Gamete cryopreservation of Australian ‘old endemic’ rodents – spermatozoa from the plains mouse (Pseudomys australis) and spinifex hopping mouse (Notomys alexis)
Kathrine M. Ferres A B C , Nicole O. McPherson B D E , Michelle Lane B D E F , Hassan W. Bakos B D , Karen L. Kind C and William G. Breed A D G H
+ Author Affiliations
- Author Affiliations
A Discipline of Anatomy and Pathology, Adelaide Medical School, Faculty of Health Sciences, The University of Adelaide, SA 5005, Australia.
B Discipline of Obstetrics and Gynaecology, Adelaide Medical School, Faculty of Health Sciences, The University of Adelaide, SA 5005, Australia.
C School of Animal and Veterinary Sciences, Faculty of Sciences, The University of Adelaide, SA 5005, Australia.
D Robinson Research Institute, University of Adelaide, Adelaide, SA 5005, Australia.
E Freemasons Centre for Men’s Health, The University of Adelaide, SA 5005, Australia.
F Repromed, 180 Fullarton Road, Dulwich, SA 5065, Australia.
G School of Biological Sciences, Faculty of Sciences, The University of Adelaide, SA 5005, Australia.
H Corresponding author. Email: bill.breed@adelaide.edu.au
Australian Mammalogy 40(1) 76-83 https://doi.org/10.1071/AM16055
Submitted: 20 November 2016 Accepted: 6 April 2017 Published: 7 July 2017
Abstract
Most of the Australian ‘old endemic’ rodents have greatly reduced distributions with several species now threatened with extinction. Application of assisted reproductive technology has the potential to assist in their conservation programs in at least a few species. Here we describe an attempt to cryopreserve spermatozoa from two of these species – those of the plains mouse (Pseudomys australis) and spinifex hopping mouse (Notomys alexis), which have dramatic differences in sperm morphology. Slow and rapid freezing and three different cryoprotectant media with either raffinose, glycerol and/or skim milk were used and the results compared with those of house mouse sperm, which were used as controls. Sperm morphology, motility, membrane integrity and DNA damage were determined. Prior to cryopreservation there was a higher percentage of morphologically normal, motile, P. australis sperm than in those from N. alexis. Following cryopreservation, regardless of treatment, the percentage of motile sperm was low but it was higher when raffinose with skim milk was used as a cryoprotectant than in raffinose with glycerol albeit that minimal differences in membrane integrity or DNA damage were evident. Raffinose with skim milk should thus be used as a cryoprotectant for storing sperm of these Australian rodents in the future.
Additional keywords: cryostorage, hydromyine rodents, male gamete.
References
An, T. Z., Iwakiri, M., Edashige, K., Sakurai, T., and Kasai, M. (2000). Factors affecting the survival of frozen-thawed mouse spermatozoa. Cryobiology 40, 237–249.| Factors affecting the survival of frozen-thawed mouse spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXktVKgsbo%3D&md5=ecfc81e1572d7aa072567f4464e95a82CAS |
Aplin, K. P. (2005). Ten million years of rodent evolution in Australasia: phylogenetic evidence and a speculative historical biogeography. In ‘Vertebrate Zoogeography and Evolution in the Australasian Region’. (Eds J. R. Merrick, M. Archer, and S. J. Hand.) Ch. 3, pp. 707–744. (Auscipub Pty Ltd: Sydney.)
