Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology

Improving the effect of incubation and oxidative stress on thawed spermatozoa from red deer by using different antioxidant treatments

Álvaro E. Domínguez-Rebolledo A , María R. Fernández-Santos A B C , Alfonso Bisbal A , José Luis Ros-Santaella A , Manuel Ramón C , Manuel Carmona D , Felipe Martínez-Pastor E F G and J. Julián Garde A B F

A Biology of Reproduction Group, National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), 02071 Albacete, Spain.

B Institute for Regional Development (IDR), 02071 Albacete, Spain.

C Regional Center of Animal Selection and Reproduction (CERSYRA), JCCM, Valdepeñas, 13300 Ciudad Real, Spain.

D Cátedra de Química Agrícola, E.T.S.I. Agrónomos (UCLM), 02071 Albacete, Spain.

E ITRA-ULE, INDEGSAL, University of León, 24071 León, Spain.

F These authors contributed equally to this study.

G Corresponding author. Email:

Reproduction, Fertility and Development 22(5) 856-870
Submitted: 19 August 2009  Accepted: 10 December 2009   Published: 15 April 2010


Antioxidants could improve sperm media, extending the viability of spermatozoa and protecting their DNA. The protective ability of lipoic acid, melatonin, Trolox and crocin was tested on red deer spermatozoa incubated at 37°C. Cryopreserved spermatozoa were thawed and incubated with 1 mM or 0.1 mM of each antioxidant, with or without oxidative stress (100 μM Fe2+). Motility (CASA), viability, mitochondrial membrane potential and acrosomal status were assessed. Lipoperoxidation (malondialdehyde production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were checked at 4 h. Incubation alone increased ROS and decreased motility. Oxidative stress intensified these effects, increasing lipoperoxidation and DNA damage. Lipoic acid had little protective effect, whereas 1 mM melatonin showed limited protection. Trolox lowered ROS and lipoperoxidation both in oxidised and non-oxidised samples. In oxidised samples, Trolox prevented DNA and acrosomal damage, and ameliorated motility. Crocin at 1 mM showed similar results to Trolox, but noticeably stimulated motility and had no effect on lipoperoxidation. In a second experiment, a broader range of crocin and melatonin concentrations were tested, confirming the effects of crocin (positive effects noticeable at 0.5–0.75 mM), but showing an increase in lipoperoxidation at 2 mM. Melatonin was increasingly effective at 2.5 and 5 mM (ROS, lipoperoxidation and DNA status). Crocin seems a promising new antioxidant, but its particular effects on sperm physiology must be further studied, especially the consequences of motility stimulation and confirming its effect on lipoperoxidation. Melatonin might be useful at relatively high concentrations, compared to Trolox.

Additional keywords: crocin, lipoic acid, melatonin, Trolox.


Acuña-Castroviejo D.Martín M.Macías M.Escames G.León J.Khaldy H.Reiter R. J.2001Melatonin, mitochondria and cellular bioenergetics.J. Pineal Res.306574doi:10.1034/J.1600-079X.2001.300201.XPubmed Abstract

Aitken R. J.Baker M. A.2004Oxidative stress and male reproductive biology.Reprod. Fertil. Dev.16581588doi:10.1071/RD03089Pubmed Abstract

Aitken R. J.Harkiss D.Buckingham D. W.1993Analysis of lipid peroxidation mechanisms in human spermatozoa.Mol. Reprod. Dev.35302315doi:10.1002/MRD.1080350313Pubmed Abstract

Askari H. A.Check J. H.Peymer N.Bollendorf A.1994Effect of natural antioxidants tocopherol and ascorbic acids in maintenance of sperm activity during freeze–thaw process.Arch. Androl.331115doi:10.3109/01485019408987797Pubmed Abstract

Bast A.Haenen G. R. M. M.2003Lipoic acid: a multifunctional antioxidant.Biofactors17207213doi:10.1002/BIOF.5520170120Pubmed Abstract

Berlinguer F.Ledda S.Rosati I.Bogliolo L.Leoni G.Naitana S.2003Superoxide dismutase affects the viability of thawed European mouflon (Ovis g. musimon) semen and the heterologous fertilization using both IVF and intracytoplasmatic sperm injection.Reprod. Fertil. Dev.151925doi:10.1071/RD02048Pubmed Abstract

