white tea as a promising antioxidant medium additive for sperm storage at room temperature: a...

White Tea as a Promising Antioxidant Medium Additive for SpermStorage at Room Temperature: A Comparative Study with Green TeaTania R. Dias, Marco G. Alves, Goncalo D. Tomas, Sílvia Socorro, Branca M. Silva,*and Pedro F. Oliveira*

CICS − UBI − Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilha, Portugal

ABSTRACT: Storage of sperm under refrigeration reduces its viability, due to oxidative unbalance. Unfermented teas presenthigh levels of catechin derivatives, known to reduce oxidative stress. This study investigated the effect of white tea (WTEA) onepididymal spermatozoa survival at room temperature (RT), using green tea (GTEA) for comparative purposes. The chemicalprofiles of WTEA and GTEA aqueous extracts were evaluated by 1H NMR. (−)-Epigallocatechin-3-gallate was the mostabundant 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 themost with WTEA supplementation, which was concomitant with the highest increase in sperm antioxidant potential and lipidperoxidation decrease. WTEA supplementation restored spermatozoa viability to values similar to those obtained at collectiontime. These findings provide evidence that WTEA extract is an excellent media additive for RT sperm storage, to facilitatetransport and avoid the deleterious effects of refrigeration.

KEYWORDS: sperm, Camellia sinensis, white tea, green tea, epigallocatechin-3-gallate, reactive oxygen species, antioxidants

■ INTRODUCTION

Tea (Camellia sinensis (L.)) is one of the world’s most widelyconsumed beverages, and its medicinal properties have beenwidely explored.1 It can be classified in three types:unfermented (green and white teas), partially fermented(oolong tea), and completely fermented (black tea).2 Toproduce green tea (GTEA), freshly harvested leaves aresteamed to inactivate polyphenol oxidase enzyme and thenrolled and dried. Its chemical composition is very similar to thatof the fresh tea leaf.1 White tea (WTEA) is exclusively preparedfrom young tea leaves or buds, harvested before being fullyopened. The tea materials are picked and immediately sent tobe steamed and dried to prevent oxidation, frequently followedby polymerization.3 Unfermented teas are known to have highpolyphenolic content, mainly catechin derivatives, (−)-epi-gallocatechin 3-gallate (EGCG) being the most abundant andpowerful antioxidant.4 With respect to processing, there arevery little differences between green and white teas, althoughseveral papers suggest that WTEA presents higher levels ofantioxidants than GTEA.5 Recently, antioxidant componentshave aroused great interest due to their ability to minimize thedeleterious effects of reactive oxygen species (ROS) on anumber of biological and pathological processes.6 ROS arenecessary for the normal physiological function of sperm,7

although their concentration must be kept under strict controlto avoid deleterious effects, such as damage to cell structures:lipids and membranes, proteins, and DNA.8 It has beenreported that ROS overproduction results in oxidative stress(OS), which is related to several problems that may end up inmale subfertility or infertility.9 In fact, spermatozoa areparticularly vulnerable to such stress because ROS readilyattack the polyunsaturated fatty acids (PUFA) of the cellsmembrane, initiating a self-propagating chain reaction. End-products of these lipid peroxidation reactions, such as

malondialdehyde (MDA), are especially dangerous for cellviability.10 Therefore, there is a growing interest in enlighteningthe role of ROS formation in sperm as they are responsible forlower sperm quality in freshly collected semen and poor qualityof sperm after processing for usage in reproductivetechnologies, such as artificial insemination (AI), in vitrofertilization (IVF), or cryopreservation.11

The maintenance of mammalian sperm at room temperature(RT) for short-term periods is advantageous as the storage ofsperm in a refrigerated environment induces a rapid decline inits viability.12 Establishment of optimal composition for spermstorage is of extreme relevance, as these cells are highlydependent on the supply of exogenous substrates and, due totheir high metabolic rates, produce elevated amounts of ROS.12

The addition of GTEA polyphenols has proven to be of greatsignificance on frozen−thawed spermatozoa motility.13 Here,we aimed to investigate the possible protective effect of WTEAextract on epididymal spermatozoa survival at RT, using GTEAas a comparison reference.14 For that purpose, the chemicalprofiles of WTEA and GTEA aqueous extracts were determinedby using 1H NMR as well as the antioxidant potential of storagemedia containing these extracts. Furthermore, the effect of bothextracts on epididymal spermatozoa maintenance at RT during24, 48, and 72 h was evaluated by determining the spermatozoaantioxidant potential, lipid peroxidation, and viability duringthat time frame.

Received: June 5, 2013Revised: December 28, 2013Accepted: December 28, 2013Published: December 28, 2013

Article

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© 2013 American Chemical Society 608 dx.doi.org/10.1021/jf4049462 | J. Agric. Food Chem. 2014, 62, 608−617

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■ MATERIALS AND METHODSChemicals. 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 - Comercio e Distribuicao Lda.,Portugal). Samples (n = 5) were subjected to infusion (1 g/100 mLdistilled water; pH 5.5) at 100 °C during 3 min, according to themanufacturer’s instructions. The resulting infusions were filtered withqualitative filter papers (catalog no. 516-0819, VWR, Leuven, France)in a vacuum system and freeze-dried overnight in a ScanVacCoolSafeFreeze-Dryer (Labogene, Lynge, Denmark). The mean extractionyields (g of lyophilized extract per 100 g of dried teas leaves) were 25%(for WTEA) and 20% (for GTEA). The lyophilized extracts were keptin a desiccator, protected from light, until analysis.Proton Nuclear Magnetic Resonance (1H NMR). 1H NMR

spectra were acquired as previously described.15 Briefly, 1H NMRspectra of WTEA and GTEA aqueous extracts dissolved in D2O wereacquired at 14.1 T, 25 °C, using a Bruker Avance 600 MHzspectrometer equipped with a 5 mm QXI probe and a z-gradient. 1HNMR spectra were acquired with solvent suppression and a sweepwidth of 6 kHz, using a delay of 14 s, a water presaturation of 3 s, apulse 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 teaextracts and was used as a reference (6.50 ppm), as routinely done inour laboratory,16,17 to quantify the extract compounds wheneverpresent in solution. The following coupling patterns, available in theliterature,18−23 were used to quantify the identifiable extractcompounds (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); (−)-epi-catechin (EC) (singlet, 7.0) (for a representative image see Figure 1).The relative areas of 1H NMR resonances were quantified using thecurve-fitting routine supplied with the NUTSproTM NMR spectralanalysis 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 animalcolony (Health Sciences Research Centre, University of Beira Interior)and maintained on ad libitum food and water in a constant roomtemperature (20 ± 2 °C) on a 12 h cycle of artificial lighting. Rats werefed a standard chow diet (4RF21 certificate, Mucedola, Italy). Allanimal experiments were performed according to the Guide for the

Care and Use of Laboratory Animals published by the U.S. NationalInstitutes of Health (NIH Publication 85-23, revised 1996) and theEuropean directives for the care and handling of laboratory animals(Directive 86/609/EEC), after approbation by the National EthicsCommittee of Animal Welfare.

Animals were anesthetized, by intraperitoneal injection of a mixtureof 90 mg/kg of ketamine and 10 mg/kg of xylazine, and euthanized bycervical displacement. Cauda epididymis were isolated and immedi-ately placed separately in 3 mL of a prewarmed (37 °C) Krebs−Ringerbicarbonate (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 streptomycinsulfate), prepared on the day of the experiment, as described byToyoda.24 The two cauda epididymis of each animal were mincedtogether with a scalpel blade, to allow sperm to disperse into themedium, 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; sodiumlactate, 23.28; HEPES, 20.85; plus 4.00 mg/mL BSA, 0.06 mg/mLpotassium penicillin G, 0.05 mg/mL streptomycin sulfate) aspreviously described.25 Moreover, the same amount of spermatozoawas placed in four other media, with the same composition of thecontrol group but containing additional concentrations of freeze-driedWTEA or GTEA aqueous extracts to a final concentration of 0.5 or 1mg/mL. Subsequently, sperm suspensions were kept at acclimatizedRT (22−23 °C) during 24, 48, and 72 h. These sperm suspensionswere used for the FRAP, TBARS, and sperm viability assays, afterbeing processed as described in each section.

Protein Quantification. After the 72 h of incubation, spermatozoawere separated from the storage media through centrifugation at 5000gfor 15 min at 4 °C. Then, sperm pellets were washed with 100 μL ofphosphate-buffered saline (PBS) solution (in mM: NaCl, 137; KCl,2.7; Na2HPO4, 4.3; KH2PO4, 1.47; pH 7.4) and centrifuged again at5000g for 15 min at 4 °C. Total proteins were isolated from thespermatozoa by the addition of an adequate amount of PBS, followedby a sonication for 15 min at 4 °C. Protein concentration wasdetermined by Bio-Rad (Hemel Hempstead, UK) Bradford microassayaccording to the manufacturer’s instructions. Sperm suspensions wereused in FRAP and TBARS assays.

Ferric Reducing Antioxidant Power (FRAP) assay. The ferricreducing antioxidant power (FRAP) of the media samples and

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.

Journal of Agricultural and Food Chemistry Article

dx.doi.org/10.1021/jf4049462 | J. Agric. Food Chem. 2014, 62, 608−617609

spermatozoa pellets was performed according to the colorimetricmethod described by Benzie and Strain.26 Briefly, working FRAPreagent 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 mixedwith 6 μL of each sample. The reduction of the Fe3+−TPTZ complexto a colored Fe2+−TPTZ complex by the samples was monitoredimmediately after addition of the sample and 40 min later, bymeasuring the absorbance at 595 nm using an Anthos 2010 microplatereader (Biochrom, Berlin, Germany). The antioxidant potential of thesamples was determined against standards of ascorbic acid, which wereprocessed in the same manner as the samples. Absorbance results werecorrected by using a blank, with water instead of sample. The changesin absorbance values of test reaction mixtures were used to calculateFRAP values as described elsewhere.26

Thiobarbituric Acid Reactive Substances (TBARS) Assay.TBARS are formed as a byproduct of lipid peroxidation, which can bedetected by the TBARS assay using thiobarbituric acid (TBA) as areagent. This peroxidation reaction produces MDA, which reacts withTBA in conditions of high temperature and low pH, generating a pinkcomplex that absorbs at 532 nm.27 The TBARS assay was carried outaccording to the method described by Iqbal et al.,28 with slightadaptations. Briefly, the reaction mixture in a total volume of 0.1 mLcontained 0.01 mL of the sample, 0.01 mL of Tris-HCl buffer (150mM, pH 7.1), 0.01 mL of ferrous sulfate (1.0 mM), 0.01 mL ofascorbic acid (1.5 mM), and 0.06 mL of H2O. This mixture wasincubated at 37 °C for 15 min. The reaction was stopped by theaddition 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 sampleswere incubated for 15 min at 100 °C. Finally, samples were subjectedto centrifugation at 3000g for 10 min at 4 °C. The amount of MDAformed in each of the samples was estimated by measuring the opticaldensity at 532 nm using a UV−vis spectrophotometer (Shimadzu,Kyoto, Japan) against a blank. The results were expressed asnanomoles 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, inaddition to allowing sperm to be readily visualized, permits assessmentof sperm membrane integrity. This method was performed with slightmodifications of a method previously described.29 A total of 5 μL ofthe sperm suspensions obtained as described above (see Isolation ofEpididymal 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. Thenumber of viable and nonviable spermatozoa was determined bycounting a total of 333 spermatozoa per slide in continuous randomfields under a light microscope, with oil immersion (×1000magnification), to determine the percentage of viable sperm, aspreviously described.16 Live sperm remained white, whereas deadsperm stained pink, because the integrity of their plasma membranewas compromised, causing an increase in membrane permeability,which led to the uptake of the dye (Figure 5A).Statistical Analysis. Statistical significance was assessed by two-

way ANOVA, followed by Bonferroni post-test using GraphPad Prism5 (GraphPad Software, San Diego, CA, USA). All data are presented asthe mean ± SEM. Differences with p < 0.05 were consideredstatistically significant. Further analysis of the statistical power (SP) ofdifferences of experimental data was evaluated with a one-tailed testassuming an α value of 0.05, which corresponds to a 0.95 confidenceinterval, as described by Levin,30 using the software provided byhttp://www.dssresearch.com/KnowledgeCenter/toolkitcalculators/statisticalpowercalculators.aspx.

■ RESULTS

White Tea Extract Presents the Highest Content inCatechin Derivatives, Especially EGCG. Tea has beencharacterized by its high content in flavonoids,31 such ascatechin derivatives and other polyphenols,32 which have

biological and pharmaceutical properties that have been linkedto beneficial effects on human health.33

The 1H NMR data obtained from the aqueous extractsallowed the assignment of the following phytochemicals: threecatechin derivatives, EC, EGC, and EGCG; one methylxan-thine, 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 catechinfamily, representing 133 ± 14 and 91 ± 7 g/kg of WTEA andGTEA extracts, respectively. Among catechins, EGCG was theone present in the highest amount in both tea extracts, althoughin WTEA extract the quantity of this compound (82 ± 7 g/kgof WTEA extract) was nearly twice that verified in GTEAextract (42 ± 2 g/kg of GTEA extract). Concerning caffeineand sucrose, WTEA extract also demonstrated a larger amount(71 ± 8 and 60 ± 4 g/kg of WTEA extract, respectively) incomparison with GTEA (21 ± 3 and 26 ± 2 g/kg of GTEAextract, correspondingly). L-Theanine, alanine, and glucosewere 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 (Table1).

In fact, (−)-epicatechin 3-gallate (ECG), (−)-catechin (C),and (−)-gallocatechin-3-gallate (GCG) were absent in ourextracts. Generally, these polyphenols are minor tea compo-nents 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 teaqualitative phytochemical profiles obtained using 1H NMR(Table 1) are in accordance with the phenolic andmethylxanthine profiles obtained by HPLC-UV, HPLC-DAD,or HPLC-MS previously reported by Carvalho et al.,34 Rusak etal.,35 and Unachukwu et al.36 The observed differences in thequantitative profile may be mainly due to the use of differentextraction conditions (solvents, temperatures, times ofextraction, and ratio leaves/water). Also, the natural variabilityof plants caused by edapho-climatic factors, harvestingtechniques, or agricultural practices may contribute to thesedifferences.

Storage Media Supplemented with White Tea ExtractHave the Highest Antioxidant Potential. As the aqueousextracts of WTEA and GTEA demonstrated to be rich in

Table 1. White (WTEA) and Green Tea (GTEA) ExtractPhytochemical Profiles As Determined by 1H NMRSpectroscopy

contenta (g of compd/kg of tea extract)

compound WTEA GTEA

glucose 6 ± 1.00 11 ± 2.00sucrose 60 ± 4.00 26 ± 2.00lactate 0.40 ± 0.01 0.46 ± 0.02alanine 0.7 ± 0.10 0.20 ± 0.04caffeine 71 ± 8.00 21 ± 3.00L-theanine 19 ± 2.00 22 ± 1.00ECb 5 ± 1.00 28 ± 2.00EGCc 46 ± 6.00 21 ± 3.00EGCGd 82 ± 7.00 42 ± 2.00

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



http://www.dssresearch.com/KnowledgeCenter/toolkitcalculators/statisticalpowercalculators.aspx

http://www.dssresearch.com/KnowledgeCenter/toolkitcalculators/statisticalpowercalculators.aspx

catechin derivatives, namely, EGCG, we performed a FRAPassay to evaluate the antioxidant potential of all experimentalmedia supplemented with these extracts in comparison with thecontrol media. The FRAP assay measures the potential of anantioxidant to reduce ferric(III) to ferrous(II) in a redox-linkedcolorimetric reaction that involves single electron transfer.37

The reducing power of a compound/extract serves as asignificant indicator of its potential antioxidant activity (FRAPvalue). The control medium showed a FRAP value of 1.64 ±0.05 μmol of antioxidant potential/L (Figure 2). On the otherhand, the FRAP value of the media supplemented with WTEAand GTEA revealed a dose-dependent reducing power (Figure2), with significantly higher FRAP values relative to the control

group during all of the experiments (SP = 100%). ComparingGTEA supplementation, the medium with 1 mg/mL of GTEAextract presented a significantly higher antioxidant potentialcompared 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 mediumsupplemented with 0.5 mg/mL of WTEA extract was 14 ±0.3 μmol of antioxidant potential/L, which was significantlyhigher compared to the medium with the same concentrationof GTEA extract (SP = 100%). Moreover, the mediumcontaining 1 mg/mL of WTEA presented a significantly higherantioxidant potential of 24 ± 0.6 μmol of antioxidant potential/L in comparison with the medium supplemented with 0.5 mg/

Figure 2. Ferric reducing antioxidant power (FRAP) of the epididymal spermatozoa storage media, control medium (CTRL), and mediasupplemented with freeze-dried WTEA or GTEA aqueous extracts to a final concentration of 0.5 or 1 mg/mL. The antioxidant power is expressed bythe 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 GTEAextract/mL.

Figure 3. Ferric reducing antioxidant power (FRAP) of the epididymal spermatozoa stored in control medium (CTRL) and media supplementedwith 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. Theantioxidant 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.



mL of WTEA extract and 1 mg/mL of GTEA extract (SP =100%).White Tea Extract Considerably Increases the Anti-

oxidant Potential of Spermatozoa. Because the mediasupplemented with WTEA extract proved to have the highestantioxidant potential, we expected to verify the same profile inthe antioxidant potential of the spermatozoa. Therefore, wealso measured the antioxidant potential of the spermatozoapellets at 24, 48, and 72 h with the FRAP assay. Accordingly,spermatozoa kept in control medium had always a lowerantioxidant potential than spermatozoa stored in the mediasupplemented with WTEA and GTEA extracts, although thesedifferences were statistically significant only after 48 and 72 h ofstorage, respectively (Figure 3). Indeed, at 48 h thespermatozoa stored in the medium with 0.5 mg of WTEAextract/mL presented a FRAP value of 2.8 ± 0.2 μmol ofantioxidant potential/μg of protein, significantly higher thanthat of spermatozoa maintained in the control medium (1.1 ±0.3) and in medium supplemented with 0.5 mg of GTEAextract/mL (1.4 ± 0.1) (SP = 100%). At 72 h, the FRAP valuesof spermatozoa maintained in media with 0.5 and 1 mg ofGTEA 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 thecontrol group. At this time of incubation, the FRAP valuedetermined for spermatozoa kept in the control medium wassignificantly 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 ofspermatozoa stored in the media supplemented with WTEAextract (0.5 and 1.0 mg of WTEA extract/mL) were remarkablyhigher than those observed at 48 h (8.4 ± 2.7 (SP = 99.9%)and 12.2 ± 3.1 (SP = 100%) μmol of antioxidant potential/μgof protein, respectively) and also than the FRAP value of thecontrol group. Moreover, at 72 h there were no significantdifferences between both concentrations of the same extract.

Spermatozoa Lipid Peroxidation Is SignificantlyReduced by White Tea Supplementation. Duringspermatozoa storage, ROS production is known to induceseveral deleterious effects38 such as lipid peroxidation. Becausespermatozoa antioxidant potential was increased after incuba-tion with the tea extracts, we hypothesized that it could alsodecrease ROS production, thus decreasing lipid peroxidation.Therefore, we performed the TBARS assay to determine thelevels of lipid peroxidation that occurred in spermatozoa duringstorage in the different media. Because ROS have extremelyshort half-lives, they are difficult to measure directly. Instead,several products induced by OS, such as TBARS, can bemeasured and used as an accurate indicator of OS.39 Using theTBARS assay, it was possible to detect a significant decrease inlipid peroxidation in spermatozoa stored in media supple-mented with WTEA and GTEA extracts. At 24 h, there was asignificant decrease in lipid peroxidation from spermatozoastored in the control medium (73 ± 4 nmol TBARS/mgprotein) relative to the media containing 0.5 mg/mL of GTEA(47 ± 7 nmol TBARS/mg protein) and 0.5 and 1 mg/mL ofWTEA (54.8 ± 4.7 and 46 ± 2 nmol TBARS/mg protein,respectively) (Figure 4) (SP = 100%). After 48 h, the lipidperoxidation significantly decreased from 81 ± 10 nmolTBARS/mg protein in the control group to 44 ± 6 and 41 ±8 nmol TBARS/mg protein (SP = 100%) in the spermatozoastored in the media supplemented with 0.5 and 1 mg of WTEAextract/mL, respectively (Figure 4). Moreover, the spermato-zoa stored in the medium with 1 mg of WTEA extract/mL alsopresented significantly less lipid peroxidation than thespermatozoa maintained in the medium with 1 mg of GTEAextract/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 nmolTBARS/mg protein that significantly decreased to 58 ± 8, 42 ±5, and 41 ± 3 nmol TBARS/mg protein (SP = 100%) afterstorage in the media supplemented with 0.5 mg of GTEAextract/mL and 0.5 and 1 mg of WTEA extract/mL,

Figure 4. Sperm TBARS values produced in epididymal spermatozoa stored in control medium (CTRL) and media supplemented with freeze-driedWTEA 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 innmol/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, 0.5mg of GTEA extract/mL; e, versus GTEA, 1 mg of GTEA extract/mL.



respectively. Additionally, lipid peroxidation in spermatozoakept in the media with 0.5 and 1 mg of WTEA extract/mL weresignificantly lower than in spermatozoa stored in the mediasupplemented with 0.5 (SP = 99.5%) and 1 mg of GTEAextract/mL (SP = 100%), correspondingly.White Tea Supplementation Increases Spermatozoa

