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Optimized protocols for sperm cryopreservation and in vitro fertilization in the rat

Lab Animal volume 51, pages 256–274 (2022)Cite this article

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Abstract

Laboratory rats have been used in biomedical research for over 170 years. Recently, genome editing technology has facilitated the generation of genetically modified rats worldwide. This development has increased the demand for efficient preservation and production of rat resources. Sperm cryopreservation is the most efficient and robust means to archive genetic resources, and this technique reduces the number of animals required for colony management. Previously, we have reported a protocol for rat sperm cryopreservation and in vitro fertilization using frozen–thawed sperm. Here we describe an improved in vitro fertilization protocol to enhance the fertilization rate of cryopreserved sperm in major strains of rats. In this optimized protocol, treatment of frozen–thawed rat sperm with a high concentration of bovine serum albumin (40 mg/ml) results in a high in vitro fertilization rate. This protocol consists of three main steps: preparation of cryopreserved sperm, in vitro fertilization using cryopreserved sperm and embryo transfer. This process takes approximately 1 month to produce live pups from cryopreserved sperm. This protocol can be easily implemented by researchers and technicians with experience in reproductive engineering technology; it can also be used, albeit with some practice, by researchers and technicians who have no experience in reproductive techniques. This sperm cryopreservation and in vitro fertilization protocol for rats will provide an efficient system for the archiving and production of genetically modified rats for the transgenic community.

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Fig. 1: Overview of procedures.
Fig. 2: Equipment and instruments required for sperm cryopreservation and embryo transfer.
Fig. 3: Preparation of the sperm suspension and freezing straw.
Fig. 4: Preparation and cryopreservation of the freezing straw containing rat sperm.
Fig. 5: Thawing and preincubation of the cryopreserved rat sperm.
Fig. 6: Preparation of oocytes and IVF procedure.
Fig. 7: Embryo transfer procedure.
Fig. 8: Motility analysis of frozen–thawed rat sperm.
Fig. 9: Fertilized oocytes derived from IVF using cryopreserved sperm from LE rats.
Fig. 10: Blastocysts and live pups (2 weeks old) derived from IVF using cryopreserved LE rat sperm.

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Data availability

The data supporting the study findings are available within the paper and its supplementary information files.

Change history

  • 21 October 2022

    In the version of this article initially published, the label “EGFP” now appearing on the second from bottom row of Table 4 was originally placed on the third from bottom row. The table has been amended in the HTML and PDF versions of the article.

References

  1. Jacob, H. J. Functional genomics and rat models. Genome Res. 9, 1013–1016 (1999).

    Article  CAS  PubMed  Google Scholar 

  2. Suckow, M. A., Hankenson, F. C. & Wilson, R. P. The Laboratory Rat 3rd edn (Academic Press, 2020).

  3. Li, D. et al. Heritable gene targeting in the mouse and rat using a CRISPR–Cas system. Nat. Biotechnol. 31, 681–683 (2013).

    Article  CAS  PubMed  Google Scholar 

  4. Li, W., Teng, F., Li, T. & Zhou, Q. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR–Cas systems. Nat. Biotechnol. 31, 684–686 (2013).

    Article  CAS  PubMed  Google Scholar 

  5. Yoshimi, K. et al. ssODN-mediated knock-in with CRISPR–Cas for large genomic regions in zygotes. Nat. Commun. 7, 10431 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Neff, E. P. Rats on the rise. Lab. Anim. https://doi.org/10.1038/s41684-021-00812-0 (2021).

  7. Aitman, T. J. et al. Progress and prospects in rat genetics: a community view. Nat. Genet. 40, 516–522 (2008).

    Article  CAS  PubMed  Google Scholar 

  8. Serikawa, T. et al. National BioResource Project-Rat and related activities. Exp. Anim. 58, 333–341 (2009).

    Article  CAS  PubMed  Google Scholar 

  9. Hart-Johnson, S. & Mankelow, K. Archiving genetically altered animals: a review of cryopreservation and recovery methods for genome edited animals. Lab. Anim. https://doi.org/10.1177/00236772211007306 (2021).

  10. Marschall, S. & Hrabé de Angelis, M. Cryopreservation of mouse spermatozoa: double your mouse space. Trends Genet. 15, 128–131 (1999).

