Register      Login
Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
RESEARCH ARTICLE

Tolerance of spermatozoa to hypotonic stress: role of membrane fluidity and correlation with cryosurvival

Harriëtte Oldenhof A F , Anna Heutelbeck A , Anne-Kathrin Blässe B , Heinrich Bollwein B E , Gunilla Martinsson C , Willem F. Wolkers D and Harald Sieme A
+ Author Affiliations
- Author Affiliations

A Clinic for Horses – Unit for Reproductive Medicine, University of Veterinary Medicine Hannover, Bünteweg 15, D-30559 Hannover, Germany.

B Clinic for Cattle, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, D-30173 Hannover, Germany.

C National Stud Lower Saxony, Spörckenstraße 10, D-29221 Celle, Germany.

D Institute of Multiphase Processes, Leibniz Universität Hannover, Callinstrasse 36, D-30167 Hannover, Germany.

E Present address: Clinic of Reproductive Medicine, Vetsuisse-Faculty, University of Zurich, Winterthurerstrasse 260, CH-8057 Zurich, Switzerland.

F Corresponding author. Email: harriette.oldenhof@tiho-hannover.de

Reproduction, Fertility and Development 27(2) 285-293 https://doi.org/10.1071/RD13177
Submitted: 6 June 2013  Accepted: 3 October 2013   Published: 11 November 2013

Abstract

The aim of this study was to evaluate inter-individual variability in osmotic properties of stallion spermatozoa and its correlation with cryosurvival. In addition, temperature dependency of hypo-osmotic tolerance and membrane fluidity were studied. Stallion sperm membranes exhibited good resistance towards hypotonic stress in the 15–30°C temperature range, whereas membrane stability was found to be decreased at 4 and 37°C. Bull spermatozoa showed greater hypo-osmotic tolerance compared with stallion spermatozoa, especially at temperatures above 30°C, which coincided with decreased membrane fluidity of bovine spermatozoa in this temperature range. The critical osmolality at 22°C, at which half of the sperm population survived exposure to hypotonic saline solution, was found to vary between 55 and 170 mOsm kg–1 among different stallions. Clear correlations were found for pre- versus post-freeze sperm motility and membrane integrity. Pre-freeze percentages of membrane-intact spermatozoa after exposure to hypotonic stress showed a weak correlation with sperm motility after cryopreservation. This correlation, however, was not found when data were corrected for initial numbers of membrane-intact spermatozoa in the sample. We thus conclude that studies on pre-freeze tolerance towards hypotonic stress cannot be used to predict sperm cryosurvival rates for individual stallions.

Additional keywords: cryopreservation, osmotic tolerance, stallion spermatozoa.


References

Akhoondi, M., Oldenhof, H., Stoll, C., Sieme, H., and Wolkers, W. F. (2011). Membrane hydraulic permeability changes during cooling of mammalian cells. Biochim. Biophys. Acta 1808, 642–648.
Membrane hydraulic permeability changes during cooling of mammalian cells.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhvFentrg%3D&md5=ddbae33efb69ff4eb172ca20474ea569CAS | 21126509PubMed |

Benson, J. D., Woods, E. J., Walters, E. M., and Critser, J. K. (2012). The cryobiology of spermatozoa. Theriogenology 78, 1682–1699.
The cryobiology of spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XhsFSlt7vK&md5=20fbbd6ab60d6ec9300d8137c8d1b880CAS | 23062722PubMed |

Blässe, A. K., Oldenhof, H., Ekhlasi-Hundrieser, M., Wolkers, W. F., Sieme, H., and Bollwein, H. (2012). Osmotic properties and intracellular ion concentrations of bovine sperm are affected by cryopreservation. Theriogenology 78, 1312–1320.
Osmotic properties and intracellular ion concentrations of bovine sperm are affected by cryopreservation.Crossref | GoogleScholarGoogle Scholar | 22819283PubMed |

Brinsko, S. P., Love, C. C., Bauer, J. E., Macpherson, M. L., and Varner, D. D. (2007). Cholesterol-to-phospholipid ratio in whole sperm and seminal plasma from fertile stallions and stallions with unexplained subfertility. Anim. Reprod. Sci. 99, 65–71.
Cholesterol-to-phospholipid ratio in whole sperm and seminal plasma from fertile stallions and stallions with unexplained subfertility.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXit1GmsLo%3D&md5=d9a46e12a6179e03e35fbdcbfa1d3ef5CAS | 16713689PubMed |

