Animal Bioscience

아세아태평양축산학회 (Asian Australasian Association of Animal Production Societies)
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Reproductive technologies needed for the generation of precise gene-edited pigs in the pathways from laboratory to farm

  • Ching-Fu Tu (Division of Animal Technology, Animal Technology Research Center, Agricultural Technology Research Institute) ;
  • Shu-Hui Peng (Division of Animal Technology, Animal Technology Research Center, Agricultural Technology Research Institute) ;
  • Chin-kai Chuang (Division of Animal Technology, Animal Technology Research Center, Agricultural Technology Research Institute) ;
  • Chi-Hong Wong (Division of Animal Technology, Animal Technology Research Center, Agricultural Technology Research Institute) ;
  • Tien-Shuh Yang (Division of Animal Technology, Animal Technology Research Center, Agricultural Technology Research Institute)
  • 투고 : 2022.10.11
  • 심사 : 2022.11.07
  • 발행 : 2023.02.01
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초록

Gene editing (GE) offers a new breeding technique (NBT) of sustainable value to animal agriculture. There are 3 GE working sites covering 5 feasible pathways to generate GE pigs along with the crucial intervals of GE/genotyping, microinjection/electroporation, induced pluripotent stem cells, somatic cell nuclear transfer, cryopreservation, and nonsurgical embryo transfer. The extension of NBT in the new era of pig breeding depends on the synergistic effect of GE and reproductive biotechnologies; the outcome relies not only on scientific due diligence and operational excellence but also on the feasibility of application on farms to improve sustainability.

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과제정보

This article was financially supported (NSTC 110-2313-B-866-002) by the National Science and Technology Council, Executive Yung, Taiwan.

참고문헌

  1. Tu CF, Chuang CK, Yang TS. The application of new breeding technology based on gene editing in pig industry. Anim Biosci 2022;35:791-803. https://doi.org/10.5713/ab.21.0390
  2. Tanihara F, Hirata M, Otoi T. Current status of the application of gene editing in pigs. J Reprod Dev 2021;67:177-87. https://doi.org/10.1262/jrd.2021-025
  3. Zhang J, Khazalwa EM, Abkallo HM, et al. The advancements, challenges, and future implications of the CRISPR/Cas9 system in swine research. J Genet Genomics 2021;48:347-60. https://doi.org/10.1016/j.jgg.2021.03.015
  4. Betermier M, Bertrand P, Lopez BS. Is non-homologous end-joining really an inherently error-prone process? PLoS Genet 2014;10:e1004086. https://doi.org/10.1371/journal.pgen.1004086
  5. Bennett EP, Petersen BL, Johansen IE, et al. INDEL detection, the 'Achilles heel' of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels. Nucleic Acids Res 2020;48:11958-81. https://doi.org/10.1093/nar/gkaa975
  6. Chuang CK, Lin WM. Points of view on the tools for genome/gene editing. Int J Mol Sci 2021;22:9872. https://doi.org/10.3390/ijms22189872
  7. Chuang CK, Lee KH, Fan CT, Su YS. Porcine type III RNA polymerase III promoters for short hairpin RNA expression. Anim Biotechnol 2009;20:34-9. https://doi.org/10.1080/10495390802603064
  8. Christou-Kent M, Dhellemmes M, Lambert E, Ray PF, Arnoult C. Diversity of RNA-binding proteins modulating posttranscriptional regulation of protein expression in the maturing mammalian oocyte. Cells 2020;9:662. https://doi.org/10.3390/cells9030662
  9. Li L, Zheng P, Dean J. Maternal control of early mouse development. Development 2010;137:859-70. https://doi.org/10.1242/dev.039487
  10. Chuang CK, Chen CH, Huang CL, et al. Generation of GGTA1 mutant pigs by direct pronuclear microinjection of CRISPR/Cas9 plasmid vectors. Anim Biotechnol 2017;28:174-81. https://doi.org/10.1080/10495398.2016.1246453
