Animal Reproduction (AR)
Animal Reproduction (AR)
Original Article

Effects of oocyte source, cell origin, and embryo reconstruction procedures on in vitro and in vivo embryo survival after goat cloning

Cristiano Feltrin, Luís Henrique de Aguiar, Carlos Enrique Méndez Calderón, Igor de Sá Carneiro, Felipe de Jesus Moraes Junior, Josiane da Silva Quetz, Aldo Ângelo Moreira Lima, Matthew Wheeler, Davide Rondina, José Luiz Rodrigues, James Donald Murray, Elizabeth Anne Maga, Luciana Relly Bertolini, Marcelo Bertolini

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The birth of cloned goats has been well documented, but the overall goat cloning efficiency by somatic cell nuclear transfer procedures is still low, which may be further intensified in extreme environments. The aim of this study was to produce cloned goats under the conditions of the Brazilian SemiArid region, in a transgenic program for the expression of human lysozyme in the milk to target childhood diarrhea and malnutrition, comparing the effects of oocyte source, cell type, and embryo reconstruction procedures on in vitro and in vivo embryo survival after cloning by micromanipulation or by handmade cloning. The use of in vitro-matured oocytes resulted in more viable embryos after cloning than in vivo-matured cytoplasts, but no differences in pregnancy rates on day 23 were seen between oocyte sources (77.5 vs. 77.8%, respectively). The presence or absence of the zona pellucida for embryo reconstruction (78.8 vs. 76.0%, respectively) did not affect pregnancy outcome after transfer. However, pregnancy rate on day 23 was higher for embryos chemically activated by a conventional than a modified protocol (88.1 vs. 50.0%), and for embryos reconstructed with mesenchymal stem cells and fetal fibroblasts (100.0 and 93.3%) than with adult fibroblasts (64.7%). Although most pregnancies were lost, the birth of a cloned female was obtained from embryos reconstructed by micromanipulation using non-transgenic control cells and in vitro-matured oocytes with intact zona pellucida, after conventional activation and transfer at the 1-cell stage.


goats, oocyte maturation, somatic cell nuclear transfer, zona-free embryos.


Akshey YS, MalakarD, De AK, Jena MK, Garg S, Dutta R, Pawar SK, Mukesh M. 2010. Hand-made cloned goat (Capra hircus) embryos - a comparison of different donor cells and culture systems. Cell Reprogram, 12:581-588. American Academy of Pediatrics. Work Group on Breastfeeding. 1997. Breastfeeding and the use of human milk. Pediatrics, 100:1035-1039.

An L, Yuan Y, Yu B, Yang T, Cheng Y. 2012. [Cloning goat producing human lactoferrin with genetically modified donor cells selected by single or dual markers]. Sheng Wu Gong Cheng Xue Bao, 28:1482-1491.

Apimeteetumrong M, Thuangsanthia A, Leingcharoen N. 2004. The effect of activation protocols on the development of cloned goat embryos. J Vet Med Sci, 66:1529-1534.

Baguisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM, Cammuso C, Williams JL, Nims SD, Porter CA, Midura P, Palacios MJ, Ayres SL, Denniston RS, Hayes ML, Ziomek CA, Meade HM, Godke RA, Gavin WG, Overstrom EW, Echelard Y. 1999. Production of goats by somatic cell nuclear transfer. Nat Biotechnol, 17:456-461.

Bai DP, Yang MM, Qu L, Chen YL. 2017. Generation of a transgenic cashmere goat using the piggyBac transposition system. Theriogenology, 93:1-6. doi: 10.1016/j.theriogenology.2017.01.036.

Baldassarre H, Wang B, Keefer CL, Lazaris A, Karatzas CN. 2004. State of the art in the production of transgenic goats. Reprod Fertil Dev, 16, 465-470.

Behboodi E, Memili E, Melican DT, Destrempes MM, Overton SA, Williams JL, Flanagan PA, Butler RE, Liem H, Chen LH, Meade HM, Gavin WG, Echelard Y. 2004. Viable transgenic goats derived from skin cells. Transgenic Res, 13:215-224.

