that observed, 67 ± 2%, at the collection time (SP = 100%). Conversely, viability corresponding to the spermatozoa stored in the media with 1 mg/mL of GTEA or WTEA (59 ± 3 (SP = 100%) and 65 ± 3% (SP = 99.5%), respectively) was significantly higher than the viability verified in the media containing half the concentration of these extracts. The same profile was verified at 72 h of storage with the only exception that spermatozoa kept in 1 mg of WTEA extract/mL medium exhibited significantly higher viability of 63 ± 3% in relation to the spermatozoa kept in the medium with 1 mg of GTEA extract/mL, which was 49 ± 2% (SP = 100%), being in its turn significantly lower compared to the viability at 0 h (SP = 100%). Remarkably, spermatozoa maintained in the medium with 1 mg of WTEA extract/mL averaged a viability of 65% 613



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compromising spermatozoa survival and fertilizing capacity.54 Epididymal spermatozoa survival and maintenance are crucial for both natural and assisted reproduction.55 The mammalian epididymis creates a unique microenvironment that helps in testicular spermatozoa maturation from an immotile immature state to fully fertile competent cells. Besides, it stores the fertile and viable spermatozoa in cauda epididymis until ejaculation. For that reason, spermatozoa from cauda epididymis are often used in assisted reproductive technologies. In fact, cryopreservation and refrigeration of spermatozoa have been highly debated, and it has been proposed that the maintenance of spermatozoa at RT for short-term periods can be an effective alternative to avoid the rapid decline of sperm viability after storage in a refrigerated environment.12,56 Hence, there is a growing interest in the establishment of an optimal composition medium for RT sperm storage. Several media intended to improve spermatozoa survival at RT have been developed, but spermatozoa viability after storage in those media is still very low and far from the ideal.12,57−60 A study using mouse sperm evaluated the effect of RT storage in various bicarbonate and phosphate-based media used for IVF or embryo culture, reporting that the low bicarbonate- and HEPES-containing media were able to better preserve spermatozoa in vitro.12 The addition of substrates, such as glucose, further enhanced sperm survival, although the authors concluded that further tests concerning the addition of preserving agents are still required.12,60 Studies using sperm from different mammalian origins preserved at RT in a salinebuffered storage medium also showed that supplementation with serum or egg yolk could greatly increase sperm survival and function.57−59 At the same time those authors concluded that there was still a crucial requirement in controlling the ROS, which are more likely to be generated in an ambient temperature system, in the media in which the spermatozoa are suspended. Indeed, some studies have been made to assess the effect of tea catechins, which are known antioxidant agents, on sperm viability and survival.13,47 In one of those studies, several concentrations of EGCG (1, 50, and 100 μM) were tested as a supplement of the storage media, and although the authors obtained encouraging results in the sperm fertilization ability of frozen−thawed sperm, they did not verify a significant increase in sperm survival after storage.13 Therefore, we hypothesized that WTEA extract could modulate some important functions such as antioxidant capacity and lipid peroxidation of spermatozoa, thus improving spermatozoa viability during RT storage. Using the FRAP assay, we verified that the media containing WTEA extract presented a higher antioxidant potential value, in a dose-dependent manner, than the media with GTEA extract, evidencing that the antioxidant potential is greatly increased by the addition of WTEA extract. The higher increase in the antioxidant potential of sperm after storage was verified in media with WTEA extract, and this is concomitant with the alteration measured for the media antioxidant potential. Indeed, the sperm antioxidant potential showed an increase over time with storage in WTEA supplemented media that is surely due to incorporation of antioxidant compounds present in the extract. The same profile was verified in sperm stored with GTEA extract in a much smaller extent, evidencing that WTEA extract is the one giving the best antioxidative potential. One of the most important deleterious effects caused by OS is lipid peroxidation because mammalian spermatozoa are rich in PUFA that are highly vulnerable to OS.10 When subjected to

throughout the experiment, a very similar value relative to viability at 0 h (Figure 5B).