  11. Tu CF, Chuang CK, Hsiao KH, et al. Lessening of porcine epidemic diarrhoea virus susceptibility in piglets after editing of the CMP-N-glycolylneuraminic acid hydroxylase gene with CRISPR/Cas9 to nullify N-glycolylneuraminic acid expression. PLoS One 2019;14:e0217236. https://doi.org/10.1371/journal.pone.0217236
  12. Hung SW, Chuang Ck. Wong CH, et al. Activated macrophages of CD 163 gene edited pigs generated by direct cytoplasmic microinjection with CRISPR gRNA/Cas9 mRNA are resistant  to PRRS virus assault. Anim Biotechnol 2022;May 4:1-14. https://doi.org/10.1080/10495398.2022.2062602
  13. Tanihara F, Hirata M, Nguyen NT, et al. Generation of PDX-1 mutant porcine blastocysts by introducing CRISPR/Cas9-system into porcine zygotes via electroporation. Anim Sci J 2019;90:55-61. https://doi.org/10.1111/asj.13129
  14. Tanihara F, Hirata M, Nguyen NT, et al. Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/ Cas9 system into in vitro-fertilized zygotes. BMC Biotechnol 2020;20:40. https://doi.org/10.1186/s12896-020-00638-7
  15. Tanihara F, Hirata M, Nguyen NT, et al. Generation of CD163-edited pig via electroporation of the CRISPR/Cas9 system into porcine in vitro-fertilized zygotes. Anim Biotechnol 2021;32:147-54. https://doi.org/10.1080/10495398.2019.1668801
  16. Ren C, Xu K, Segal DJ, Zhang Z. Strategies for the enrichment and selection of genetically modified cells. Trends Biotechnol 2019;37:56-71. https://doi.org/10.1016/j.tibtech.2018.07.017
  17. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663-76. https://doi.org/10.1016/j.cell.2006.07.024
  18. Esteban MA, Xu J, Yang J, et al. Generation of induced pluripotent stem cell lines from Tibetan miniature pig. J Biol Chem 2009;284:17634-40. https://doi.org/10.1074/jbc.M109.008938
  19. Ezashi T, Telugu BP, Alexenko AP, et al. Derivation of induced pluripotent stem cells from pig somatic cells. Proc Natl Acad Sci USA 2009;106:10993-8. https://doi.org/10.1073/pnas.0905284106
  20. Wu Z, Chen J, Ren J, et al. Generation of pig induced pluripotent stem cells with a drug-inducible system. J Mol Cell Biol 2009;1:46-54. https://doi.org/10.1093/jmcb/mjp003
  21. West FD, Terlouw SL, Kwon DJ, et al. Porcine induced pluripotent stem cells produce chimeric offspring. Stem Cells Dev 2010;19:1211-20. https://doi.org/10.1089/scd.2009.0458
  22. West FD, Uhl EW, Liu Y, et al. Brief report: chimeric pigs produced from induced pluripotent stem cells demonstrate germline transmission and no evidence of tumor formation in young pigs. Stem Cells 2011;29:1640-3. https://doi.org/10.1002/stem.713
  23. Liu K, Ji G, Mao J, et al. Generation of porcine-induced pluripotent stem cells by using OCT4 and KLF4 porcine factors. Cell Reprogram 2012;14:505-13. https://doi.org/10.1089/cell.2012.0047
  24. Du X, Feng T, Yu D, et al. Barriers for deriving transgene-free pig iPS cells with episomal vectors. Stem Cells 2015;33:3228-38. https://doi.org/10.1002/stem.2089
  25. Chen CH, Su YH, Lee KH, Chuang CK. Germline competent pluripotent mouse stem cells generated by plasmid vectors. Anim Biotechnol 2016;27:157-65. https://doi.org/10.1080/10495398.2016.1140056
  26. Kim JY, Nam Y, Rim YA, Ju JH. Review of the current trends in clinical trials involving induced pluripotent stem cells. Stem Cell Rev Rep 2022;18:142-54. https://doi.org/10.1007/s12015-021-10262-3
  27. Howden SE, Maufort JP, Duffin BM, Elefanty AG, Stanley EG, Thomson JA. Simultaneous reprogramming and gene correction of patient fibroblasts. Stem Cell Rep 2015;5:1109-18. https://doi.org/10.1016/j.stemcr.2015.10.009
  28. Cheng D, Guo Y, Li Z, et al. Porcine induced pluripotent stem cells require LIF and maintain their developmental potential in early stage of embryos. PLoS One 2012;7:e51778. https://doi.org/10.1371/journal.pone.0051778