Bertolini M, Beam SW, Shim H, Bertolini LR, Moyer AL, Famula TR, Anderson GB. 2002. Growth, development, and gene expression by in vivo- and in vitro-produced day 7 and 16 bovine embryos. Mol Reprod Dev, 63:318-328.

Bertolini M. 2009. Advances in reproductive technologies in cattle: from artificial insemination to cloning. Rev Fac Med Vet Zootec, 56:184-194.

Boccolini CS, Boccolini PMM, Carvalho ML, Couto OMI. 2012. Padrões de aleitamento materno exclusivo e internação por diarreia entre 1999 e 2008 em capitais brasileiras. Cienc Saude Colet, 17:1857-1863.

Booth PJ, Tan SJ, Reipurth R, Holm P, Callesen H. 2001. Simplification of bovine somatic cell nuclear transfer by application of a zona-free manipulation technique. Cloning Stem Cells, 3:139-150.

Carneiro GF. 2008. Biotécnicas da reprodução assistida em pequenos ruminantes. Tecnol Cienc Agropec, 2:23-28.

Cecconi S, Mauro A, Capacchietti G, Berardinelli P, Bernabò N, Di Vincenzo AR, Mattioli M, Barboni B. 2007. Meiotic maturation of incompetent prepubertal sheep oocytes is induced by paracrine factor(s) released by gonadotropin-stimulated oocyte-cumulus cell complexes and involves mitogen-activated protein kinase activation. Endocrinology, 149:100-107.

Chavatte-Palmer P, Lee R, Bertolini M, Jammes H, Schmidt M, Callesen H. 2013. Pregnancy and neonatal care of SCNT animals. In: Cibelli J, Wilmut IS, Jaenisch R, Gurdon J, Lanza R, West M, Campbell KHS (Ed.). Principles of Cloning. 2nd ed. London: Academic Press. pp. 107-126.

Chaves RM, Aguiar FC, Santos JE, Almeida FJM, Lima PF, Oliveira MAL. 2010. Efeito do diâmetro folicular sobre a qualidade dos oócitos obtidos de ovários de ovelhas (Ovis aries) e cabras (Capra hircus). Cienc Anim Bras, 11:683-688.

Chaves RM, Bezerra FQG, Lima PF, Rabelo MC, jLopes FP, Oliveira MAO. 2011. Maturação Nuclear in vitro e morte celular por apoptose em oócitos de caprinos nos períodos seco e chuvoso. Cienc Anim Bras, 12:593-601.

Chen DY, Jiang MX, Zhao ZJ, Wang HL, Sun Q, Zhang LS, Li RC, Cao H, Zhang Q, Ma DL. 2007. Cloning of Asian Yellow Goat (C. hircus) by somatic cell nuclear transfer: telophase enucleation combined with whole cell intracytoplasmic injection. Mol Reprod Dev, 74;28-34.

Chow BD, Reardon JL, Perry EO, Laforce-Nesbitt SS, Tucker R, Bliss JM. 2016. Host defense proteins in breast milk and neonatal yeast colonization. J Hum Lact, 32:168-173.

Colato C, Albornoz M, Mellano ML, Mellano PH, Mellano JI, Meltsas A, Mellano MA, Mellano JC, Bordignon V, Baldassarre H. 2011. Production of cloned boer goats and dorper sheep in Argentina. Reprod Fertil Dev Suppl, 23:123. (abstract).

Cooper CA, Maga EA, Murray JD. 2015. Production of human lactoferrin and lysozyme in the milk of transgenic dairy animals: past, present, and future. Transgenic Res, 24:605-614.

Dominko T, Ramalho-Santos J, Chan A, Moreno RD, Luetjens CM, Simerly C, Hewitson L, Takahashi D, Martinovich C, White JM, Schatten G. 1999. Optimization strategies for production of mammalian embryos by nuclear transfer. Cloning, 1:143-152.