Burnaugh, L., Ball, B. A., Sabeur, K., Thomas, A. D., and Meyers, S. A. (2010). Osmotic stress stimulates generation of superoxide anion by spermatozoa in horses. Anim. Reprod. Sci. 117, 249–260.
Osmotic stress stimulates generation of superoxide anion by spermatozoa in horses.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhsVGltrbM&md5=a988792ec87c7f8fc25609846bfa215aCAS | 19553037PubMed |

Chan, S. Y. W., Craft, I. L., Chan, Y. M., Leong, M. K. H., and Leung, C. K. M. (1990). The hypo-osmotic swelling test and cryosurvival of human spermatozoa. Hum. Reprod. 5, 715–718.
| 1:STN:280:DyaK3M%2Fnt1Gktg%3D%3D&md5=2b7a261c9964c853d22bff5b3bf06249CAS |

Christova, Y., James, P. S., Cooper, T. G., and Jones, R. (2002). Lipid diffusion in the plasma membrane of mouse spermatozoa: changes during epididymal maturation, effects of pH, osmotic pressure and knockout of the c-ros gene. J. Androl. 23, 384–392.
| 1:CAS:528:DC%2BD38XjvVKmtL0%3D&md5=fbc4dc16241f055c621b29c521a030deCAS | 12002440PubMed |

Comizzoli, P., Songsasen, N., Hagedorn, M., and Wildt, D. E. (2012). Comparative cryobiological traits and requirements for gametes and gonadal tissues collected from wildlife species. Theriogenology 78, 1666–1681.
Comparative cryobiological traits and requirements for gametes and gonadal tissues collected from wildlife species.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC38jit1yntQ%3D%3D&md5=8f84d952bee97e4f41a2cc7655cb90deCAS | 22704386PubMed |

Crowe, J. H., Hoekstra, F. A., Crowe, L. M., Anchordoguy, T. J., and Drobnis, E. (1989). Lipid phase transitions measured in intact cells with Fourier transform infrared spectroscopy. Cryobiology 26, 76–84.
Lipid phase transitions measured in intact cells with Fourier transform infrared spectroscopy.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaL1M7mvFGquw%3D%3D&md5=062c5d9247d0fcc996ac2ad6d3e52798CAS | 2924595PubMed |

Drevius, L. O., and Eriksson, H. (1966). Osmotic swelling of mammalian spermatozoa. Exp. Cell Res. 42, 136–156.
Osmotic swelling of mammalian spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaF287itVaksA%3D%3D&md5=9f5f8542ecdca42515fb2a3939032ac2CAS | 5948776PubMed |

Drobnis, E. Z., Crowe, L. M., Berger, T., Anchordoguy, T. J., Overstreet, J. W., and Crowe, J. H. (1993). Cold-shock damage is due to lipid phase transitions in cell membranes: a demonstration using sperm as a model. J. Exp. Zool. 265, 432–437.
Cold-shock damage is due to lipid phase transitions in cell membranes: a demonstration using sperm as a model.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3sXksVKisrc%3D&md5=3689ef48b97a58427dc7eedb82d4adddCAS | 8463792PubMed |

Druart, X., Gatti, J. L., Huet, S., Dacheux, J. L., and Humblot, P. (2009). Hypotonic resistance of boar spermatozoa: sperm subpopulations and relationship with epididymal maturation and fertility. Reproduction 137, 205–213.
Hypotonic resistance of boar spermatozoa: sperm subpopulations and relationship with epididymal maturation and fertility.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXovV2ks7k%3D&md5=523b6b5459c3c6f135607a8b68366875CAS | 18996973PubMed |

Flesch, F. M., and Gadella, B. M. (2000). Dynamics of the mammalian sperm plasma membrane in the process of fertilization. Biochim. Biophys. Acta 1469, 197–235.
Dynamics of the mammalian sperm plasma membrane in the process of fertilization.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXovFKnsL4%3D&md5=2fc373271520107543cd42adc5986eb5CAS | 11063883PubMed |

