Skip to main content Accessibility help

Effect of serum on the mitochondrial active area on developmental days 1 to 4 in in vitro-produced bovine embryos

  • M. Crocco (a1) (a2) (a3), R.H. Alberio (a3), L. Lauria (a2) and M.I. Mariano (a4) (a2)


Certain morphological changes at the subcellular level caused by the current techniques for in vitro embryo production seem to affect mitochondria. Many of these, including dysfunctional changes, have been associated with the presence of serum in the culture medium. Thus, the aim of the present work was to assess the mitochondrial dynamics occurring in embryos during the first 4 days of development, in order to analyze the most appropriate time for adding the serum. We used transmission electron microscopy (TEM) micrographs to calculate the embryo area occupied by the different morphological types of mitochondria, and analyzed them with Image Pro Plus analyzer. The results showed hooded mitochondria as the most representative type in 1- to 4-day-old embryos. Swollen, on-fusion, orthodox and vacuolated types were also present. When analyzed in embryos cultured without serum, the dynamics of the different mitochondrial types appeared to be similar, a fact that may provide evidence that the developmental changes control the mitochondrial dynamics, and that swollen mitochondria may not be completely inactive. In contrast, in culture medium supplemented with serum from estrous cows, we observed an increased area of hooded mitochondria by developmental day 4, a fact that may indicate an increased production of energy compared with previous days. According to these results, the bovine serum added to the culture medium seems not to be responsible for the functional changes in mitochondria.


Corresponding author

All correspondence to: Melisa Crocco. Instituto Nacional de Parasitología ‘M.F. Chaben’ ANLIS MALBRÁN, Av. Paseo Colón 568, Ciudad de Buenos Aires, Argentina. Tel/Fax: +54 114331 4016/7142. e-mail:


Hide All
Abe, H., Yamahita, S., Itoh, T., Satoh, T. & Hoshi, H. (1999). Ultrastructure of bovine embryos developed from in vitro-matured and -fertilized oocytes: comparative morphological evaluation of embryos cultured either in serum-free medium or in serum-supplemented medium. Mol. Reprod. Dev. 53, 325–35.
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.
Barnett, D.K. & Bavister, B.D. (1996). Inhibitory effect of glucose and phosphate on the second cleavage division of hamster embryos: is it linked to metabolism? Human Reprod. 11, 177–83.
Barni, S., Sciola, L., Spano, A. & Pippia, P. (1996). Static cytofluorometry and fluorescence morphology of mitochondria and DNA in proliferating fibroblasts. Biotech. Histochem. 71, 6670.
Bavister, B.D. (1995). Culture of preimplantation embryos: facts and artifacts. Hum. Reprod. Update 1, 91148.
Betteridge, K.J. & Fléchon, J.E. (1988). The anatomy and physiology of pre-attachment bovine embryos. Theriogenology 29, 155–87.
Chen, H., Detmer, S.A., Ewald, A.J., Griffin, E.E., Fraser, S.E. & Chan, D.C. (2003). Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development. J. Cell Biol. 160, 189200.
Chi, M.M-Y., Hoehn, A. & Moley, K.H. (2002). Metabolic changes in the glucose-induced apoptotic blastocyst suggest alterations in mitochondrial physiology. Am. J. Physiol. Endocrinol. Metab. 283, E22632.
Crosier, A.E., Farin, P.W., Dykstra, M.J., Alexander, J.E. & Farin, C.E. (2001). Ultrastructural morphometry of bovine blastocysts produced in vivo or in vitro. Biol. Reprod. 64, 1375–85.
Dorland, M., Gardner, D.K. & Trounson, A.O. (1994). Serum in synthetic oviduct fluid causes mitochondrial degeneration in ovine embryos. J. Reprod. Fertil. 13, 17.
Dumollard, R., Carroll, J., Duchen, M.R., Campbell, K. & Swann, K. (2009). Mitochondrial function and redox state in mammalian embryos. Sem. Cell Dev. Biol. 20, 346–53.
Fair, T., Lonergan, P., Dinnyes, A., Cottell, D.C., Hyttel, P., Ward, F.A. & Boland, M.P. (2001). Ultrastructure of bovine blastocysts following cryopreservation: effect of method of blastocyst production. Mol. Reprod. Dev. 58, 186–95.
Ferreirinha, F., Quattrini, A., Pirozzi, M., Valsecchi, V., Dina, G., Broccoli, V., Auricchio, A., Piemonte, F., Tozzi, G., Gaeta, L., Casari, G., Ballabio, A. & Rugarli, E. (2004). Axonal degeneration in paraplegin-deficient mice is associated with abnormal mitochondria and impairment of axonal transport. J. Clin. Invest. 113, 231–43.
Gardner, D.K. (1998). Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture. Theriogenology 49, 83102.
Gomez, E. & Diez, C. (2000). Effects of glucose and protein sources on bovine embryo development in vitro. Anim. Reprod. Sci. 58, 2337.
Han, Z., Vassena, R., Chi, M.M.Y., Potireddy, S., Sutovsky, M., Moley, K.H., Sutovsky, P. & Latham, K.E. (2008). Role of glucose in cloned mouse embryo development. Am. J. Physiol. Endocrinol. Metab. 295, E798809.
Karbowski, M., Spodnik, J.H., Teranishi, M., Wozniak, M., Nishizawa, Y., Usukura, J. & Wakabayashi, T. (2001). Opposite effects of microtubule-stabilizing and microtubule-destabilizing drugs on biogenesis of mitochondria in mammalian cells. J. Cell Sci. 114, 281–91.
Krisher, R.L. & Bavister, B.D. (1998). Responses of oocytes and embryos to the culture environment. Theriogenology 49, 103–14.
Lindner, G.M. & Wright, R.W. (1983). Bovine embryo morphology and evaluation. Theriogenology 20, 407–16.
Logan, D.C. (2003). Mitochondrial dynamics. New Phytologist 160, 463–78.
Logan, D.C. (2006). The mitochondrial compartment. J. Exp. Bot. 57, 1225–43.
Mannella, C.A. (2008). Structural diversity of mitochondria, functional implications. Ann. N.Y. Acad. Sci. 1147, 171–9.
Margineantu, D.H., Cox, W.G., Sundell, L., Sherwood, S.W., Beechem, J.M. & Capaldi, R.A. (2002). Cell cycle dependent morphology changes and associated mitochondrial DNA redistribution in mitochondria of human cell lines. Mitochondrion 1, 425–35.
Maurer, H.R. (1992). Towards serum-free, chemical defined media for mammalian cell culture. In: Animal Cell Culture: A Practical Approach. 2nd edn (ed. Freshney, R.I.). Oxford: Oxford University Press. pp. 1546.
Mohr, L.R. & Trounson, A.O. (1981). Structural changes associated with freezing of bovine embryos. Biol. Reprod. 25, 1009–25.
Mollenhauer, H.H. (1964). Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111.
Mucci, N., Aller, J., Kaiser, G.G., Hozbor, F., Cabodevila, J. & Alberio, R.H. (2006). Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification. Theriogenology 65, 1551–62.
Okamoto, K. & Shaw, J.M. (2005). Mitochondrial morphology and dynamics in yeast and multicellular eukaryotes. Ann. Rev. Genet. 39, 503–36.
Palma, G.A. (2001). Producción in vitro de embriones bovinos. In: Biotecnología de la Reproducción. Argentina: Instituto Nacional de Tecnología Agropecuaria. (ed. Palma, G.) pp. 225–94.
Peng, T.I. & Jou, M.J. (2004). Mitochondrial swelling and generation of reactive oxygen species induced by photoirradiation are heterogeneously distributed. Ann. N.Y. Acad. Sci. 1011, 112–22.
Pikó, L. & Taylor, K.D. (1987). Amounts of mitochondrial DNA and abundance of some mitochondrial gene transcripts in early mouse embryos. Dev. Biol. 123, 364–74.
Pinyopummintr, T. & Bavister, B.D. (1991. In vitro-matured/in vitro-fertilized bovine oocytes can develop into morulae/blastocysts in chemically defined protein-free cultured media. Biol. Reprod. 45, 736–42.
Ram, P.T. & Schultz, R.M. (1993). Reporter gene expression in G2 of the 1-cell mouse embryo. Dev. Biol. 156, 552–6.
Rexroad, C.E. Jr. (1989). Co-culture of domestic animal embryos. Theriogenology 31, 105–14.
Reynolds, E.W. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208–12.
Shamsuddin, M. & Rodriguez-Martinez, H. (1994). Fine structure of bovine blastocysts developed either in serum-free medium or in conventional co-culture with oviduct epithelial cells. J. Vet. Med. 41, 307–16.
Sheahan, M.B., McCurdy, D.W. & Rose, R.J. (2005). Mitochondria as a connected population: ensuring continuity of the mitochondrial genome during plant cell dedifferentiation through massive mitochondrial fusion. Plant J. 44, 744–55.
Skulachev, V.P., Bakeeva, L.E., Chernyak, B.V., Domnina, L.V., Minin, A.A., Pletjushkina, O.Y., Saprunova, V.B., Skulachev, I.V., Tsyplenkova, V.G., Vasiliev, J.M., Yaguzhinsky, L.S. & Zorov, D.B. (2004). Thread-grain transition of mitochondrial reticulum as a step of mitoptosis and apoptosis. Mol. Cell. Biochem. 256/752 85143.
Smith, L.C., Bordignon, V., García, J.M. & Meirelles, F.V. (2000). Mitochondrial genotype segregation and effects during mammalian development: applications to biotechnology. Theriogenology 53, 3546.
Taylor, K.D. & Pikó, L. (1995). Mitochondrial biogenesis in early mouse embryos: expression of the mRNAs for subunits IV, Vb, and VIIc of cytochrome c oxidase and subunit 9 (P1) of H+-ATP synthase. Mol. Reprod. Dev. 40, 2935.
Trimarchi, J.R., Liu, L., Portereld, D.M., Smith, P.J. & Keefe, D.L. (2000). Oxidative phosphorylation-dependent and -independent oxygen consumption by individual preimplantation mouse embryos. Biol. Reprod. 62, 1866–74.
Van Blerkom, J. (2004). Mitochondria in human oogenesis and preimplantation embryogenesis: engines of metabolism, ionic regulation and developmental competence. Reproduction 128, 269–80.
Van Blerkom, J., Davis, P.W. & Lee, J. (1995). ATP content of human oocytes and developmental potential and outcome after in-vitro fertilization and embryo transfer. Hum. Reprod. 10, 415–24.
Van Langendonckt, A., Donnay, N., Shuurbiers, P., Auquier, C., Carolan, C., Massip, A. & Dessy, F. (1997). Effects of supplementation with fetal calf serum on development of bovine embryos in synthetic oviduct fluid medium. J. Reprod. Fertil. 109, 8793.
Watson, A.J., De Sousa, P., Caveney, A., Barcroft, L.C., Natale, D., Urquhart, J. & Westhusin, M.E. (2000). Impact of bovine oocyte maturation media on oocyte transcript levels, blastocyst development, cell number, and apoptosis. Biol. Reprod. 62, 355–64.


Effect of serum on the mitochondrial active area on developmental days 1 to 4 in in vitro-produced bovine embryos

  • M. Crocco (a1) (a2) (a3), R.H. Alberio (a3), L. Lauria (a2) and M.I. Mariano (a4) (a2)


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed