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

Effect of sperm pretreatment with sodium hydroxide and dithiothreitol on the efficiency of bovine intracytoplasmic sperm injection

M. E. Arias A , R. Sánchez A , J. Risopatrón A , L. Pérez A and R. Felmer A B C
+ Author Affiliations
- Author Affiliations

A Laboratorio de Reproducción, Centro de Biotecnología de La Reproducción (BIOREN-CEBIOR), Facultad de Medicina, Universidad de La Frontera, Montevideo 0870, PO box 54-D, Temuco, Chile.

B Departamento de Ciencias Agronómicas y Recursos Naturales, Facultad de Ciencias Agropecuarias y Forestales, Universidad de La Frontera, Montevideo 0870, PO box 54-D, Temuco, Chile.

C Corresponding author. Email: rfelmerd@gmail.com

Reproduction, Fertility and Development 26(6) 847-854 https://doi.org/10.1071/RD13009
Submitted: 21 January 2013  Accepted: 20 May 2013   Published: 18 June 2013

Abstract

The efficiency of intracytoplasmic sperm injection (ICSI) in bovines is lower than in other species due, in part, to a lack of optimal conditions for its implementation; this has hindered the achievement of high rates of embryonic development and the birth of live offspring. The aim of the present study was to evaluate the effects of pretreatment of bovine spermatozoa with NaOH and dithiothreitol (DTT) on the viability, plasma membrane integrity, DNA fragmentation and in vitro developmental potential of embryos generated by ICSI. Following pretreatment of spermatozoa with 5 mM DTT for 20 min and a low concentration of NaOH (1 mM for 60 min), there were fewer live and acrosome reacted spermatozoa (44% and 34%, respectively) than in the control group without treatment (82%). Spermatozoa subjected to higher alkali concentrations (10–50 mM) were mostly dead and reacted. However, pronuclear formation, cleavage, blastocyst rate and embryo quality did not differ between these pretreatment groups and the untreated control group. In conclusion, we have described, for the first time, the effects of NaOH treatment on bovine spermatozoa and subsequent in vitro embryonic development after ICSI, and have demonstrated that pretreatment of bovine spermatozoa with NaOH or DTT is not necessary for an appropriate in vitro embryo development in this species.

Additional keywords: cattle, embryo.


References

Abdalla, H., Shimoda, M., Hirabayashi, M., and Hochi, S. (2009). A combined treatment of ionomycin with ethanol improves blastocyst development of bovine oocytes harvested from stored ovaries and microinjected with spermatozoa. Theriogenology 72, 453–460.
A combined treatment of ionomycin with ethanol improves blastocyst development of bovine oocytes harvested from stored ovaries and microinjected with spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXptl2nsrw%3D&md5=719e408bae58b4272690ec1f01ab28f5CAS | 19464048PubMed |

Bedford, S. J., Kurokawa, M., Hinrichs, K., and Fissore, R. A. (2003). Intracellular calcium oscillations and activation in horse oocytes injected with stallion sperm extracts or spermatozoa. Reproduction 126, 489–499.
Intracellular calcium oscillations and activation in horse oocytes injected with stallion sperm extracts or spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXpt1Clu7Y%3D&md5=469d22a0c20c383092e814f2238a3bdeCAS | 14525531PubMed |

Chen, S. H., and Seidel, G. E. (1997). Effects of oocyte activation and treatment of spermatozoa on embryonic development following intracytoplasmic sperm injection in cattle. Theriogenology 48, 1265–1273.
Effects of oocyte activation and treatment of spermatozoa on embryonic development following intracytoplasmic sperm injection in cattle.Crossref | GoogleScholarGoogle Scholar |

Cheng, W. M., An, L., Wu, Z. H., Zhu, Y. B., Liu, J. H., Gao, H. M., Li, X. H., Zheng, S. J., Chen, D. B., and Tian, J. H. (2009). Effects of disulfide bond reducing agents on sperm chromatin structural integrity and developmental competence of in vitro matured oocytes after intracytoplasmic sperm injection in pigs. Reproduction 137, 633–643.
Effects of disulfide bond reducing agents on sperm chromatin structural integrity and developmental competence of in vitro matured oocytes after intracytoplasmic sperm injection in pigs.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXosl2ntLY%3D&md5=91f0adb3d1a1b60e7bf8c19814c14567CAS | 19155332PubMed |

