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Вавиловский журнал генетики и селекции

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Биотехнологические основы получения клонированных эмбрионов свиней

https://doi.org/10.18699/VJ19.521

Аннотация

Термин «клон» в биотехнологии животных обозначает организм, полученный в результате неполового размножения, который одновременно является прямым потомком и генетической копией родительского организма. На сегодняшний день домашняя свинья (Sus scrofa domestica) представляется наиболее интересным объектом в исследованиях по клонированию. Клонирование свиней имеет широкий спектр потенциальных возможностей использования в различных областях научной и хозяйственной деятельности человека. Тем не менее эффективность получения клонированных эмбрионов свиней все еще остается ниже, чем других видов сельскохозяйственных животных, в частности лошадей и крупного рогатого скота. Соматическое клонирование – сложная многоступенчатая технология, на каждом этапе которой более восприимчивые к изменениям окружающих условий ооциты свиней испытывают неблагоприятные воздействия различных по своей природе факторов (механические, физические, химические). На этапе созревания ооцитов происходят изменения клеточных ультраструктур ооплазмы, которые играют важную роль в последующем репрограммировании ядра пересаженной донорской клетки. Донорские соматические клетки перед переносом в ооцит синхронизируют в стадии G0/G1 клеточного цикла с целью обеспечения нормальной плоидности клонированного эмбриона. При удалении ядра у созревших in vitro ооцитов свиней следует обращать внимание на проблему сохранения жизнеспособности клеток после извлечения собственного ядерного материала. В ходе реконструирования соматическую клетку с помощью микроинструментов помещают в перивителлиновое пространство, где ранее находилось первое полярное тельце, или в цитоплазму энуклеированного ооцита. Метод ручного клонирования (handmade cloning) предполагает удаление ядра ооцита с последующим слиянием с донорской клеткой без помощи микроманипуляционной техники. Повышенная чувствительность ооцитов к факторам окружающей среды обусловливает особые требования к выбору системы in vitro культивирования клонированных эмбрионов свиней. В рамках настоящего обзора проведен мониторинг современных методов, используемых при получении клонированных эмбрионов, выявлены технологические особенности, препятствующие повышению эффективности метода соматического клонирования свиней.

Об авторах

А. В. Лопухов
Федеральный научный центр животноводства – ВИЖ им. академика Л.К. Эрнста
Россия
Москва


Г. Н. Сингина
Федеральный научный центр животноводства – ВИЖ им. академика Л.К. Эрнста
Россия
Москва


Н. А. Зиновьева
Федеральный научный центр животноводства – ВИЖ им. академика Л.К. Эрнста
Россия
Москва


Список литературы

1. Betthauser J., Forsberg E., Augenstein M., Childs L., Eilertsen K., Enos J., Forsythe T., Golueke P., Jurgella G., Koppang R., Lesmeister T., Mallon K., Mell G., Misica P., Pace M., Pfister-Genskow M., Strelchenko N., Voelker G., Watt S., Thompson S., Bishop M. Production of cloned pigs from in vitro systems. Nat. Biotechnol. 2000; 18:1055-1059. DOI 10.1038/80242.

2. Boquest A.C., Day B.N., Prather R.S. Flow cytometric cell cycle analysis of cultured porcine fetal fibroblast cells. Biol. Reprod. 1999;60: 1013-1019. DOI 10.1095/biolreprod60.4.1013.

3. Campbell K.H. Nuclear transfer in farm animal species. Semin. Cell Dev. Biol. 1999;10(3):245-252. DOI 10.1006/scdb.1999.0310.

4. Campbell K.H., Loi P., Otaegui P.J., Wilmut I. Cell cycle co-ordination in embryo cloning by nuclear transfer. Rev. Reprod. 1996;1(1):4046. DOI 10.1530/ror.0.0010040.

5. Cao Y., Yang J., Yin Z.Q., Luo H.Y., Yang M., Hu N., Yang J., Huo D.Q., Hou C.J., Jiang Z.Z., Zhang R.Q., Xu R., Zheng X.L. Study of high-throughput cell electrofusion in a microelectrodearray chip. Microfluid. Nanofluidics. 2008;5:669-675. DOI 10.1007/s10404-008-0289-1.