Gilmore, J. A., Du, J., Tao, J., Peter, A. T., and Critser, J. K. (1996). Osmotic properties of boar spermatozoa and their relevance to cryopreservation. J. Reprod. Fertil. 107, 87–95.
Osmotic properties of boar spermatozoa and their relevance to cryopreservation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK28XmtFOlur8%3D&md5=7356c43dc9229d90cff90c1eba96fdc5CAS | 8699439PubMed |

Gilmore, J. A., Liu, J., Peter, A. T., and Critser, J. K. (1998). Determination of plasma membrane characteristics of boar spermatozoa and their relevance to cryopreservation. Biol. Reprod. 58, 28–36.
Determination of plasma membrane characteristics of boar spermatozoa and their relevance to cryopreservation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXhsl2gsA%3D%3D&md5=d70253380a13b4c6c794c0232921a52aCAS | 9472919PubMed |

Glazar, A. I., Mullen, S. F., Liu, J., Benson, J. D., Critser, J. K., Squires, E. L., and Graham, J. K. (2009). Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol. Cryobiology 59, 201–206.
Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtVKgu7nE&md5=9d061122fb26c2a30b76dc8e28054dccCAS | 19646432PubMed |

Guthrie, H. D., and Welch, G. R. (2012). Effects of reactive oxygen species on sperm function. Theriogenology 78, 1700–1708.
Effects of reactive oxygen species on sperm function.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XosFCgsLw%3D&md5=f3776f3c5d543dbcfe9d0da1735935ffCAS | 22704396PubMed |

Hammerstedt, R. H., Graham, J. K., and Nolan, J. P. (1990). Cryopreservation of mammalian sperm: what we ask them to survive. J. Androl. 11, 73–88.
| 1:CAS:528:DyaK3cXhtlKgs7c%3D&md5=32c0a3c393ca409727a8d7a7001b2b7aCAS | 2179184PubMed |

Hoffmann, E. K., Lambert, I. A., and Pedersen, S. F. (2009). Physiology of cell volume regulation in vertebrates. Physiol. Rev. 89, 193–277.
Physiology of cell volume regulation in vertebrates.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXit1Cqtrs%3D&md5=0a000c7f5ccc91b612cc337e3b0eb461CAS | 19126758PubMed |

Jeyendran, R. S., Van der Ven, H. H., Perez-Pelaez, M., Crabo, B. G., and Zaneveld, L. J. D. (1984). Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J. Reprod. Fertil. 70, 219–228.
Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaL2c7hsVKntA%3D%3D&md5=6fa952dfa1fcc77f2ebb9e713ce3c695CAS | 6694140PubMed |

Jeyendran, R. S., Van der Ven, H. H., and Zaneveld, L. J. D. (1992). The hypo-osmotic swelling test: an update. Arch. Androl. 29, 105–116.
The hypo-osmotic swelling test: an update.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK3s%2FovF2qsA%3D%3D&md5=a03e346ca72a06c07f5223853a98a761CAS | 1456832PubMed |

Kayser, J. P., Amann, R. P., Shideler, R. K., Squires, E. L., Jasko, D. J., and Pickett, B. W. (1992). Effects of linear cooling rate on motion characteristics of stallion spermatozoa. Theriogenology 38, 601–614.
Effects of linear cooling rate on motion characteristics of stallion spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD283pvF2htw%3D%3D&md5=0965cba0d57ac1de6138f7d2c1459e88CAS | 16727163PubMed |

Khalil, A. A. Y., Petrunkina, A. M., Sahin, E., Waberski, D., and Töpfer-Petersen, E. (2006). Enhanced binding of sperm with superior volume regulation to oviductal epithelium. J. Androl. 27, 754–765.
Enhanced binding of sperm with superior volume regulation to oviductal epithelium.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD28nksF2jsA%3D%3D&md5=8b430ca9e364d1e905927631e0069099CAS |

Loomis, P. R., and Graham, J. K. (2008). Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols. Anim. Reprod. Sci. 105, 119–128.
Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXislSmtLk%3D&md5=e3ed54d386e5a51bcc84e5689deb1fb9CAS | 18178040PubMed |