Cheuquemán, C., Bravo, P., Treulen, F., Giojalas, L., Villegas, J., Sanchez, R., and Risopatron, J. (2012). Sperm membrane functionality in the dog assessed by flow cytometry. Reprod. Domest. Anim. 47, 39–43.
Sperm membrane functionality in the dog assessed by flow cytometry.Crossref | GoogleScholarGoogle Scholar | 21535242PubMed |

Choi, J. Y., Lee, E. Y., Cheong, H. T., Yoon, B. K., Bae, D. S., and Choi, D. S. (2004). Effects of activation timing on the fertilization rate and early embryo development in porcine ROSI procedure. J. Assist. Reprod. Genet. 21, 329–334.
Effects of activation timing on the fertilization rate and early embryo development in porcine ROSI procedure.Crossref | GoogleScholarGoogle Scholar | 15587146PubMed |

Chung, J. T., Keefer, C. L., and Downey, B. R. (2000). Activation of bovine oocytes following intracytoplasmic sperm injection (ICSI). Theriogenology 53, 1273–1284.
Activation of bovine oocytes following intracytoplasmic sperm injection (ICSI).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXktVOmsb8%3D&md5=121fbd0021e5e4f07430cf5447274e2aCAS | 10832752PubMed |

Fouladi-Nashta, A. A., Alberio, R., Kafi, M., Nicholas, B., Campbell, K. H., and Webb, R. (2005). Differential staining combined with TUNEL labelling to detect apoptosis in preimplantation bovine embryos. Reprod. Biomed. Online 10, 497–502.
Differential staining combined with TUNEL labelling to detect apoptosis in preimplantation bovine embryos.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD2M3mtFKgtw%3D%3D&md5=e0ec2b07a01cf1085627ced3fdd3d302CAS | 15901458PubMed |

Galli, C., Vassiliev, I., Lagutina, I., Galli, A., and Lazzari, G. (2003). Bovine embryo development following ICSI: effect of activation, sperm capacitation and pre-treatment with dithiothreitol. Theriogenology 60, 1467–1480.
Bovine embryo development following ICSI: effect of activation, sperm capacitation and pre-treatment with dithiothreitol.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXns12kt7s%3D&md5=7ef35a5fdcdacfc3af63a9ca052db9f0CAS | 14519468PubMed |

García-Vázquez, F. A., Ruiz, S., Matás, C., Izquierdo-Rico, M. J., Grullón, L. A., De Ondiz, A., Vieira, L., Avilés-López, K., Gutiérrez-Adán, A., and Gadea, J. (2010). Production of transgenic piglets using ICSI-sperm-mediated gene transfer in combination with recombinase RecA. Reproduction 140, 259–272.
Production of transgenic piglets using ICSI-sperm-mediated gene transfer in combination with recombinase RecA.Crossref | GoogleScholarGoogle Scholar | 20501790PubMed |

Goto, K. (1993). Bovine microfertilization and embryo transfer. Mol. Reprod. Dev. 36, 288–290.
Bovine microfertilization and embryo transfer.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK2c%2FnvFOnuw%3D%3D&md5=9b9841e2ae4634408adcbe1ecfde0f81CAS | 8257590PubMed |

Goto, K., Kinoshita, A., Takuma, Y., and Ogawa, K. (1990). Fertilisation of bovine oocytes by the injection of immobilised, killed spermatozoa. Vet. Rec. 127, 517–520.
| 1:STN:280:DyaK3M7is12nsg%3D%3D&md5=76f78e3a1687290e0a506b25e4ecc21aCAS | 2281585PubMed |

Hamano, K., Li, X., Qian, X. Q., Funauchi, K., Furudate, M., and Minato, Y. (1999). Gender preselection in cattle with intracytoplasmically injected, flow cytometrically sorted sperm heads. Biol. Reprod. 60, 1194–1197.
Gender preselection in cattle with intracytoplasmically injected, flow cytometrically sorted sperm heads.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXislegtb8%3D&md5=9517622ac0cb98b515d5680a58556d35CAS | 10208983PubMed |

Horiuchi, T., and Numabe, T. (1999). Intracytoplasmic sperm injection (ICSI) in cattle and other domestic animals: problems and improvements in practical use. J. Mamm. Ova Res. 16, 1–9.
Intracytoplasmic sperm injection (ICSI) in cattle and other domestic animals: problems and improvements in practical use.Crossref | GoogleScholarGoogle Scholar |