6. Cervera R.P., Marti-Gutierrez N., Escorihuela E., Moreno R., Stojkovic M. Trichostatin A affects histone acetylation and gene expression in porcine somatic cell nucleus transfer embryos. Theriogenology. 2009;72:10971110. DOI 10.1016/j.theriogenology.2009.06.030.

7. Cervera R.P., Silvestre M.A., Marti N., García-Mengual E., Moreno R., Stojkovic M. Effects of different oocyte activation procedures on development and gene expression of porcine pre-implantation embryos. Reprod. Domest. Anim. 2010;45:12-20. DOI 10.1111/j.14390531.2009.01509.x.

8. Cheng W.M., Sun X.L., An L., Zhu S.E., Li X.H., Li Y., Tian J.H. Effect of different parthenogenetic activation methods on the developmental competence of in vitro matured porcine oocytes. Anim. Biotechnol. 2007;18:131-141. DOI 10.1080/10495390601096148.

9. Cordova A., King W.A., Mastromonaco G.F. Choosing a culture medium for SCNT and iSCNT reconstructed embryos: from domestic to wildlife species. J. Anim. Sci. Technol. 2017;59:24. DOI 10.1186/s40781-017-0149-1.

10. Dobrinsky J.R., Johnson L.A., Rath D. Development of a culture medium (BECM-3) for porcine embryos: effects of bovine serum albumin and fetal bovine serum on embryo development. Biol. Reprod. 1996;55(5):1069-1074.

11. Enright B.P., Kubota C., Yang X., Tian X.C. Epigenetic characteristics and development of embryos cloned from donor cells treated by Trichostatin A or 5-aza-2′-deoxycytidine. Biol. Reprod. 2003;69: 896-901. DOI 10.1095/biolreprod.103.017954.

12. Ferreira E.M., Vireque A.A., Adona P.R., Meirelles F.V., Ferriani R.A., Navarro P.A. Cytoplasmic maturation of bovine oocytes: structural and biochemical modifications and acquisition of developmental competence. Theriogenology. 2009;71:836-848. DOI 10.1016/j.theriogenology.2008.10.023.

13. Fissore R.A., Long C.R., Duncan R.P., Robl J.M. Initiation and organization of events during the first cell cycle in mammals: applications in cloning. Cloning. 1999;1(2):89-100. DOI 10.1089/15204559950019979.

14. Fissore R.A., Robl J.M. Intracellular Сa2+ response of rabbit oocytes to electrical stimulation. Mol. Reprod. Dev. 1992;32:9-16. DOI 10.1002/mrd.1080320103.

15. Fulka J., Moor R.M. Noninvasive chemical enucleation of mouse oocytes. Mol. Reprod. Dev. 1993;34:427-430. DOI 10.1002/mrd.1080340412.

16. Garcia-Mengual E., Alfonso J., Salvador I., Duque C.C., Silvestre M.A. Oocyte activation procedures and influence of serum on porcine oocyte maturation and subsequent parthenogenetic and nuclear transfer embryo development. Zygote. 2008;16:279-284. DOI 10.1017/S0967199408004796.

17. Gil M.A., Martinez C.A., Nohalez A., Parrilla I., Roca J., Wu J., Ross P.J., Cuello C., Izpisua J.C., Martinez E.A. Developmental competence of porcine genome-edited zygotes. Mol. Reprod. Dev. 2017;84(9):814-821. DOI 10.1002/mrd.22829.

18. Grupen C., Mau J.C., McIlfatrick S.M., Maddocks S., Nottle M.B. Effect of 6-dimethylaminopurine on electrically activated in vitro matured porcine oocytes. Mol. Reprod. Dev. 2002;62:387-96. DOI 10.1002/mrd.10126.

19. Hardarson T., Lundin K., Hamberger L. The position of the metaphase II spindle cannot be predicted by the location of the first polar body in the human oocyte. Hum. Reprod. 2000;15(6):1372-1376. DOI 10.1093/humrep/15.6.1372.

20. Heindryckx B., Rybouchkin A., Van Der Elst J., Dhont M. Effect of culture media on in vitro development of cloned mouse embryos. Cloning. 2001;3(2):41-50. DOI 10.1089/15204550152475545.

