, Volume 68, Issue 4, pp 593–608 | Cite as

A comparative study on efficiency of adult fibroblasts and amniotic fluid-derived stem cells as donor cells for production of hand-made cloned buffalo (Bubalus bubalis) embryos

  • Sadeesh EMEmail author
  • Meena Kataria
  • Fozia Shah
  • P. S. Yadav
Original Research


The efficiency of two cell types, namely adult fibroblasts, and amniotic fluid stem (AFS) cells as nuclear donor cells for somatic cell nuclear transfer by hand-made cloning in buffalo (Bubalus bubalis) was compared. The in vitro expanded buffalo adult fibroblast cells showed a typical “S” shape growth curve with a doubling time of 40.8 h and stained positive for vimentin. The in vitro cultured undifferentiated AFS cells showed a doubling time of 33.2 h and stained positive for alkaline phosphatase, these cells were also found positive for undifferentiated embryonic stem cell markers like OCT-4, NANOG and SOX-2, which accentuate their pluripotent property. Further, when AFS cells were exposed to corresponding induction conditions, these cells differentiated into osteogenic, adipogenic and chondrogenic lineages which was confirmed through alizaran, oil red O and alcian blue staining, respectively. Cultured adult fibroblasts and AFS cells of passages 10–15 and 8–12, respectively, were used as nuclear donors. A total of 94 embryos were reconstructed using adult fibroblast as donor cells with cleavage and blastocyst production rate of 62.8 ± 1.8 and 19.1 ± 1.5, respectively. An overall cleavage and blastocyst formation rate of 71.1 ± 1.2 and 29.9 ± 2.2 was obtained when 97 embryos were reconstructed using AFS cells as donor cells. There were no significant differences (P > 0.05) in reconstructed efficiency between the cloned embryos derived from two donor cells, whereas the results showed that there were significant differences (P < 0.05) in cleavage and blastocyst rates between the cloned embryos derived from two donor cell groups. Average total cell numbers for blastocyst generated using AFS cells (172.4 ± 5.8) was significantly (P < 0.05) higher than from adult fibroblasts (148.2 ± 6.1). This study suggests that the in vitro developmental potential of the cloned embryos derived from AFS cells were higher than that of the cloned embryos derived from adult fibroblasts in buffalo hand-made cloning.


Buffalo Adult fibroblasts Amniotic fluid stem cells Hand-made cloning Embryos 



The authors would like to thank Dr. Inderjeet Singh, Director, ICAR-Central Institute for Research on Buffaloes, for providing the necessary facilities for carrying out this work. We are also thankful to Dr. B S Punia for reviewing the manuscript. Funding support from Indian Council of Agricultural Research (ICAR), New Delhi is gratefully acknowledged.

Conflict of interest

The authors declare no conflict of interest.


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Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Sadeesh EM
    • 1
    Email author
  • Meena Kataria
    • 2
  • Fozia Shah
    • 3
  • P. S. Yadav
    • 1
  1. 1.Division of Animal Physiology and ReproductionICAR-Central Institute for Research on BuffaloesHisarIndia
  2. 2.Division of BiochemistryICAR-Indian Veterinary Research InstituteBareillyIndia
  3. 3.Department of Veterinary Physiology and BiochemistryLala Lajpat Rai University of Veterinary and Animal SciencesHisarIndia

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