Outlining adequate protocols for Lidia bull epididymal storage and sperm cryopreservation: use of glycerol, dimethylformamide and N-acetylcysteine

  • Elvira Matilla Jesús Usón Minimally Invasive Surgery Centre (CCMIJU), Assisted Reproduction Unit, N. 521 Km 41.8. 10071 Cáceres
  • Lauro González-Fernández - University of Porto, Centro de Estudos de Ciência Animal/ Instituto de Ciências, Tecnologias e Agroambiente; Campus Agrario de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, Portugal. - University of Extremadura, School of Veterinary Medicine, Research Group of Intracellular Signaling and Technology of Reproduction. (SINTREP), Av. de la Universidad s/n. 10003 Cáceres, Spain.
  • Felipe Martínez-Pastor University of León, Institute for Animal Health and Cattle Development (INDEGSAL) and Molecular Biology (Cell Biology), Campus de Vegazana s/n. 24007 León
  • Nuria Hernández Jesús Usón Minimally Invasive Surgery Centre (CCMIJU), Assisted Reproduction Unit, N. 521 Km 41.8. 10071 Cáceres
  • Carolina Tobajas Jesús Usón Minimally Invasive Surgery Centre (CCMIJU), Assisted Reproduction Unit, N. 521 Km 41.8. 10071 Cáceres
  • Violeta Calle-Guisado University of Extremadura, School of Veterinary Medicine, Research Group of Intracellular Signaling and Technology of Reproduction. (SINTREP), Av. de la Universidad s/n. 10003 Cáceres
  • José Mijares Jesús Usón Minimally Invasive Surgery Centre (CCMIJU), Assisted Reproduction Unit, N. 521 Km 41.8. 10071 Cáceres
  • Francisco M. Sánchez-Margallo Jesús Usón Minimally Invasive Surgery Centre (CCMIJU), Assisted Reproduction Unit, N. 521 Km 41.8. 10071 Cáceres
  • Ignacio S. Álvarez University of Extremadura, School of Biology, Cell Biology Dept., Av/ de Elvas s/n. 06006 Badajoz
  • Beatriz Macías-García Jesús Usón Minimally Invasive Surgery Centre (CCMIJU), Assisted Reproduction Unit, N. 521 Km 41.8. 10071 Cáceres
Keywords: fighting bull, epididymal sperm, sperm freezing extenders, antioxidants, cooling


The Lidia bovine breed is an important hallmark of the Spanish cattle industry. Bulls are selected based upon aggressiveness and epididymal sperm cryopreservation is the way to obtain and store their genetics. There are not specifically designed protocols yet to perform Lidia bull sperm cryopreservation. The present study aimed to determine if a tris-fructose-citrate-egg yolk (20% v/v; TFY) extender supplemented with 7% glycerol (TFY1) or 3.5% glycerol plus 3.5% dimethylformamide (DMF; TFY2) are suitable media for cryopreservation of epididymal Lidia bull sperm. Moreover, the effect of N-acetylcysteine (NAC), a potent antioxidant, was evaluated. The epididymis were stored at 4°C for 24, 48, 72 or 96 h, and both freezing media were tested as such or supplemented with 1 or 2.5 mM of NAC. Our data demonstrated that post-thaw viability was well maintained (TFY1: 50.8% ± 1.9 at 24 h and 52.4% ± 0.8 at 96 h and TFY2: 52.6% ± 1.6 at 24 h and 56.1% ± 1.8 at 96 h; mean % ± SEM; p>0.05) as also were total and progressive sperm motility, high mitochondrial membrane potential, ROS production, DNA status and acrosomal intactness of Lidia bull sperm up to 96 h of epididymal storage, all extender variations being similar (p>0.05). In conclusion, the use of TFY medium supplemented either with 7% glycerol alone or the combination of 3.5% glycerol and 3.5% DMF were equally safe choices for epididymal Lidia bull sperm cryopreservation, and NAC addition did not significantly improve sperm post-thaw quality.


Download data is not yet available.