21. Hölker M., Petersen B., Hassel P., Kues W.A., Lemme E., LucasHahn A., Niemann H. Duration of in vitro maturation of recipient oocytes affects blastocyst development of cloned porcine embryos. Cloning Stem Cells. 2005;7:35-44. DOI 10.1089/clo.2005.7.35.

22. Hyun H., Lee S.E., Son Y.J., Shin M.Y., Park Y.G., Kim E.Y., Park S.P. Cell synchronization by rapamycin improves the developmental competence of porcine SCNT embryos. Cell. Reprogram. 2016; 18(3):195-205. DOI 10.1089/cell.2015.0090.

23. Hyun S., Lee G., Kim D., Kim H., Lee S., Nam D., Jeong Y., Kim S., Yeom S., Kang S., Han J., Lee B., Hwang W. Production of nuclear transfer-derived piglets using porcine fetal fibroblasts transfected with the enhanced green fluorescent protein. Biol. Reprod. 2003;69: 1060-1068. DOI 10.1095/biolreprod.102.014886.

24. Im G.S., Lai L., Liu Z., Hao Y., Wax D., Bonk A., Prather R.S. In vitro development of preimplantation porcine nuclear transfer embryos cultured in different media and gas atmospheres. Theriogenology. 2004;61(6):1125-1135. DOI 10.1016/j.theriogenology.2003.06.006.

25. Im G.S., Samuel M., Lai L., Hao Y., Prather R.S. Development and calcium level changes in pre-implantation porcine nuclear transfer embryos activated with 6-DMAP after fusion. Mol. Reprod. Dev. 2007;74:1158-1164. DOI 10.1002/mrd.20492.

26. Im G.S., Seo J.S., Hwang I.S., Kim D.H., Kim S.W., Yang B.C., Yang B.S., Lai L., Prather R.S. Development and apoptosis of preimplantation porcine nuclear transfer embryos activated with different combination of chemicals. Mol. Reprod. Dev. 2006;73:10941101. DOI 10.1002/mrd.20455.

27. Jaenisch R. Nuclear cloning and direct reprogramming: the long and the short path to Stockholm. Cell Stem Cell. 2012;11(6):744-747. DOI 10.1016/j.stem.2012.11.005.

28. Jin Y., Zhang M., Ju X., Liang S., Xiong Q., Zhao L., Nie X., Hou D., Liu Q., Wang J., Wang C., Li X., Zhang L., Liu X., Wang Y., Yang H., Dai Y., Li R. Factors influencing the somatic cell nuclear transfer efficiency in pigs. Front. Agr. Sci. Eng. 2019;6(1):73-83. (2018. Epub ahead of print). DOI 10.15302/J-FASE-2018220.

29. 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(1):49-59. DOI 10.1007/s10815-009-9378-7.

30. Kawakami M., Tani T., Yabuuchi A., Kobayashi T., Murakami H., Fujimura T., Kato Y., Tsunoda Y. Effect of demecolcine and nocodazole on the efficiency of chemically assisted removal of chromosomes and the developmental potential of nuclear transferred porcine oocytes. Cloning Stem Cells. 2003;5(4):379-387. DOI 10.1089/153623003772032871.

31. Kim Y.S., Lee S.L., Ock S.A., Balasubramanian S., Choe S.Y., Rho G.J. Development of cloned pig embryos by nuclear transfer following different activation treatments. Mol. Reprod. Dev. 2005;70:308-313. DOI 10.1002/mrd.20211.

32. Kong Q.R., Luo Y.B., Tian J.T., Wang Z.K., Zhang L., Liu Z.H. Production of porcine reconstructed embryos by whole-cell intracytoplasmic microinjection (Article in Chinese). Fen Zi Xi Bao Sheng Wu Xue Bao. 2008;41(1):70-74.

33. Koo D.B., Chae J.I., Kim J.S., Wee G., Song B.S., Lee K.K., Han Y.M. Inactivation of MPF and MAP kinase by single electrical stimulus for parthenogenetic development of porcine oocytes. Mol. Reprod. Dev. 2005;72:542-549. DOI 10.1002/mrd.20382.

34. Kragh P.M., Vajta G., Corydon T.J., Purup S., Bolund L., Callesen H. Production of transgenic porcine blastocysts by hand-made cloning. Reprod. Fertil. Dev. 2004;16:315-318. DOI 10.10371/RD04007.

