Reducing the cytoplasmic volume during hand-made cloning adversely affects the developmental competence and quality, and alters relative abundance of mRNA transcripts and epigenetic status of buffalo (Bubalus bubalis) embryos.


Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute, Karnal 132001, Haryana, India. Electronic address: [Email]


Hand-made cloning (HMC) is a method of choice for somatic cell nuclear transfer (SCNT). There is 20% to 50% of cytoplasm lost during manual enucleation of oocytes with HMC. To compensate, two enucleated demicytoplasts, instead of one, are fused with each donor cell, which leads to cytoplasm pooling from two different demicytoplasts. In this study, effects of using one, instead of two demicytoplasts (controls) was examined, for production of embryos using HMC. Use of one demicytoplast decreased blastocyst development (12.7 ± 1.98% compared with 47.6 ± 3.49%, P < 0.001), total cell number (TCN, 167.6 ± 14.66 compared with 335.9 ± 58.96, P < 0.01), apoptotic index (2.11 ± 0.38 compared with 3.43±0.38, P < 0.05) but did not significantly alter inner cell mass:trophectoderm cell number ratio (0.17 ± 0.01 compared with 0.19 ± 0.02) and the global content of H3K9ac and H3K27me3 of blastocysts, compared to controls. There were gene expression alterations in pluripotency- (SOX2 and NANOG but not OCT4), epigenetic- (DNMT1 but not DNMT3a and HDAC1), apoptosis- (CASPASE3 but not BCL-2 and BAX), trophectoderm- (CDX2), development- (G6PD but not GLUT1) and cell cycle check point control-related related genes (P53) compared with controls. Transfer of cloned blastocysts from one demicytoplast (n = 8) to recipients resulted in a live calf birth that after 12 days died whereas, with transfer of control blastocysts (n = 14) there was birth of a healthy calf. In conclusion, use of one, instead of two demicytoplasts for HMC, compromises in vitro developmental competence, and alters expression of several important genes affecting embryo development.


Hand-made cloning,IVF,Nuclear transfer,SCNT,

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