Macías García, B., González Fernández, L., Ortega Ferrusola, C., Salazar-Sandovali, C., Morillo Rodríguez, A., Rodríguez Martinez, H., Tapia, J. A., Morcuende, D., and Peña, F. J. (2011). Membrane lipids of the stallion spermatozoon in relation to sperm quality and susceptibility to lipid peroxidation. Reprod. Domest. Anim. 46, 141–148.
Membrane lipids of the stallion spermatozoon in relation to sperm quality and susceptibility to lipid peroxidation.Crossref | GoogleScholarGoogle Scholar |

Mazur, P. (1984). Freezing of living cells: mechanisms and implications. Am. J. Physiol. 247, C125–C142.
| 1:CAS:528:DyaL2cXls1Ors7w%3D&md5=82736d16825d5f229f8558a85f392abeCAS | 6383068PubMed |

Mazur, P. (2004). Principles of cryobiology. In ‘Life in the Frozen State’. (Eds B. J. Fuller, N. L. Lane and E. E. Benson.) pp. 3–65. (CRC Press LLC: Boca Raton, FL.)

Mazur, P., Leibo, S. P., and Seidel, G. E. (2008). Cryopreservation of the germplasm of animals used in biological and medical research: importance, impact, status and future directions. Biol. Reprod. 78, 2–12.
Cryopreservation of the germplasm of animals used in biological and medical research: importance, impact, status and future directions.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXls1I%3D&md5=248a87c4d3a5a97cc8ce34c5750808cdCAS | 17901073PubMed |

Mocé, E., Blanch, E., Tomás, C., and Graham, J. K. (2010). Use of cholesterol in sperm cryopreservation: present moment and perspectives to future. Reprod. Domest. Anim. 45, 57–66.
Use of cholesterol in sperm cryopreservation: present moment and perspectives to future.Crossref | GoogleScholarGoogle Scholar | 20591066PubMed |

Moore, A. I., Squires, E. L., and Graham, J. K. (2005). Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival. Cryobiology 51, 241–249.
Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhtlShsLbP&md5=a3a3ba1b939134242be63409d75d9a1dCAS | 16122725PubMed |

Müller, K., Müller, P., Pincemy, G., Kurz, A., and Labbe, C. (2008). Characterization of sperm plasma membrane properties after cholesterol modification: consequences for cryopreservation of rainbow trout spermatozoa. Biol. Reprod. 78, 390–399.
Characterization of sperm plasma membrane properties after cholesterol modification: consequences for cryopreservation of rainbow trout spermatozoa.Crossref | GoogleScholarGoogle Scholar | 18003944PubMed |

Neild, D. M., Chaves, M. G., Flores, M., Miragaya, M. H., Gonzalez, E., and Agüero, A. (2000). The HOS test and its relationship to fertility in the stallion. Andrologia 32, 351–355.
The HOS test and its relationship to fertility in the stallion.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3M7hsFSgsQ%3D%3D&md5=7b80044ac2f7b171d45a35cfe272bf0bCAS | 11131844PubMed |

Nolan, J. P., and Hammerstedt, R. H. (1997). Regulation of membrane stability and the acrosome reaction in mammalian sperm. FASEB J. 11, 670–682.
| 1:CAS:528:DyaK2sXkvFKjt78%3D&md5=ed3ab41f94a0ea63872af5d489dea339CAS | 9240968PubMed |

Oldenhof, H., Friedel, K., Akhoondi, M., Gojowsky, M., Wolkers, W. F., and Sieme, H. (2012). Membrane phase behaviour during cooling of stallion sperm and its correlation with freezability. Mol. Membr. Biol. 29, 95–106.
Membrane phase behaviour during cooling of stallion sperm and its correlation with freezability.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38Xoslals7g%3D&md5=6ffc6a55fdc3d581f157ddb19128e871CAS | 22480267PubMed |

Oldenhof, H., Akhoondi, M., Sieme, H., and Wolkers, W. F. (2013a). Use of Fourier transform infrared spectroscopy to study membrane properties of cells at subzero temperatures. Biomedical Spectroscopy and Imaging 2, 83–90.