Bakos, H. W., Thompson, J. G., Feil, D., and Lane, M. (2008). Sperm DNA damage is associated with assisted reproductive technology and pregnancy. International Journal of Andrology 31, 518–526.
| Sperm DNA damage is associated with assisted reproductive technology and pregnancy.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXht1aqsbbN&md5=0ab5e16ddeba205d150858bec216356dCAS |
Bath, M. L. (2011). Optimized cryopreservation of mouse sperm based on fertilization rate. The Journal of Reproduction and Development 57, 92–98.
| Optimized cryopreservation of mouse sperm based on fertilization rate.Crossref | GoogleScholarGoogle Scholar |
Bauer, M., and Breed, W. G. (2006). Variation of sperm head shape and tail length in a species of Australian hydromyine rodent: the spinifex hopping mouse, Notomys alexis. Reproduction, Fertility and Development 18, 797–805.
| Variation of sperm head shape and tail length in a species of Australian hydromyine rodent: the spinifex hopping mouse, Notomys alexis.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD28ngt1Sgsw%3D%3D&md5=18d1b2d4ce516877b8290c5b5656c022CAS |
Binder, N. K., Hannan, N. J., and Gardner, D. K. (2012). Paternal diet-induced obesity retards early mouse embryo development, mitochondrial activity and pregnancy health. PLoS One 7, e52304.
| Paternal diet-induced obesity retards early mouse embryo development, mitochondrial activity and pregnancy health.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXmvFGhsA%3D%3D&md5=f01bb80e3897e157dc77f2a6b9aa3f75CAS |
Breed, W. G. (1986). Comparative morphology and evolution of the male reproductive tract in the Australian hydromyine rodents (Muridae). Journal of Zoology 209, 607–629.
| Comparative morphology and evolution of the male reproductive tract in the Australian hydromyine rodents (Muridae).Crossref | GoogleScholarGoogle Scholar |
Breed, W. G. (1997). Evolution of the spermatozoon in Australasian rodents. Australian Journal of Zoology 45, 459–478.
| Evolution of the spermatozoon in Australasian rodents.Crossref | GoogleScholarGoogle Scholar |
Breed, B., and Ford, F. (2007). ‘Native Mice and Rats.’ (CSIRO Publishing: Melbourne.)
Breed, W. G., and Sarafis, V. (1983). Variation in sperm head morphology in the Australian rodent Notomys alexis. Australian Journal of Zoology 31, 313–316.
| Variation in sperm head morphology in the Australian rodent Notomys alexis.Crossref | GoogleScholarGoogle Scholar |
Breed, W. G., and Taylor, J. (2000). Body mass, testes mass, and sperm size in murine rodents. Journal of Mammalogy 81, 758–768.
| Body mass, testes mass, and sperm size in murine rodents.Crossref | GoogleScholarGoogle Scholar |
Breed, W. G., Idriss, D., and Oko, R. J. (2000). Protein composition of the ventral processes on the sperm head of Australian hydromyine rodents. Biology of Reproduction 63, 629–634.
| Protein composition of the ventral processes on the sperm head of Australian hydromyine rodents.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXltl2gurY%3D&md5=e078cc0bb97a2df15625c40f892741cbCAS |
Breed, W. G., Leigh, C. M., and Speight, N. (2013). Coevolution of the male and female reproductive tracts in an old endemic rodent of Australia. Journal of Zoology 289, 94–100.
| Coevolution of the male and female reproductive tracts in an old endemic rodent of Australia.Crossref | GoogleScholarGoogle Scholar |
Burbidge, A. A., McKenzie, N. L., Brennan, K. E. C., Woinarski, J. C. Z., Dickman, C. R., Baynes, A., Gordon, G., Menkhorst, P. W., and Robinson, A. C. (2008). Conservation status and biogeography of Australia’s terrestial mammals. Australian Journal of Zoology 56, 411–422.
| Conservation status and biogeography of Australia’s terrestial mammals.Crossref | GoogleScholarGoogle Scholar |
Drew, S., Leigh, C. M., and Breed, W. G. (2014). Spermatozoa of the old endemic rodents of Australia – the possible functional significance of their ventral processes. Reproduction, Fertility and Development 26, 1183–1187.