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DISCUSSION Despite the similarities between GTEA and WTEA, the number of investigations studying the health benefits of WTEA is negligible compared to GTEA. Recently, WTEA aroused great interest among investigators due to its higher concentrations of tea polyphenols when compared to GTEA.41 Thus, WTEA has been proven to have higher antioxidant activity than any other type of tea.2,5 As expected, the WTEA extract we obtained was richer in this class of flavonoids than the GTEA extract, predominantly in EGCG (82 ± 7 g/kg of WTEA extract vs 42 ± 2 g/kg of GTEA extract), which is known as one of the most powerful antioxidants and the most pharmacologically active catechin derivative.42 There are several pieces of evidence that EGCG has a positive impact in a variety of human diseases that is dependent on its concentration.2,43,44 EGCG has been proven to have protective action against several deleterious effects of diseases by minimizing OS.45 Recently, it was reported that EGCG therapy protects against testicular ischemia-reperfusion injury through its antioxidant activity.46 Importantly, it has been reported that for human spermatozoa maintained at 37 °C during 30 min, motility and viability can be improved by the addition of EGCG to the extracellular media at low concentrations.47 In fact, our results showed that WTEA is very rich in EGCG, which represents 61.7% of the total catechin content, and the lower content in EGCG of GTEA may be reflected in the antioxidant properties of the extract.41 In addition, caffeine content is also known to be higher in WTEA,41 and our extract presented a remarkably high caffeine content (71 ± 8 g/kg of WTEA extract) when compared with the results obtained by other authors.48 This phytochemical content difference, as well as the variation found between our GTEA extract composition and the ones verified by other authors,34 could be due to the natural variability of the plants (caused by the influence of geographical origin, climate, and/or agricultural practices) and to differences in the extraction procedures (solvents, temperatures, times of extraction, and ratio of leaves to water) and analytical techniques.35,49,50 Our WTEA extract also presented higher caffeine content compared to our GTEA extract (21 ± 3 g/kg of GTEA extract). This should be due to differences in leaf processing (e.g., the collection date and/or the type of leaves) to produce WTEA or GTEA, which have already been proven to influence the tea chemical composition.41 Nevertheless, caffeine is described to stimulate lipolysis and interfere with glucose and fatty acid metabolism, thus having an important role in cellular metabolism.51 The WTEA extract was also richer in the content of total sugars, which are known to be important substrates to cellular metabolism. Overall, our 1H NMR results concerning WTEA and GTEA are in accordance with the phenolic and methylxanthine profiles obtained by HPLC-UV, HPLC-DAD, or HPLC-MS previously reported by Carvalho et al.,34 Rusak et al.,35 and Unachukwu et al.36 Possible differences found in our quantitative profile compared to previous ones are certainly due to the reasons above-mentioned. Spermatozoa present high metabolic rates that are in close association with elevated amounts of OS,52 which is known as one of most relevant factors responsible for poor semen quality.53 OS is associated with uncontrolled ROS production that, when it exceeds spermatozoa antioxidant capacity, becomes harmful, inducing membrane lipid peroxidation, 614



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responsible for the overall better results observed with this concentration. In conclusion, WTEA is richer in antioxidants than other teas (black, oolong, and green teas),2 and the addition of WTEA aqueous extract to the media can be a good, simple, and inexpensive strategy for sperm storage at RT. Our results strongly indicate that WTEA extract improves spermatozoa viability by increasing the spermatozoa and storage media antioxidant potential and decreasing spermatozoa lipid peroxidation. Moreover, the first hours of sperm preservation is crucial and marked by an increase in OS that can be counteracted by WTEA polyphenols. More studies will be needed to fully disclose the molecular mechanisms behind the results now reported. Nevertheless, the addition of WTEA aqueous extract to the standard sperm storage medium proved to be an excellent strategy for sperm storage at RT, which can reduce or even eliminate the limitations of the in vitro spermatozoa storage in a refrigerated environment and enable sperm transport for IVF or AI use.