  29. Yuan Y, Lee K, Park KW, et al. Cell cycle synchronization of leukemia inhibitory factor (LIF)-dependent porcine-induced pluripotent stem cells and the generation of cloned embryos. Cell Cycle 2014;13:1265-76. https://doi.org/10.4161/cc.28176
  30. Kim E, Hwang SU, Yoo H, et al. Putative embryonic stem cells derived from porcine cloned blastocysts using induced pluripotent stem cells as donors. Theriogenology 2016;85: 601-16. https://doi.org/10.1016/j.theriogenology.2015.09.051
  31. Fan N, Chen J, Shang Z, et al. Piglets cloned from induced pluripotent stem cells. Cell Res 2013;23:162-6. https://doi.org/10.1038/cr.2012.176
  32. Yu D, Wang J, Zou H, et al. Silencing of retrotransposonderived imprinted gene RTL1 is the main cause for postimplantational failures in mammalian cloning. Proc Natl Acad Sci 2018;115:E11071-80. https://doi.org/10.1073/pnas.1814514115
  33. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KHS. Viable offspring derived from fetal and adult mammalian cells. Nature 1997;385:810-3. https://doi.org/10.1038/385810a0
  34. Polejaeva IA, Chen SH, Vaught TD, et al. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 2000;407:86-90. https://doi.org/10.1038/35024082
  35. Betthauser J, Forsberg E, Augenstein M, et al. Production of cloned pigs from in vitro systems. Nat Biotechnol 2000;18:1055-9. https://doi.org/10.1038/80242
  36. Onishi A, Iwamoto M, Akita T, et al. Pig cloning by microinjection of fetal fibroblast nuclei. Science 2000;289:1188-90. https://doi.org/10.1126/science.289.5482.1188
  37. Hua Z, Xu G, Liu X, et al. Impact of different sources of donor cells upon the nuclear transfer efficiency in Chinese indigenous Meishan pig. Pol J Vet Sci 2016;19:205-12. https://doi.org/10.1515/pjvs-2016-0029
  38. Richter A, Kurome M, Kessler B, et al. Potential of primary kidney cells for somatic cell nuclear transfer mediated transgenesis in pig. BMC Biotechnol 2012;12:84. https://doi.org/10.1186/1472-6750-12-84
  39. Fahrudin M, Kikuchi K, Kurniani Karja NW, et al. Development to the blastocyst stage of porcine somatic cell nuclear transfer embryos reconstructed by the fusion of cumulus cells and cytoplasts prepared by gradient centrifugation. Cloning Stem Cells 2007;9:216-28. https://doi.org/10.1089/clo.2006.0048
  40. Li X, Zhang P, Jiang S, et al. Aging adult porcine fibroblasts can support nuclear transfer and transcription factor-mediated reprogramming. Anim Sci J 2018;89:289-297. https://doi.org/10.1111/asj.12871
  41. Li Z, He X, Chen L, et al. Bone marrow mesenchymal stem cells are an attractive donor cell type for production of cloned pigs as well as genetically modified cloned pigs by somatic cell nuclear transfer. Cell Reprogram 2013;15:459-70. https://doi.org/10.1089/cell.2013.0010
  42. Park CH, Jeoung YH, Uh KJ, et al. Extraembryonic endoderm (XEN) cells capable of contributing to embryonic chimeras established from pig embryos. Stem Cell Rep 2021;16:212-223. https://doi.org/10.1016/j.stemcr.2020.11.011
  43. Dang-Nguyen TQ, Wells D, Haraguchi S, et al. Combined refinements to somatic cell nuclear transfer methods improve porcine embryo development. J Reprod Dev 2020;66:281-6. https://doi.org/10.1262/jrd.2019-156
  44. Jiao D, Cheng W, Zhang X, et al. Improving porcine SCNT efficiency by selecting donor cells size. Cell Cycle 2021;20: 2264-77. https://doi.org/10.1080/15384101.2021.1980983
  45. Yin XJ, Tani T, Yonemura I, et al. Production of cloned pigs from adult somatic cells by chemically assisted removal of maternal chromosomes. Biol Reprod 2002;67:442-6. https://doi.org/10.1095/biolreprod67.2.442
  46. Du Y, Kragh PM, Zhang Y, et al. Piglets born from handmade cloning, an innovative cloning method without micromanipulation. Theriogenology 2007;68:1104-10. https://doi.org/10.1016/j.theriogenology.2007.07.021