| Spermatozoa of the old endemic rodents of Australia – the possible functional significance of their ventral processes.Crossref | GoogleScholarGoogle Scholar |
Fernández-Gonzalez, R., Moreira, P. N., Pérez-Crespo, M., Sánchez-Martín, M., Ramirez, M. A., Pericuesta, E., Bilbao, A., Bermejo-Alvarez, P., Hourcade, J. D., de Fonseca, F. R., and Gutiérrez-Adán, A. (2008). Long-term effects of mouse intracytoplasmic sperm injection with DNA-fragmented sperm on health and behavior of adult offspring. Biology of Reproduction 78, 761–772.
| Long-term effects of mouse intracytoplasmic sperm injection with DNA-fragmented sperm on health and behavior of adult offspring.Crossref | GoogleScholarGoogle Scholar |
Flaherty, S. P., and Breed, W. G. (1983). The sperm head of the plains mouse, Pseudomys australis: ultrastructure and effects of chemical treatments. Gamete Research 8, 231–244.
| The sperm head of the plains mouse, Pseudomys australis: ultrastructure and effects of chemical treatments.Crossref | GoogleScholarGoogle Scholar |
Gardner, D. K., and Lane, M. (2004). Culture of the mammalian preimplantation embryo. In ‘A Laboratory Guide to the Mammalian Embryo’. (Eds D. K. Gardner, M. Lane, and A. J. Watson.) pp. 41–61. (Oxford University Press: New York.)
Geffen, E., Rowe, K. C., and Yom-Tov, Y. (2011). Reproductive rates in Australian rodents are related to phylogeny. PLoS One 6, e19199.
| Reproductive rates in Australian rodents are related to phylogeny.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXlslGmtrc%3D&md5=dfe74c2e6c568294387904f20b82e11eCAS |
Holt, W. V., and Pickard, A. R. (1999). Role of reproductive technologies and genetic resource banks in animal conservation. Reviews of Reproduction 4, 143–150.
| Role of reproductive technologies and genetic resource banks in animal conservation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXmsVClsr8%3D&md5=413c41505152fedfab29d6f2eb9a9d10CAS |
Holt, W. V., Pichard, A. R., and Prather, R. S. (2004). Wildlife conservation and reproductive cloning. Reproduction (Cambridge, England) 127, 317–324.
| Wildlife conservation and reproductive cloning.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXjt1Ols7w%3D&md5=e672043e589373a127fb6842975567c6CAS |
Immler, S., Moore, H. D. M., Breed, W. G., and Birkhead, T. R. (2007). By hook or by crook? Morphometry, competition and cooperation in rodent sperm. PLoS One 2, .
| By hook or by crook? Morphometry, competition and cooperation in rodent sperm.Crossref | GoogleScholarGoogle Scholar |
Kaneko, T. (2016). Sperm freeze-drying and micro-insemination for biobanking and maintenance of genetic diversity in mammals. Reproduction, Fertility and Development 28, 1079–1087.
| Sperm freeze-drying and micro-insemination for biobanking and maintenance of genetic diversity in mammals.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC28XhtVWgtbvI&md5=7c8ea5c36350cf1869a73a46d0a00998CAS |
Kaneko, T., Yamamura, A., Ide, Y., Ogi, M., Yanagita, T., and Nakagata, N. (2006). Long-term cryopreservation of mouse sperm. Theriogenology 66, 1098–1101.
| Long-term cryopreservation of mouse sperm.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XotleqsLs%3D&md5=a88acfa694049bfae5d4fa65fdd65edbCAS |
Kaneko, T., Ito, H., Sakamoto, H., Onuma, M., and Inoue-Murayama, M. (2014). Sperm preservation by freeze – drying for the conservation of wild animals. PLoS One 9, e113381.
| Sperm preservation by freeze – drying for the conservation of wild animals.Crossref | GoogleScholarGoogle Scholar |
Kimura, Y., and Yanagimachi, R. (1995). Intracytoplasmic sperm injection in the mouse. Biology of Reproduction 52, 709–720.
| Intracytoplasmic sperm injection in the mouse.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2MXksFWhtrk%3D&md5=a1de3da5a982df4e8ecd1ef1e33ab25bCAS |
Kruger, T., Ackerman, S. B., Simmons, K. F., Swanson, R. J., Brugo, S. S., and Acosta, A. A. (1987). A quick, reliable staining technique for human sperm morphology. Archives of Andrology 18, 275–277.