35. Kues W.A., Carnwath J.W., Paul D., Niemann H. Cell cycle synchronization of porcine fetal fibroblasts by serum deprivation initiates a nonconventional form of apoptosis. Cloning Stem Cells. 2002;4(3): 231-243. DOI 10.1089/15362300260339511.

36. Lee E., Estrada J., Piedrahita J. Comparative study on the efficiency of two enucleation methods in pig somatic cell nuclear transfer: effects of the squeezing and the aspiration methods. Anim. Biotechnol. 2008;19(2):71-79. DOI 10.1080/10495390701839264.

37. Lee J.W., Wu S.C., Tian X.C., Barber M., Hoaqland T., Riesen J., Lee K.H., Tu C.F., Cheng W.T., Yang X. Production of cloned pigs by whole-cell intracytoplasmic microinjection. Biol. Reprod. 2003; 69:995-1001. DOI 10.1095/biolreprod.103.015917.

38. Liu Y., Zeng B.H., Shang H.T., Cen Y.Y., Wei H. Bama miniature pigs (Sus scrofa domestica) as a model for drug evaluation for humans: comparison of in vitro metabolism and in vivo pharmacokinetics of lovastatin. Comp. Med. 2008;58:580-587.

39. Marteil G., Richard-Parpaillon L., Kubiak J.Z. Role of oocyte quality in meiotic maturation and embryonic development. Reprod. Biol. 2009;9(3):203-224. DOI 10.1016/S1642-431X(12)60027-8.

40. McGrath J., Solter D. Nuclear transplantation in the mouse embryo by microsurgery and cell fusion. Science. 1983;220:1300-1302. DOI 10.1002/jez.1402280218.

41. Miao Y.L., Kikuchi K., Sun Q.Y., Schatten H. Oocyte aging: cellular and molecular changes, developmental potential and reversal possibility. Hum. Reprod. Update. 2009;15(5):573-585. DOI 10.1093/humupd/dmp014.

42. Miao Y., Ma S., Liu X., Miao D., Chang Z., Luo M., Tan J. Fate of the first polar bodies in mouse oocytes. Mol. Reprod. Dev. 2004;69: 66-76. DOI 10.1002/mrd.20148.

43. Nickoloff J.A. (Ed.) Animal Cell Electroporation and Electrofusion Protocols. In: Ser. “Methods in Molecular Biology”. Vol. 48. Totowa, NJ: Humana Press, 1995.

44. Niemann H., Kues W.A., Lucas-Hahn A., Carnwath J.W. Somatic cloning and epigenetic reprogramming in mammals. In: Atala A., Lanza R., Thompson J., Nerem R. (Eds.) Principles in Regenerative Medicine. 2nd edn. Academic Press, 2011:148-167.

45. Oback B., Wiersema A.T., Gaynor P., Laible G., Tucker F.C., Oliver J.E., Miller A.L., Troskie H.E., Wilson K.L., Forsyth J.T., Berg M.C., Cockrem K., McMillan V., Tervit H.R., Wells D.N. Cloned cattle derived from a novel zona-free embryo reconstruction system. Cloning Stem Cells. 2003;5(1):3-12. DOI 10.1089/153623003321512111.

46. Onishi A., Iwamoto M., Akita T., Mikawa S., Takeda K., Awata T., Hanada H., Perry A.C. Pig cloning by microinjection of fetal fibroblast nuclei. Science. 2000;289:1188-1190. DOI 10.1126/science.289.5482.1188.

47. Park H.J., Koo O.J., Kwon D.K., Kang J.T., Jang G., Lee B.C. Effect of roscovitine-treated donor cells on development of porcine cloned embryos. Reprod. Domest. Anim. 2010;45(6):1082-1088. DOI 10.1111/j.1439-0531.2009.01499.x.

48. Peng H., Liu F.J., Zhang X.F., Zhuang Y.F., Wang X.A., Li H.X., Hong Z.Y., Lin X.J., Zhang W.C. Study of electro-fusion/activation in somatic cell nuclear transfer to obtain cloned putian black pig embryos. J. Anim. Vet. Adv. 2013;12(4):497-503. DOI 10.3923/javaa.2013.497.503.