Oldenhof, H., Gojowsky, M., Wang, S., Henke, S., Yu, C., Rohn, K., Wolkers, W. F., and Sieme, H. (2013b). Osmotic stress and membrane phase changes during freezing of stallion sperm: mode of action of cryoprotective agents. Biol. Reprod. 88, 68.
Osmotic stress and membrane phase changes during freezing of stallion sperm: mode of action of cryoprotective agents.Crossref | GoogleScholarGoogle Scholar | 23325813PubMed |

Parks, J. E., and Lynch, D. V. (1992). Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes. Cryobiology 29, 255–266.
Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK38Xitlaktr0%3D&md5=7530a88f44058630bcd4c9b8c2781c98CAS | 1582232PubMed |

Petrunkina, A. M., and Töpfer-Petersen, E. (2000). Heterogeneous osmotic behaviour in boar sperm populations and its relevance for detection of changes in plasma membrane. Reprod. Fertil. Dev. 12, 297–305.
Heterogeneous osmotic behaviour in boar sperm populations and its relevance for detection of changes in plasma membrane.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3MzpvF2jsw%3D%3D&md5=503afc79afcd8c4d8bed98c1ee91cb3dCAS | 11451021PubMed |

Petrunkina, A. M., Gröpper, B., Günzel-Apel, A. R., and Töpfer-Petersen, E. (2004a). Functional significance of the cell volume for detecting sperm membrane changes and predicting freezability in dog semen. Reproduction 128, 829–842.
Functional significance of the cell volume for detecting sperm membrane changes and predicting freezability in dog semen.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhtFSqtg%3D%3D&md5=9b201aba1553faebef677a65b3fe43d7CAS | 15579601PubMed |

Petrunkina, A. M., Harrison, R. A. P., Ekhlasi-Hundrieser, M., and Töpfer-Petersen, E. (2004b). Role of volume-stimulated osmolyte and anion channels in volume regulation by mammalian sperm. Mol. Hum. Reprod. 10, 815–823.
Role of volume-stimulated osmolyte and anion channels in volume regulation by mammalian sperm.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXovVOis78%3D&md5=40f6dcc0c28596142a3180f7f924c92dCAS | 15361553PubMed |

Petrunkina, A. M., Jebe, E., and Töpfer-Petersen, E. (2005). Regulatory and necrotic volume increase in boar spermatozoa. J. Cell. Physiol. 204, 508–521.
Regulatory and necrotic volume increase in boar spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXlvFCrsLg%3D&md5=42c2e2cee90e983f3e3437440892cc5bCAS | 15744775PubMed |

Purdy, P. H., and Graham, J. K. (2004). Effect of cholesterol-loaded cyclodextrin on the cryosurvival of bull sperm. Cryobiology 48, 36–45.
Effect of cholesterol-loaded cyclodextrin on the cryosurvival of bull sperm.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXht1yiur8%3D&md5=4ff950f7ff0c30224af6f0372aa36ff0CAS | 14969680PubMed |

Ramu, S., and Jeyendran, R. S. (2013). The hypo-osmotic swelling test for evaluation of sperm membrane integrity. Methods Mol. Biol. 927, 21–25.
The hypo-osmotic swelling test for evaluation of sperm membrane integrity.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXit1Snurs%3D&md5=9e9be34cf45bf91a6827dfb5ecc88914CAS | 22992900PubMed |

Ricker, J. V., Linfor, J. J., Delfino, W. J., Kysar, P., Scholtz, E. L., Tablin, F., Crowe, J. H., Ball, B. A., and Meyers, S. A. (2006). Equine sperm membrane phase behaviour: the effects of lipid-based cryoprotectants. Biol. Reprod. 74, 359–365.
Equine sperm membrane phase behaviour: the effects of lipid-based cryoprotectants.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xot1Kksw%3D%3D&md5=dbd1dff40f7cdbd13ecf42a259eff162CAS | 16251500PubMed |

Röpke, T., Oldenhof, H., Leiding, C., Sieme, H., Bollwein, H., and Wolkers, W. F. (2011). Liposomes for cryopreservation of bovine sperm. Theriogenology 76, 1465–1472.
Liposomes for cryopreservation of bovine sperm.Crossref | GoogleScholarGoogle Scholar | 21820724PubMed |