  47. Betthauser J, Forsberg E, Augenstein M, et al. Production of cloned pigs from in vitro systems. Nat Biotechnol 2000;18:1055-9. https://doi.org/10.1038/80242
  48. Du Y, Kragh PM, Zhang Y, et al. Piglets born from handmade cloning, an innovative cloning method without micromanipulation. Theriogenology 2007;68:1104-10. https://doi.org/10.1016/j.theriogenology.2007.07.021
  49. de Macedo MP, Glanzner WG, Rissi VB, et al. A fast and reliable protocol for activation of porcine oocytes. Theriogenology 2019;123:22-9. https://doi.org/10.1016/j.theriogenology.2018.09.021
  50. Lee K, Davis A, Zhang L, et al. Pig oocyte activation using a Zn2+ chelator, TPEN. Theriogenology 2015;84:1024-32. https://doi.org/10.1016/j.theriogenology.2015.05.036
  51. Wang X, Qu J, Li J, et al. Epigenetic reprogramming during somatic cell nuclear transfer: recent progress and future directions. Front Genet 2020;11:205. https://doi.org/10.3389/fgene.2020.00205
  52. Zeng Y, Chen T. DNA methylation reprogramming during mammalian development. Genes 2019;10:257. https://doi.org/10.3390/genes10040257
  53. Ju S, Rui R, Lu Q, Lin P, Guo H. Analysis of apoptosis and methyltransferase mRNA expression in porcine cloned embryos cultured in vitro. J Assist Reprod Genet 2010;27:49-59. https://doi.org/10.1007/s10815-009-9378-7
  54. Park HB, Park YR, Kim MJ, Jung BD, Park CK, Cheong HT. Endoplasmic reticulum (ER) stress inhibitor or antioxidant treatments during micromanipulation can inhibit both ER and oxidative stresses in porcine SCNT embryos. Dev Reprod 2020;24:31-41. https://doi.org/10.12717/DR.2020.24.1.31
  55. Park J, Lai L, Samuel MS, et al. Disruption of mitochondrionto-nucleus interaction in deceased cloned piglets. PLoS One 2015;10:e0129378. https://doi.org/10.1371/journal.pone.0129378
  56. Srirattana K, Kaneda M, Parnpai R. Strategies to improve the efficiency of somatic cell nuclear transfer. Int J Mol Sci 2022;23:1969. https://doi.org/10.3390/ijms23041969
  57. Chen Z, Zhang Y. Role of mammalian DNA methyltransferases in development. Annu Rev Biochem 2019;89:135-58. https://doi.org/10.1146/annurev-biochem-103019-102815
  58. Gu TP, Guo F, Yang H, et al. The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature 2011;477:606-10. https://doi.org/10.1038/nature10443
  59. Inoue A, Zhang Y. Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos. Science 2011;334:194. https://doi.org/10.1126/science.1212483
  60. Huang Y, Tang X, Xie W, et al. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos. Biochem Biophys Res Commun 2011;411:397-401. https://doi.org/10.1016/j.bbrc.2011.06.160
  61. Kere M, Siriboon C, Lo NW, Nguyen NT, Ju JC. Ascorbic acid improves the developmental competence of porcine oocytes after parthenogenetic activation and somatic cell nuclear transplantation. J Reprod Dev 2013;59:78-84. https://doi.org/10.1262/jrd.2012-114
  62. Yin R, Mao SQ, Zhao B, et al. Ascorbic acid enhances Tetmediated 5-methylcytosine oxidation and promotes DNA demethylation in mammals. J Am Chem Soc 2013;135:10396-403. https://doi.org/10.1021/ja4028346
  63. Zhao M, Hur TY, No J, et al. Ascorbic acid increases demethylation in somatic cell nuclear transfer embryos of the pig (Sus scrofa). Asian-Australas J Anim Sci 2017;30:944-9. https://doi.org/10.5713/ajas.16.0818
  64. Lee K, Hamm J, Whitworth K, et al. Dynamics of TET family expression in porcine preimplantation embryos is related to zygotic genome activation and required for the maintenance of NANOG. Dev Biol 2014;386:86-95. https://doi.org/10.1016/j.ydbio.2013.11.024
  65. Guo Z, Lv L, Liu D, Fu B. Effects of trichostatin A on pig SCNT blastocyst formation rate and cell number: A metaanalysis. Res Vet Sci 2018;117:161-6. https://doi.org/10.1016/j.rvsc.2017.12.011