| A quick, reliable staining technique for human sperm morphology.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaL1c%2FkvVKrtw%3D%3D&md5=0a22c906e5dbbc890587f54cd06e2e1aCAS |
McLennan, H. J., Lüpold, S., Smissen, P., Rowe, K. C., and Breed, W. G. (2017). Greater sperm complexity in the Australasian old endemic rodents (Tribe: Hydromyini) is associated with increased levels of intermale sperm competition. Reproduction, Fertility and Development 29, 921–930.
| Greater sperm complexity in the Australasian old endemic rodents (Tribe: Hydromyini) is associated with increased levels of intermale sperm competition.Crossref | GoogleScholarGoogle Scholar |
Moore, H., Dvorakova, K., Jenkins, N., and Breed, W. (2002). Exceptional sperm cooperation in the wood mouse. Nature 418, 174–177.
| Exceptional sperm cooperation in the wood mouse.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XltFGlsrw%3D&md5=cbc8dbbd1f2c3e9ad167d6408786918fCAS |
Nakagata, N. (2011). Cryopreservation of mouse spermatozoa and in vitro fertilisation. Methods in Molecular Biology (Clifton, N.J.) 693, 57–73.
| Cryopreservation of mouse spermatozoa and in vitro fertilisation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXhs1Wmu7nN&md5=cac80f0fce39d318b4a31c7cdd5adb04CAS |
Nakagata, N., Ueda, S., Yamanuchi, K., Okamoto, M., Matsuda, Y., Tsuchiya, K., Nishimura, M., Oda, S., Koyasu, K., and Azuma, Y. (1995). Cryopreservation of wild mouse spermatozoa. Theriogenology 43, 635–643.
| Cryopreservation of wild mouse spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD28zgtVCisA%3D%3D&md5=dae1e87b39326b21a8ffe49887143887CAS |
Ostermeier, G. C., Wiles, M. V., Farley, J. S., and Taft, R. A. (2008). Conserving, distributing and managing genetically modified mouse lines by sperm cryopreservation. PLoS One 3, .
| Conserving, distributing and managing genetically modified mouse lines by sperm cryopreservation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhtVWrs7zO&md5=13cbbcae3d34e2e1e0cc402d33e6d6f0CAS |
Parker, G. A., and Pizzari, T. (2010). Sperm competition and ejaculate economics. Biological Reviews of the Cambridge Philosophical Society 85, 897–934.
Parker, G. A., Ball, M. A., Stockley, P., and Gage, M. J. G. (1997). Sperm competition games. A prospective analysis of risk assessment. Proceedings of The Royal Society of London, Biological Sciences 264, 1793–1802.
Peirce, E. J., and Breed, W. G. (2001). A comparative study of sperm production in two species of Australian arid zone rodents (Pseudomys australis, Notomys alexis) with marked differences in testis size. Reproduction (Cambridge, England) 121, 239–247.
| A comparative study of sperm production in two species of Australian arid zone rodents (Pseudomys australis, Notomys alexis) with marked differences in testis size.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXhslSisb4%3D&md5=6109a8d2a8aec5d7ee3de02178960502CAS |
Peirce, E. J., Moore, H. D. M., Leigh, C. M., and Breed, W. G. (2003). Studies on sperm storage in the vas deferens of the spinifex hopping mouse (Notomys alexis). Reproduction (Cambridge, England) 125, 233–240.
| Studies on sperm storage in the vas deferens of the spinifex hopping mouse (Notomys alexis).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXisVKlt7o%3D&md5=3e5a756f6929b3377947ef5ce9b589b4CAS |
Pitnick, S., Hosken, D. J., and Birkhead, T. R. (2009). Sperm morphological diversity. In ‘Sperm Biology: An Evolutionary Perspective’. (Eds T. R. Birkhead, D. J. Hosken, and S. Pitnick.) pp. 69–149. (Academic Press: Burlington.)
