Purpose: Bovine embryos and zygotes are known to be sensitive to “temperature shock” when cooled to temperatures near 0°C. The effect of chilling on in vitro derived embryos at various cleavage stages was investigated.
Methods: Cumulus-oocyte-complexes (COCs) were matured in IVM medium with serum. Presumptive zygotes were cultured in serum free in vitro culture (IVC) medium. Embryos were used as chilled or control samples at the 2-cell, 4-cell, 8-cell, morula, and blastocyst stages. Embryos in 0.2 mL PBS in plastic straws were cooled rapidly in ethanol baths at 0°C for 30 min. Embryo viability was assessed by in vitro development.
Results: The percentage of control embryos that hatched as blastocysts increased the later stage at which they were selected. Relative proportion of embryos increased from 28% to 48% to 68% when chilled at the 8-cell, morula or blastocyst stages.
Conclusions: IVF-produced embryos are differentially susceptible to cooling injury. Cell counts made of those blastocysts formed from chilled embryos indicated subtle effects of chilling.
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REFERENCES
Leibo SP: Cryobiology-Preservation of mammalian embryos. In Genetic Engineering of Animals, JW Evans and Hollaender A (eds.), New York, Plenum, 1986; pp. 251–272
Gordon I: Laboratory Production of Cattle Embryos, 1st edn. UK, CAB International Wallingford Oxon, 1994; pp. 295–297
Watson PF, Morris GJ: Cold shock injury in animal cells. Symp Soc Exp Biol 1987;41:311–340
Morris GJ, Watson PF: Cold shock injury—A comprehensive bibliography. CryoLetters 1984;5:352–372
Mazur P, Schneider U, Mahowald P: Characteristics and Kinetics of subzero chilling injury in Drosophila embryos. Cryobiology 1992;29:39–68
Zhang T, Rowson DM: Studies of chilling sensitivity of Zebra fish (Brachydanio rerio) embryos. Cryobiology 1995;32: 239–246
Gordon I: Laboratory Production of Cattle Embryos, 2nd edn. UK, CAB International Wallingford Oxon, 2003; pp. 277
Kasai M: Nonfreezing technique for short-term storage of mouse embryos. J. In Vitro Fert Embryo Transf 1986;3: 10–14
Leibo SP, Winniger D: Production of bovine pregnancies from embryos transported at 0°C by air. Theriogenology 1986;25:165 (Abstrract)
Leibo SP, Loskutoff NM: Cryobiology of in vitro derived bovine embryos. Theriogenology 1993;39:81–94
Trounson AO, Willadsen SM, Rowson LEA: The storage of cows eggs at room temperature and at low temperatures. J Reprod Fertil 1976a;146:173–178
Trounson AO, Willadsen SM, Rowson LEA: The influence of in vitro culture and cooling on the survival and development of cow embryos. J Reprod Fertil 1976b;47:367–370
Richards DW, Sibes JD, Murphy CN: Non surgical transfer and survival of frozen-thawed bovine embryos supplemented with raffinose. Theriogenology 1988;21: 138–149
Mohr LR, Trounson AO: Structural changes associated with freezing of bovine embryos. Biol Reprod 1981;25:1009–1025
Pollard JW, Leibo SP: Chilling sensitivity of mammalian embryos. Theriogenology 1994;41:101–106
Rieger D, Grisart B, Semple E, Van Langendonckt A, Betteridge KJ, Dessy F: Comparison of effects of oviductal cell co-culture and oviductal cell-conditioned medium on the development and metabolic activity of cattle embryos. J Reprod Fertil 1995;105:91–98
Hoshi H: In vitro production of bovine embryos and their application for embryo transfer. Theriogenology 2003;59:675–685
Looney CR, Westhusin ME, Bondioli KR: Effect of cooling temperatures on precompacted bovine embryos. Theriogenology 1989;31:218 (Abstract)
Enders AC: The fine structure of the blastocyst. In The Biology of the Blastocyst, Blandau RJ (ed), Chicago, The University of Chicago Press, 1971; pp. 71– 94
Norberg HS: Ultrastructural aspects of the pre-attached pig embryo: cleavage and early blastocyst stages. Zeitschrift für Anatomische Entwicklungsgeschichte 1973;143:95–114
Hasler JF, Henderson WB, Hurtgen PJ, Jin ZQ, McCauly AD, Mower SA: Production, freezing and transfer of bovine IVF embryos and subsequent calving results. Theriogenology 1995;43:141–152
Ushijima H, Yamakawa H, Nagashima H: Cryopreservation of bovine pre-morula stage in vitro maturation/in vitro fertilized embryos after delipidation and before use in nuclear transfer. Biol Reprod 1999;60:535–539
Wilmut I, Polge C, Rowson LEA: The effect on cow embryos of cooling to 20°, 0° and −196°C. J Reprod Fertil 1975;45:409–411
Martino A, Pollard JW, Leibo SP: Effect of chilling bovine oocytes on their developmental competence. Mol Reprod Dev 1996;45:503–512
Abe H, Hoshi H: Evaluation of bovine embryos produced in high performance serum-free media. J Reprod Dev 2003;49:193–202
Nagashima H, Kashiwazaki N, Ashman RJ, Grupen CG, Nottle MB: Cryopreservation of porcine embryos. Nature 1995;374:416
Papaionnou VE, Ebert KM: Development of fertilized embryos transferred to oviducts of immature mice. J Reprod Fertil 1986;76:603–608
Wurth YA, Van der zee-kotting WI, Dieleman SJ, Bevers MM, ThAM. Kruip. Presence of mitotic cells: A parameter of embryo quality. In Bovine Embryo Production In Vitro Influencing Factors. 1994; pp. 27–36
Shea BF: Evaluating the bovine embryo. Theriogenology 1981;15:31–42
ACKNOWLEDGMENTS
The first author is grateful to Dr. S. P. Leibo, Ph.D., Research Professor, Animal Biotechnology Embryo Laboratory, Department of Biomedical Sciences, University of Guelph, Ontario, Canada N1G 2W1 for the Post-doctoral position to pursue this research and to the authorities of Tamil Nadu Veterinary and Animal Sciences University, Madhavaram Milk Colony, Chennai 600 051, India for financial support under Agricultural Human Resources Development Project.
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Balasubramanian, S., Rho, GJ. Effect of chilling on the development of in vitro produced bovine embryos at various cleavage stages. J Assist Reprod Genet 23, 55–61 (2006). https://doi.org/10.1007/s10815-005-9018-9
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DOI: https://doi.org/10.1007/s10815-005-9018-9