  66. Zhao J, Ross JW, Hao Y, et al. Significant improvement in cloning efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer. Biol Reprod 2009;81:525-30. https://doi.org/10.1095/biolreprod.109.077016
  67. Zhao J, Hao Y, Ross JW, et al. Histone deacetylase inhibitors improve in vitro and in vivo developmental competence of somatic cell nuclear transfer porcine embryos. Cell Reprogram 2010;12:75-83. https://doi.org/10.1089/cell.2009.0038
  68. Liang S, Zhao MH, Choi JW, et al. Scriptaid treatment decreases DNA methyltransferase 1 expression by induction of microRNA-152 expression in porcine somatic cell nuclear transfer embryos. PLoS One 2015;10:e0134567. https://doi.org/10.1371/journal.pone.0134567
  69. Jeong PS, Sim BW, Park SH, et al. Chaetocin improves pig cloning efficiency by enhancing epigenetic reprogramming and autophagic activity. Int J Mol Sci 2020;21:4836. https://doi.org/10.3390/ijms21144836
  70. Kobayashi W, Kurumizaka H. Structural transition of the nucleosome during chromatin remodeling and transcription. Curr Opin Struct Biol 2019;59:107-14. https://doi.org/10.1016/j.sbi.2019.07.011
  71. Antony J, Oback F, Chamley LW, et al. Transient JMJD2Bmediated reduction of H3K9me3 levels improves reprogramming of embryonic stem cells into cloned embryos. Mol Cell Biol 2013;33:974-83. https://doi.org/10.1128/MCB.01014-12
  72. Matoba S, Liu Y, Lu F, et al. Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation. Cell 2014;159:884-95. https://doi.org/10.1016/j.cell.2014.09.055
  73. Chung YG, Matoba S, Liu Y, et al. Histone demethylase expression enhances human somatic cell nuclear transfer effciency and promotes derivation of pluripotent stem cells. Cell Stem Cell 2015;17:758-66. https://doi.org/10.1016/j.stem.2015.10.001
  74. Liu Z, Cai Y, Wang Y, et al. Cloning of macaque monkeys by somatic cell nuclear transfer. Cell 2018;172:881-7.e7. https://doi.org/10.1016/j.cell.2018.01.020
  75. Weng XG, Cai MM, Zhang YT, et al. Improvement in the in vitro development of cloned pig embryos after kdm4a overexpression and an H3K9me3 methyltransferase inhibitor treatment. Theriogenology 2020;146:162-70. https://doi.org/10.1016/j.theriogenology.2019.11.027
  76. Greiner D, Bonaldi T, Eskeland R, Roemer E, Imhof A. Identification of a specific inhibitor of the histone methyltransferase SU(VAR)3-9. Nat Chem Biol 2005;1:143-5. https://doi.org/10.1038/nchembio721
  77. Jeong PS, Sim BW, Park SH, et al. Chaetocin improves pig cloning efficiency by enhancing epigenetic reprogramming and autophagic activity. Int J Mol Sci 2020;21:4836. https://doi.org/10.3390/ijms21144836
  78. Jeong PS, Yang HJ, Park SH, et al. Combined chaetocin/trichostatin A treatment improves the epigenetic modification and developmental competence of porcine somatic cell nuclear transfer embryos. Front Cell Dev Biol 2021;9:709574. https://doi.org/10.3389/fcell.2021.709574
  79. Yoshino J, Kojima T, Shimizu M, Tomizuka T. Cryopreservation of porcine blastocysts by vitrification. Cryobiology 1993;30:413-22. https://doi.org/10.1006/cryo.1993.1041
  80. Katayama KP, Stehlik J, Kuwayama M, Kato O, Stehlik Ed. High survival rate of vitrified human oocytes results in clinical pregnancy. Fertil Steril 2003;80:223-4. https://doi.org/10.1016/s0015-0282(03)00551-x
  81. Esaki R, Ueda H, Kurome M, et al. Cryopreservation of porcine embryos derived from in vitro-matured oocytes. Biol Reprod 2004;71:432-7. https://doi.org/10.1095/biolreprod.103.026542
  82. Cuello C, Gil MA, Parrilla I, et al. Vitrification of porcine embryos at various developmental stages using different ultra-rapid cooling procedures. Theriogenology 2004;62: 353-61. https://doi.org/10.1016/j.theriogenology.2003.10.007
