Frog (Unspecified specie) |
Semen (n = unspecified) |
Spermatozoa mounted on a mica film |
Sucrose used. Spermatozoa immersed in liquid air for 10 seconds. Warmed in pond water at +20 °C a. |
Vitrification with 1 M sucrose resulted in sperm motility recovery of 20%. At concentrations between 1 M and 2 M, intermediate results were obtained. |
Luyet and Hodapp (1938)Luyet BJ, Hodapp EL. Revival of Frog’s Spermatozoa Vitrified in Liquid Air. Exp Biol Med (Maywood). 1938;39(3):433-4. http://dx.doi.org/10.3181/00379727-39-10229P. http://dx.doi.org/10.3181/00379727-39-10...
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Chicken (Gallus domesticus) |
Semen pools (n = unspecified) |
Spermatozoa placed in a test tube |
Fructose 0.75 M used. Spermatozoa were placed in a test tube and subjected to quick frozen at -76 °C. Warmed at 42 to 45 °Ca. |
30% of spermatozoa resumed motion. No fertile eggs have been produced after artificial insemination. |
Shaffner et al. (1941)Shaffner CS, Henderson EW, Card CG. Viability of spermatozoa of the chicken under various environmental conditions. Poult Sci. 1941;20(3):259-65. http://dx.doi.org/10.3382/ps.0200259. http://dx.doi.org/10.3382/ps.0200259...
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Rabbit (Unspecified specie) |
Spermatozoa from vas deferens (n=31) and ejaculated semen samples (n=8) |
Suspension of sperm smeared on cellophane |
With or without Ringer solution. Suspension of sperm was smeared on cellophane and partially dried in air before immersing in liquid nitrogen. Warmed in 37 °C medium a. |
Recoverable of 0.5% motile sperm on untreated and partially dried suspension of sperm. Semen treated with hypertonic Ringer solution gave a recoverable yield of 0.1% |
Hoagland and Pincus (1942)Hoagland H, Pincus G. Revival of mammalian sperm after immersion in liquid nitrogen. J Gen Physiol. 1942;25(3):337-44. http://dx.doi.org/10.1085/jgp.25.3.337. PMid:19873277. http://dx.doi.org/10.1085/jgp.25.3.337...
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Human |
Semen (n = 30) |
Samples of spermatozoa were located onto copper loop or into 0,25 mL straw |
Cryoprotectant free. Fresh and swim-up samples used. 20 μl of sample onto copper loop or in 0.25 mL straw. Plunged into LN2. Warmed in 37 °C medium, 5–10 minutes. |
Swim-up prepared spermatozoa without cryoprotectant onto cooper loops resulted in highest motility (49.5%) and the highest normal morphology spermatozoa when compared with other vitrification groups. |
Nawroth et al. (2002)Nawroth F, Isachenko V, Dessole S, Rahimi G, Farina M, Vargiu N, Mallmann P, Dattena M, Capobianco G, Peters D, Orth I, Isachenko E. Vitrification of human spermatozoa without cryoprotectants. Cryo Lett. 2002;23(2):93-102. PMid:12050777.
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Human |
Semen (n = 18) |
Samples of spermatozoa were located onto copper loop |
Cryoprotectant free. Swim-up samples used. 20 μl of sample onto copper loop. Plunged into LN2. Warmed in 37 °C medium, 5–10 minutes. |
Swim-up prepared spermatozoa without cryoprotectant onto cooper loops resulted in highest motility (51.5%). No significant differences in the DNA integrity of prepared spermatozoa related to presence of a cryoprotectant |
Isachenko et al. (2004a)Isachenko E, Isachenko V, Katkov II, Rahimi G, Schöndorf T, Mallmann P, Dessole S, Nawroth F. DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification. Hum Reprod. 2004a;19(4):932-9. http://dx.doi.org/10.1093/humrep/deh194. PMid:15016773. http://dx.doi.org/10.1093/humrep/deh194...
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Human |
Semen (n = 38) |
Samples of spermatozoa were located onto copper loop |
Cryoprotectant free. Swim-up samples used. 20 μl plunged into LN2. Warmed in 37 °C medium, under intense agitation, 5–10 minutes. |
40% reduction of motility of spermatozoa in comparison with swim-up-treated control. |
Isachenko et al. (2004b)Isachenko V, Isachenko E, Katkov II, Montag M, Dessole S, Nawroth F, Van Der Ven H. Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability. Biol Reprod. 2004b;71(4):1167-73. http://dx.doi.org/10.1095/biolreprod.104.028811. PMid:15175233. http://dx.doi.org/10.1095/biolreprod.104...
