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Splitting of IVP bovine blastocyst affects morphology and gene expression of resulting demi-embryos during in vitro culture and in vivo elongation

  • Alejandra E. Velasquez (a1), Fidel O. Castro (a1), Daniel Veraguas (a1), Jose F. Cox (a1), Evelyn Lara (a1), Mario Briones (a1) and Lleretny Rodriguez-Alvarez (a2)...


Embryo splitting might be used to increase offspring yield and for molecular analysis of embryo competence. How splitting affects developmental potential of embryos is unknown. This research aimed to study the effect of bovine blastocyst splitting on morphological and gene expression homogeneity of demi-embryos and on embryo competence during elongation. Grade I bovine blastocyst produced in vitro were split into halves and distributed in nine groups (3 × 3 setting according to age and stage before splitting; age: days 7–9; stage: early, expanded and hatched blastocysts). Homogeneity and survival rate in vitro after splitting (12 h, days 10 and 13) and the effect of splitting on embryo development at elongation after embryo transfer (day 17) were assessed morphologically and by RT-qPCR. The genes analysed were OCT4, SOX2, NANOG, CDX2, TP1, TKDP1, EOMES, and BAX. Approximately 90% of split embryos had a well conserved defined inner cell mass (ICM), 70% of the halves had similar size with no differences in gene expression 12 h after splitting. Split embryos cultured further conserved normal and comparable morphology at day 10 of development; this situation changes at day 13 when embryo morphology and gene expression differed markedly among demi-embryos. Split and non-split blastocysts were transferred to recipient cows and were recovered at day 17. Fifty per cent of non-split embryos were larger than 100 mm (33% for split embryos). OCT4, SOX2, TP1 and EOMES levels were down-regulated in elongated embryos derived from split blastocysts. In conclusion, splitting day-8 blastocysts yields homogenous demi-embryos in terms of developmental capability and gene expression, but the initiation of the filamentous stage seems to be affected by the splitting.


Corresponding author

All correspondence to: Lleretny Rodriguez-Alvarez. Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepcion . Avenida Vicente Mendez 595, Chillan, Chile. Tel: +56 042 2208835. e-mail address:


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Abolfazl, S., Sara, B., Hassan, N., Ebrahim, A., Banafsheh, H. & Amin, B. (2010). Effects of timing on cell biopsy from pre-compacted morula stage bovine embryos on subsequent embryonic development. J. Reprod. Infertil. 11, 2532.
Albihn, A., Rodriguez-Martinez, H. & Gustafsson, H. (1990). Morphology of day 7 bovine demi-embryos during in vitro reorganization. Acta Anat. (Basel) 138, 42–9.
Alexopoulos, N.I. & French, A.J. (2009). The prevalence of embryonic remnants following the recovery of post-hatching bovine embryos produced in vitro or by somatic cell nuclear transfer. Anim. Reprod. Sci. 114, 4353.
Bazer, F.W., Spencer, T.E. & Ott, T.L. (1997). Interferon tau: a novel pregnancy recognition signal. Am. J. Reprod. Immunol. 37, 412–20.
Bertolini, M., Beam, S.W., Shim, H., Bertolini, L.R., Moyer, A.L., Famula, T.R. & Anderson, G.B. (2002). Growth, development, and gene expression by in vivo- and in vitro-produced day 7 and 16 bovine embryos. Mol. Reprod. Dev. 63, 318–28.
Boiani, M., Eckardt, S., Schöler, H. & McLaughlin, K.J. (2002). Oct4 distribution and level in mouse clones: consequences for pluripotency. Genes and Development. 16, 1209–19.
Clemente, M., de la Fuente, J., Fair, T., Al Naib, A., Gutierrez-Adan, A., Roche, J.F., Rizos, D. & Lonergan, P. (2009). Progesterone and conceptus elongation in cattle: a direct effect on the embryo or an indirect effect via the endometrium? Reproduction 138, 507–17.
de Armas, R., Solano, R., Riego, E., Pupo, C.A., Aguilar, A., Ramos, B., Aguirre, A., de la Fuente, J. & Castro, F.O. (1994). Use of F1 progeny of Holstein × Zebu cross cattle as oocyte donors for in vitro embryo production and gene microinjection. Theriogenology 42, 977–85.
Degrelle, S.A., Campion, E., Cabau, C., Piumi, F., Reinaud, P., Richard, C., Renard, J.P. & Hue, I. (2005). Molecular evidence for a critical period in mural trophoblast development in bovine blastocysts. Dev. Biol. 288, 448–60.
El-Sayed, A., Hoelker, M., Rings, F., Salilew, D., Jennen, D., Tholen, E., Sirard, M.A., Schellander, K. & Tesfaye, D. (2006). Large-scale transcriptional analysis of bovine embryo biopsies in relation to pregnancy success after transfer to recipients. Physiol. Genomics 28, 8496.
Gardner, D.K., Lane, M., Stevens, J. & Schoolcraft, W.B. (2000). Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil. Steril. 73, 1155–58.
Heyman, Y., Vignon, X., Chesné, P., Le Bourhis, D., Marchal, J. & Renard, J.P. (1998). Cloning in cattle: from embryo splitting to somatic nuclear transfer. Reprod. Nutr. Dev. 38, 595603.
Hoelker, M., Schmoll, F., Schneider, H., Rings, F., Gilles, M., Tesfaye, D., Jennen, D., Tholen, E., Griese, J. & Schellander, K. (2006). Bovine blastocyst diameter as a morphological tool to predict embryo cell counts, embryo sex, hatching ability and developmental characteristics after transfer to recipients. Reprod. Fertil. Dev. 18, 551–7.
Illmensee, K., Levanduski, M., Vidali, A., Husami, N. & Goudas, V.T. (2010). Human embryo twinning with applications in reproductive medicine. Fertil. Steril. 93, 423–7.
Kirkegaard, K., Hindkjaer, J.J., Ingerslev, H.J. (2012). Human embryonic development after blastomere removal: a time-lapse analysis. Hum. Reprod. 27, 97105.
Machado, G.M., Ferreira, A.R., Pivato, I., Fidelis, A., Spricigo, J.F., Paulini, F., Lucci, C.M., Franco, M.M. & Dode, M.A. (2013). Post-hatching development of in vitro bovine embryos from day 7 to 14 in vivo versus in vitro . Mol. Reprod. Dev. 80, 936–47.
Maddox-Hyttel, P., Alexopoulos, N.I., Vajta, G., Lewis, I., Rogers, P., Cann, L., Callesen, H., Tveden-Nyborg, P. & Trounson, A. (2003). Immunohistochemical and ultrastructural characterization of the initial post-hatching development of bovine embryos. Reproduction 125, 607–23.
Niwa, H., Miyazaki, J., Nichols, J., Zevnik, B., Anastassiadis, K., Klewe-Nebenius, D., et al. (2000). Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24, 372–6.
Peterson, A.J. & Lee, R.S. (2003). Improving successful pregnancies after embryo transfer. Theriogenology 59, 687–97.
Picard, L., King, W.A. & Betteridge, K.J., Production of sexed calves from frozen-thawed embryos. (1985) Vet. Rec. 117, 603–8.