Cao G.Cutler R. G.1993High concentrations of antioxidants may not improve defence against oxidative stress.Arch. Gerontol. Geriatr.17189201doi:10.1016/0167-4943(93)90050-RPubmed Abstract

Carmona M.Zalacain A.Pardo J. E.López E.Alvarruiz A.Alonso G. L.2005Influence of different drying and ageing conditions on saffron constituents.J. Agric. Food Chem.5339743979doi:10.1021/JF0404748Pubmed Abstract

Cassani P.Beconi M. T.O’Flaherty C.2005Relationship between total superoxide dismutase activity with lipid peroxidation, dynamics and morphological parameters in canine semen.Anim. Reprod. Sci.86163173doi:10.1016/J.ANIREPROSCI.2004.06.006Pubmed Abstract

Chen H.Chow P. H.Cheng S. K.Cheung A. L. M.Cheng L. Y. L.O W. S.2003Male genital tract antioxidant enzymes: their source, function in the female, and ability to preserve sperm DNA integrity in the golden hamster.J. Androl.24704711Pubmed Abstract

de Lamirande E.O’Flaherty C.2008Sperm activation: role of reactive oxygen species and kinases.Biochim. Biophys. Acta1784106115Pubmed Abstract

Domínguez-Rebolledo A. E.Fernández-Santos M. R.García-Alvarez O.Maroto-Morales A.Garde J. J.Martínez-Pastor F.2009Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa.Theriogenology7210731084doi:10.1016/J.THERIOGENOLOGY.2009.06.027Pubmed Abstract

Donnelly E. T.McClure N.Lewis S. E.1999aAntioxidant supplementation in vitro does not improve human sperm motility.Fertil. Steril.72484495doi:10.1016/S0015-0282(99)00267-8Pubmed Abstract

Donnelly E. T.McClure N.Lewis S. E.1999bThe effect of ascorbate and alpha-tocopherol supplementation in vitro on DNA integrity and hydrogen peroxide-induced DNA damage in human spermatozoa.Mutagenesis14505512doi:10.1093/MUTAGE/14.5.505Pubmed Abstract

Fernández-Santos M. R.Martínez-Pastor F.García-Macías V.Esteso M. C.Soler A. J.Paz P.Anel L.Garde J. J.2007Sperm characteristics and DNA integrity of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa frozen in the presence of enzymatic and nonenzymatic antioxidants.J. Androl.28294305doi:10.2164/JANDROL.106.000935Pubmed Abstract

Fernández-Santos M. R.Domínguez-Rebolledo A. E.Esteso M. C.Garde J. J.Martínez-Pastor F.2009aCatalase supplementation on thawed bull spermatozoa abolishes the detrimental effect of oxidative stress on motility and DNA integrity.Int. J. Androl.32353359doi:10.1111/J.1365-2605.2008.00871.XPubmed Abstract

Fernández-Santos M.Domínguez-Rebolledo A.Esteso M.Garde J.Martínez-Pastor F.2009bRefrigerated storage of red deer epididymal spermatozoa in the epididymis, diluted and with vitamin C supplementation.Reprod. Domest. Anim.44212220doi:10.1111/J.1439-0531.2007.01032.XPubmed Abstract

Gadea J.Garcia-Vazquez F.Matas C.Gardon J. C.Canovas S.Gumbao D.2005aCooling and freezing of boar spermatozoa: supplementation of the freezing media with reduced glutathione preserves sperm function.J. Androl.26396404doi:10.2164/JANDROL.04155Pubmed Abstract

Gadea J.Gumbao D.Matas C.Romar R.2005bSupplementation of the thawing media with reduced glutathione improves function and the in vitro-fertilizing ability of boar spermatozoa after cryopreservation.J. Androl.26749756doi:10.2164/JANDROL.05057Pubmed Abstract

García-Álvarez O.Maroto-Morales A.Martínez-Pastor F.Fernández-Santos M. R.Esteso M. C.Pérez-Guzmán M. D.Soler A. J.2009aHeterologous in vitro fertilization is a good procedure to assess the fertility of thawed ram spermatozoa.Theriogenology71643650doi:10.1016/J.THERIOGENOLOGY.2008.09.036Pubmed Abstract

García-Álvarez O.Maroto-Morales A.Martínez-Pastor F.Garde J. J.Ramón M.Fernández-Santos M. R.Esteso M. C.Pérez-Guzmán M. D.Soler A. J.2009bSperm characteristics and in vitro-fertilization ability of thawed spermatozoa from Black Manchega ram: electroejaculation and postmortem collection.Theriogenology72160168doi:10.1016/J.THERIOGENOLOGY.2009.02.002Pubmed Abstract

Garde J. J.Martinez-Pastor F.Gomendio M.Malo A. F.Soler A. J.Fernandez-Santos M. R.Esteso M. C.Garcia A. J.Anel L.Roldan E. R. S.2006The application of reproductive technologies to natural populations of red deer.Reprod. Domest. Anim.41Suppl. 293102doi:10.1111/J.1439-0531.2006.00773.XPubmed Abstract

Gavella M.Lipovac V.2000Antioxidative effect of melatonin on human spermatozoa.Arch. Androl.442327doi:10.1080/014850100262371Pubmed Abstract

González-Fernández L.Ortega-Ferrusola C.Macias-Garcia B.Salido G. M.Peña F. J.Tapia J. A.2009Identification of protein tyrosine phosphatases and dual-specificity phosphatases in mammalian spermatozoa and their role in sperm motility and protein tyrosine phosphorylation.Biol. Reprod.8012391252doi:10.1095/BIOLREPROD.108.073486Pubmed Abstract

Grunewald S.Sharma R.Paasch U.Glander H.Agarwal A.2009Impact of caspase activation in human spermatozoa.Microsc. Res. Tech.72878888doi:10.1002/JEMT.20732

Hardeland R.2005Antioxidative protection by melatonin: multiplicity of mechanisms from radical detoxification to radical avoidance.Endocrine27119130doi:10.1385/ENDO:27:2:119Pubmed Abstract

Heidary M.Vahhabi S.Reza Nejadi J.Delfan B.Birjandi M.Kaviani H.Givrad S.2008Effect of saffron on semen parameters of infertile men.Urol. J.5255259Pubmed Abstract

Hughes C. M.Lewis S. E.McKelvey-Martin V. J.Thompson W.1998The effects of antioxidant supplementation during Percoll preparation on human sperm DNA integrity.Hum. Reprod.1312401247doi:10.1093/HUMREP/13.5.1240Pubmed Abstract

Idziorek T.Estaquier J.De Bels F.Ameisen J. C.1995YOPRO-1 permits cytofluorometric analysis of programmed cell death (apoptosis) without interfering with cell viability.J. Immunol. Methods185249258doi:10.1016/0022-1759(95)00172-7Pubmed Abstract

Jang H., Kim Y., Kim B., Park I., Cheong H., Kim J., Park C., Kong H., Lee H., and Yang B. (2009). Ameliorative effects of melatonin against hydrogen peroxide-induced oxidative stress on boar sperm characteristics and subsequent in vitro embryo development. Reprod. Domest. Anim., in press. doi:10.1111/J.1439-0531.2009.01466.XPubmed Abstract

Kumaresan A.Kadirvel G.Bujarbaruah K. M.Bardoloi R. K.Das A.Kumar S.Naskar S.2009Preservation of boar semen at 18 degrees C induces lipid peroxidation and apoptosis-like changes in spermatozoa.Anim. Reprod. Sci.110162171doi:10.1016/J.ANIREPROSCI.2008.01.006Pubmed Abstract

Leon J.Acuna-Castroviejo D.Sainz R. M.Mayo J. C.Tan D. X.Reiter R. J.2004Melatonin and mitochondrial function.Life Sci.75765790doi:10.1016/J.LFS.2004.03.003Pubmed Abstract

Martínez-Pastor F.Anel L.Guerra C.Álvarez M.Soler A. J.Garde J. J.Chamorro Paz P.2006aSeminal plasma improves cryopreservation of Iberian red deer epididymal sperm.Theriogenology6618471856doi:10.1016/J.THERIOGENOLOGY.2006.04.036Pubmed Abstract

Martínez-Pastor F.Martínez F.García-Macias V.Esteso M.Anel E.Fernández-Santos M.Soler Paz P.Garde J.Anel L.2006bA pilot study on post-thawing quality of Iberian red deer spermatozoa (epididymal and electroejaculated) depending on glycerol concentration and extender osmolality.Theriogenology6611651172doi:10.1016/J.THERIOGENOLOGY.2006.03.027Pubmed Abstract

Martínez-Pastor F.Fernández-Santos M. R.del Olmo E.Domínguez-Rebolledo A. E.Esteso M. C.Montoro V.Garde J. J.2008Mitochondrial activity and forward scatter vary in necrotic, apoptotic and membrane-intact spermatozoan subpopulations.Reprod. Fertil. Dev.20547556doi:10.1071/RD08002Pubmed Abstract

Martínez-Pastor F.Aisen E.Fernández-Santos M. R.Esteso M. C.Maroto-Morales A.Garcia-Álvarez O.Garde J. J.2009aReactive oxygen species generators affect quality parameters and apoptosis markers differently in red deer spermatozoa.Reproduction137225235doi:10.1530/REP-08-0357Pubmed Abstract

Martínez-Pastor F.Martínez F.Álvarez M.Maroto-Morales A.Garcia-Alvarez O.Soler A. J.Garde J. Paz P.Anel L.2009bCryopreservation of Iberian red deer (Cervus elaphus hispanicus) spermatozoa obtained by electroejaculation.Theriogenology71628638doi:10.1016/J.THERIOGENOLOGY.2008.09.033Pubmed Abstract

Mickle D. A.Weisel R. D.1993Future directions of vitamin E and its analogues in minimizing myocardial ischemia-reperfusion injury and its analogues in minimizing myocardial ischemia-reperfusion injury.Can. J. Cardiol.98993Pubmed Abstract

Mortimer D.Serres C.Mortimer S. T.Jouannet P.1988Influence of image sampling frequency on the perceived movement characteristics of progressively motile human spermatozoa.Gamete Res.20313327doi:10.1002/MRD.1120200307

Mortimer S. T.Maxwell W. M. C.1999Kinematic definition of ram sperm hyperactivation.Reprod. Fertil. Dev.112530doi:10.1071/RD99019Pubmed Abstract

Nagy S.Jansen J.Topper E. K.Gadella B. M.2003A triple-stain flow cytometric method to assess plasma- and acrosome-membrane integrity of cryopreserved bovine sperm immediately after thawing in presence of egg-yolk particles.Biol. Reprod.6818281835doi:10.1095/BIOLREPROD.102.011445Pubmed Abstract

Navari-Izzo F.Quartacci M. F.Sgherri C.2002Lipoic acid: a unique antioxidant in the detoxification of activated oxygen species.Plant Physiol. Biochem.40463470doi:10.1016/S0981-9428(02)01407-9

Ochiai T.Shimeno H.Mishima K. I.Iwasaki K.Fujiwara M.Tanaka H.Shoyama Y.Toda A.Eyanagi R.Soeda S.2007Protective effects of carotenoids from saffron on neuronal injury in vitro and in vivo.Biochim. Biophys. Acta1770578584Pubmed Abstract

Ortega Ferrusola C.González Fernández L.Macías García B.Salazar-Sandoval C.Morillo Rodríuez A.Rodríguez Martinez H.Tapia J. A.Peña F. J.2009Effect of cryopreservation on nitric oxide production by stallion spermatozoa.Biol. Reprod.8111061111doi:10.1095/BIOLREPROD.109.078220Pubmed Abstract

Packer L.Witt E. H.Tritschler H. J.1995α-lipoic acid as a biological antioxidant.Free Radic. Biol. Med.19227250doi:10.1016/0891-5849(95)00017-RPubmed Abstract

Peña F. J.Johannisson A.Wallgren M.Rodriguez Martinez H.2003Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate.Anim. Reprod. Sci.788598doi:10.1016/S0378-4320(03)00049-6Pubmed Abstract

Peña F. J.Johannisson A.Wallgren M.Martinez H. R.2004Antioxidant supplementation of boar spermatozoa from different fractions of the ejaculate improves cryopreservation: changes in sperm membrane lipid architecture.Zygote12117124doi:10.1017/S096719940400262XPubmed Abstract

Peña F. J.Saravia F.Johannisson A.Walgren M.Rodríguez-Martínez H.2005A new and simple method to evaluate early membrane changes in frozen–thawed boar spermatozoa.Int. J. Androl.28107114doi:10.1111/J.1365-2605.2005.00512.XPubmed Abstract

Petrunkina A. M.Harrison R. A. P.2009Systematic mis-estimation of cell subpopulations by flow cytometry: a mathematical analysis.Theriogenologydoi:10.1016/J.THERIOGENOLOGY.2009.09.007Pubmed Abstract

Prior R. L.Wu X.Schaich K.2005Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements.J. Agric. Food Chem.5342904302doi:10.1021/JF0502698Pubmed Abstract

R Development Core Team (2008). ‘R: A Language and Environment for Statistical Computing.’ (R Foundation for Statistical Computing: Vienna.)

