Animal Reproduction (AR)
https://animal-reproduction.org/article/doi/10.21451/1984-3143-AR2019-0081
Animal Reproduction (AR)
Original Article

Effects of the cryopreservation process on dog sperm integrity

Carmen Cecilia Sicherle; Fabiana Ferreira de Souza; Camila de Paula Freitas-Dell’Aqua; Gabriele Barros Mothé; Carlos Roberto Padovani; Frederico Ozanam Papa; Maria Denise Lopes

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Abstract

Abstract: Sperm cryopreservation has become an indispensable tool in reproductive biology. However, frozen/thawed semen has a short lifespan due to loss of sperm cell integrity. To better understand which sperm cell structures are compromised by the cryopreservation process and apoptosis markers, the sperm of five healthy mature dogs was analyzed in this study. Analysis was performed after collection, cooling, and thawing via computer assisted sperm analyzer (CASA) and evaluation of membrane fluidity and permeability, phosphatidylserine translocation (Annexin V), membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation (LPO) and activity of the apoptotic markers caspases 3 and 7 by flow cytometry. Cryopreservation decreased total and progressive motility and the percentage of rapid sperm (P < 0.01). Damage to sperm cells was confirmed by Annexin V (P <0.01), indicating that capacitation-like changes were induced by the cryopreservation procedures. An increase in sperm membrane fluidity was also noted in frozen/thawed samples (P < 0.01). Plasma and acrosomal cell membranes were affected (P < 0.01), with decreases in the subpopulation displaying high membrane potential (P < 0.01). Membrane LPO was increased in thawed sperm compared to cooled sperm (P < 0.05) but was not different from that in fresh sperm. No differences were observed in caspase 3 and 7 activity after cooling, freezing, or thawing. In conclusion, total and progressive motility, plasma membrane integrity and mitochondrial membrane potential suffered from the deleterious effects caused by cryopreservation, unlike the activity of caspases that remained stable during the freezing process.

Keywords

dog, spermatozoa, cryopreservation, sperm quality, cell integrity

References

Agarwal A, Nallella KP, Allamaneni SS, Said TM. Role of antioxidants in treatment of male infertility: an overview of the literature. Reprod Biomed Online. 2004;8(6):616-27. http://dx.doi.org/10.1016/S1472-6483(10)61641-0. PMid:15169573.

Aitken RJ, Wingate JK, De Iuliis GN, McLaughlin EA. Analysis of lipid peroxidation in human spermatozoa using BODIPY C11. Mol Hum Reprod. 2007;13(4):203-11. http://dx.doi.org/10.1093/molehr/gal119. PMid:17327268.

Alhaider AK, Watson PF. Cryopreservation of dog semen: the effects of Equex STM paste on plasma membrane fluidity and the control of intracellular free calcium. Anim Reprod Sci. 2009;110(1-2):147-61. http://dx.doi.org/10.1016/j.anireprosci.2008.01.011. PMid:18295987.

Anzar M, He L, Buhr MM, Kroetsch TG, Pauls KP. Sperm apoptosis in fresh and cryopreserved bull semen detected by flow cytometry and its relationship with fertility. Biol Reprod. 2002;66(2):354-60. http://dx.doi.org/10.1095/biolreprod66.2.354. PMid:11804948.

Burgess CM, Clutterbuck AL, England GCW. The effect of cryopreservation on the capacitation status and epithelial cell attachment capability of dog spermatozoa. Vet J. 2012;192(3):398-402. http://dx.doi.org/10.1016/j.tvjl.2011.08.026. PMid:21958721.

Câmara DR, Guerra MMP. Mitocôndria espermática: além da síntese de adenosina trifosfato (ATP). Rev. Bras. Reprod. Anim. 2008;32:93-9.

Chirinéa VH, Martins MIM, de Souza FF, Tebet JM, Papa FO, Lopes MD. Características morfofuncionais do sêmen canino refrigerado e congelado usando dois diferentes meios diluentes. Cienc Anim Bras. 2006;7:407-15.

Donnelly ET, O’Connell M, McClure N, Lewis SEM. Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa. Hum Reprod. 2000;15(7):1552-61. http://dx.doi.org/10.1093/humrep/15.7.1552. PMid:10875865.

Duru NK, Morshedi M, Schuffner A, Oehninger S. Cryopreservation-thawing of fractionated human spermatozoa and plasma membrane translocation of phosphatidylserine. Fertil Steril. 2001;75(2):263-8. http://dx.doi.org/10.1016/S0015-0282(00)01694-0. PMid:11172825.

Farstad W. Customizing semen preservation protocols for individual dogs and individual species: sperm preservation beyond the state of the art. Reprod Domest Anim. 2012;47(Suppl 6):269-73. http://dx.doi.org/10.1111/rda.12020. PMid:23279516.