    Article  CAS  PubMed  Google Scholar 

  11. Agca, Y. & Agca, C. Cryopreservation of mouse sperm for genome banking. Methods Mol. Biol. 2180, 401–412 (2021).

    Article  CAS  PubMed  Google Scholar 

  12. Chulavatnatol, M. Motility initiation of quiescent spermatozoa from rat caudal epididymis: effects of pH, viscosity, osmolality and inhibitors. Int. J. Androl. 5, 425–436 (1982).

    Article  CAS  PubMed  Google Scholar 

  13. Si, W., Benson, J. D., Men, H. & Critser, J. K. Osmotic tolerance limits and effects of cryoprotectants on the motility, plasma membrane integrity and acrosomal integrity of rat sperm. Cryobiology 53, 336–348 (2006).

    Article  CAS  PubMed  Google Scholar 

  14. Kim, S., Agca, C. & Agca, Y. Changes in rat spermatozoa function after cooling, cryopreservation and centrifugation processes. Cryobiology 65, 215–223 (2012).

    Article  PubMed  Google Scholar 

  15. Nakatsukasa, E., Inomata, T., Ikeda, T., Shino, M. & Kashiwazaki, N. Generation of live rat offspring by intrauterine insemination with epididymal spermatozoa cryopreserved at −196 degrees C. Reproduction 122, 463–467 (2001).

    Article  CAS  PubMed  Google Scholar 

  16. Seita, Y., Sugio, S., Ito, J. & Kashiwazaki, N. Generation of live rats produced by in vitro fertilization using cryopreserved spermatozoa. Biol. Reprod. 80, 503–510 (2009).

    Article  CAS  PubMed  Google Scholar 

  17. Yamashiro, H. et al. Extracellular ATP and dibutyryl cAMP enhance the freezability of rat epididymal sperm. J. Am. Assoc. Lab. Anim. Sci. 49, 167–172 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Nakagata, N., Mikoda, N., Nakao, S., Nakatsukasa, E. & Takeo, T. Establishment of sperm cryopreservation and in vitro fertilisation protocols for rats. Sci. Rep. 10, 93 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Jiang, J. Y. & Tsang, B. K. Optimal conditions for successful in vitro fertilization and subsequent embryonic development in Sprague–Dawley rats. Biol. Reprod. 71, 1974–1979 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Popova, E., Bader, M. & Krivokharchenko, A. Strain differences in superovulatory response, embryo development and efficiency of transgenic rat production. Transgenic Res. 14, 729–738 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Davis, B. K. Influence of serum albumin on the fertilizing ability in vitro of rat spermatozoa. Proc. Soc. Exp. Biol. Med. 151, 240–243 (1976).

    Article  CAS  PubMed  Google Scholar 

  22. Visconti, P. E. et al. Cholesterol efflux-mediated signal transduction in mammalian sperm. beta-cyclodextrins initiate transmembrane signaling leading to an increase in protein tyrosine phosphorylation and capacitation. J. Biol. Chem. 274, 3235–3242 (1999).

    Article  CAS  PubMed  Google Scholar 

  23. Takeo, T. et al. Methyl-β-cyclodextrin improves fertilizing ability of C57BL/6 mouse sperm after freezing and thawing by facilitating cholesterol efflux from the cells. Biol. Reprod. 78, 546–551 (2008).

    Article  CAS  PubMed  Google Scholar 

  24. Kikuchi, K. et al. Reproduction in pigs using frozen–thawed spermatozoa from epididymis stored at 4 C. J. Reprod. Dev. 45, 345–350 (1999).

    Article  Google Scholar 

  25. Nakata, M. et al. Successful production of offspring using cryopreserved sperm via nonsurgical artificial insemination in rats. J. Reprod. Dev. 58, 501–504 (2012).

    Article  PubMed  Google Scholar 

  26. Quinn, P., Kerin, J. F. & Warnes, G. M. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil. Steril. 44, 493–498 (1985).

    Article  CAS  PubMed  Google Scholar 

  27. Quinn, P., Warnes, G. M., Kerin, J. F. & Kirby, C. Culture factors affecting the success rate of in vitro fertilization and embryo transfer. Ann. N. Y. Acad. Sci. 442, 195–204 (1985).