Rao M. V.Gangadharan B.2008Antioxidative potential of melatonin against mercury-induced intoxication in spermatozoa in vitro.Toxicol. In Vitro22935942doi:10.1016/J.TIV.2008.01.014Pubmed Abstract

Roca J.Rodriguez M. J.Gil M. A.Carvajal G.Garcia E. M.Cuello C.Vazquez J. M.Martinez E. A.2005Survival and in vitro fertility of boar spermatozoa frozen in the presence of superoxide dismutase and/or catalase.J. Androl.261524Pubmed Abstract

Scott B. C.Aruoma O. I.Evans P. J.O’Neill C.Van der Vliet A.Cross C. E.Tritschler H.Halliwell B.1994Lipoic and dihydrolipoic acids as antioxidants. A critical evaluation.Free Radic. Res.20119133doi:10.3109/10715769409147509Pubmed Abstract

Selvakumar E.Prahalathan C.Sudharsan P. T.Varalakshmi P.2006Chemoprotective effect of lipoic acid against cyclophosphamide-induced changes in the rat sperm.Toxicology2177178doi:10.1016/J.TOX.2005.08.020Pubmed Abstract

Shang X.Huang Y.Ye Z.Yu X.Gu W.2004Protection of melatonin against damage of sperm mitochondrial function induced by reactive oxygen species.Zhonghua Nan Ke Xue10604607Pubmed Abstract

Siems W.Salerno C.Crifò C.Sommerburg O.Wiswedel I.2009Beta-carotene degradation products – formation, toxicity and prevention of toxicity.Forum Nutr.617586doi:10.1159/000212740Pubmed Abstract

Silva P. F. N.Gadella B. M.2006Detection of damage in mammalian sperm cells.Theriogenology65958978doi:10.1016/J.THERIOGENOLOGY.2005.09.010Pubmed Abstract

Storey B. T.1997Biochemistry of the induction and prevention of lipoperoxidative damage in human spermatozoa.Mol. Hum. Reprod.3203213doi:10.1093/MOLEHR/3.3.203Pubmed Abstract

Upreti G. C.Jensen K.Oliver J. E.Duganzich D. M.Munday R.Smith J. F.1997Motility of ram spermatozoa during storage in a chemically-defined diluent containing antioxidants.Anim. Reprod. Sci.48269278doi:10.1016/S0378-4320(97)00054-7Pubmed Abstract

Venkatraman M.Konga D.Peramaiyan R.Ganapathy E.Dhanapal S.2008Reduction of mitochondrial oxidative damage and improved mitochondrial efficiency by administration of crocetin against benzo[a]pyrene-induced experimental animals.Biol. Pharm. Bull.3116391645doi:10.1248/BPB.31.1639Pubmed Abstract

Vijayaraghavan S.Stephens D.Trautman K.Smith G.Khatra B.da Cruz e Silva E. F.Greengard P.1996Sperm motility development in the epididymis is associated with decreased glycogen synthase kinase-3 and protein phosphatase 1 activity.Biol. Reprod.54709718doi:10.1095/BIOLREPROD54.3.709Pubmed Abstract

Wolf A. M., Asoh S., Hiranuma H., Ohsawa I., Iio K., Satou A., Ishikura M., and Ohta S. (2009). Astaxanthin protects mitochondrial redox state and functional integrity against oxidative stress. J. Nutr. Biochem., in press. doi:10.1016/J.JNUTBIO.2009.01.011Pubmed Abstract

Zanotto-Filho A.Schröder R.Moreira J. C. F.2008Differential effects of retinol and retinoic acid on cell proliferation: a role for reactive species and redox-dependent mechanisms in retinol supplementation.Free Radic. Res.42778788doi:10.1080/10715760802385702Pubmed Abstract

Zheng Y. Q.Liu J. X.Wang J. N.Xu L.2007Effects of crocin on reperfusion-induced oxidative/nitrative injury to cerebral microvessels after global cerebral ischemia.Brain Res.11388694doi:10.1016/J.BRAINRES.2006.12.064Pubmed Abstract

Export Citation Cited By (44)