Abstract
It is surprising that so little attention is currently given to in vitro culture of preimplantation rabbit embryos, even though the rabbit is the only laboratory animal in which there is very considerable embryo growth before implantation, resulting in a 300-fold increase in protein content of embryonic cells during the preimplantation period and the formation of more than a 100,000 cells in the blastocyst. This growth pattern explains why blastocyst formation in vitro has an absolute requirement for amino acids, and vitamins, particularly inositol, are esssential for blastocyst growth. A semi-defined medium supplemented with 1.5% BSA (variously known as BSM II or modified F10) was developed at Cornell University at the end of the 1960s and allowed the systematic investigation of the requirements for development of 1-cell rabbit embryos to blastocysts. However, the requirements for in vitro blastocyst growth comparable to in vivo growth still remain an unsolved problem. Citrate, often found as a contaminant in serum albumin, may have an essential role in rabbit blastocyst growth, which would fit in with its role in the development of serum-free media for culture of various types of mammalian cells.
A comprehensive account of the methodology is given to enable a researcher with experience culturing embryos of a different species to work on the rabbit embryo. This account covers medium preparation, hormonal stimulation of superovulation, natural breeding/artificial insemination, and collection of embryos of different stages from 1-cell to blastocyst either after euthanasia or under anesthesia. Peculiarities of the rabbit embryo such as the presence of the mucoprotein coat and its effects on behavior of cultured and transferred embryos are described. Suggestions are made for future avenues of research.
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References
Brachet A (1912) Développement in vitro de blastodermes et jeunes embryons de mammifères. C R Acad Sci Paris 155:1191–1193
Lewis WH, Gregory PW (1929) Cinematographs of living developing rabbit-eggs. Science 69:226–229
Whitten WK (1957) Culture of tubal ova. Nature 179:1081–1082
McLaren A, Biggers JD (1958) Successful development and birth of mice cultivated in vitro as early embryos. Nature 182:877–878
Kane MT, Foote RH (1970) Culture of two-and four-cell rabbit embryos to the expanding blastocyst stage in synthetic media. Proc Soc Exp Biol Med 133:921–925
Carney EW, Foote RH (1991) Improved development of rabbit one-cell embryos to the hatching blastocyst stage by culture in a defined, protein-free medium. J Reprod Fertil 91:113–123
Hogan B, Costantini F, Lacy E (1986) Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor
Brinster RL (1967) Protein content of the mouse embryo during the first five days of development. J Reprod Fertil 13:413–420
Turner K, Goldstein DJ, Rogers AW (1992) Variation in the dry mass of mouse embryos throughout the preimplantation period. Hum Reprod 7:112–116
Denker H-W (1977) Implantation: the role of proteinases, and blockage of implantation by proteinase inhibitors. Adv Anat Embryol Cell Biol 53:1–123
Fujimoto S, Pahlavan N, Woody HD, Dukelow WR (1975) Cell numbers in rabbit pre-implantation blastocysts. Cytologia (Tokyo) 40:307–311
Daniel JC Jr (1964) Early growth of rabbit trophoblast. Am Naturalist 98:85–97
Morgan PM, Kane MT (1993) Protein content of rabbit embryos: one cell to peri-implantation blastocyst. J Reprod Fertil 97:101–106
Kane MT (1969) In vitro culture of two- to four-cell rabbit embryos to expanding blastocysts in serum extracts and synthetic media. PhD Thesis, Cornell University
Brinster RL (1963) A method for in vitro cultivation of mouse ova from two-cell to blastocyst. Exp Cell Res 32:205–208
Ham RG (1963) An improved nutrient solution for diploid Chinese hamster and human cell lines. Exp Cell Res 29:515–526
Hegele-Hartung C, Fischer B, Beier HM (1988) Development of preimplantation rabbit embryos after in-vitro culture and embryo transfer: an electron microscopic study. Anat Rec 220:31–42
Lindenau A, Fischer B (1996) Embryotoxicity of polychlorinated biphenyls (PCBS) for preimplantation embryos. Reprod Toxicol 10:227–230
Kane MT (1988) The effects of water soluble vitamins on the expansion of rabbit blastocysts in vitro. J Exp Zool 245:220–222
Kane MT (1989) Effects of the putative phospholipid precursors, inositol, choline, serine and ethanolamine, on formation and expansion of rabbit blastocysts in vitro. J Reprod Fertil 87:275–279
Fahy MM, Kane MT (1992) Inositol stimulates DNA and protein synthesis, and expansion by rabbit blastocysts in vitro. Hum Reprod 7:550–552