Viability after Room Temperature Storage. Spermviability is one of the most important parameters to assesssperm quality.40 Once the WTEA and GTEA supplementationsignificantly increased the antioxidant potential of storagemedia and spermatozoa and decreased spermatozoa lipidperoxidation, we hypothesized that sperm viability could beimproved by these extracts. Therefore, we evaluated the spermviability 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 weobserved that viability was continuously decreasing, presentingvalues of 31 ± 2, 19 ± 2, and 14 ± 1% at 24, 48, and 72 h,respectively (Figure 5B) and being significantly lowercompared to viability at collection time (SP = 100%). Therewas a dose-dependent significant increase in viability, during

the 3 day storage, in spermatozoa stored in mediasupplemented with both tea extracts when compared with thecontrol. At 24 h, viability of spermatozoa kept in the mediasupplemented with any of the tea extracts was very similar tothat observed at 0 h. At 48 h, viability of spermatozoa kept inthe media with 0.5 mg/mL of GTEA or WTEA significantlydecreased (49 ± 2 and 53 ± 5%, respectively) in comparisonwith that observed, 67 ± 2%, at the collection time (SP =100%). Conversely, viability corresponding to the spermatozoastored in the media with 1 mg/mL of GTEA or WTEA (59 ± 3(SP = 100%) and 65 ± 3% (SP = 99.5%), respectively) wassignificantly higher than the viability verified in the mediacontaining half the concentration of these extracts. The sameprofile was verified at 72 h of storage with the only exceptionthat spermatozoa kept in 1 mg of WTEA extract/mL mediumexhibited significantly higher viability of 63 ± 3% in relation tothe spermatozoa kept in the medium with 1 mg of GTEAextract/mL, which was 49 ± 2% (SP = 100%), being in its turnsignificantly lower compared to the viability at 0 h (SP =100%). Remarkably, spermatozoa maintained in the mediumwith 1 mg of WTEA extract/mL averaged a viability of 65%

Figure 5. (A) Spermatozoa staining using eosin/nigrosin showing a viable (left) and a nonviable (right) cell. (B) Spermatozoa viability at collectiontime (0 h) and during the 3 day storage in control medium (CTRL) and media supplemented with freeze-dried WTEA or GTEA aqueous extracts toa 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 GTEAextract/mL.



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

■ DISCUSSIONDespite the similarities between GTEA and WTEA, thenumber of investigations studying the health benefits ofWTEA is negligible compared to GTEA. Recently, WTEAaroused great interest among investigators due to its higherconcentrations of tea polyphenols when compared to GTEA.41

Thus, WTEA has been proven to have higher antioxidantactivity than any other type of tea.2,5 As expected, the WTEAextract we obtained was richer in this class of flavonoids thanthe GTEA extract, predominantly in EGCG (82 ± 7 g/kg ofWTEA extract vs 42 ± 2 g/kg of GTEA extract), which isknown as one of the most powerful antioxidants and the mostpharmacologically active catechin derivative.42 There are severalpieces of evidence that EGCG has a positive impact in a varietyof human diseases that is dependent on its concentration.2,43,44

EGCG has been proven to have protective action againstseveral deleterious effects of diseases by minimizing OS.45