Author Biography

Beatriz Macías-García, Jesús Usón Minimally Invasive Surgery Centre (CCMIJU), Assisted Reproduction Unit, N. 521 Km 41.8. 10071 Cáceres
Postdoctoral Researcher. Assisted Reproduction Unit, Minimally Invasive Surgery Center


Agarwal A, Virk G, Ong C, du Plessis SS, 2014. Effect of oxidative stress on male reproduction. World J Mens Health 32 (1): 1-17. https://doi.org/10.5534/wjmh.2014.32.1.1

Agnihotri SK, Agrawal AK, Hakim BA, Vishwakarma AL, Narender T, Sachan R, Sachdev M, 2016. Mitochondrial membrane potential (MMP) regulates sperm motility. In Vitro Cell Dev Biol Anim 52 (9): 953-960. https://doi.org/10.1007/s11626-016-0061-x

Almeida FC, Silva SV, Souza HM, Gomes WA, Lima Filho JAC, Wicke AA, Batista AM, Guerra MMP, 2017. Effects of glycerol, equilibration time and antioxidants on post-thaw functional integrity of bovine spermatozoa directly obtained from epididymis. Andrologia 49 (3): e12623. https://doi.org/10.1111/and.12623

Alvarenga MA, Papa FO, Landim-Alvarenga FC, Medeiros A, 2005. Amides as cryoprotectants for freezing stallion semen: A review. Anim Reprod Sci 89 (1-4): 105-113. https://doi.org/10.1016/j.anireprosci.2005.07.001

Álvarez C, Gil L, González N, Olaciregui M, Luño V, 2014. Equine sperm post-thaw evaluation after the addition of different cryoprotectants added to INRA 96® extender. Cryobiology 69 (1): 144-148. https://doi.org/10.1016/j.cryobiol.2014.06.008

Amann RP, Waberski D, 2014. Computer-assisted sperm analysis (CASA): Capabilities and potential developments. Theriogenology 81 (1): 5-17. https://doi.org/10.1016/j.theriogenology.2013.09.004

Bilodeau J F, Blanchette S, Gagnon C, Sirard M A, 2001. Thiols prevent H2O2-mediated loss of sperm motility in cryopreserved bull semen. Theriogenology 56 (2): 275-286. https://doi.org/10.1016/S0093-691X(01)00562-3

Canon J, Tupac-Yupanqui I, Garcia-Atance M A, Corte, O, Garcia D, Fernandez J, Dunner S, 2008. Genetic variation within the Lidia bovine breed. Anim Genet 39 (4): 439-445. https://doi.org/10.1111/j.1365-2052.2008.01738.x

Chatterjee S, Gagnon C, 2001. Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing. Mol Reprod Dev 59 (4): 451-458. https://doi.org/10.1002/mrd.1052

Chaudhari DV, Dhami AJ, Hadiya KK, Patel JA, 2015. Relative efficacy of egg yolk and soya milk-based extenders for cryopreservation (-196 degrees C) of buffalo semen. Vet World 8 (2): 239-244. https://doi.org/10.14202/vetworld.2015.239-244

Chaveiro A, Machado L, Frijters A, Engel B, Woelders H, 2006. Improvement of parameters of freezing medium and freezing protocol for bull sperm using two osmotic supports. Theriogenology 65 (9): 1875-1890. https://doi.org/10.1016/j.theriogenology.2005.10.017

Ciftci H, Verit A, Savas M, Yeni E, Erel O, 2009. Effects of N-acetylcysteine on semen parameters and oxidative/antioxidant status. Urology 74 (1): 73-76. https://doi.org/10.1016/j.urology.2009.02.034

Chen, H, Chow, P H, Cheng, S K, Cheung, A L M, Cheng, LYL, O, WS, 2003. Male genital tract antioxidant enzymes: Their source, function in the female, and ability to preserve sperm DNA integrity in the golden hamster. J Androl 24 (5): 704-711. https://doi.org/10.1002/j.1939-4640.2003.tb02730.x

Evenson DP, Larson KL, Jost, LK, 2002. Sperm chromatin structure assay: Its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J Androl 23 (1): 25-43. https://doi.org/10.1002/j.1939-4640.2002.tb02599.x

Evenson DP, Wixon R, 2006. Clinical aspects of sperm DNA fragmentation detection and male infertility. Theriogenology 65 (5): 979-991. https://doi.org/10.1016/j.theriogenology.2005.09.011

Fernández-Santos, M R, Domínguez-Rebolledo, AE, Esteso MC, Garde JJ, Martínez-Pastor F, 2009a. Catalase supplementation on thawed bull spermatozoa abolishes the detrimental effect of oxidative stress on motility and DNA integrity. Int J Androl 32 (4): 353-359. https://doi.org/10.1111/j.1365-2605.2008.00871.x