49. Polejaeva I.A., Chen S.H., Vaught T.D., Page R.L., Mullins J., Ball S., Dai Y., Boone J., Walker S., Ayares D.L., Colman A., Campbell K.H. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature. 2000;407:8690. DOI 10.1038/35024082.

50. Popova E., Bader M., Krivokharchenko A. Efficient production of nuclear transferred rat embryos by modified methods of reconstruction. Mol. Reprod. Dev. 2009;76:208-216. DOI 10.1002/mrd.20944.

51. Presicce G.A., Yang X.Z. Parthenogenetic development of bovine oocytes matured in vitro for 24 hr and activated by ethanol and cycloheximide. Mol. Reprod. Dev. 1994;38:380-385. DOI 10.1002/mrd.1080380405.

52. Rim C.H., Fu Z., Bao L., Chen H., Zhang D., Luo Q., Ri H.C., Huang H., Luan Z., Zhang Y., Cui C., Xiao L., Jong U.M. The effect of the number of transferred embryos, the interval between nuclear transfer and embryo transfer, and the transfer pattern on pig cloning efficiency. Anim. Reprod. Sci. 2013;143(1-4):91-96. DOI 10.1016/j.anireprosci.2013.10.004.

53. Robl J.M., Prather R.S., Branes F., Eyestone W., Northey D., Gilligan B., First N.L. Nuclear transplantation in bovine embryos. J. Anim. Sci. 1987;64:642-647.

54. Samiec M., Skrzyszowska M. The use of different methods of oocyte activation for generation of porcine fibroblast cell nuclear transferred embryos. Ann. Anim. Sci. 2010;10(4):399-411.

55. Savard C., Novak S., Saint-Cyr A., Moreau M., Pothier F., Sirard M.A. Comparison of bulk enucleation methods for porcine oocytes. Mol. Reprod. Dev. 2004;67(1):70-76. DOI 10.1002/mrd.20011.

56. Shi J., Zhou R., Luo L., Mai R., Zeng H., He X., Liu D., Zeng F., Cai G., Ji H., Tang F., Wang Q., Wu Z., Li Z. Influence of embryo handling and transfer method on pig cloning efficiency. Anim. Reprod. Sci. 2015;154:121-127. DOI 10.1016/j.anireprosci.2015.01.006.

57. Simões R., Santos A.R., Jr. Factors and molecules that could impact cell differentiation in the embryo generated by nuclear transfer. Organogenesis. 2017;13(4):156-178. DOI 10.1080/15476278.2017.1389367.

58. Singina G.N., Volkova N.A., Bagirov V.A., Zinovieva N.A. Сryobanking of somatic cells in conservation of animal genetic resources: prospects and successes. Selskokhozyaistvennaya Biologiya = Agricultural Biology. 2014;6:3-14. DOI 10.15389/agrobiology.2014.6.3eng.

59. Skrzyszowska M., Samiec M., Słomski R., Lipiński D., Mały E. Development of porcine transgenic nuclear-transferred embryos derived from fibroblast cells transfected by the novel technique of nucleofection or standard lipofection. Theriogenology. 2008;70:248-259. DOI 10.1016/j.theriogenology.2008.04.007.

60. Sugimura S., Yamanaka K., Kawahara M., Wakai T., Yokoo M., Sato E. Early metaphase II oocytes treated with dibutyryl cyclic adenosine monophosphate provide suitable recipient cytoplasm for the production of miniature pig somatic cell nuclear transfer embryos. Anim. Sci. J. 2010;81(1):48-57. DOI 10.1111/j.1740-0929.2009.00705.x.

61. Suzuki C., Iwamura S., Yoshioka K. Birth of piglets through the nonsurgical transfer of blastocysts produced in vitro. Reprod. Dev. 2004; 50(4):487-491.

62. Tatham B.G., Dowsing A.T., Trounson A.O. Enucleation by centrifugation of in vitro-matured bovine oocytes for use in nuclear transfer. Biol. Reprod. 1995;53:1088-1094. DOI 10.1095/biolreprod53.5.1088. Uhm S.J., Gupta M.K., Chung H.J., Kim J.H., Park C., Lee H.T. Relationship between developmental ability and cell number of Day 2 porcine embryos produced by parthenogenesis or somatic cell nuclear transfer. Asian-Australas. J. Anim. Sci. 2009;22(4):483-491. DOI 10.5713/ajas.2009.80362.