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DNA integrity of cryopreserved spermatozoa was found to be unaffected. |
Human |
Semen (n = 23) |
Sperm suspension dropped into LN2 to form spheres |
0.25M sucrose, HSA 1% and HTFb used. Swim-up samples used. 30 μl aliquots of spermatozoa suspension were dropped directly into the LN2, a sphere immediately forms and floats to the surface. Warmed in 37 °C medium, accompanied by gentle vortexing for 5–10 seconds. |
The number of progressively motile spermatozoa was significantly higher in the sucrose-supplemented medium group (57.1± 3.2%) when compared with controls (19.4±1.9%). Supplementation of HSA and sucrose (65.2±2.6%) has a stronger cryoprotective effect on the integrity of mitochondrial membrane potential compared with HSA alone (32.6± 4.7%). |
Isachenko et al. (2008)Isachenko E, Isachenko V, Weiss JM, Kreienberg R, Katkov II, Schulz M, Lulat A, Risopatrón MJ, Sánchez R. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008;136(2):167-73. http://dx.doi.org/10.1530/REP-07-0463. PMid:18483075. http://dx.doi.org/10.1530/REP-07-0463...
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Channel catfish (Ictalurus punctatus) |
Semen (n = 4) |
Sperm loaded into straws and nichrome loops |
Cryoprotectant-free vitrification. 2 vitrification devices: 20µl of sperm suspension were loaded into the cut end of five straws and 15µl of sperm on nichrome loops. Plunged into LN2. Warmed in a water bath at 40 °Ca. |
Some twitching and vibration of sperm was observed after thawing, but no true progressive post-devitrification motility was observed. Cryoprotectant-free vitrification in nichrome loops did not yield fertilization, and in cut standard straws yielded low levels (<2%) of fertilization. |
Cuevas-Uribe et al. (2011)Cuevas-Uribe R, Leibo SP, Daly J, Tiersch TR. Production of channel catfish with sperm cryopreserved by rapid non-equilibrium cooling. Cryobiology. 2011;63(3):186-97. http://dx.doi.org/10.1016/j.cryobiol.2011.06.004. PMid:21896271. http://dx.doi.org/10.1016/j.cryobiol.201...
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Rainbow trout (Oncorhynchus mykiss) |
Semen (n = 10) |
Sperm suspension dropped into LN2 to form spheres |
Sucrose 0.125M, BSA 1% and 40% seminal plasma used. 20 µl of sperm suspension was dropped directly into LN2. Warmed in 37 °C medium with intense agitation 5-10 min. |
Vitrification using sucrose 0.125M, BSA 1% and seminal plasma resulted in higher motility (82%) than other treatment groups. No cytoplasmic membrane integrity difference was found between groups. Mitochondrial membrane potentials of spermatozoa in all groups were decreased significantly comparatively with non-treated spermatozoa. |
Merino et al. (2011)Merino O, Risopatrón J, Sánchez R, Isachenko E, Figueroa E, Valdebenito I, Isachenko V. Fish (Oncorhynchus mykiss) spermatozoa cryoprotectant-free vitrification: stability of mitochondrion as criterion of effectiveness. Anim Reprod Sci. 2011;124(1-2):125-31. http://dx.doi.org/10.1016/j.anireprosci.2011.02.023. PMid:21392903. http://dx.doi.org/10.1016/j.anireprosci....
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Dog (German Shepherd, Golden Retriever, Labrador Retriever and Rottweiler) |
Semen (n = 24) |
Sperm suspension dropped into LN2 to form spheres |
Sucrose (0.1, 0.25 and 0.4 M) and HTF–BSAb 1% used. Swim-up samplesc. 30 µl of sperm suspension were dropped directly into LN2. Post-thaw sperm suspension was maintained at 37 °C/5% CO2 for 10 min and was then centrifuged at 300 g for 5 minutes. |
Vitrification resulted in higher progressive motility, increased integrity of the mitochondrial membrane potential and decrease DNA fragmentation of the sperm by the addition of 0.25M sucrose when compared with sperm vitrified only with HTF medium. |
Sánchez et al. (2011)Sánchez R, Risopatrón J, Schulz M, Villegas J, Isachenko V, Kreinberg R, Isachenko E. Canine sperm vitrification with sucrose: effect on sperm function. Andrologia. 2011;43(4):233-41. http://dx.doi.org/10.1111/j.1439-0272.2010.01054.x. PMid:21486402. http://dx.doi.org/10.1111/j.1439-0272.20...