Šandera, M., Albrecht, T., and Stopka, P. (2013). Variation in apical hook length reflects the intensity of sperm competition in murine rodents. PLoS One 8, e68427.
| Variation in apical hook length reflects the intensity of sperm competition in murine rodents.Crossref | GoogleScholarGoogle Scholar |
Simmons, L. W., and Fitzpatrick, J. L. (2012). Sperm wars and the evolution of male fertility. Reproduction (Cambridge, England) 144, 519–534.
| Sperm wars and the evolution of male fertility.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XhslClt73L&md5=64ea32d3ec9b2adc68f9613de23c590bCAS |
Songsasen, N., and Leibo, S. P. (1997). Cryopreservation of mouse spermatozoa. II. Relationship between survival after cryopreservation and osmotic tolerance of spermatozoa from three strains of mice. Cryobiology 35, 255–269.
| Cryopreservation of mouse spermatozoa. II. Relationship between survival after cryopreservation and osmotic tolerance of spermatozoa from three strains of mice.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK1c%2FjsFKqug%3D%3D&md5=1baa14682e9861354ec3c30cead0ef58CAS |
Storey, B. T., Noiles, E. E., and Thompson, K. A. (1998). Comparison of glycerol, other polyols, trehalose, and raffinose to provide a defined cryoprotectant medium for mouse sperm cryopreservation. Cryobiology 37, 46–58.
| Comparison of glycerol, other polyols, trehalose, and raffinose to provide a defined cryoprotectant medium for mouse sperm cryopreservation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXlslWrs70%3D&md5=0513a670183d271ed8535ab0dc585cd4CAS |
Suttle, J. M., Moore, H. D. M., Peirce, E. J., and Breed, W. G. (1988). Quantitative studies on variation in sperm head morphology of the hopping mouse Notomys alexis. The Journal of Experimental Zoology 247, 166–171.
| Quantitative studies on variation in sperm head morphology of the hopping mouse Notomys alexis.Crossref | GoogleScholarGoogle Scholar |
Szczygiel, M. A., Kusakabe, H., Yanagimachi, R., and Whittingham, D. G. (2002a). Intracytoplasmic sperm injection is more efficient than in vitro fertilization for generating mouse embryos from cryopreserved spermatozoa. Biology of Reproduction 67, 1278–1284.
| Intracytoplasmic sperm injection is more efficient than in vitro fertilization for generating mouse embryos from cryopreserved spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XnsV2rtLw%3D&md5=f05287d5dea282865017f4bf3c703dbdCAS |
Szczygiel, M. A., Kusakabe, H., Yanagimachi, R., and Whittingham, D. G. (2002b). Separation of motile populations of spermatozoa prior to freezing is beneficial for subsequent fertilization in vitro: a study with various mouse strains. Biology of Reproduction 67, 287–292.
| Separation of motile populations of spermatozoa prior to freezing is beneficial for subsequent fertilization in vitro: a study with various mouse strains.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XkvV2iur4%3D&md5=ea5d1e31c8a4d0cc5eb3b0cc58eb1ce6CAS |
Sztein, J. M., Farley, J. S., and Mobraaten, L. E. (2000). In vitro fertilization with cryopreserved inbred mouse sperm. Biology of Reproduction 63, 1774–1780.
| In vitro fertilization with cryopreserved inbred mouse sperm.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXosVKht7Y%3D&md5=db5889f04e33f609a73a3b7d3398acf1CAS |
Sztein, J. M., Noble, K., Farley, J. S., and Mobraaten, L. E. (2001). Comparison of permeating and nonpermeating cryoprotectants for mouse sperm cryopreservation. Cryobiology 42, 28–39.