Dutta R, Malakar D, Khate K, Sahu S, Akshey Y, Mukesh M. 2011. A comparative study on efficiency of adult fibroblast, putative embryonic stem cell and lymphocyte as donor cells for production of handmade cloned embryos in goat and characterization of putative ntES cells obtained from these embryos. Theriogenology, 76:851-863.

Edwards JL, Hansen PJ. 1996. Elevated temperature increases heat shock protein 70 synthesis in bovine twocell embryos and compromises function of maturing oocytes. Biol Reprod, 55:340-346.

Fatehi AN, Roelen BAJ, Colenbrander B, Schoevers EJ, Gadella BM, Bevers MM, Van Den Hurk R. 2005. Presence of cumulus cells during in vitro fertilization protects the bovine oocyte against oxidative stress and improves first cleavage but does not affect further development. Zygote, 13:177-185.

Feltrin C, Forell F, Santos LC, Rodrigues JL. 2006. In vitro bovine embryo development after nuclear transfer by HMC using a modified WOW culture system. Reprod Fertil Dev Suppl, 18:126. (abstract).

Feng X, Cao S, Wang H, Meng C, Li J, Jiang J, Qian Y, Su L, He Q, Zhang Q. 2015. Production of transgenic dairy goat expressing human α-lactalbumin by somatic cell nuclear transfer. Transgenic Res, 24:73- 85.

Fissore RA, Long CR, Duncan RP, Robl JM. 1999. Initiation and organization of events during the first cell cycle in mammals: applications in cloning. Cloning, 1:88-100.

Folch J, Cocero MJ, Chesne´c P, Alabart JL, Domınguez V, Cognie´d V, Roche A, FernandezArias A, Martıa I, Sanchez P, Echegoyen E, Beckers JF, Sanchez AB, Vignon X. 2009. First birth of an animal from an extinct subspecies. Theriogenology, 71:1026-1034.

Gavin W, Chen L, Schofield M, Masiello N, Meade H, Echelard Y. 2013. Transgenic cloned goats and the production of recombinant therapeutic proteins. In: Cibelli J, Wilmut IS, Jaenisch R, Gurdon J, Lanza R, West M, Campbell KHS. (Ed.). Principles of Cloning. London: Academic Press. pp. 329-342.

Gerger RP, Ribeiro ES, Forell F, Bertolini LR, Rodrigues JL, Ambrósio CE, Miglino MA, Mezzalira A, Bertolini M. 2010. In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence. Genet Mol Res, 9:295-302.

Guimarães FC. 2006. Situação atual e perspectivas da caprinocultura no Vale do São Francisco. In: Congresso Nordestino de Produção Animal, 6, 2006, Petrolina, PE. Petrolina, PE: SNPA. 14 pp. CD-ROM.

Hassiotou F, Geddes DT. 2015. Immune cell-mediated protection of the mammary gland and the infant during breastfeeding. Adv Nutr, 62:67-75.

Holm P, Booth PJ, Schmidt MH, Greve T, Callesen H. 1999. High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins. Theriogenology, 52:683-700.

Hosseini SM, Hajianm M, Forouzanfar M, Ostadhosseini S, Moulavi F, Ghanaei HR, Gourbai H, Shahverdi AH, Vosough AD, Nasr-Esfahani MH. 2015. Chemically assisted somatic cell nuclear transfer without micromanipulator in the goat: effects of demecolcine, cytochalasin-B, and MG-132 on the efficiency of a manual method of oocyte enucleation using a pulled Pasteur pipette. Anim Reprod Sci, 158:11- 18.

Jordan ER. 2003. Effects of heat stress on reproduction. J Dairy Sci, 86:104-114.

Keefer CL, Baldassarre H, Keyston R, Wang B, Bhatia B, Bilodeau AS, Zhou JF, Leduc M, Dwney BR, Lazaris A, Karatzas CN. 2001. Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes. Biol Reprod, 64:849-856.