Gadella BM, Harrison R. Capacitation induces cyclic adenosine 3′,5′-monophosphate-dependent, but apoptosis-unrelated, exposure of aminophospholipids at the apical head plasma membrane of boar sperm cells. Biol Reprod. 2002;67(1):340-50. http://dx.doi.org/10.1095/biolreprod67.1.340. PMid:12080038.

Guasti PN, Freitas-Dell’Aqua CP, Maziero RRD, Hartwig FP, Monteiro GA, Lisboa FP, Papa FO. Validation of flow cytometry for assessment of membrane lipid peroxidation of equine spermatozoa. Anim Reprod. 2012;9:929.

Harrison RAP, Ashworth PJC, Miller NGA. Bicarbonate/CO2, an effector of capacitation, induces a rapid and reversible change in the lipid architecture of boar sperm plasma membranes. Mol Reprod Dev. 1996;45(3):378-91. http://dx.doi.org/10.1002/(SICI)1098-2795(199611)45:3<378::AID-MRD16>3.0.CO;2-V. PMid:8916050.

Hossain MS, Johannisson A, Wallgren M, Nagy S, Siqueira AP, Rodriguez-Martinez H. Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art. Asian J Androl. 2011;13(3):406-19. http://dx.doi.org/10.1038/aja.2011.15. PMid:21478895.

Johnson RA, Wichern DW. Applied multivariate statistical analysis. New Jersey: Prentice-Hall; 1988.

Kim SH, Yu DH, Kim YJ. Apoptosis-like change, ROS, and DNA status in cryopreserved canine sperm recovered by glass wool filtration and Percoll gradient centrifugation techniques. Anim Reprod Sci. 2010a;119(1-2):106-14. http://dx.doi.org/10.1016/j.anireprosci.2009.11.002. PMid:19942382.

Kim SH, Yu DH, Kim YJ. Effects of cryopreservation on phosphatidylserine translocation, intracellular hydrogen peroxide, and DNA integrity in canine sperm. Theriogenology. 2010b;73(3):282-92. http://dx.doi.org/10.1016/j.theriogenology.2009.09.011. PMid:19883935.

Lucio CF, Regazzi FM, Silva LCG, Angrimani DSR, Nichi M, Vannucchi CI. Oxidative stress at different stages of two-step semen cryopreservation procedures in dogs. Theriogenology. 2016;85(9):1568-75. http://dx.doi.org/10.1016/j.theriogenology.2016.01.016. PMid:26879999.

Martin G, Sabido O, Durand P, Levy R. Cryopreservation induces an apoptosis-like mechanism in bull sperm. Biol Reprod. 2004;71(1):28-37. http://dx.doi.org/10.1095/biolreprod.103.024281. PMid:14973261.

Mason SJ, Rous NR. Comparison of endoscopic-assisted transcervical and laparotomy insemination with frozen-thawed dog semen: aretrospective clinical study. Theriogenology. 2014;82(6):844-50. http://dx.doi.org/10.1016/j.theriogenology.2014.06.014. PMid:25082020.

Maxwell WMC, Watson PF. Recent progress in the preservation of ram semen. Anim Reprod Sci. 1996;42(1-4):55-65. http://dx.doi.org/10.1016/0378-4320(96)01544-8.

Morton DB. Artificial insemination with frozen semen in the dog: principles of DNA fingerprinting. In: Jones DE, Joshua JO, editors. Reproductive clinical problems in the dog. Bristol: Wrigth and Sons; 1998. p. 169-86.

Mothé GB, Scott C, Sicherle CC, Guaitolini CR. Sperm sexing with density gradient centrifugation in dogs. Anim Reprod Sci. 2018;199:84-92. http://dx.doi.org/10.1016/j.anireprosci.2018.11.003. PMid:30455095.

Niżański W, Partyka A, Prochowska S. Evaluation of spermatozoal function: useful tools or just science. Reprod Domest Anim. 2016;51(Suppl 1):37-45. http://dx.doi.org/10.1111/rda.12786. PMid:27670939.

Niżański W, Partyka A, Rijsselaere T. Use of fluorescent stainings and flow cytometry for canine semen assessment. Reprod Domest Anim. 2012;47(Suppl 6):215-21. http://dx.doi.org/10.1111/rda.12048. PMid:23279503.

Nunez-Martínez I, Moran JM, Peña FJ. A three-step statistical procedure to identify sperm kinematic subpopulations in canine ejaculates: changes after cryopreservation. Reprod Domest Anim. 2006;41(5):408-15. http://dx.doi.org/10.1111/j.1439-0531.2006.00685.x. PMid:16984346.

Ortega-Ferrusola C, Anel-López L, Martín-Muñoz P, Ortíz-Rodríguez JM, Gil MC, Alvarez M, de Paz P, Ezquerra LJ, Masot AJ, Redondo E, Anel L, Peña FJ. Computational flow cytometry reveals that cryopreservation induces spermptosis but subpopulations of spermatozoa may experience capacitation-like changes. Reproduction. 2017;153(3):293-304. http://dx.doi.org/10.1530/REP-16-0539. PMid:27965398.