Horiuchi, T., Emuta, C., Yamauchi, Y., Oikawa, T., Numabe, T., and Yanagimachi, R. (2002). Birth of normal calves after intracytoplasmic sperm injection of bovine oocytes: a methodological approach. Theriogenology 57, 1013–1024.
Birth of normal calves after intracytoplasmic sperm injection of bovine oocytes: a methodological approach.Crossref | GoogleScholarGoogle Scholar |

Katayose, H., Yanagida, K., Shinoki, T., Kawahara, T., Horiuchi, T., and Sato, A. (1999). Efficient injection of bull spermatozoa into oocytes using a Piezo-driven pipette. Theriogenology 52, 1215–1224.
Efficient injection of bull spermatozoa into oocytes using a Piezo-driven pipette.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3c7pvFaktg%3D%3D&md5=e369fd7bc23d745498ef06b75bfc7bf0CAS | 10735099PubMed |

Keefer, C. L., Younis, A. I., and Brackett, B. G. (1990). Cleavage development of bovine oocytes fertilized by sperm injection. Mol. Reprod. Dev. 25, 281–285.
Cleavage development of bovine oocytes fertilized by sperm injection.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK3c3jsVChtQ%3D%3D&md5=a1d492e0816c965780871116b5ff2b59CAS | 2109991PubMed |

Kim, N. H., Jun, S. H., Do, J. T., Uhm, S. J., Lee, H. T., and Chung, K. S. (1999). Intracytoplasmic injection of porcine, bovine, mouse, or human spermatozoon into porcine oocytes. Mol. Reprod. Dev. 53, 84–91.
Intracytoplasmic injection of porcine, bovine, mouse, or human spermatozoon into porcine oocytes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXitleisr0%3D&md5=6efd1607cd91953da6d7b65853e2dc70CAS | 10230820PubMed |

Kimura, Y., and Yanagimachi, R. (1995). Intracytoplasmic sperm injection in the mouse. Biol. Reprod. 52, 709–720.
Intracytoplasmic sperm injection in the mouse.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2MXksFWhtrk%3D&md5=a49592d3c3d48f3c47c31a5aa0328453CAS | 7779992PubMed |

Koo, D. B., Kang, Y. K., Choi, Y. H., Park, J. S., Kim, H. N., Oh, K. B., Son, D. S., Park, H., Lee, K. K., and Han, Y. M. (2002). Aberrant allocations of inner cell mass and trophectoderm cells in bovine nuclear transfer blastocysts. Biol. Reprod. 67, 487–492.
Aberrant allocations of inner cell mass and trophectoderm cells in bovine nuclear transfer blastocysts.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XlsFKqt7g%3D&md5=775cf3875fe8e315dac6a728bfa25bc5CAS | 12135886PubMed |

Kurokawa, M., Sato, K., Wu, H., He, C., Malcuit, C., Black, S. J., Fukami, K., and Fissore, R. A. (2005). Functional, biochemical, and chromatographic characterization of the complete [Ca2+]i oscillation-inducing activity of porcine sperm. Dev. Biol. 285, 376–392.
Functional, biochemical, and chromatographic characterization of the complete [Ca2+]i oscillation-inducing activity of porcine sperm.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhtVymsLvJ&md5=a4f926504aaae7a7c45c65367e33efc0CAS | 16098961PubMed |

Li, X., Imasaki, S., and Nakahara, T. (1993). Investigation of various conditions in microfertilisation of bovine oocytes and subsequent changes in nuclei and development to embryos. J. Reprod. Dev. 39, j49–j55.
Investigation of various conditions in microfertilisation of bovine oocytes and subsequent changes in nuclei and development to embryos.Crossref | GoogleScholarGoogle Scholar |

Li, X., Hamano, K., Qian, X. Q., Funauchi, K., Furudate, M., and Minato, Y. (1999). Oocyte activation and parthenogenetic development of bovine oocytes following intracytoplasmic sperm injection. Zygote 7, 233–237.
Oocyte activation and parthenogenetic development of bovine oocytes following intracytoplasmic sperm injection.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXltVKmt7s%3D&md5=1940d962c39898663f96d736edeef0cdCAS | 10533706PubMed |

Li, G. P., Seidel, G. E., and Squires, E. L. (2004). Improved cleavage of bovine ICSI ova cultured in heparin-containing medium. Theriogenology 61, 1077–1084.
Improved cleavage of bovine ICSI ova cultured in heparin-containing medium.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXht1yqsrs%3D&md5=f5595a69566ef754f3d3c69bc8812b18CAS | 15036996PubMed |