Keefer CL, Keyston R, Lazaris A, Bhatia B, Begin I, Bilodeau AS, Zhou FJ, Kafidi N, Wang B, Baldassarre H, Karatzas CN. 2002. Production of cloned goats after nuclear transfer using adult somatic cells. Biol Reprod, 66:199-203.

Kelly JM, Kleeman DO, Rudiger SR, Walker SK. 2007. Effects of grade of oocyte-cummulus complex and the interactions between grades on the blastocysts in the cow, ewe and lamb. Reprod Domest Anim, 42:577- 582.

Król JZ, Litwińczuk A, Brodziak J, Barłowska JZ. 2010. Lactoferrin, lysozyme and immunoglobulin G content in milk of four breeds of cows managed under intensive production system. Pol J Vet Sci, 13:357-361.

Lan GC, Chang ZL, Luo MJ, Jiang YL, Han D, Wu YG. 2006. Production of cloned goats by nuclear transfer of cumulus cells and long-term cultured fetal fibroblast cells into abattoir-derived oocytes. Mol Reprod Dev, 73:834-840.

Lechner BE, Vohr BR. 2017. Neurodevelopmental outcomes of preterm infants fed human milk: a systematic review. Clin Perinatol, 44:69-83.

Leibfried L, First NL. 1979. Characterization of bovine follicular oocytes and their ability to mature in vitro. J Anim Sci, 48:76-86.

Levy J. 1998. Immunonutrition: The pediatric experience. Nutrition, 14:641-647.

Liu J, Li LL, Du S, Bai XY, Zhang HD, Tang S, Zhao MT, Ma BH, Quan FS, Zhao XE, Zhang Y. 2011. Effects of interval between fusion and activation, cytochalasin B treatment, and number of transferred embryos, on cloning efficiency in goats. Theriogenology, 76:1076-1083.

Lönnerdal B. 2003. Nutritional and physiologic significance of human milk proteins. Am J Clin Nutr, 77:1537-1543.

Lönnerdal B. 2016. Human milk: bioactive proteins/peptides and functional properties. Nestle Nutr Inst Workshop Ser, 86:97-107.

Maga EA, Murray JD. 1995. Mammary gland expression of transgenes and the potential for altering the properties of milk. Biotechnology, 13:1452-1457.

Maga EA, Cullor JS, Smith W, Anderson GB, Murray JD. 2006a. Human lysozyme expressed in the mammary gland of transgenic dairy goats can inhibit the growth of bacteria that cause mastitis and the coldspoilage of milk. Foodborne Pathog Dis, 3:384-392.

Maga EA, Walker RL, Anderson GB, Murray JD. 2006b. Consumption of milk from transgenic goats expressing human lysozyme in the mammary gland results in the modulation of intestinal microflora. Transgenic Res, 15:515-519.

Martin L, Besch-Williford C, Lai L, Cheong HT, Im GS, Park KW, Murphy C, Hao Y, Ellersieck MR, Keisler DH, Schatten H, Green JA, Prather RS. 2007. Morphologic and histologic comparisons between in vivo and nuclear transfer derived porcine embryos. Mol Reprod Dev, 74:952-960.

Martins LT, Gaudêncio Neto S, Tavares KC, Calderón CE, Aguiar LH, Lazzarotto CR, Ongaratto FL, Rodrigues VH, Carneiro IS, Rossetto R, Almeida AP, Fernandes CC, Rondina D, Dias AC, Chies JM, Polejaeva IA, Rodrigues JL, Forell F, Bertolini LR, Bertolini M. 2016. Developmental outcome and related abnormalities in goats: comparison between somatic cell nuclear transfer- and in vivoderived concepti during pregnancy through term. Cell Reprogram,18:264-279.

Melican D, Gavin W. 2008. Repeat superovulation, non-surgical embryo recovery, and surgical embryo transfer in transgenic dairy goats. Theriogenology, 69:197-203.

