Recrystallization damages occur when a frozen sample is held at high subzero temperatures and when the warming process is too slow.
In this work, ram semen diluted in two different concentrations of sugar solutions (Lyo A consisted of 0.4 M sorbitol and 0.25 M trehalose, and the second, Lyo B composed of 0.26 M sorbitol and 0.165 M trehalose) in egg yolk and Tris medium were compared after freezing 10 μL samples to: (1) − 10, − 25, and − 35 °C and thawing. (2) Freezing to − 10 and − 25 °C, holding for 1 h and then thawing, and (3) freezing to − 10 and − 25 °C and drying for 1 h at these temperatures at a vacuum of 80 mTorr, prior thawing. For drying, we used a new freeze-drying apparatus (Darya, FertileSafe, Israel) having a condensation temperature below − 110 °C and a vacuum pressure of 10–100 mTorr that is reached in less than 10s.
Results showed that samples in Lyo B solution frozen at − 25 °C had significantly higher sperm motility in partially freeze-dried samples than frozen samples (46.6 ± 2.8% vs 1.2 ± 2.5%, P < 0.001). Moreover, partially dried samples in Lyo B showed higher motility than Lyo A at − 25 °C (46.6 ± 2.8% vs 35 ± 4%). Cryomicroscopy and low-temperature/low-pressure environmental scanning electronic microscope demonstrated that the amount of the ice crystals present in partially dried samples was lower than in the frozen samples.
Holding the sperm at high subzero temperatures is necessary for the primary drying of cells during the freeze-drying process. Rapid freeze-drying can be achieved using this new device, which enables to reduce recrystallization damages.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
Arav A, Saragusty J. Directional freezing of sperm and associated derived technologies. Anim Reprod Sci. 2016;169:6–13.
Bielanski A. A review of the risk of contamination of semen and embryos during cryopreservation and measures to limit cross-contamination during banking to prevent disease transmission in ET practices. Theriogenology. 2012;77(3):467–82.
Crowe JH, Crowe LM, Wolkers WF, Oliver AE, Ma X, Auh J-H, et al. Stabilization of dry mammalian cells: lessons from nature. Integr Comp Biol. 2005;45(5):810–20.
Gil L, Olaciregui M, Luño V, Malo C, González N, Martínez F. Current status of freeze-drying technology to preserve domestic animals sperm. Reprod Domest Anim. 2014;49(Suppl 4):72–81.
Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature. 1949;164(4172):666.
Sherman JK. Freezing and freeze-drying of human spermatozoa. Fertil Steril. 1954;5(4):357–71.
Sherman JK. Freezing and freeze-drying of bull spermatozoa. Am J Phys. 1957;190(2):281–6.
Bialy G, Smith VR. Freeze-drying of bovine spermatozoa. J Dairy Sci. 1957;40(7):739–45.
Meryman HT, Kafig E. Survival of spermatozoa following drying. Nature. 1959;184(4684):470–1.
Saacke RG, Almquist JO. Freeze-drying of bovine spermatozoa. Nature. 1961;192(4806):995–6.
Sherman JK. Improved methods of preservation of human spermatozoa by freezing and freeze-drying. Fertil Steril. 1963;14(1):49–64.
Moisan AE, Leibo SP, Lynn JW, Gómez MC, Pope CE, Dresser BL, et al. Embryonic development of felid oocytes injected with freeze-dried or air-dried spermatozoa. Cryobiology. 2005;51:373. (abstract)
Watanabe H, Asano T, Abe Y, Fukui Y, Suzuki H. Pronuclear formation of freeze-dried canine spermatozoa microinjected into mouse oocytes. J Assist Reprod Genet. 2009;26(9–10):531–6.
Martins CF, Bao SN, Dode MN, Correa GA, Rumpf R. Effects of freeze-drying on cytology, ultrastructure, DNA fragmentation, and fertilizing ability of bovine sperm. Theriogenology. 2007;67(8):1307–15.
Kusakabe H, Yanagimachi R, Kamiguchi Y. Mouse and human spermatozoa can be freeze-dried without damaging their chromosomes. Hum Reprod. 2008;23(2):233–9.
Hara H, Abdalla H, Morita H, Kuwayama M, Hirabayashi M, Hochi S. Procedure for bovine ICSI, not sperm freeze-drying, impairs the function of the microtubule-organizing center. J Reprod Dev. 2011;57(3):428–32.
Gianaroli L, Magli MC, Stanghellini I, Crippa A, Crivello AM, Pescatori ES, et al. DNA integrity is maintained after freeze-drying of human spermatozoa. Fertil Steril. 2012;97(5):1067–73.
Garcia A, Gil L, Malo C, Martinez F, Kershaw-Young C, de Blas I. Effect of different disaccharides on the integrity and fertilising ability of freeze-dried boar spermatozoa: a preliminary study. Cryo Letters. 2014;35(4):277–85.
Olaciregui M, Luño V, Gonzalez N, De Blas I, Gil L. Freeze-dried dog sperm: dynamics of DNA integrity. Cryobiology. 2015;71(2):286–90.