  83. Cuello C, Sanchez-Osorio J, Alminana C, et al. Effect of the cryoprotectant concentration on the in vitro embryo development and cell proliferation of OPS-vitrified porcine blastocysts. Cryobiology 2008;56:189-94. https://doi.org/10.1016/j.cryobiol.2008.02.005
  84. Du Y, Li J, Kragh PM, et al. Piglets born from vitrified cloned blastocysts produced with a simplified method of delipation and nuclear transfer. Cloning Stem Cells 2007;9:469-76. https://doi.org/10.1089/clo.2007.0037
  85. Nakano K, Matsunari H, Nakayama N, et al. Cloned porcine embryos can maintain developmental ability after cryopreservation at the morula stage. J Reprod Dev 2011;57:312-6. https://doi.org/10.1262/jrd.10-142a
  86. Jia B, Xiang D, Guo J, et al. Successful vitrification of earlystage porcine cloned embryos. Cryobiology 2020;97:53-9. https://doi.org/10.1016/j.cryobiol.2020.10.009
  87. Du X, Zhuan Q , Cheng K, et al. Cryopreservation of porcine embryos: recent updates and progress. Biopreserv Biobank 2021;19:210-8. https://doi.org/10.1089/bio.2020.0074
  88. Kamoshita M, Kato T, Fujiwara K, et al. Successful vitrification of pronuclear-stage pig embryos with a novel cryoprotective agent, carboxylated epsilon-poly-L-lysine. PLoS One 2017;12:e0176711. https://doi.org/10.1371/journal.pone.0176711
  89. Li J, Rieke A, Day BN, Prather RS. Technical note: porcine non-surgical embryo transfer. J Anim Sci 1996;74:2263-8. https://doi.org/10.2527/1996.7492263x
  90. Suzuki C, Iwamura S, Yoshioka K. Birth of piglets through the non-surgical transfer of blastocysts produced in vitro. J Reprod Dev 2004;50:487-91. https://doi.org/10.1262/jrd.50.487
  91. Ducro-Steverink DW, Peters CG, Maters CC, Hazeleger W, Merks JWM. Reproduction results and offspring performance after non-surgical embryo transfer in pigs. Theriogenology 2004;62:522-31. https://doi.org/10.1016/j.theriogenology.2003.11.010
  92. Cuello C, Berthelot F, Martinat-Botte F, et al. Piglets born after non-surgical deep intrauterine transfer of vitrified blastocysts in gilts. Anim Reprod Sci 2005;85:275-86. https://doi.org/10.1016/j.anireprosci.2004.04.039
  93. Yoshioka K, Noguchi M, Suzuki C. Production of piglets from in vitro-produced embryos following non-surgical transfer. Anim Reprod Sci 2012;131:23-9. https://doi.org/10.1016/j.anireprosci.2012.01.018
  94. Gomis J, Cuello C, Sanchez-Osorio J, et al. Non-surgical deep intrauterine transfer of superfine open pulled straw (SOPS)-vitrified porcine embryos-evaluation of critical steps of the procedure. Theriogenology 2012;78:1339-49. https://doi.org/10.1016/j.theriogenology.2012.05.035
  95. Martinez EA, Nohalez1 A, Martinez CA, et al. The recipients' parity does not influence their reproductive performance following non-surgical deep uterine porcine embryo transfer. Reprod Dom Anim 2016;51:123-9. https://doi.org/10.1111/rda.12654
  96. Angel MA, Gil MA, Cuello C, et al. An earlier uterine environment favors the in vivo development of fresh pig morulae and blastocysts transferred by a nonsurgical deep-uterine method. J Reprod Dev 2014;60:371-6. https://doi.org/10.1262/jrd.2014-022
  97. Tajima S, Uchikura K, Kurita T, et al. Insemination of recipient sows improves the survival to term of vitrified and warmed porcine expanded blastocysts transferred non-surgically. Anim Sci J 2020;91:e13453. https://doi.org/10.1111/asj.13453
  98. Hirayama Y, Takishita R, Misawa H, et al. Non-surgical transfer of vitrified porcine embryos using a catheter designed for a proximal site of the uterus. Anim Sci J 2020;91:e13457. https://doi.org/10.1111/asj.13457
  99. Tajima S, Motoyama S, Wakiya Y, et al. Piglet production by non-surgical transfer of vitrified embryos, transported to commercial swine farms and warmed on site. Anim Sci J 2020;91:e13476. https://doi.org/10.1111/asj.13476