Schmid, S., Henning, H., Oldenhof, H., Wolkers, W. F., Petrunkina, A. M., and Waberski, D. (2013). The specific response to capacitating stimuli is a sensitive indicator of chilling injury in hypothermically stored boar spermatozoa. Andrology 1, 376–386.
The specific response to capacitating stimuli is a sensitive indicator of chilling injury in hypothermically stored boar spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXmt1KqtLc%3D&md5=6f508ff2e2d99a803899d6576cfd2506CAS | 23427145PubMed |

Sieme, H., Harrison, R. A. P., and Petrunkina, A. M. (2008). Cryobiological determinants of frozen semen quality, with special reference to stallion. Anim. Reprod. Sci. 107, 276–292.
Cryobiological determinants of frozen semen quality, with special reference to stallion.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXoslWktL8%3D&md5=6cd354d04e217af0e68d1a5440704a0aCAS | 18585878PubMed |

Sikka, S. C. (2004). Role of oxidative stress and antioxidants in andrology and assisted reproductive technology. J. Androl. 25, 5–18.
Role of oxidative stress and antioxidants in andrology and assisted reproductive technology.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXkslCguw%3D%3D&md5=00eade7414fdfaa497d82cd1ea317630CAS | 14662779PubMed |

Van der Ven, H. H., Jeyendran, R. S., Al-Hasani, S., Perez-Pelaez, M., Diedrich, K., and Zanefeld, L. J. (1986). Correlation between human sperm swelling in hypo-osmotic medium (hypo-osmotic swelling test) and in vitro fertilization. J. Androl. 7, 190–196.
| 1:STN:280:DyaL283ltVeguw%3D%3D&md5=1cbf904a42444ceabdd72d91b5da82f4CAS | 3722001PubMed |

Vidament, M., Cognard, E., Yvon, J. M., Sattler, M., Palmer, E., and Magistrini, M. (1998). Evaluation of stallion semen before and after freezing. Reprod. Domest. Anim. 33, 271–277.
Evaluation of stallion semen before and after freezing.Crossref | GoogleScholarGoogle Scholar |

Wolf, D. E., Maynard, V. M., McKinnon, C. A., and Melchior, D. L. (1990). Lipid domains in the ram sperm plasma membrane demonstrated by differential scanning calorimetry. Proc. Natl. Acad. Sci. USA 87, 6893–6896.
Lipid domains in the ram sperm plasma membrane demonstrated by differential scanning calorimetry.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3cXls12mt7s%3D&md5=52b83bdd64c83390e2d2dbca7c7347c9CAS | 2395884PubMed |

Wolkers, W. F., Crowe, L. M., Tsvetkova, N. M., Tablin, F., and Crowe, J. H. (2002). In situ assessment of erythrocyte membrane properties during cold storage. Mol. Membr. Biol. 19, 59–65.
In situ assessment of erythrocyte membrane properties during cold storage.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XjtFKgsrc%3D&md5=ea174f78f6b8bc554c3761d1af05acf8CAS | 11989823PubMed |

Wolkers, W. F., Balasubramanian, S. K., Ongstad, E. L., and Bischof, J. C. (2007). Effects of freezing on membranes and proteins in LNCaP prostate tumor cells. Biochim. Biophys. Acta 1768, 728–736.
Effects of freezing on membranes and proteins in LNCaP prostate tumor cells.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXit1Kjt7w%3D&md5=bc0cc53701a6fd7f795acef11e8ee46bCAS | 17239814PubMed |

Woods, E. J., Benson, J. D., Agca, Y., and Critser, J. K. (2004). Fundamental cryobiology of reproductive cells and tissues. Cryobiology 48, 146–156.
Fundamental cryobiology of reproductive cells and tissues.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXjt1amurw%3D&md5=49627b7c52dbbb0caafdf716a4e4e853CAS | 15094091PubMed |

Yeung, C. H. (2010). Aquaporins in spermatozoa and testicular germ cells: identification and potential role. Asian J. Androl. 12, 490–499.
Aquaporins in spermatozoa and testicular germ cells: identification and potential role.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXotlSksrw%3D&md5=ad198b3829859d5ef8f60ce6e1dea80dCAS | 20562895PubMed |