Abstract: Background: We previously demonstrated how mouse spermatozoa can be efficiently stored for two years in a -80°C freezer, maintaining their ability to fertilize mouse eggs.
Objectives: The main objective here was to evaluate the effects of five years at -80°C compared to liquid nitrogen storage (LN 2 , control condition) on mouse sperm viability, physiological parameters, and fertilization capacity.
Materials and Methods: Three different strains were used: C57BL/6N, C57BL/6J and CD1. Flow cytometry experiments were performed to analyze sperm viability (SYBR-14 + Propidium Iodide +Hoechst33342), the intracellular calcium concentration (Fluo 3-AM), the membrane lipid disorder (Merocyanine 540), and the mitochondrial activity (MitoTracker Red) in live spermatozoa. The in vitro fertilization (IVF) was used to evaluate the sperm fertilizing ability.
Results: Flow cytometry analysis showed that the percentage of live cells are reduced in B6N and B6J, but not in CD1 mice. However, in the live population no differences in terms of intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity were reported when comparing both biobanking methods. Spermatozoa stored at -80°C for 5 years successfully fertilized the eggs and developed mouse embryo normally both in culture and in vivo, generating live pups with no differences compared to control samples stored in LN 2 .
Discussion: Long-term mouse sperm storage at -80°C (five years) could be considered an ideal alternative to the most common LN 2 approach, giving economical and logistic advantages. Moreover, the precise information originated from the flow cytometry analysis stands up this technique as an optimal strategy to evaluate the sperm quality and ranking.
Conclusion: It is demonstrated here the possibility to store mouse spermatozoa for up to five years in a -80°C freezer with no significant differences compared to the storage in LN 2 in terms of fertilizing ability, sperm viability, intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity.
(© 2021 American Society of Andrology and European Academy of Andrology.)
References: Nakagata N. Cryopreservation of mouse spermatozoa. Mamm Genome. 2000;11(7):572-576. https://doi.org/10.1007/s003350010109.
Sztein JM, Noble K, Farley JS, Mobraaten LE. Comparison of permeating and nonpermeating cryoprotectants for mouse sperm cryopreservation. Cryobiology. 2001;42(1):28-39. https://doi.org/10.1006/cryo.2001.2300.
Tada N, Sato M, Yamanoi J, Mizorogi T, Kasai K, Ogawa S. Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol. J Reprod Fertil. 1990;89(2):511-516. https://doi.org/10.1530/jrf.0.0890511.
Sztein JM, Farley JS, Young AF, Mobraaten LE. Motility of cryopreserved mouse spermatozoa affected by temperature of collection and rate of thawing. Cryobiology. 1997;35(1):46-52. https://doi.org/10.1006/cryo.1997.2024.
Ostermeier GC, Wiles MV, Farley JS, Taft RA. Conserving, distributing and managing genetically modified mouse lines by sperm cryopreservation. PLoS One. 2008;3(7):e2792. https://doi.org/10.1371/journal.pone.0002792.
Takeo T, Nakagata N. Reduced glutathione enhances fertility of frozen/thawed C57BL/6 mouse sperm after exposure to methyl-beta-cyclodextrin. Biol Reprod. 2011;85(5):1066-1072. https://doi.org/10.1095/biolreprod.111.092536.
Raspa M, Guan MO, Paoletti R, et al. Dry ice is a reliable substrate for the distribution of frozen mouse spermatozoa: A multi-centric study. Theriogenology. 2017;96:49-57. https://doi.org/10.1016/j.theriogenology.2017.04.003.
Mähler (Convenor) M, Berard M, Feinstein R, et al. FELASA recommendations for the health monitoring of mouse, rat, hamster, guinea pig and rabbit colonies in breeding and experimental units. Lab Anim. 2014;48(3):178-192. https://doi.org/10.1177/0023677213516312.
Torres MA, Díaz R, Boguen R, et al. Novel flow cytometry analyses of boar sperm viability: can the addition of whole sperm-rich fraction seminal plasma to frozen-thawed boar sperm affect it? PLoS One. 2016;11(8):e0160988. https://doi.org/10.1371/journal.pone.0160988.
Nagy S, Jansen J, Topper EK, Gadella BM. A 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. 2003;68(5):1828-1835. https://doi.org/10.1095/biolreprod.102.011445.
Garner DL, Dobrinsky JR, Welch GR, Johnson LA. Porcine sperm viability, oocyte fertilization and embryo development after staining spermatozoa with SYBR-14. Theriogenology. 1996;45(6):1103-1113. https://doi.org/10.1016/0093-691x(96)00067-2.
Raspa M, Fray M, Paoletti R, et al. Long term maintenance of frozen mouse spermatozoa at -80 degrees C. Theriogenology. 2018;107:41-49. https://doi.org/10.1016/j.theriogenology.2017.10.036.