Warner SM, Conlon FV, Kane MT (2003) Inositol transport in preimplantation rabbit embryos: effects of embryo stage, sodium, osmolality and metabolic inhibitors. Reproduction 125:479–493
Fahy MM, Kane MT (1993) Incorporation of [3H]inositol into phosphoinositides and inositol phosphates by rabbit blastocysts. Mol Reprod Dev 34:391–395
Kane MT, Foote RH (1971) Factors affecting blastocyst expansion of rabbit zygotes and young embryos in defined media. Biol Reprod 4:41–47
Kane MT (1987) Minimal nutrient requirements for culture of one-cell rabbit embryos. Biol Reprod 37:775–778
Kane MT (1972) Energy substrates and culture of single-cell rabbit ova to blastocysts. Nature 238:468–469
Naglee DL, Maurer RR, Foote RH (1969) Effect of osmolarity on in vitro development of rabbit embryos in a chemically defined medium. Exp Cell Res 58:331–333
Kane MT (1974) The effects of pH on culture of one-cell rabbit ova to blastocysts in bicarbonate buffered medium. J Reprod Fertil 38:477–480
Cross MH (1974) Rabbit blastocoele bicarbonate: accumulation rate. Biol Reprod 11:654–666
Kane MT (1975) Bicarbonate requirements for culture of one-cell rabbit ova to blastocysts. Biol Reprod 12:552–555
Hallden K, Li JM, Carney EW, Foote RH (1992) Increasing carbon dioxide from five percent to ten percent improves rabbit blastocyst development from cultured zygotes. Mol Reprod Dev 33:276–280
Farrell PB, Foote RH (1995) Beneficial effects of culturing rabbit zygotes to blastocysts in 5% oxygen and 10% carbon dioxide. J Reprod Fertil 103:127–130
Lindenau A, Fischer B (1994) Effect of oxygen concentration in the incubator’s gas phase on the development of cultured preimplantation rabbit embryos. Theriogenology 41:889–898
Li J, Foote RH (1993) Culture of rabbit zygotes into blastocysts in protein-free medium with one to twenty per cent oxygen. J Reprod Fertil 98:163–167
Seidel GE Jr, Bowen RA, Kane MT (1976) In vitro fertilization, culture and transfer of rabbit ova. Fert Steril 27:861–869
Denker HW (2000) Structural dynamics and function of early embryonic coats. Cells Tissues Organs 166:180–207
Fasano M, Curry S, Terreno E et al (2005) The extraordinary ligand binding properties of human serum albumin. IUBMB Life 57:787–796. https://doi.org/10.1080/15216540500404093
Westphal U (1970) Corticosteroid-binding globulin and other steroid hormone carriers in the blood stream. J Reprod Fertil Suppl 10:15–38
Blundell TL (1977) The binding of heavy metals to proteins. Chem Soc Rev 6:131–171
Kane MT (1983) Variability in different lots of commercial bovine serum albumin affects both cell multiplication and hatching of rabbit blastocysts in culture. J Reprod Fertil 69:555–558
Kane MT (1979) Fatty acids as energy sources for culture of one-cell rabbit ova to viable morulae. Biol Reprod 20:323–332
Kane MT, Headon DR (1980) The role of commercial bovine serum albumin preparations in the culture of one-cell rabbit embryos to blastocysts. J Reprod Fertil 60:469–475
Kane MT (1985) A low molecular weight extract of bovine serum albumin stimulates rabbit blastocyst cell division and expansion. J Reprod Fertil 73:147–150
Gray CW, Morgan PM, Kane MT (1992) Purification of an embryotrophic factor from commercial bovine serum albumin and its identification as citrate. J Reprod Fertil 94:471–480
Rorie RW, Miller GF, Nasti KB, McNew RW (1994) In vitro development of bovine embryos as affected by different lots of bovine serum albumin and citrate. Theriogenology 42:397–403
Wassarman PM, Litscher ES (2013) Biogenesis of the mouse egg’s extracellular coat, the zona pellucida. Curr Top Dev Biol 102:243–266. https://doi.org/10.1016/B978-0-12-416024-8.00009-X
Denker H-W, Gerdes H-J (1979) The dynamic structure of rabbit blastocyst coverings. I. Transformation during regular preimplantation development. Anat Embryol 157:15–34
Lin SP, Lee RK, Tsai YJ (2001) In vivo hatching phenomenon of mouse blastocysts during implantation. J Assist Reprod Genet 18:341–345
Kane MT (1975) Inhibition of zona shedding of rabbit blastocysts in culture by the presence of a mucin coat. J Reprod Fertil 44:539–542
Murakami H, Imai H (1996) Successful implantation of in vitro cultured rabbit embryos after uterine transfer: a role for mucin. Mol Reprod Dev 43:167–170
Koerber S, Santos AN, Tetens F, Küchenhoff A, Fischer B (1998) Increased expression of NADH-ubiquinone oxidoreductase chain 2 (ND2) in preimplantation rabbit embryos cultured with 20% oxygen concentration. Mol Reprod Dev 49:394–399
Navarrete Santos A, Tonack S, Kirstein M, Pantaleon M, Kaye P, Fischer B (2004) Insulin acts via mitogen-activated protein kinase phosphorylation in rabbit blastocysts. Reproduction 128:517–526