    Article  CAS  PubMed  Google Scholar 

  28. Takeo, T. & Nakagata, N. Cryobanking and recovery of genetically modified mice. Methods Mol. Biol. 2066, 195–209 (2020).

    Article  CAS  PubMed  Google Scholar 

  29. Takeo, T., Sztein, J. & Nakagata, N. The CARD method for mouse sperm cryopreservation and in vitro fertilization using frozen–thawed sperm. Methods Mol. Biol. 1874, 243–256 (2019).

    Article  CAS  PubMed  Google Scholar 

  30. Nakagata, N. [Cryopreservation of unfertilized rat oocytes by ultrarapid freezing]. Jikken Dobutsu 41, 443–447 (1992).

    CAS  PubMed  Google Scholar 

  31. Aoto, T., Takahashi, R. & Ueda, M. A protocol for rat in vitro fertilization during conventional laboratory working hours. Transgenic Res. 20, 1245–1252 (2011).

    Article  CAS  PubMed  Google Scholar 

  32. Honda, A. et al. Efficient derivation of knock-out and knock-in rats using embryos obtained by in vitro fertilization. Sci. Rep. 9, 11571 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Kito, S. et al. Improved in vitro fertilization and development by use of modified human tubal fluid and applicability of pronucleate embryos for cryopreservation by rapid freezing in inbred mice. Comp. Med. 54, 564–570 (2004).

    CAS  PubMed  Google Scholar 

  34. Biggers, J. D., Whitten, W. K., Whittingham, D. G. The Culture of Mouse Embryos In Vitro pp. 86–116 (San Francisco: W. H. Freeman & Co., 1971).

  35. Miyamoto, H. & Chang, M. C. In vitro fertilization of rat eggs. Nature 241, 50–52 (1973).

    Article  CAS  PubMed  Google Scholar 

  36. Niwa, K., Imai, H., Kim, C. I. & Iritani, A. Fertilization in vitro of hamster and mouse eggs in a chemically defined medium. J. Reprod. Fertil. 58, 109–114 (1980).

    Article  CAS  PubMed  Google Scholar 

  37. Toyoda, Y. & Chang, M. C. Capacitation of epididymal spermatozoa in a medium with high K–Na ratio and cyclic AMP for the fertilization of rat eggs in vitro. J. Reprod. Fertil. 36, 125–134 (1974).

    Article  CAS  PubMed  Google Scholar 

  38. Toyoda, Y. & Chang, M. C. Fertilization of rat eggs in vitro by epididymal spermatozoa and the development of eggs following transfer. J. Reprod. Fertil. 36, 9–22 (1974).

    Article  CAS  PubMed  Google Scholar 

  39. Miyoshi, K., Tanaka, N. & Niwa, K. Penetration in vitro of naturally ovulated rat eggs and the development of eggs in a chemically defined medium. J. Mamm. Ova Res. 12, 35–39 (1995).

    Article  Google Scholar 

  40. Miyoshi, K., Abeydeera, L. R., Okuda, K. & Niwa, K. Effects of osmolarity and amino acids in a chemically defined medium on development of rat one-cell embryos. J. Reprod. Fertil. 103, 27–32 (1995).

    Article  CAS  PubMed  Google Scholar 

  41. Oh, S. H., Miyoshi, K. & Funahashi, H. Rat oocytes fertilized in modified rat 1-cell embryo culture medium containing a high sodium chloride concentration and bovine serum albumin maintain developmental ability to the blastocyst stage. Biol. Reprod. 59, 884–889 (1998).

    Article  CAS  PubMed  Google Scholar 

  42. Hirabayash, M. et al. Offspring derived from intracytoplasmic injection of transgenic rat sperm. Transgenic Res. 11, 221–228 (2002).

    Article  PubMed  Google Scholar 

  43. Kaneko, T., Kimura, S. & Nakagata, N. Offspring derived from oocytes injected with rat sperm, frozen or freeze-dried without cryoprotection. Theriogenology 68, 1017–1021 (2007).