Li, C., Mizutani, E., Ono, T., and Wakayama, T. (2009). Production of normal mice from spermatozoa denatured with high alkali treatment before ICSI. Reproduction 137, 779–792.
Production of normal mice from spermatozoa denatured with high alkali treatment before ICSI.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXovVKgtLs%3D&md5=1004c15b965424e87d493135b887daaeCAS | 19251742PubMed |

Li, C., Mizutani, E., Ono, T., and Wakayama, T. (2010). An efficient method for generating transgenic mice using NaOH-treated spermatozoa. Biol. Reprod. 82, 331–340.
An efficient method for generating transgenic mice using NaOH-treated spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXhsVSnsLY%3D&md5=4345f341c772e4d293046d137485d870CAS | 19812303PubMed |

Morozumi, K., and Yanagimachi, R. (2005). Incorporation of the acrosome into the oocyte during intracytoplasmic sperm injection could be potentially hazardous to embryo development. Proc. Natl Acad. Sci. USA 102, 14 209–14 214.
Incorporation of the acrosome into the oocyte during intracytoplasmic sperm injection could be potentially hazardous to embryo development.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhtFChu7bJ&md5=49a8358fdf85da352d7ca903ecf3db77CAS |

Morozumi, K., Shikano, T., Miyazaki, S., and Yanagimachi, R. (2006). Simultaneous removal of sperm plasma membrane and acrosome before intracytoplasmic sperm injection improves oocyte activation/embryonic development. Proc. Natl Acad. Sci. USA 103, 17 661–17 666.
Simultaneous removal of sperm plasma membrane and acrosome before intracytoplasmic sperm injection improves oocyte activation/embryonic development.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xht1Klur7N&md5=676fe127e9a71453ab12e9fa06180307CAS |

Ock, S. A., Bhak, J. S., Balasubramanian, S., Lee, H. J., Choe, S. Y., and Rho, G. J. (2003). Different activation treatments for successful development of bovine oocytes following intracytoplasmic sperm injection. Zygote 11, 69–76.
Different activation treatments for successful development of bovine oocytes following intracytoplasmic sperm injection.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXht1Wgs7w%3D&md5=cde5d5b95f33cf89ca7e01a1dd7144b3CAS | 12625531PubMed |

Oikawa, T., Takada, N., Kikuchi, T., Numabe, T., Takenaka, M., and Horiuchi, T. (2005). Evaluation of activation treatments for blastocyst production and birth of viable calves following bovine intracytoplasmic sperm injection. Anim. Reprod. Sci. 86, 187–194.
Evaluation of activation treatments for blastocyst production and birth of viable calves following bovine intracytoplasmic sperm injection.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXit1CjtLc%3D&md5=05f7913ac6cd26af47bdfd0040afc01eCAS | 15766799PubMed |

Perry, A. C., Wakayama, T., Kishikawa, H., Kasai, T., Okabe, M., Toyoda, Y., and Yanagimachi, R. (1999). Mammalian transgenesis by intracytoplasmic sperm injection. Science 284, 1180–1183.
Mammalian transgenesis by intracytoplasmic sperm injection.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXjtlajsbk%3D&md5=dde2fb89af1b151d18050b19235f8872CAS | 10325231PubMed |

Probst, S., and Rath, D. (2003). Production of piglets using intracytoplasmic sperm injection (ICSI) with flow cytometrically sorted boar semen and artificially activated oocytes. Theriogenology 59, 961–973.
Production of piglets using intracytoplasmic sperm injection (ICSI) with flow cytometrically sorted boar semen and artificially activated oocytes.Crossref | GoogleScholarGoogle Scholar | 12517397PubMed |

Qian, X. Q., Inagaki, H., Sasada, H., and Sugawara, S. (1996). Decondensation and pronuclear formation in bovine oocytes after microinjection of bovine sperm pre-treated by with disulfide bond reducing agents. J. Mamm. Ova Res. 13, 118–121.
Decondensation and pronuclear formation in bovine oocytes after microinjection of bovine sperm pre-treated by with disulfide bond reducing agents.Crossref | GoogleScholarGoogle Scholar |

Rho, G. J., Kawarsky, S., Johnson, W. H., Kochhar, K., and Betteridge, K. J. (1998a). Sperm and oocyte treatments to improve the formation of male and female pronuclei and subsequent development following intracytoplasmic sperm injection into bovine oocytes. Biol. Reprod. 59, 918–924.
Sperm and oocyte treatments to improve the formation of male and female pronuclei and subsequent development following intracytoplasmic sperm injection into bovine oocytes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXmsVGrtr0%3D&md5=b921619c1cd82f9d1307845ffaf49627CAS | 9746744PubMed |