| Comparison of permeating and nonpermeating cryoprotectants for mouse sperm cryopreservation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXjt1Wgsb4%3D&md5=a8189ae4d254b09be8c1a9b7453d2371CAS |
Tada, N., Sato, M., Yamanoi, J., Mizorogi, T., Kasai, K., and Ogawa, S. (1990). Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol. Journal of Reproduction and Fertility 89, 511–516.
| Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3cXlsV2msbY%3D&md5=9ce44fadd1f04217e2a909b1114e644aCAS |
Takeo, T., and Nakagata, N. (2010). Combination medium of cryoprotective agents containing L-glutamine and methyl-β-cyclodextrin in a preincubation medium yields a high fertilization rate for cryopreserved C57BL/6J mouse sperm. Laboratory Animals 44, 132–137.
| Combination medium of cryoprotective agents containing L-glutamine and methyl-β-cyclodextrin in a preincubation medium yields a high fertilization rate for cryopreserved C57BL/6J mouse sperm.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3c3ivVCjsQ%3D%3D&md5=d08e91bcf1fc05d3a6fd580b160c674aCAS |
Takeo, T., and Nakagata, N. (2011). Reduced glutathione enhances fertility of frozen/thawed C57BL/6 mouse sperm after exposure to methyl-beta-cyclodextrin. Biology of Reproduction 85, 1066–1072.
| Reduced glutathione enhances fertility of frozen/thawed C57BL/6 mouse sperm after exposure to methyl-beta-cyclodextrin.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhtl2is7fL&md5=165e45ebf00d55f3e890fd337fb2218bCAS |
Van Dyck, S., and Strahan, R. (2008). ‘The Mammals of Australia.’ 3rd edn. (Reed New Holland: Sydney.)
Wakayama, T., Whittingham, D. G., and Yanagimachi, R. (1998). Production of normal offspring from mouse oocytes injected with spermatozoa cryopreserved with or without cryoprotection. Journal of Reproduction and Fertility 112, 11–17.
| Production of normal offspring from mouse oocytes injected with spermatozoa cryopreserved with or without cryoprotection.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXitVKqsbg%3D&md5=f23c7be4dab186051907c269864875ccCAS |
Walters, E. M., Men, H. S., Agca, Y., Mullen, S. F., Critser, E. S., and Critser, J. K. (2005). Osmotic tolerance of mouse spermatozoa from various genetic backgrounds: acrosome integrity, membrane integrity, and maintenance of motility. Cryobiology 50, 193–205.
| Osmotic tolerance of mouse spermatozoa from various genetic backgrounds: acrosome integrity, membrane integrity, and maintenance of motility.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXjsVGks7o%3D&md5=85e950dfc2352d5afe0485f8f98b23c3CAS |
Watts, C. H. S., and Aslin, H. J. (1981). ‘The Rodents of Australia.’ (Angus and Robertson Press: Melbourne.)
Woinarski, J. C. Z., Burbidge, A. A., and Harrison, P. L. (2014). ‘The Action Plan for Australian Mammals 2012.’ (CSIRO Publishing: Melbourne.)
Wood, M. J., Cancy, C. J., and Holt, W. V. (2001). Gamete and embryo cryopreservation in rodents. In ‘Cryobanking the Genetic Resource – Wildlife Conservation for the Future’. (Eds P. F. Watson and W. V. Holt.) pp. 228–266. (Taylor and Francis: London & New York.)
World Health Organisation (2010). WHO laboratory manual for the examination and processing of human semen. 5th edn. Available at: http://whqlibdoc.who.int/publications/2010/9789241547789_eng.pdf [accessed 4 October 2012].
Yildiz, C., Ottaviani, P., Law, N., Ayearst, R., Liu, L., and McKerlie, C. (2007). Effects of cryopreservation on sperm quality, nuclear DNA integrity, in vitro fertilization, and in vitro embryo development. Reproduction (Cambridge, England) 133, 585–595.
| Effects of cryopreservation on sperm quality, nuclear DNA integrity, in vitro fertilization, and in vitro embryo development.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXls1yguro%3D&md5=377dd6a938b683d58e989fa8adecc9baCAS |