    Article  CAS  PubMed  Google Scholar 

  44. Seita, Y., Ito, J. & Kashiwazaki, N. Removal of acrosomal membrane from sperm head improves development of rat zygotes derived from intracytoplasmic sperm injection. J. Reprod. Dev. 55, 475–479 (2009).

    Article  PubMed  Google Scholar 

  45. Said, S., Han, M. S. & Niwa, K. Development of rat oocytes following intracytoplasmic injection of sperm heads isolated from testicular and epididymal spermatozoa. Theriogenology 60, 359–369 (2003).

    Article  CAS  PubMed  Google Scholar 

  46. Whittingham, D. G. Survival of rat embryos after freezing and thawing. J. Reprod. Fertil. 43, 575–578 (1975).

    Article  CAS  PubMed  Google Scholar 

  47. Kono, T., Suzuki, O. & Tsunoda, Y. Cryopreservation of rat blastocysts by vitrification. Cryobiology 25, 170–173 (1988).

    Article  CAS  PubMed  Google Scholar 

  48. Anzai, M. et al. [Cryopreservation of in vitro fertilized embryos from transgenic rat by ultrarapid freezing]. Jikken Dobutsu 43, 247–250 (1994).

    CAS  PubMed  Google Scholar 

  49. Hirabayashi, M. & Chaya, N. Low temperature storage of rat 2-cell embryos by vitrification. J. Mamm. Ova Res. 7, 72–77 (1990).

    Google Scholar 

  50. Tada, N., Sato, M., Mizorogi, T., Kasai, K. & Ogawa, S. Efficient cryopreservation of hairless mutant (bald) and normal Wistar rat embryos by vitrification. Lab. Anim. Sci. 45, 323–325 (1995).

    CAS  PubMed  Google Scholar 

  51. Sato, M., Yokokawa, K., Kasai, K. & Tada, N. Successful vitrification of stroke-prone spontaneously hypertensive and normal Wistar rat 2-cell embryos. Lab. Anim. 30, 132–137 (1996).

    Article  CAS  PubMed  Google Scholar 

  52. Jiang, J. Y., Umezu, M. & Sato, E. Vitrification of two-cell rat embryos derived from immature hypothyroid rdw rats by in vitro fertilization in ethylene glycol-based solutions. Cryobiology 38, 160–164 (1999).

    Article  CAS  PubMed  Google Scholar 

  53. Han, M. S., Niwa, K. & Kasai, M. Vitrification of rat embryos at various developmental stages. Theriogenology 59, 1851–1863 (2003).

    Article  PubMed  Google Scholar 

  54. Hirabayashi, M., Takahashi, R., Sekiguchi, J. & Ueda, M. Viability of transgenic rat embryos after freezing and thawing. Exp. Anim. 46, 111–115 (1997).

    Article  CAS  PubMed  Google Scholar 

  55. Pfaff, R. T. et al. Cryobiology of rat embryos I: determination of zygote membrane permeability coefficients for water and cryoprotectants, their activation energies, and the development of improved cryopreservation methods. Biol. Reprod. 63, 1294–1302 (2000).

    Article  CAS  PubMed  Google Scholar 

  56. Takahashi, R., Hirabayashi, M. & Ueda, M. Production of transgenic rats using cryopreserved pronuclear-stage zygotes. Transgenic Res. 8, 397–400 (1999).

    Article  CAS  PubMed  Google Scholar 

  57. Isachenko, V. V., Isachenko, E. F., Ostashko, F. I. & Grishchenko, V. I. Ultrarapid freezing of rat embryos with rapid dilution of permeable cryoprotectants. Cryobiology 34, 157–164 (1997).

    Article  CAS  PubMed  Google Scholar 

  58. Rall, W. F. et al. Factors affecting the efficiency of embryo cryopreservation and rederivation of rat and mouse models. ILAR J. 41, 221–227 (2000).

    Article  CAS  PubMed  Google Scholar 

  59. Taketsuru, H. & Kaneko, T. Efficient collection and cryopreservation of embryos in F344 strain inbred rats. Cryobiology 67, 230–234 (2013).

    Article  CAS  PubMed  Google Scholar 

  60. Taketsuru, H. & Kaneko, T. Tolerance to vitrification of rat embryos at various developmental stages. Cryobiology 84, 1–3 (2018).