Rho, G. J., Wu, B., Kawarsky, S., Leibo, S. P., and Betteridge, K. J. (1998b). Activation regimens to prepare bovine oocytes for intracytoplasmic sperm injection. Mol. Reprod. Dev. 50, 485–492.
Activation regimens to prepare bovine oocytes for intracytoplasmic sperm injection.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXktlKrurk%3D&md5=85be8c52ef94a77bc2993704e03e27f1CAS | 9669532PubMed |

Rho, G. J., Lee, S. L., Kim, Y. S., Yeo, H. J., Ock, S. A., Balasubramanian, S., and Choe, S. Y. (2004). Intracytoplasmic sperm injection of frozen–thawed bovine oocytes and subsequent embryo development. Mol. Reprod. Dev. 68, 449–455.
Intracytoplasmic sperm injection of frozen–thawed bovine oocytes and subsequent embryo development.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXlslWiu78%3D&md5=e0ebad9ba6542f072c30b2b81ac4c837CAS | 15236329PubMed |

Rodger, J. C., Paris, D. B., Czarny, N. A., Harris, M. S., Molinia, F. C., Taggart, D. A., Allen, C. D., and Johnston, S. D. (2009). Artificial insemination in marsupials. Theriogenology 71, 176–189.
Artificial insemination in marsupials.Crossref | GoogleScholarGoogle Scholar | 18950846PubMed |

Seita, Y., Ito, J., and Kashiwazaki, N. (2009). Removal of acrosomal membrane from sperm head improves development of rat zygotes derived from intracytoplasmic sperm injection. J. Reprod. Dev. 55, 475–479.
Removal of acrosomal membrane from sperm head improves development of rat zygotes derived from intracytoplasmic sperm injection.Crossref | GoogleScholarGoogle Scholar | 19444004PubMed |

Sekhavati, M. H., Shadanloo, F., Hosseini, M. S., Tahmoorespur, M., Nasiri, M. R., Hajian, M., and Nasr-Esfahani, M. H. (2012). Improved bovine ICSI outcomes by sperm selected after combined heparin–glutathione treatment. Cell Reprogram. 14, 295–304.
| 1:CAS:528:DC%2BC38XhtFCrsLnE&md5=ef3a675c1985e0c79fa679fcae5de054CAS | 22862150PubMed |

Seshagiri, P., and Bavister, B. (1989). Phosphate is required for inhibition by glucose of development of hamster 8-cell embryos in vitro. Biol. Reprod. 40, 607–614.
| 1:CAS:528:DyaL1MXit12is7c%3D&md5=d28fa58c6d95b6b26a1bec40a23790d8CAS | 2758091PubMed |

Stricker, S. A. (1999). Comparative biology of calcium signaling during fertilization and egg activation in animals. Dev. Biol. 211, 157–176.
Comparative biology of calcium signaling during fertilization and egg activation in animals.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXkt1Ghu78%3D&md5=77f7bae9baf3800b8174fa3f361492e4CAS | 10395780PubMed |

Suttner, R., Zakhartchenko, V., Stojkovic, P., Muller, S., Alberio, R., Medjugorac, I., Brem, G., Wolf, E., and Stojkovic, M. (2000). Intracytoplasmic sperm injection in bovine: effects of oocyte activation, sperm pretreatment and injection technique. Theriogenology 54, 935–948.
Intracytoplasmic sperm injection in bovine: effects of oocyte activation, sperm pretreatment and injection technique.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3M%2Fot1Cjug%3D%3D&md5=f81bdc7f14d49c5407056c107036a288CAS | 11097046PubMed |

Szczygiel, M. A., and Ward, W. S. (2002). Combination of dithiothreitol and detergent treatment of spermatozoa causes paternal chromosomal damage. Biol. Reprod. 67, 1532–1537.
Combination of dithiothreitol and detergent treatment of spermatozoa causes paternal chromosomal damage.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38Xot1Kjs7c%3D&md5=9c08cb3edd5702a5ef8beff2105a50d1CAS | 12390885PubMed |