Ortega-Ferrusola C, Macias Garcia B, Suarez Rama V, Gallardo-Bolaños J, González-Fernández L, Tapia J, Rodríguez-Martinez H, Peña F. Identification of sperm subpopulations in stallion ejaculates: changes after cryopreservation and comparison with traditional statistics. Reprod Domest Anim. 2009;44(3):419-23. http://dx.doi.org/10.1111/j.1439-0531.2008.01097.x. PMid:19055563.

Parrish JJ, Susko-Parrish J, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod. 1988;38:1171-1180.

Peña A, Johannisson A, Linde-Forsberg C. Post-thaw evaluation of dog spermatozoa using new triple fluorescent staining and flow cytometry. Theriogenology. 1999;52(6):965-80. http://dx.doi.org/10.1016/S0093-691X(99)00186-7. PMid:10735104.

Peña FJ, Johannisson A, Wallgren M, Rodríguez-Martínez H. Assessment of fresh and frozen-thawed boar semen using an Annexin-V assay: A new method of evaluating sperm membrane integrity. Theriogenology. 2003;60(4):677-89. http://dx.doi.org/10.1016/S0093-691X(03)00081-5. PMid:12832017.

Peña FJ, Saravia F, García-Herreros M, Núñes-Martínez I, Tapia JA, Johannisson A, Walgreen M, Rodríguez-Martínez H. Identification of sperm morphometric subpopulations in two different portions of the boar ejaculate and its relation to postthaw quality. J Androl. 2005;26(6):716-23. http://dx.doi.org/10.2164/jandrol.05030. PMid:16291966.

Petrunkina A, Harrison RAP. Fluorescence technologies for evaluating male gamete (dys)function. Reprod Domest Anim. 2013;48(Suppl 1):11-24. http://dx.doi.org/10.1111/rda.12202. PMid:23962211.

Petrunkina AM, Harrison RAP. Cytometric solutions in veterinary andrology: Developments, advantages, and limitations. Citometry A. 2011;79(5):338-48. http://dx.doi.org/10.1002/cyto.a.21044. PMid:21448977.

Ramón M, Pérez-Guzmán MD, Jiménez-Rabadán P, Esteso MC, García-Álvarez O, Maroto-Morales A, Anel-López L, Soler AJ, Fernández-Santos MR, Garde JJ. Sperm cell population dynamics in ram semen during the cryopreservation process. PLoS One. 2013;8(3):e59189. http://dx.doi.org/10.1371/journal.pone.0059189. PMid:23544054.

Rodríguez-Martínez H. Laboratory semen assessment and prediction of fertility: still Utopia? Reprod Domest Anim. 2003;38(4):312-8. http://dx.doi.org/10.1046/j.1439-0531.2003.00436.x. PMid:12887570.

Rota A, Milani C, Romagnoli S, Zucchini P, Mollo A. Pregnancy and conception rate after two intravaginal inseminations with dog semen frozen either with 5% glycerol or 5% ethylene glycol. Anim Reprod Sci. 2010;118(1):94-7. http://dx.doi.org/10.1016/j.anireprosci.2009.06.007. PMid:19577869.

Sokolowska A, García BM, Fernández LG, Ortega-Ferrusola C, Tapia JA, Peña FJ. Activated caspases are present in frozen-thawed canine sperm and may be related to post thaw sperm quality. Zygote. 2009;17(4):297-305. http://dx.doi.org/10.1017/S0967199409005401. PMid:19416559.

Sousa AP, Amaral A, Baptista M, Tavares R, Campo PC, Peregrín PC, Freitas A, Paiva A, Almeida-Santos T, Ramalho-Santos J. Not all sperm are equal: functional mitochondria characterize a subpopulation of human sperm with better fertilization potential. PLoS One. 2011;6(3):e18112. http://dx.doi.org/10.1371/journal.pone.0018112. PMid:21448461.

Steckler D, Stout TAE, Durandt C, Nothling JO. Validation of merocyanine 540 staining as a technique for assessing capacitation-related membrane destabilization of fresh dog sperm. Theriogenology. 2015;83(9):1451-60. http://dx.doi.org/10.1016/j.theriogenology.2015.01.019. PMid:25796286.

Turner RM. Tales from the tail: what do we really know about sperm motility? J Androl. 2003;24(6):790-803. http://dx.doi.org/10.1002/j.1939-4640.2003.tb03123.x. PMid:14581499.

Verstegen J, Iguer-Ouada M, Onclin K. Computer assisted semen analyzers in andrology research and veterinary practice. Theriogenology. 2002;57(1):149-79. http://dx.doi.org/10.1016/S0093-691X(01)00664-1. PMid:11775967.

Volpe S, Leoci R, Aiudi G, Lacalandra GM. Relationship between motility and mitochondrial functional status in canine spermatozoa. Reprod Domest Anim. 2009;44(Suppl 2):275-8. http://dx.doi.org/10.1111/j.1439-0531.2009.01457.x. PMid:19754585.
 

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