Fernández-Santos MR, Domínguez-Rebolledo AE, Esteso MC, Garde JJ and Martínez-Pastor F, 2009b. Refrigerated storage of red deer epididymal spermatozoa in the epididymis, diluted and with vitamin C supplementation. Reprod Domest Anim 44 (2): 212-220. https://doi.org/10.1111/j.1439-0531.2007.01032.x

Forero-Gonzalez RA, Celeghini ECC, Raphael CF, Andrade AFC, Bressan FF, Arruda RP, 2012. Effects of bovine sperm cryopreservation using different freezing techniques and cryoprotective agents on plasma, acrosomal and mitochondrial membranes. Andrologia 44: 154-159. https://doi.org/10.1111/j.1439-0272.2010.01154.x

Garner DL, Thomas CA, 1999. Organelle-specific probe JC-1 identifies membrane potential differences in the mitochondrial function of bovine sperm. Mol Reprod Dev 53 (2): 222-229. https://doi.org/10.1002/(SICI)1098-2795(199906)53:2<222::AID-MRD11>3.0.CO;2-L

Guijarro RA, Katchicualula A, Alvarez J, Fernández L, de la Varga S, Carbajo MT, 2014. Effect of freezing extender on quality after thawing in Lidia bulls epididymal sperm cell for conservation postmortem. Reprod Domest Anim 49 (S4): 102, P5.

Gürler H, Malama E, Heppelmann M, Calisici O, Leiding C, Kastelic JP, Bollwein H, 2016. Effects of cryopreservation on sperm viability, synthesis of reactive oxygen species, and DNA damage of bovine sperm. Theriogenology 86 (2): 562-571. https://doi.org/10.1016/j.theriogenology.2016.02.007

Hurtado de Llera A, Martin-Hidalgo D, Rodriguez-Gil JE, Gil MC, Garcia-Marin LJ, Bragado, MJ, 2013. AMP-activated kinase, AMPK, is involved in the maintenance of plasma membrane organization in boar spermatozoa. Biochimica et Biophysica Acta (BBA) - Biomembranes 1828 (9): 2143-2151.

Katska-Ksiazkiewicz L, Lechniak-Cieslak D, Korwin-Kossakowska A, Alm H, Rynska B, Warzych E, Sosnowski J, Sender G, 2006. Genetical and biotechnological methods of utilization of female reproductive potential in mammals. Reprod Biol 6 (S1): 21-36.

Kulaksiz R, Ari Ç, Daşkin A, Üner AG, 2013. The effect of different glycerol concentrations on freezability of semen from Angora, Kilis and Saanen goats. Slovak J Anim Sci 46: 5.

Long JA, Purdy PH, Zuidberg K, Hiemstra SJ, Velleman SG, Woelders H, 2014 Cryopreservation of turkey semen: Effect of breeding line and freezing method on post-thaw sperm quality, fertilization, and hatching. Cryobiology 68 (3): 371-378. https://doi.org/10.1016/j.cryobiol.2014.04.003

Lopes G, Soares L, Ferreira P, Rocha A, 2015. Tris–egg yolk–glycerol (TEY) extender developed for freezing dog semen is a good option to cryopreserve bovine epididymal sperm cells. Reprod Domest Anim 50 (1): 97-103. https://doi.org/10.1111/rda.12454

Macias Garcia B, Ortega Ferrusola C, Aparicio IM, Miro-Moran A, Morillo Rodriguez A, Gallardo Bolanos JM, Gonzalez Fernandez L, Balao da Silva CM, Rodriguez Martinez H, Tapia JA, Pena FJ, 2012. Toxicity of glycerol for the stallion spermatozoa: effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential. Theriogenology 77 (7): 1280-1289. https://doi.org/10.1016/j.theriogenology.2011.10.033

Macias-Garcia B, Gonzalez-Fernandez L, Loux SC, Rocha AM, Guimaraes T, Pena FJ, Varner DD, Hinrichs K, 2015. Effect of calcium, bicarbonate, and albumin on capacitation-related events in equine sperm. Reproduction 149 (1): 87-99. https://doi.org/10.1530/REP-14-0457

Malcotti V, Pelufo V, Bergamo N, Aisen E, 2012. Recovery of epididymal spermatozoa from bull and red deer, stored at different times and temperatures before freezing–thawing. Anim Sci 52 (8): 741-745. https://doi.org/10.1071/AN11366