Nakagata N, Takeo T, Fukumoto K, et al. Rescue in vitro fertilization method for legacy stock of frozen mouse sperm. J Reprod Develop. 2014;60(2):168-171. https://doi.org/10.1262/jrd.2013-141.
Li MW, Glass OC, Zarrabi J, Baker LN, Lloyd KC. Cryorecovery of mouse sperm by different IVF methods using MBCD and GSH. J Fertili Vitro. 2016;04(02): https://doi.org/10.4172/2375-4508.1000175.
Nakagata N. Cryopreservation of mouse spermatozoa and in vitro fertilization. Methods Mol Biol. 2011;693:57-73. https://doi.org/10.1007/978-1-60761-974-1_4.
Ho Y, Wigglesworth K, Eppig JJ, Schultz RM. Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression. Mol Reprod Dev. 1995;41(2):232-238. https://doi.org/10.1002/mrd.1080410214.
Garner DL, Johnson LA. Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol Reprod. 1995;53(2):276-284. https://doi.org/10.1095/biolreprod53.2.276.
Bernabò N, Ordinelli A, Di Agostino R, Mattioli M, Barboni B. Network analyses of sperm-egg recognition and binding: ready to rethink fertility mechanisms? OMICS. 2014;18(12):740-753. https://doi.org/10.1089/omi.2014.0128.
Avella MA, Dean J. Fertilization with acrosome-reacted mouse sperm: Implications for the site of exocytosis. Proc Natl Acad Sci. 2011;108(50):19843-19844. https://doi.org/10.1073/pnas.1118234109.
Thomas P, Meizel S. An influx of extracellular calcium is required for initiation of the human sperm acrosome reaction induced by human follicular fluid. Gamete Res. 1988;20(4):397-411.
Blackmore PF, Beebe SJ, Danforth DR, Alexander N. Progesterone and 17 alpha-hydroxyprogesterone. Novel stimulators of calcium influx in human sperm. J Biol Chem. 1990;265(3):1376-1380.
Baldi E, Luconi M, Bonaccorsi L, Muratori M, Forti G. Intracellular events and signaling pathways involved in sperm acquisition of fertilizing capacity and acrosome reaction. Front Biosci. 2000;5:E110-E123. https://doi.org/10.2741/baldi.
Kumaresan A, Siqueira AP, Hossain MS, Bergqvist AS. Cryopreservation-induced alterations in protein tyrosine phosphorylation of spermatozoa from different portions of the boar ejaculate. Cryobiology. 2011;63(3):137-144. https://doi.org/10.1016/j.cryobiol.2011.08.002.
Januskauskas A, Gil J, Söderquist L, et al. Effect of cooling rates on post-thaw sperm motility, membrane integrity, capacitation status and fertility of dairy bull semen used for artificial insemination in Sweden. Theriogenology. 1999;52(4):641-658. https://doi.org/10.1016/S0093-691X(99)00159-4.
Hallap T, Nagy S, Jaakma Ü, Johannisson A, Rodriguez-Martinez H. Usefulness of a triple fluorochrome combination merocyanine 540/Yo-Pro 1/Hoechst 33342 in assessing membrane stability of viable frozen-thawed spermatozoa from Estonian Holstein AI bulls. Theriogenology. 2006;65(6):1122-1136. https://doi.org/10.1016/j.theriogenology.2005.07.009.
Harrison RA, Ashworth PJ, Miller NG. 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):378-391.
Green CE, Watson PF. Comparison of the capacitation-like state of cooled boar spermatozoa with true capacitation. Reproduction (Cambridge, England). 2001;122(6):889-898.
Rathi R, Colenbrander B, Bevers MM, Gadella BM. Evaluation of in vitro capacitation of stallion spermatozoa. Biol Reprod. 2001;65(2):462-470.
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):1451-1460. https://doi.org/10.1016/j.theriogenology.2015.01.019.
Amaral A, Lourenço B, Marques M, Ramalho-Santos J. Mitochondria functionality and sperm quality. Reproduction. 2013;146(5):R163-R174. https://doi.org/10.1530/REP-13-0178.
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):406-419. https://doi.org/10.1038/aja.2011.15.
Huntriss J, Balen AH, Sinclair KD, Brison DR, Picton HM. Epigenetics and reproductive medicine. BJOG. 2018;125(13):e43-e54. https://doi.org/10.1111/1471-0528.15240.
Denomme MM, Mann MR. Genomic imprints as a model for the analysis of epigenetic stability during assisted reproductive technologies. Reproduction. 2012;144(4):393-409. https://doi.org/10.1530/REP-12-0237.
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