63. Vacková I., Engelová M., Marinov I., Tománek M. Cell cycle synchronization of porcine granulosa cells in G1 stage with mimosine. Anim. Reprod. Sci. 2003;77(3-4):235-245. DOI 10.1016/S03784320(03)00034-4.

64. Vajta G., Kragh P.M., Mtango N.R., Callesen H. Hand-made cloning approach: potentials and limitations. Reprod. Fertil. Dev. 2005; 17(1-2):97-112. DOI 10.1071/RD04116.

65. Vajta G., Lewis I.M., Hyttel P., Thouas G., Trounson A. Somatic cell cloning without micromanipulators. Cloning. 2001;3:89-95. DOI 10.1089/15204550152475590.

66. Verma P.J., Du Z.T., Crocker L., Faast R., Grupen C.G., McIlfatrick S.M., Ashman R.J., Lyons I.G., Nottle M.B. In vitro development of porcine nuclear transfer embryos constructed using fetal fibroblasts. Mol. Reprod. Dev. 2000;57(3):262-226. DOI 10.1002/1098-2795(200011)57:3<262::AID-MRD8>3.0.CO;2-X.

67. Whitworth K.M., Li R., Spate L.D., Wax D.M., Rieke A., Whyte J.J., Manandhar G., Sutovsky M., Green J.A., Sutovsky P., Prather R.S. Method of oocyte activation affects cloning efficiency in pigs. Mol. Reprod. Dev. 2009;76:490-500. DOI 10.1002/mrd.20987.

68. Wilmut I., Schnieke A.E., McWhir J., Kind A.J., Campbell K.H. Viable offspring derived from fetal and adult mammalian cells. Nature. 1997;385:810-813. DOI 10.1089/clo.2006.0002.

69. Wimmers K., Murani E., Ponsuksili S. Functional genomics and genetical genomics approaches towards elucidating networks of genes affecting meat performance in pigs. Brief. Funct. Genomics. 2010;9: 251-258. DOI 10.1093/bfgp/elq003.

70. Yamanaka K., Sugimura S., Wakai T., Kawahara M., Sato E. Difference in sensitivity to culture condition between in vitro fertilized and somatic cell embryos in pigs. Reprod. Dev. 2009;55(3):299-304. DOI 10.1262/jrd.20174.

71. Yang F., Hao R., Kessler B., Brem G., Wolf E., Zakhartchenko V. Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and hymeric embryo-complementation. Reproduction. 2007;133(1):219-230. DOI 101530/rep.1.01206.

72. Yin X.J., Tani T., Yonemura I., Kawakami M., Miyamoto K., Hasegaw R., Kato Y., Tsunoda Y. Production of cloned pigs from adult somatic cells by chemically assisted removal of maternal chromosomes. Biol. Reprod. 2002;67:442-446. DOI 10.1095/biolreprod67.2.442.

73. Yoshioka K., Suzuki C., Tanaka A., Anas I.M., Iwamura S. Birth of piglets derived from porcine zygotes cultured in a chemically defined medium. Biol. Reprod. 2002;66(1):112-119.

74. Zhang Y., Pan D., Sun X., Sun G., Wang X., Liu X., Li Y., Dai Y., Li N. Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer. Sci. China C. Life Sci. 2006;49(2):164-171. DOI 10.1007/s11427-005-0071-5.

75. Zheng Y.M., Zhao H.Y., Zhao X.E., Quan F.S., Hua S., He X.Y., Liu J., He X.N., Lin H. Development of cloned embryos from porcine neural stem cells and amniotic fluid-derived stem cells transfected with enhanced green fluorescence protein gene. Reproduction. 2009; 137(5):793-801. DOI 10.1530/REP-08-0469.

76. Zhu J., Telfer E.E., Fletcher J., Springbett A., Dobrinsky J.R., De Sousa P.A., Wilmut I. Improvement of an electrical activation protocol for porcine oocytes. Biol. Reprod. 2002;66(3):635-641. DOI 10.1095/biolreprod66.3.635.


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