Malo C, Gil L, Cano R, Martínez F, García A, Jerez RA, 2012. Dimethylformamide is not better than glycerol for cryopreservation of boar semen. Andrologia 44: 605-610. https://doi.org/10.1111/j.1439-0272.2011.01237.x

Marchetti C, Obert G, Deffosez A, Formstecher P, Marchetti P, 2002. Study of mitochondrial membrane potential, reactive oxygen species, DNA fragmentation and cell viability by flow cytometry in human sperm. Hum Reprod 17 (5): 1257-1265. https://doi.org/10.1093/humrep/17.5.1257

Martínez-Pastor F, Anel L, Guerra C, Álvarez M, Soler AJ, Garde JJ, Chamorro C, de Paz P, 2006. Seminal plasma improves cryopreservation of Iberian red deer epididymal sperm. Theriogenology 66 (8): 1847-1856. https://doi.org/10.1016/j.theriogenology.2006.04.036

Martínez-Pastor F, Del Rocío Fernández-Santos M, Domínguez-Rebolledo A, Esteso MC, Garde JJ, 2009. DNA status on thawed semen from fighting bull: A comparison between the SCD and the SCSA tests. Reprod Domest Anim 44 (3): 424-431. https://doi.org/10.1111/j.1439-0531.2008.01098.x

Martins CF, Rumpf R, Pereira DC, Dode MN, 2007. Cryopreservation of epididymal bovine spermatozoa from dead animals and its uses in vitro embryo production. Anim Reprod Sci 101 (3-4): 326-331. https://doi.org/10.1016/j.anireprosci.2007.01.018

Martins CF, Silva AEDF, Dode MN, Rumpf R, Cumpa HCB, Silva CG, Pivato I, 2015. Morphological characterization and conservation of bovine spermatogenic cells by refrigeration at 4 °C and freezing using different cryoprotective molecules. Cryobiology 71 (1): 47-53. https://doi.org/10.1016/j.cryobiol.2015.06.003

Mata-Campuzano M, Álvarez-Rodríguez M, Álvarez M, Anel L, de Paz P, Garde JJ, Martínez-Pastor F, 2012. Effect of several antioxidants on thawed ram spermatozoa submitted to 37°C up to four hours. Reprod Domes Anim 47 (6): 907-914. https://doi.org/10.1111/j.1439-0531.2012.01990.x

Matilla E, González-Fernández L, Hernández N, Tobajas C, Calle-Guisado V, Mijares J, Sánchez-Margallo F, Álvarez I, Macías-García B, 2017, N-Acetylcysteine does not improve sperm motility of Lidia bull after prolonged epididymal storage. J Vet Androl 2 (1): 23-29.

Minervini F, Guastamacchia R, Pizzi F, Dell'Aquila ME, Barile VL, 2013. Assessment of different functional parameters of frozen–thawed buffalo spermatozoa by using cytofluorimetric determinations. Reprod Domest Anim 48 (2): 317-324. https://doi.org/10.1111/j.1439-0531.2012.02152.x

Morillo Rodriguez A, Balao da Silva C, Macías-García B, Gallardo Bolaños JM, Tapia JA, Aparicio IM, Ortega-Ferrusola C, Peña FJ, 2012. Dimethylformamide improves the in vitro characteristics of thawed stallion spermatozoa reducing sublethal damage. Reprod Domest Anim 47 (6): 995-1002. https://doi.org/10.1111/j.1439-0531.2012.02005.x

Nichi M, Goovaerts IGF, Cortada CNM, Barnabe VH, De Clercq JBP, Bols PEJ, 2007. Roles of lipid peroxidation and cytoplasmic droplets on in vitro fertilization capacity of sperm collected from bovine epididymides stored at 4 and 34 °C. Theriogenology 67 (2): 334-340. https://doi.org/10.1016/j.theriogenology.2006.08.002

Nichi M, Rijsselaere T, Losano JDA, Angrimani DSR, Kawai GKV, Goovaerts IGF, Van Soom A, Barnabe VH, De Clercq JBP, Bols PEJ, 2016. Evaluation of epididymis storage temperature and cryopreservation conditions for improved mitochondrial membrane potential, membrane integrity, sperm motility and in vitro fertilization in bovine epididymal sperm. Reprod Domest Anim 52 (2): 257-263. https://doi.org/10.1111/rda.12888

Oeda T, Henkel R, Ohmori H, Schill WB, 1997. Scavenging effect of N-acetyl-L-cysteine against reactive oxygen species in human semen: a possible therapeutic modality for male factor infertility? Andrologia 29 (3): 125-131. https://doi.org/10.1111/j.1439-0272.1997.tb00305.x