    Article  CAS  PubMed  Google Scholar 

  61. Fukuda, Y. et al. Small-volume vitrification and rapid warming yield high survivals of one-cell rat embryos in cryotubes. Biol. Reprod. https://doi.org/10.1093/biolre/ioab059 (2021).

  62. Varisli, O., Uguz, C., Agca, C. & Agca, Y. Various physical stress factors on rat sperm motility, integrity of acrosome, and plasma membrane. J. Androl. 30, 75–86 (2009).

    Article  PubMed  Google Scholar 

  63. Takeo, T., Nakao, S., Nakagawa, Y., Sztein, J. M. & Nakagata, N. Cryopreservation of mouse resources. Lab. Anim. Res. 36, 33 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  64. Hino, C., Ueda, J., Funakoshi, H. & Matsumoto, S. Defined oocyte collection time is critical for reproducible in vitro fertilization in rats of different strains. Theriogenology 144, 146–151 (2020).

    Article  CAS  PubMed  Google Scholar 

  65. Nakagata, N. [Embryo transfer through the wall of the fallopian tube in mice]. Jikken Dobutsu 41, 387–388 (1992).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank K. Uchiyama (Livestock Improvement Association of Japan, Inc.), H. Yamashiro (Graduate School of Science and Technology, Niigata University) and A. Takizawa (Human & Molecular Genetic Centre, Medical College of Wisconsin) for invaluable advice and discussions. We are grateful to our staff: T. Kondo, Y. Haruguchi, K. Yamashita, E. Ishida, M. Sakaguchi and Y. Deshimaru, for their technical support and helpful discussions. We would like to thank Enago (www.enago.jp) for English language review. This work was supported by a grant from Kyudo Company.

Author information

Author notes

  1. These authors contributed equally: Toru Takeo, Satohiro Nakao.

Authors and Affiliations

  1. Division of Reproductive Engineering, Center for Animal Resources and Development, Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan

    Toru Takeo, Satohiro Nakao, Katsuma Yamaga, Ryusei Maeda & Shuuji Tsuchiyama

  2. Division of Reproductive Biotechnology and Innovation, Center for Animal Resources and Development, Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan

    Nobuyuki Mikoda & Naomi Nakagata

  3. Department of Animal Model Development, Brain Research Institute, Niigata University, Niigata, Japan

    Ena Nakatsukasa

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  1. Toru Takeo
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Contributions

N.N. designed the work, and N.N., N.M., S.N. and K.Y. contributed to the acquisition of the data. N.N., N.M., S.N. and T.T. wrote the initial draft of the manuscript. S.T. contributed to the illustrations of the procedures. E.N. contributed to the interpretation of the data. T.T. contributed to the analysis, interpretation of the data and revision of the manuscript. R.M. produced the video abstract. All authors approved the final version of the manuscript, and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Corresponding author

Correspondence to Naomi Nakagata.

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Competing interests

The authors declare competing financial interests. The present study was partially supported by a grant of Kyudo Co. Ltd. N.M. is a member of Kyudo Co. Ltd. K.Y. received a grant from Kyudo Co. Ltd. N.N. is a member of collaboration laboratory between Kyudo Co. Ltd and Kumamoto University. T.T., S.N., R.M., S.T. and E.N. declare no financial interests.

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Lab Animal thanks Elizabeth Bryda and Martha Koerner for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, and Supplementary Fig. 1.

Reporting Summary

Supplementary Video 1

Animation abstract. The animation abstract introduces the optimized IVF protocol of cryopreserved rat sperm by combining preincubation of sperm with increased concentration of BSA and the removal of cumulus cells that surround the oocytes. This cryopreservation and IVF protocol has the potential to be implemented in several rat strains and will provide an efficient system for the archiving and production of genetically modified rats for the transgenic community.

Supplementary Video 2

Motility of frozen–thawed rat sperm after 120 min preincubation. Frozen–thawed rat sperm recovered motility during the preincubation. Sperm with good motility were observed at the edge of the drop.

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Takeo, T., Nakao, S., Mikoda, N. et al. Optimized protocols for sperm cryopreservation and in vitro fertilization in the rat. Lab Anim 51, 256–274 (2022). https://doi.org/10.1038/s41684-022-01053-5

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