Meng Q, Hall J, Rutigliano H, Zhou X, Sessions BR, Stott R, Panter K, Davies CJ, Ranjan R, Dosdall D, MacLeod R, Marrouche N, White KL, Wang Z, Polejaeva IA. 2012. Generation of cloned transgenic goats with cardiac specific overexpression of transforming growth factor β1. Reprod Fertil Dev Suppl, 25:162-163.

Mohapatra SK, Sandhu A, Neerukattu VS, Singh KP, Selokar NL, Singla SK, Chauhan MS, Manik RS, Palta P. 2015. Buffalo embryos produced by handmade cloning from oocytes selected using brilliant cresyl blue staining have better developmental competence and quality and are closer to embryos produced by in vitro fertilization in terms of their epigenetic status and gene expression pattern. Cell Reprogram, 17:141-150.

Monaco E, Lima AS, Bionaz M, Maki A, Wilson S, Hurley WL, Wheeler MB. 2009. Morphological and transcriptomic comparison of adipose and bone marrow derived porcine stem cells. Open Tissue Eng Regen Med J, 2:20-33.

Mountzouris KC, McCartney AL, Gibson GR. 2002. Intestinal microflora of human infants and current trends for its nutritional modulation. Br J Nutr, 87:405- 420.

Nasr-Esfahani MH, Hosseini SM, Hajian M, Forouzanfar M, Ostadhosseini S, Abedi P, Khazaie Y, Dormiani K, Ghaedi K, Forozanfar M, Gourabi H, Shahverdi AH, Vosough AD, Vojgani H. 2011.

Development of an optimized zona-free method of somatic cell nuclear transfer in the goat. Cell Reprogram, 13:157-170.

Oback B, Wiersema AT, Gaynor P, Laible G, Tucker FC, Oliver JE, Miller AL, Troskie HE, Wilson KL, Forsyth JT, Berg MC, Cockrem K, McMillan V, Tervit HR, Wells DN. 2003. Cloned cattle derived from a novel zona-free embryo reconstruction system. Cloning, 5:3-12.

Oddy WH. 2017. Breastfeeding, childhood asthma, and allergic disease. Ann Nutr Metab,70:26-36.

Ohkoshi K, Takahashi S, Koyama S, Akagi S, Adachi N, Furusawa TD, Fujimoto J, Takeda K, Kubo M, Izaike Y, Tokunaga T. 2003. In vitro oocyte culture and somatic cell nuclear transfer used to produce a live-born cloned goat. Cloning Stem Cells, 5:109-115.

Pereira AF, Feltrin C, Almeida KC, Carneiro IS, Avelarm SRG, Neto AS, Alcântara SFC, Melo CHS, Moura RR, Teixeira DIA, Bertolini LR, Freitas VJF, Bertolini M. 2013. Analysis of factors contributing to the efficiency of the in vitro production of transgenic goat embryos (Capra hircus) by handmade cloning (HMC). Small Rumin Res, 109:163-172.

Peura TT, Lewis IM, Trounson AO. 1998. The effect of recipient oocyte volume on nuclear transfer in cattle. Mol Reprod Dev, 50:185-191.

Peura TT. 2003. Improved in vitro development rates of sheep somatic cell nuclear transfer embryos by using a reverse-order zona-free cloning methods. Cloning Stem Cells, 5:13-24.

Reggio BC, James AN, Green HL, Gavin W, Behboodi E, Echelard Y, Godke RA. 2001. Cloned transgenic offspring resulting from somatic cell nuclear transfer in the goat: oocytes derived from both folliclestimulating hormone-stimulated and nonstimulated abattoir-derived ovaries. Biol Reprod, 65:1528-1533.

Ribeiro ES, Gerger RPC, Ohlweiler LU, Ortigari JI, Mezzalira JC, Forell F, Bertolini LR, Rodrigues JL, Ambrósio CR, Miglino MA, Mezzalira A, Bertolini M. 2009. Developmental potential of bovine hand-made clone embryos reconstructed by aggregation or fusion with distinct cytoplasmic volumes. Cloning Stem Cells, 11:377-386.