high levels of OS, spermatozoa lipid membranes are oxidized, and the end-product of these reactions, MDA, can be measured by the TBARS assay. Our results showed that during the 3 day sperm storage at RT, WTEA extract was the most effective in decreasing the lipid peroxidation in spermatozoa. As WTEA extract presented a higher concentration of polyphenols, which are known to decrease lipid peroxidation in cells, it was expected that the media with WTEA extract presented a higher antioxidant potential and, consequently, a higher capacity to prevent lipid peroxidation. However, at 24 h, although there was a significant decrease in lipid peroxidation relative to the control group, there were no significant differences between storage with WTEA or GTEA. This is concordant with the sperm FRAP assay at 24 h that also showed no differences in the antioxidant potential of sperm stored with both tea extracts. This could be explained by ROS production, which is expected to increase over time and needs to be accompanied by the antioxidant defenses increase to control the amount of ROS. After 48 and 72 h, lipid peroxidation of sperm stored in the media supplemented with WTEA extract remained constant and similar to that observed at 24 h, whereas in sperm stored in the media containing GTEA extract the lipid peroxidation increased over time. As our WTEA and GTEA extracts presented EGCG contents of 82 ± 7 g/kg of WTEA extract and 42 ± 2 g/kg of GTEA extract, these amounts reflect EGCG concentrations in the ranges of about 90−180 μM for WTEA and 45−90 μM for GTEA supplemented sperm storage media. Therefore, as our EGCG concentrations are in the same order of magnitude of those used in the study of Kaedei et al.13 and, conversely to the data reported by those authors who were not able to verify a significant increase in the survival of sperm kept in the EGCG supplemented media, our results showed that tea supplementation highly improved spermatozoa survival, and this supports our idea that the synergistic effect between the tea components may be responsible for the positive effects observed in sperm viability. Additionally, our results support the effectiveness of WTEA extract in detriment to GTEA extract. Male infertility is relatively common and affects about 50% of couples with fertility problems.61 In most male patients with subfertility or infertility, the condition is due to loss of sperm function rather than the number of spermatozoa.62 Therefore, spermatozoa viability is an essential parameter to evaluate sperm quality and male factor infertility. Supplementation of the storage media with tea extracts significantly increased sperm viability in a dose-dependent manner. OS is known to play a crucial role in the loss of functional competence, and when ROS production is significantly elevated, dysfunctional spermatozoa are produced.63 A negative correlation between ROS production and sperm movement, evidencing the importance of ROS control in spermatozoa function, has also been reported.64 High ROS levels are reported to be detrimental to fertility potential in natural and assisted conception,65 and sperm capacitation is also known to be lost in the presence of high OS levels.66 Therefore, the higher increase of sperm viability in spermatozoa stored in WTEAsupplemented media compared to spermatozoa stored in GTEA-supplemented media is certainly due to the higher polyphenolic content in WTEA extract and, consequently, to its higher antioxidant potential. Moreover, because the viability improvement was more effective with the highest dose of WTEA extract (1 mg/mL), we suggest that the best protection attained with the highest dose of WTEA extract may be

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AUTHOR INFORMATION

Corresponding Authors

*(B.M.S.) Phone: +351 275 329 077. Fax: +351 275 329 099. E-mail: [email protected]. *(P.F.O.) Phone: +351 275 329 077. Fax: +351 275 329 099. E-mail: [email protected]. Funding

This work was supported by the Fundaçaõ para a Ciência e a Tecnologia − FCT (PTDC/QUI-BIQ/121446/2010 and PEstC/SAU/UI0709/2011) cofunded by Fundo Europeu de Desenvolvimento Regional − FEDER via Programa Operacional Factores de Competitividade − COMPETE/QREN. T.R.D. was funded by Programa Operacional Regional do Centro 2007−2013 QREN (Programa Mais Centro). M.G.A. (SFRH/BPD/80451/2011) was funded by FCT. P.F.O. was funded by FCT through FSE and POPH funds (Programa Ciência 2008). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED AI, artificial insemination; BSA, bovine serum albumin; C, (−)-catechin; EC, (−)-epicatechin; ECG, (−)-epicatechin-3gallate; EGC, (−)-epigallocatechin; EGCG, (−)-epigallocatechin-3-gallate; GCG, (−)-gallocatechin-3-gallate; FRAP, ferric reducing antioxidant power; GTEA, green tea; IVF, in vitro fertilization; MDA, malondialdehyde; OS, oxidative stress; PBS, phosphate-buffered saline; PUFA, polyunsaturated fatty acids; ROS, reactive oxygen and nitrogen species; RT, room temperature; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substances; TPTZ, 2,4,6-tripyridyl-s-triazine; TYH, Krebs−Ringer bicarbonate medium; WTEA, white tea

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