Recently, it was reported that EGCG therapy protects againsttesticular ischemia-reperfusion injury through its antioxidantactivity.46 Importantly, it has been reported that for humanspermatozoa maintained at 37 °C during 30 min, motility andviability can be improved by the addition of EGCG to theextracellular media at low concentrations.47 In fact, our resultsshowed that WTEA is very rich in EGCG, which represents61.7% of the total catechin content, and the lower content inEGCG of GTEA may be reflected in the antioxidant propertiesof the extract.41 In addition, caffeine content is also known tobe higher in WTEA,41 and our extract presented a remarkablyhigh caffeine content (71 ± 8 g/kg of WTEA extract) whencompared with the results obtained by other authors.48 Thisphytochemical content difference, as well as the variation foundbetween our GTEA extract composition and the ones verifiedby other authors,34 could be due to the natural variability of theplants (caused by the influence of geographical origin, climate,and/or agricultural practices) and to differences in theextraction procedures (solvents, temperatures, times ofextraction, and ratio of leaves to water) and analyticaltechniques.35,49,50 Our WTEA extract also presented highercaffeine content compared to our GTEA extract (21 ± 3 g/kgof GTEA extract). This should be due to differences in leafprocessing (e.g., the collection date and/or the type of leaves)to produce WTEA or GTEA, which have already been provento influence the tea chemical composition.41 Nevertheless,caffeine is described to stimulate lipolysis and interfere withglucose and fatty acid metabolism, thus having an importantrole in cellular metabolism.51 The WTEA extract was also richerin the content of total sugars, which are known to be importantsubstrates to cellular metabolism. Overall, our 1H NMR resultsconcerning WTEA and GTEA are in accordance with thephenolic and methylxanthine profiles obtained by HPLC-UV,HPLC-DAD, or HPLC-MS previously reported by Carvalho etal.,34 Rusak et al.,35 and Unachukwu et al.36 Possible differencesfound in our quantitative profile compared to previous ones arecertainly 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 asone of most relevant factors responsible for poor semenquality.53 OS is associated with uncontrolled ROS productionthat, when it exceeds spermatozoa antioxidant capacity,becomes harmful, inducing membrane lipid peroxidation,

compromising spermatozoa survival and fertilizing capacity.54

Epididymal spermatozoa survival and maintenance are crucialfor both natural and assisted reproduction.55 The mammalianepididymis creates a unique microenvironment that helps intesticular spermatozoa maturation from an immotile immaturestate to fully fertile competent cells. Besides, it stores the fertileand viable spermatozoa in cauda epididymis until ejaculation.For that reason, spermatozoa from cauda epididymis are oftenused in assisted reproductive technologies. In fact, cryopre-servation and refrigeration of spermatozoa have been highlydebated, and it has been proposed that the maintenance ofspermatozoa at RT for short-term periods can be an effectivealternative to avoid the rapid decline of sperm viability afterstorage in a refrigerated environment.12,56 Hence, there is agrowing interest in the establishment of an optimalcomposition medium for RT sperm storage. Several mediaintended to improve spermatozoa survival at RT have beendeveloped, but spermatozoa viability after storage in thosemedia is still very low and far from the ideal.12,57−60 A studyusing mouse sperm evaluated the effect of RT storage in variousbicarbonate and phosphate-based media used for IVF orembryo culture, reporting that the low bicarbonate- andHEPES-containing media were able to better preservespermatozoa in vitro.12 The addition of substrates, such asglucose, further enhanced sperm survival, although the authorsconcluded that further tests concerning the addition ofpreserving agents are still required.12,60 Studies using spermfrom different mammalian origins preserved at RT in a saline-buffered storage medium also showed that supplementationwith serum or egg yolk could greatly increase sperm survivaland function.57−59 At the same time those authors concludedthat there was still a crucial requirement in controlling the ROS,which are more likely to be generated in an ambienttemperature system, in the media in which the spermatozoaare suspended. Indeed, some studies have been made to assessthe 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) weretested as a supplement of the storage media, and although theauthors obtained encouraging results in the sperm fertilizationability of frozen−thawed sperm, they did not verify a significantincrease in sperm survival after storage.13

Therefore, we hypothesized that WTEA extract couldmodulate some important functions such as antioxidantcapacity and lipid peroxidation of spermatozoa, thus improvingspermatozoa viability during RT storage. Using the FRAP assay,we verified that the media containing WTEA extract presenteda higher antioxidant potential value, in a dose-dependentmanner, than the media with GTEA extract, evidencing that theantioxidant potential is greatly increased by the addition ofWTEA extract. The higher increase in the antioxidant potentialof sperm after storage was verified in media with WTEA extract,and this is concomitant with the alteration measured for themedia antioxidant potential. Indeed, the sperm antioxidantpotential showed an increase over time with storage in WTEAsupplemented media that is surely due to incorporation ofantioxidant compounds present in the extract. The same profilewas verified in sperm stored with GTEA extract in a muchsmaller extent, evidencing that WTEA extract is the one givingthe best antioxidative potential.One of the most important deleterious effects caused by OS

is lipid peroxidation because mammalian spermatozoa are richin PUFA that are highly vulnerable to OS.10 When subjected to