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Human |
Semen (n = 68 oligoasthenoteratozoospermic samples) |
Capillary filled with sperm suspension and inserted into 0,25 mL straws |
0.5M sucrose, HSA 1% and HTF used. Swim-up samples used. 50 mL plastic capillaries were manufactured from hydrophobic material. The capillary was filled with 10 μl of spermatozoa suspension by aspiration. After aspiration, the capillary was inserted into a 0.25 mL straw and plunged into LN2. Warmed in 37 °C medium for 20 seconds. |
Vitrification in the absence of permeable cryoprotectants when compared with slow conventional freezing resulted in higher levels of motility (28.0 vs 18.0 respectively), membrane integrity (56.0 vs 22.0%, respectively) and acrossomal integrity (55% vs 21%). |
Isachenko et al. (2012a)Isachenko V, Maettner R, Petrunkina AM, Sterzik K, Mallmann P, Rahimi G, Sanchez R, Risopatron J, Damjanoski I, Isachenko E. Vitrification of human ICSI/IVF spermatozoa without cryoprotectants: new capillary technology. J Androl. 2012a;33(3):462-8. http://dx.doi.org/10.2164/jandrol.111.013789. PMid:21719694. http://dx.doi.org/10.2164/jandrol.111.01...
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Human |
Semen (n = 1) |
Spermatozoa suspension deposited on the end of cut standard straws (CSS) |
0.5M sucrose, HSA 1% and HTF used. Swim-up samples used. A 10 μl aliquot of spermatozoa suspension was deposited on the end of the inner part of the cut standard straws (CSS) and plunged into LN2. Warmed in 37 °C medium in a 2-mL tube fast warming (~30 000 °C min-1) a.. |
Vitrified spermatozoa resulted in 60% progressive motility (vs 90% in freshly spermatozoa). 63% of spermatozoa were classified as having high mitochondrial membrane potential (vs 96% of freshly prepared spermatozoa). |
Isachenko et al. (2012b)Isachenko V, Isachenko E, Petrunkina AM, Sanchez R. Human spermatozoa vitrified in the absence of permeable cryoprotectants: birth of two healthy babies. Reprod Fertil Dev. 2012b;24(2):323-6. http://dx.doi.org/10.1071/RD11061. PMid:22281078. http://dx.doi.org/10.1071/RD11061...
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Rabbit (Hybrid rabbit buck and commercial line) |
Semen (n = 216) |
Sperm suspension dropped into LN2 to form spheres |
Cryoprotectant free or added sucrose or trehalose (0, 0.05, 0.1, or 0.25 M) with 0,5% BSA. 5 replicates of pooled sperm were immediately vitrified by dropping 30 mL semen aliquots directly in a LN2 bath to form frozen spheres. Warmed in a water bath at 38 °C accompanied by gentle vortexing for 10-12 seconds. |
Cryoprotectant-free vitrification resulted in the null or low post devitrification recovery of motile (0%–1%) or membrane intact sperm (1%–5%), whereas DNA integrity ranged from 93% to 97%. Vitrification with BSA alone or with BSA/sucrose (0.1/0.25 M) or BSA/ trehalose (0.25 M) resulted in higher numbers of motile and membrane-intact cells. |
Rosato and Iaffaldano (2013)Rosato MP, Iaffaldano N. Cryopreservation of rabbit semen: comparing the effects of different cryoprotectants, cryoprotectant-free vitrification, and the use of albumin plus osmoprotectants on sperm survival and fertility after standard vapor freezing and vitrification. Theriogenology. 2013;79(3):508-16. http://dx.doi.org/10.1016/j.theriogenology.2012.11.008. PMid:23218394. http://dx.doi.org/10.1016/j.theriogenolo...
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Human |
Semen (n = 10) |
Sperm suspension dropped into LN2 to form spheres |
0.5M sucrose and 10mg/ml of HSA used. Swim-up samples used. 30 μl directly dropped into LN2 (spheres). Warmed in 42 °C medium for 10 seconds. |
Vitrified spermatozoa resulted in 74.7% of total motility (vs 94.3% in freshly spermatozoa), progressive motility was 68% and membrane viability of spermatozoa was 77.21%. |
Dupesh et al. (2019)Dupesh S, Rasappan, Shila, Gunasekaran K. A simple method of human sperm vitrification. MethodsX. 2019;6:2198-204. http://dx.doi.org/10.1016/j.mex.2019.09.022. http://dx.doi.org/10.1016/j.mex.2019.09....
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Donkey (Andalusian donkey) |
Semen (n=4) |
Sperm suspension dropped into LN2 to form spheres |
0.1 M, 0.2 M and 0.3 M sucrose or 1%, 5% and 10% BSA. 30 μl directly dropped into LN2 (spheres). Warmed in 42 °C medium extender |
Sucrose 0.1M result highest total motility and progressive motility (21.67% vs 13.42) when compared to other sucrose concentrations. The addition of different concentrations of BSA to the vitrification extender resulted in no significant differences (p > 0.05) in any of the sperm parameters assessed |
Hidalgo et al. (2020)Hidalgo M, Diaz-Jimenez M, Consuegra C, Pereira B, Dorado J. Vitrification of donkey sperm: is it better using permeable cryoprotectants? Animals. 2020;10(9):1462. http://dx.doi.org/10.3390/ani10091462. PMid:32825370. http://dx.doi.org/10.3390/ani10091462...
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