Successful cloning of a superior buffalo bull

Abstract

Somatic cell nuclear transfer (SCNT) technology provides an opportunity to multiply superior animals that could speed up dissemination of favorable genes into the population. In the present study, we attempted to reproduce a superior breeding bull of Murrah buffalo, the best dairy breed of buffalo, using donor cells that were established from tail-skin biopsy and seminal plasma. We studied several parameters such as cell cycle stages, histone modifications (H3K9ac and H3K27me3) and expression of developmental genes in donor cells to determine their SCNT reprogramming potentials. We successfully produced the cloned bull from an embryo that was produced from the skin-derived cell. Growth, blood hematology, plasma biochemistries, and reproductive organs of the produced cloned bull were found normal. Subsequently, the bull was employed for semen production. Semen parameters such as CASA (Computer Assisted Semen Analysis) variables and in vitro fertilizing ability of sperms of the cloned bull were found similar to non-cloned bulls, including the donor bull. At present, we have 12 live healthy progenies that were produced using artificial insemination of frozen semen of the cloned bull, which indicate that the cloned bull is fertile and can be utilized in the buffalo breeding schemes. Taken together, we demonstrate that SCNT can be used to reproduce superior buffalo bulls.

Figure 1

Culture and characterization of skin- and semen-derived somatic cells of the buffalo breeding bull. Skin-derived cells have spindle-shaped morphology; whereas, semen-derived cells have polygonal-shaped morphology and form a flat monolayer (A), both the type of cells were used to produce cloned embryos that developed to blastocysts (A). Skin-derived cells expressed vimentin, a fibroblast cell type marker; whereas, semen-derived cells expressed cytokeratin, an epithelial cell type marker, which were confirmed by mRNA expression using PCR (B) and immunofluorescence staining (C). Semen-derived epithelial cells have lower cell proliferation than that of skin-derived fibroblast cells (D). Cell images were captured at 100X total magnification; whereas, blastocyst images were captured at 40X total magnification.

Figure 2

Distribution of cell cycle stages of donor cells, and mRNA expression of some developmental genes in donor cells and embryos. Somatic cells were cultured under identical culture conditions such as the same culture media, culture time, and culture surface. Cell cycle stages distribution of skin-derived somatic cells and semen-derived somatic cells (A), mRNA expression of epigenetic-related genes (HDAC1, DNMT1, and DNMT3a), apoptosis-related genes (Caspase 3 and Caspase 9) in both cell types (B). mRNA expression of pluripotency genes (OCT 3/4, SOX2, NANOG, and CDX2) in cloned blastocysts and IVF embryos (C). mRNA expression of the skin group was set to 1 in the case of somatic cells; whereas, the IVF embryo group was set to 1 for embryos. Bars with different superscript letters are significantly different (P < 0.05).

Figure 3

Epigenetic staining of somatic cells and their respective cloned embryos. Mean pixel intensity of histone H3 lysine 9 acetylation (H3K9ac) and Histone H3 lysine 27 trimethylation (H3K27me3) in somatic cells (A,B) and their respective cloned blastocysts (C,D), which was determined by the confocal microscopy. Somatic cell images were captured at 400X total magnification; whereas, blastocysts images were captured at 100X total magnification. Bars with different superscripts are significantly different (P < 0.01).

Figure 4

Donor bull and its cloned. Cloned bull has no physical abnormalities on the body, including face. White tail-switch color mark is identical to its donor bull (indicated by arrow).