Olaciregui M, Gil L, Montón A, Luño V, Jerez RA, Martí JI, 2014. Cryopreservation of epididymal stallion sperm. Cryobiology 68 (1): 91-95. https://doi.org/10.1016/j.cryobiol.2013.12.009

Partyka A, Niżański W, Bajzert J, Łukaszewicz E, Ochota M, 2013. The effect of cysteine and superoxide dismutase on the quality of post-thawed chicken sperm. Cryobiology 67 (2): 132-136. https://doi.org/10.1016/j.cryobiol.2013.06.002

Pérez L, Arias ME, Sánchez R, Felmer R, 2015. N-acetyl-L-cysteine pre-treatment protects cryopreserved bovine spermatozoa from reactive oxygen species without compromising the in vitro developmental potential of intracytoplasmic sperm injection embryos. Andrologia 47 (10): 1196-1201. https://doi.org/10.1111/and.12412

Posado R, Hernández M, García J, Bartolomé D, Olmedo S, Rodríguez L, López-Fernández C, Gosálvez J, 2008. DNA fragmentation in frozen semen samples of fighting bulls. 24th Ann Meeting Assoc Eur Embryo Transfer, PAU, France, 12-13 Sept, p. 220.

Rocha A, Oliveira E, Vilhena MJ, Diaz J, Sousa M, 2006. A novel apical midpiece defect in the spermatozoa of a bull without an apparent decrease in motility and fertility. Theriogenology 66 (4): 913-922. https://doi.org/10.1016/j.theriogenology.2006.02.032

Squires EL, Keith SL, Graham JK, 2004. Evaluation of alternative cryoprotectants for preserving stallion spermatozoa. Theriogenology 62 (6): 1056-1065. https://doi.org/10.1016/j.theriogenology.2003.12.024

Swegen A, Lambourne SR, Aitken RJ, Gibb Z, 2016. Rosiglitazone improves stallion sperm motility, ATP content, and mitochondrial function. Biol Reprod 95 (5): 107. https://doi.org/10.1095/biolreprod.116.142687

Takahashi T, Itoh R, Nishinomiya H, Katoh M, Manabe N, 2012. Effect of linoleic acid albumin in a dilution solution and long-term equilibration for freezing of bovine spermatozoa with poor freezability. Reprod Domest Anim 47 (1): 92-97. https://doi.org/10.1111/j.1439-0531.2011.01806.x

Van Wagtendonk-de Leeuw AM, Haring RM, Kaal-Lansbergen LMTE, den Daas JHG, 2000. Fertility results using bovine semen cryopreserved with extenders based on egg yolk and soy bean extract. Theriogenology 54 (1): 57-67. https://doi.org/10.1016/S0093-691X(00)00324-1

Waterhouse KE, Haugan T, Kommisrud E, Tverdal A, Flatberg G, Farstad W, Evenson DP, De Angelis PM, 2006. Sperm DNA damage is related to field fertility of semen from young Norwegian Red bulls. Reprod Fertil Dev 18 (7): 781-788. https://doi.org/10.1071/RD06029

Windsor DP, 1997. Mitochondrial function and ram sperm fertility. Reprod Fertil Dev 9 (3): 279-284. https://doi.org/10.1071/R96109

Wu W, Goldstein G, Adams C, Matthews RH, Ercal N, 2006. Separation and quantification of N-acetyl-l-cysteine and N-acetyl-cysteine-amide by HPLC with fluorescence detection. Biomed Chromatogr 20 (5): 415-422. https://doi.org/10.1002/bmc.583

Yu I, Leibo SP, 2002. Recovery of motile, membrane-intact spermatozoa from canine epididymides stored for 8 days at 4°C. Theriogenology 57 (3): 11. https://doi.org/10.1016/S0093-691X(01)00711-7

How to Cite
MatillaE., González-FernándezL., Martínez-PastorF., HernándezN., TobajasC., Calle-GuisadoV., MijaresJ., Sánchez-MargalloF. M., ÁlvarezI. S., & Macías-GarcíaB. (2017). Outlining adequate protocols for Lidia bull epididymal storage and sperm cryopreservation: use of glycerol, dimethylformamide and N-acetylcysteine. Spanish Journal of Agricultural Research, 15(3), e0405. https://doi.org/10.5424/sjar/2017153-11463
Animal breeding, genetics and reproduction