Magalhães MJ Jr, Martins LF, Senra RL, Santos TF, Okano DS, Pereira PR, et al. Differential abundances of four forms of binder of sperm in the seminal plasma of Bos taurus indicus bulls with different patterns of semen freezability. Theriogenology. 2016;86(3):766–77.
Wakayama T, Yanagimachi R. Development of normal mice from oocytes injected with freeze-dried spermatozoa. Nat Biotechnol. 1998;16(7):639–41.
Liu JL, Kusakabe H, Chang CC, Suzuki H, Schmidt DW, Julian M, et al. Freeze-dried sperm fertilization leads to full-term development in rabbits. Biol Reprod. 2004;70(6):1776–81.
Hirabayashi M, Kato M, Ito J, Hochi S. Viable rat offspring derived from oocytes intracytoplasmically injected with freeze-dried sperm heads. Zygote. 2005;13(1):79–85.
Hochi S, Watanabe K, Kato M, Hirabayashi M. Live rats resulting from injection of oocytes with spermatozoa freeze-dried and stored for one year. Mol Reprod Dev. 2008;75(5):890–4.
Arav A, Natan D. Freeze drying (lyophilization) of red blood cells. J Trauma Acute Care Surg. 2011;70(5):S61–4.
Goodrich RP, Sowemimo-Coker SO, Zerez CR, Tanaka KR. Preservation of metabolic activity in lyophilized human erythrocytes. Proc Nat Acad Sci USA. 1992;89(3):967–71.
Natan D, Nagler A, Arav A. Freeze-drying of mononuclear cells derived from umbilical cord blood followed by colony formation. PLoS One. 2009;4(4):e5240.
Das ZC, Gupta MK, Uhm SJ, Lee HT. Lyophilized somatic cells direct embryonic development after whole cell intracytoplasmic injection into pig oocytes. Cryobiology. 2010;61(2):220–4.
Zhang M, Oldenhof H, Sydykov B, Bigalk J, Sieme H, Wolkers WF. Freeze-drying of mammalian cells using trehalose: preservation of DNA integrity. Sci Rep. 2017;7(1):6198.
Loi P, Matsukawa K, Ptak G, Clinton M, Fulka J Jr, Natan Y, et al. Freeze-dried somatic cells direct embryonic development after nuclear transfer. PLoS One. 2008;3(8):e2978.
Li S, Chakraborty N, Borcar A, Menze MA, Toner M, Hand SC. Late embryogenesis abundant proteins protect human hepatoma cells during acute desiccation. Proc Nat Acad Sci USA. 2012;109(51):20859–64.
Arav A, Gavish Z, Elami A, Silber S, Patrizio P. Ovarian survival 6 years after whole organ cryopreservation and transplantation. Fertil Steril. 2007;88(Supplement 1):S352. (abstract)
Arav A. Large tissue freezing. J Assist Reprod Genet. 2003;20(9):351.
Mazur P, Cole KW. Roles of unfrozen fraction, salt concentration, and changes in cell volume in the survival of frozen human erythrocytes. Cryobiology. 1989;26(1):1–29.
Wolkers WF, Tablin F, Crowe JH. From anhydrobiosis to freeze-drying of eukaryotic cells. Comp Biochem Physiol A Mol Integr Physiol. 2002;131(3):535–43.
Guo N, Puhlev I, Brown DR, Mansbridge J, Levine F. Trehalose expression confers desiccation tolerance on human cells. Nat Biotechnol. 2000;18(2):168–71.
Sitaula R, Fowler A, Toner M, Bhowmick S. A study of the effect of sorbitol on osmotic tolerance during partial desiccation of bovine sperm. Cryobiology. 2010;60(3):331–6.
Curry MR, Watson PF. Osmotic effects on ram and human sperm membranes in relation to thawing injury. Cryobiology. 1994;31(1):39–46.
Rindler V, Lüneberger S, Schwindke P, Heschel I, Rau G. Freeze-drying of red blood cells at ultra-low temperatures. Cryobiology. 1999 Feb;38(1):2–15.
Hara H, Tagiri M, Hirabayashi M, Hochi S. Effect of cake collapse on the integrity of freeze-dried bull spermatozoa. Reprod Fertil Dev. 2013;26(1):144. (Abstract)
Watson PF, Duncan AE. Effect of salt concentration and unfrozen water fraction on the viability of slowly frozen ram spermatozoa. Cryobiology. 1988;25(2):131–42.
Saragusty J, Gacitua H, Rozenboim I, Arav A. Do physical forces contribute to cryodamage? Biotechnol Bioeng. 2009;104(4):719–28.
We thank Dr. Monica Strina for the proofreading and editing of the manuscript. This work was supported by Progetto RAS MIGLIOVIGENSAR 2016/2018 and by Programma visiting professor RAS 2016. This work was also supported by FertileSafe Ltd.
About this article
Cite this article
Arav, A., Idda, A., Nieddu, S.M. et al. High post-thaw survival of ram sperm after partial freeze-drying. J Assist Reprod Genet 35, 1149–1155 (2018). https://doi.org/10.1007/s10815-018-1145-1