Roth Z, Hansen PJ. 2004. Sphingosine 1-phosphate protects bovine oocytes from heat shock during maturation. Biol Reprod, 71:2072-2078.

Stenfors LE, Bye HM, Räisänen S. 2002. Immunocytochemical localization of lysozyme and lactoferrin attached to surface bacteria of the palatine tonsils during infectious mononucleosis. J Laryngol Otol, 116:264-268.

Tang S, Liu J, Du S, Li LL, Zheng CY, Zhao MT, Wang YS, Zhang Y. 2011. Optimization of embryo culture conditions in the production of cloned goat embryos, following somatic cell nuclear transfer. Small Rumin Res, 96:64-69.

Vajta G, Peura TT, Holm P, Paldi A, Greve T, jTrounson AO, Callesen H. 2000. New method for culture of zona-included or zona-free embryos: the well of the well (WOW) system. Mol Reprod Dev, 55:256- 264.

Verduci E, Banderali G, Barberi S, Radaelli G, Lops A, Betti F, Riva E, Giovannini M. 2014. Epigenetic effects of human breast milk. Nutrients, 6:1711-1724.

Victora C, Horta BL, Mola CL, Quevedo L, Pinheiro RT, Gigante DP, Gonçalves H, Barros FC. 2015. Association between breastfeeding and intelligence, educational attainment, and income at 30 years of age: a prospective birth cohort study from Brazil. Lancet Global Health, 3:e199-205.

Vinoles C, Forsberg M, Banchero G, Rubianes E. 2002. Ovarian follicular dynamics and endocrine profiles in Polwarth ewes with high and low body condition. J Anim Sci, 74:539-545.

Wells DN, Berg MC, Cole SAE, Cullum AA, Oback FC, Oliver JE, Gavin WG, Laible G. 2011. Effect of activation method on in vivo development following somatic cell nuclear transfer in goats. Reprod Fertil Dev Suppl, 24, 127-128.

Westhusin ME, Collas P, Marek D. 1996. Reducing the amount of cytoplasm available for early embryonic development decreases the quality but not the quantity of embryos produced by in vitro fertilization and nuclear transplantation. Theriogenology, 46:243-252.

Yang M, Hall J, Fan Z, Regouski M, Meng Q, Rutigliano HM, Stott R, Rood KA, Panter KE, Polejaeva IA. 2016a. Oocytes from small and large follicles exhibit similar development competence following goat cloning despite their differences in meiotic and cytoplasmic maturation. Theriogenology, 86:2302-2311.

Yang Z, Vajta G, Xu Y, Luan J, Lin M, Liu C, Tian J, Dou H, Li Y, Liu T, Zhang Y, Li L, Yang W, Bolund L, Yang H, Du Y. 2016b. Production of pigs by hand-made cloning using mesenchymal stem cells and fibroblasts. Cell Reprogram, 18:256-263.

Yuan YG, An L, Yu B, Song S, Zhou F, Zhang L, Gu Y, Yu M, Cheng Y. 2014. Expression of recombinant human alpha-lactalbumin in the milk of transgenic goats using a hybrid promoter/enhancer. J Anal Methods Chem, 2014:281031. doi: 10.1155/2014/281031.

Zhang L, Wan ZY, Wang D, Xu XS, Pang L, Meng LH, Wang ZFW. 2010. Production of dairy goat embryos, by nuclear transfer, transgenic for human acid β– glucosidase. Theriogenology, 73:681-690.

Zhang YL, Zhang GM, Wan YJ, Jia RX, Li PZ, Han L, Wang F, Huang MR. 2015. Identification of transgenic cloned dairy goats harboring human lactoferrin and methylation status of the imprinted gene IGF2R in their lungs. Genet Mol Res, 14:11099-11109 Zhou ZR, Zhong BS, Jia RX, Wan YJ, Zhang YL, Fan YX, Wang LZ, You JH, Wang ZY, Wang F. 2013. Production of myostatin-targeted goat by nuclear transfer from cultured adult somatic cells. Theriogenology, 79:225-233.

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