high levels of OS, spermatozoa lipid membranes are oxidized,and the end-product of these reactions, MDA, can be measuredby the TBARS assay. Our results showed that during the 3 daysperm storage at RT, WTEA extract was the most effective indecreasing the lipid peroxidation in spermatozoa. As WTEAextract presented a higher concentration of polyphenols, whichare known to decrease lipid peroxidation in cells, it wasexpected that the media with WTEA extract presented a higherantioxidant potential and, consequently, a higher capacity toprevent lipid peroxidation. However, at 24 h, although therewas a significant decrease in lipid peroxidation relative to thecontrol group, there were no significant differences betweenstorage with WTEA or GTEA. This is concordant with thesperm FRAP assay at 24 h that also showed no differences inthe antioxidant potential of sperm stored with both tea extracts.This could be explained by ROS production, which is expectedto increase over time and needs to be accompanied by theantioxidant defenses increase to control the amount of ROS.After 48 and 72 h, lipid peroxidation of sperm stored in themedia supplemented with WTEA extract remained constantand similar to that observed at 24 h, whereas in sperm stored inthe media containing GTEA extract the lipid peroxidationincreased 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 GTEAextract, these amounts reflect EGCG concentrations in theranges of about 90−180 μM for WTEA and 45−90 μM forGTEA supplemented sperm storage media. Therefore, as ourEGCG concentrations are in the same order of magnitude ofthose used in the study of Kaedei et al.13 and, conversely to thedata reported by those authors who were not able to verify asignificant increase in the survival of sperm kept in the EGCGsupplemented media, our results showed that tea supplementa-tion highly improved spermatozoa survival, and this supportsour idea that the synergistic effect between the tea componentsmay be responsible for the positive effects observed in spermviability. Additionally, our results support the effectiveness ofWTEA 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 withsubfertility or infertility, the condition is due to loss of spermfunction rather than the number of spermatozoa.62 Therefore,spermatozoa viability is an essential parameter to evaluatesperm quality and male factor infertility. Supplementation ofthe storage media with tea extracts significantly increased spermviability in a dose-dependent manner. OS is known to play acrucial role in the loss of functional competence, and whenROS production is significantly elevated, dysfunctionalspermatozoa are produced.63 A negative correlation betweenROS production and sperm movement, evidencing theimportance of ROS control in spermatozoa function, has alsobeen reported.64 High ROS levels are reported to bedetrimental to fertility potential in natural and assistedconception,65 and sperm capacitation is also known to be lostin the presence of high OS levels.66 Therefore, the higherincrease of sperm viability in spermatozoa stored in WTEA-supplemented media compared to spermatozoa stored inGTEA-supplemented media is certainly due to the higherpolyphenolic content in WTEA extract and, consequently, to itshigher antioxidant potential. Moreover, because the viabilityimprovement was more effective with the highest dose ofWTEA extract (1 mg/mL), we suggest that the best protectionattained with the highest dose of WTEA extract may be

responsible for the overall better results observed with thisconcentration.In conclusion, WTEA is richer in antioxidants than other teas

(black, oolong, and green teas),2 and the addition of WTEAaqueous extract to the media can be a good, simple, andinexpensive strategy for sperm storage at RT. Our resultsstrongly indicate that WTEA extract improves spermatozoaviability by increasing the spermatozoa and storage mediaantioxidant potential and decreasing spermatozoa lipidperoxidation. Moreover, the first hours of sperm preservationis crucial and marked by an increase in OS that can becounteracted by WTEA polyphenols. More studies will beneeded to fully disclose the molecular mechanisms behind theresults now reported. Nevertheless, the addition of WTEAaqueous extract to the standard sperm storage medium provedto be an excellent strategy for sperm storage at RT, which canreduce or even eliminate the limitations of the in vitrospermatozoa storage in a refrigerated environment and enablesperm transport for IVF or AI use.

■ AUTHOR INFORMATIONCorresponding 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].

FundingThis work was supported by the Fundacao para a Ciencia e aTecnologia − FCT (PTDC/QUI-BIQ/121446/2010 and PEst-C/SAU/UI0709/2011) cofunded by Fundo Europeu deDesenvolvimento Regional − FEDER via Programa Oper-acional Factores de Competitividade − COMPETE/QREN.T.R.D. was funded by Programa Operacional Regional doCentro 2007−2013 QREN (Programa Mais Centro). M.G.A.(SFRH/BPD/80451/2011) was funded by FCT. P.F.O. wasfunded by FCT through FSE and POPH funds (ProgramaCiencia 2008).

NotesThe authors declare no competing financial interest.

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

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white tea as a promising antioxidant medium additive for sperm storage at room temperature: a...

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