Tian, J. H., Wu, Z. H., Liu, L., Cai, Y., Zeng, S. M., Zhu, S. E., Liu, G. S., Li, Y., and Wu, C. X. (2006). Effects of oocyte activation and sperm preparation on the development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection. Theriogenology 66, 439–448.
Effects of oocyte activation and sperm preparation on the development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xmt1Gktr0%3D&md5=5b71db8ba0dec20664f3e3647011a9f4CAS | 16426671PubMed |

Van Steirteghem, A. C., Nagy, Z., Joris, H., Liu, J., Staessen, C., Smitz, J., Wisanto, A., and Devroey, P. (1993). High fertilization and implantation rates after intracytoplasmic sperm injection. Hum. Reprod. 8, 1061–1066.
| 1:STN:280:DyaK2c%2Fhtleisw%3D%3D&md5=c9cd5fa452ca28c58af6f50de540eabbCAS | 8408487PubMed |

Watanabe, H., and Fukui, Y. (2006). Effects of dithiothreitol and boar on pronuclear formation and embryonic development following intracytoplasmic sperm injection in pigs. Theriogenology 65, 528–539.
Effects of dithiothreitol and boar on pronuclear formation and embryonic development following intracytoplasmic sperm injection in pigs.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XhvFGruw%3D%3D&md5=dd14a1cd790ecf9bee3aca8c28556864CAS | 16009412PubMed |

Watanabe, H., Akiyama, J., Musharraf, M., Bhuiyan, U., and Fukui, Y. (2009). Enhanced oocyte activation by intracytoplasmic injection of porcine spermatozoa pre-treated with dithiothreitol. J. Mamm. Ova Res. 26, 54–60.
Enhanced oocyte activation by intracytoplasmic injection of porcine spermatozoa pre-treated with dithiothreitol.Crossref | GoogleScholarGoogle Scholar |

Watanabe, H., Suzuki, H., and Fukui, Y. (2010). Fertilizability, developmental competence, and chromosomal integrity of oocytes microinjected with pre-treated spermatozoa in mice. Reproduction 139, 513–521.
Fertilizability, developmental competence, and chromosomal integrity of oocytes microinjected with pre-treated spermatozoa in mice.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXjtV2lsbw%3D&md5=eaa29c1db5fe9f6467dbeba1210014afCAS | 19955207PubMed |

Wei, H., and Fukui, Y. (1999). Effects of bull, sperm type and sperm pretreatment on male pronuclear formation after intracytoplasmic sperm injection in cattle. Reprod. Fertil. Dev. 11, 59–65.
Effects of bull, sperm type and sperm pretreatment on male pronuclear formation after intracytoplasmic sperm injection in cattle.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3c7ktlagug%3D%3D&md5=85eeb6abd488769f2a82dc065fbbdf46CAS | 10681003PubMed |

Yan, W., Morozumi, K., Zhang, J., Ro, S., Park, C., and Yanagimachi, R. (2008). Birth of mice after intracytoplasmic injection of single purified sperm nuclei and detection of messenger RNAs and MicroRNAs in the sperm nuclei. Biol. Reprod. 78, 896–902.
Birth of mice after intracytoplasmic injection of single purified sperm nuclei and detection of messenger RNAs and MicroRNAs in the sperm nuclei.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXltVOnsrk%3D&md5=0cd2b723f0a7c421835197a8e576a7e6CAS | 18256326PubMed |

Yanagimachi, R. (1994). Mammalian fertilization. In ‘The Physiology of Reproduction’, 2nd edn. (Eds E. Knobil, J. D. Neill, G. S. Greenwald, C. L. Markert, D. W. Pfaff.) pp. 189–317. (Raven Press: New York.)

Yanagimachi, R. (2005). Intracytoplasmic injection of spermatozoa and spermatogenic cells: its biology and applications in humans and animals. Reprod. Biomed. Online 10, 247–288.
Intracytoplasmic injection of spermatozoa and spermatogenic cells: its biology and applications in humans and animals.Crossref | GoogleScholarGoogle Scholar | 15823233PubMed |

Yong, H. Y., Hong, J. Y., Kang, S. K., Lee, B. C., Lee, E. S., and Hwang, W. S. (2005). Sperm movement in the ooplasm, dithiothreitol pretreatment and sperm freezing are not required for the development of porcine embryos derived from injection of head membrane-damaged sperm. Theriogenology 63, 783–794.
Sperm movement in the ooplasm, dithiothreitol pretreatment and sperm freezing are not required for the development of porcine embryos derived from injection of head membrane-damaged sperm.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXjsVWn&md5=b631d274d7dccb88fc0911274d15bd55CAS | 15629797PubMed |