Roberts, R.M., Leaman, D.W. & Cross, J.C. (1992). Role of interferons in maternal recognition of pregnancy in ruminants. Proc. Soc. Exp. Biol. Med. 200, 718.
Roberts, R.M., Chen, Y., Ezashi, T. & Walker, A.M. (2008). Interferons and the maternal-conceptus dialog in mammals. Semin. Cell. Dev. Biol. 19, 170–7.
Rodríguez, L., Navarrete, F.I., Tovar, H., Cox, J.F. & Castro, F.O. (2008). High developmental potential in vitro and in vivo of cattle embryos cloned without micromanipulators. J. Assist. Reprod. Genet. 25, 13–6.
Rodríguez-Alvarez, L., Cox, J., Tovar, H., Einspanier, R. & Castro, F.O. (2010a).Changes in the expression of pluripotency-associated genes during preimplantation and peri-implantation stages in bovine cloned and in vitro produced embryos. Zygote 18, 269–79.
Rodríguez-Alvarez, L., Sharbati, J., Sharbati, S., Cox, J.F., Einspanier, R. & Castro, F.O. (2010b). Differential gene expression in bovine elongated (Day 17) embryos produced by somatic cell nucleus transfer and in vitro fertilization. Theriogenology 74, 4559.
Rodríguez-Alvarez, L., Manriquez, J., Velasquez, A. & Castro, F.O. (2013). Constitutive expression of the embryonic stem cell marker OCT4 in bovine somatic donor cells influences blastocysts rate and quality after nucleus transfer. In Vitro Cell. Dev. Biol. Anim. 49, 657–67.
Russ, A.P., Wattler, S., Colledge, W.H., Aparicio, S.A., Carlton, M.B., Pearce, J.J., Barton, S.C., Surani, M.A., Ryan, K., Nehls, M.C., Wilson, V. & Evans, M.J. (2000). Eomesodermin is required for mouse trophoblast development and mesoderm formation. Nature 404, 9599.
Schramm, R.D. & Paprocki, A.M. (2004). Strategies for the production of genetically identical monkeys by embryo splitting. Reprod. Biol. Endocrinol. 2, 38.
Seike, N., Teranishi, M., Yamada, S., Takakura, R., Nagao, Y. & Kanagawa, H. (1989). Increase in calf production by the transfer of bisected bovine embryos. Nihon Juigaku Zasshi 51, 1193–9.
Skrzyszowska, M., Smorag, Z. & Katska, L. (1997). Demi-embryo production from hatching of zona-drilled bovine and rabbit blastocysts. Theriogenology 48, 551–7.
Solter, D. (2000). Mammalian cloning: advances and limitations. Nat. Rev. Genet. 1, 199207.
Stojkovic, M., Büttner, M., Zakhartchenko, V., Riedl, J., Reichenbach, H.D., Wenigerkind, H., Brem, G. & Wolf, E. (1999). Secretion of interferon-tau by bovine embryos in long-term culture: comparison of in vivo derived, in vitro produced, nuclear transfer and demi-embryos. Anim. Reprod. Sci. 55, 151–62.
Tang, H.H., Tsai, Y.C. & Kuo, C.T. (2012). Embryo splitting can increase the quantity but not the quality of blastocysts. Taiwan J. Obstet. Gynecol. 51, 236–9.
Vajta, G., Holm, P., Greve, T. & Callesen, H. (1997). Comparison of two manipulation methods to produce in vitro fertilized, biopsied and vitrified bovine embryos. Theriogenology 47, 501–9.
Viebahn, C. (1999). The anterior margin of the mammalian gastrula: comparative and phylogenetic aspects of its role in axis formation and head induction. Curr. Top. Dev. Biol. 46, 63103.
Voelkel, S.A., Viker, S.D., Johnson, C.A., Hill, K.G., Humes, P.E. & Godke, R.A. (1985). Multiple embryo-transplant offspring produced from quartering a bovine embryo at the morula stage. Vet. Rec. 117, 528–30.
Williams, T.J. & Moore, L. (1988) Quick-splitting of bovine embryos. Theriogenology 29, 477–84.
Williams, T.J., Elsden, R.P. & Seidel, J.G. (1984). Pregnancy rates with bisected bovine embryos. Theriogenology 22, 521–31.
Wrenzycki, C., Herrmann, D., Lucas-Hahn, A., Korsawe, K., Lemme, E. & Niemann, H. (2005). Messenger RNA expression patterns in bovine embryos derived from in vitro procedures and their implications for development. Reprod. Fertil. Dev. 17, 2335.
Zoheir, K.M. & Allam, A.A.(2010). A rapid method for sexing the bovine embryo. Anim Reprod Sci. 119, 92–6.


Splitting of IVP bovine blastocyst affects morphology and gene expression of resulting demi-embryos during in vitro culture and in vivo elongation

  • Alejandra E. Velasquez (a1), Fidel O. Castro (a1), Daniel Veraguas (a1), Jose F. Cox (a1), Evelyn Lara (a1), Mario Briones (a1) and Lleretny Rodriguez-Alvarez (a2)...


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