Ramin N, Thieme R, Fischer S, Schindler M, Schmidt T, Fischer B, Navarrete Santos A (2010) Maternal diabetes impairs gastrulation and insulin and IGF-I receptor expression in rabbit blastocysts. Endocrinology 151:4158–4167. https://doi.org/10.1210/en.2010-0187
Liu Z, Foote RH, Simkin ME (1996) Effect of amino acids and α-amanitin on the development of rabbit embryos in modified protein-free KSOM with HEPES. Mol Reprod Dev 45:157–162
Bai Y, Wu C, Zhao J, Liu YH, Ding W, Ling WL (2011) Role of iron and sodium citrate in animal protein-free CHO cell culture medium on cell growth and monoclonal antibody production. Biotechnol Prog 27:209–219. https://doi.org/10.1002/btpr.513
Baker E, Baker SM, Morgan EH (1998) Characterisation of non-transferrin-bound iron (ferric citrate) uptake by rat hepatocytes in culture. Biochim Biophys Acta 1380:21–30
Keen MJ, Rapson NT (1995) Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line. Cytotechnology 17:153–163. https://doi.org/10.1007/BF00749653
Graham RM, Morgan EH, Baker E (1998) Characterisation of citrate and iron citrate uptake by cultured rat hepatocytes. J Hepatol 29:603–613
Hanson RW, Ballard FJ (1968) Citrate, pyruvate, and lactate contaminants of commercial serum albumin. J Lipid Res 9:667–668
Krisher RL, Prather RS (2012) A role for the Warburg effect in preimplantation embryo development: metabolic modification to support rapid cell proliferation. Mol Reprod Dev 79:311–320. https://doi.org/10.1002/mrd.22037
Kane MT (1987) In vitro growth of preimplantation rabbit embryos. In: Bavister BD (ed) The mammalian preimplantation embryo: regulation of growth and differentiation in vitro. Plenum, New York, pp 193–217
Close B, Banister K, Baumans V et al (1996) Recommendations for euthanasia of experimental animals: Part 2. Lab Anim 31:1–32
Green CJ (1975) Neuroleptanalgesic drug combinations in the anaesthetic management of small laboratory animals. Lab Anim 9:161–178
Blandau RJ (1961) Biology of eggs and implantation. In: Young WC (ed) Sex and internal secretions, vol II, 3rd edn. The Williams and Wilkins Co., Baltimore, pp 797–882
Kladakis S (2014) Choosing sutures in small animal surgery. J Dairy Vet Anim Res 1:68–71. https://doi.org/10.15406/jdvar.2014.01.00015
Chen PH, White CE (2006) Comparison of rectal, microchip transponder, and infrared thermometry techniques for obtaining body temperature in the laboratory rabbit (Oryctolagus cuniculus). J Am Assoc Lab Anim Sci 45:57–63
Wenger RH, Kurtcuoglu V, Scholz CC, Marti HH, Hoogewijs D (2015) Frequently asked questions in hypoxia research. Hypoxia (Auckl) 3:35–43. https://doi.org/10.2147/HP.S92198
Pursel VG, Wall RJ, Rexroad CE, Hammer RE, Brinster RL (1985) A rapid whole-mount staining procedure for nuclei of mammalian embryos. Theriogenology 24:687–691
Grayson K (1978) An improved method for staining mammalian oocytes. Stain Technol 53:115–116
Giles JR, Foote RH (1995) Rabbit blastocyst: allocation of cells to the inner cell mass and trophectoderm. Mol Reprod Dev 41:204–211
Kennelly JJ, Foote RH (1965) Superovulatory response of pre- and post-pubertal rabbits to commercially available gonadotrophins. J Reprod Fertil 9:177–188
Varian NB, Maurer RR, Foote RH (1967) Ovarian response and cleavage rate of ova in control and FSH-primed rabbits receiving varying levels of luteinizing hormone. J Reprod Fertil 13:67–73
Daniel N, Renard JP (2010) Artificial insemination in rabbits. Cold Spring Harb Protoc 2010:pdb prot5358. https://doi.org/10.1101/pdb.prot5358
Bredderman PJ, Foote RH, Yassen AM (1964) An improved artificial vagina for collecting rabbit semen. J Reprod Fertil 7:401–403
Amann RP, Foote RH (2004) Artificial vagina for rabbits. J Androl 25:184
Flecknell PA, Mitchell M (1984) Midazolam and fentanyl-fluanisone: assessment of anaesthetic effects in laboratory rodents and rabbits. Lab Anim 18:143–146
Alliston CW, Pardee NR (1973) Variability of embryonic development in the rabbit at 19 to 168 hours after mating. Lab Anim Sci 23:665–670
Acknowledgments
Thanks are due to Dr. G.E. Seidel, Jr., Colorado State University, Dr. L.R. Quinlan, National University of Ireland, Galway for very helpful criticism of the manuscript and to Ms. B. Coen, National University of Ireland, Galway for advice on equipment.
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Kane, M.T. (2019). Culture of Preimplantation Rabbit Embryos. In: Herrick, J. (eds) Comparative Embryo Culture. Methods in Molecular Biology, vol 2006. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9566-0_5
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