Tiger, Bengal and Domestic Cat Embryos Produced by Homospecific and Interspecific Zona‐Free Nuclear Transfer

Abstract

The aim of this study was to evaluate three different cloning strategies in the domestic cat (Felis silvestris) and to use the most efficient to generate wild felid embryos by interspecific cloning (iSCNT) using Bengal (a hybrid formed by the cross of Felis silvestris and Prionailurus bengalensis) and tiger (Panthera tigris) donor cells. In experiment 1, zona-free (ZP-free) cloning resulted in higher fusion and expanded blastocyst rates with respect to zona included cloning techniques that involved fusion or injection of the donor cell. In experiment 2, ZP-free iSCNT and embryo aggregation (2X) were assessed. Division velocity and blastocyst rates were increased by embryo aggregation in the three species. Despite fewer tiger embryos than Bengal and cat embryos reached the blastocyst stage, Tiger 2X group increased the percentage of blastocysts with respect to Tiger 1X group (3.2% vs 12.1%, respectively). Moreover, blastocyst cell number was almost duplicated in aggregated embryos with respect to non-aggregated ones within Bengal and tiger groups (278.3 ± 61.9 vs 516.8 ± 103.6 for Bengal 1X and Bengal 2X groups, respectively; 41 vs 220 ± 60 for Tiger 1X and Tiger 2X groups, respectively). OCT4 analysis also revealed that tiger blastocysts had higher proportion of OCT4-positive cells with respect to Bengal blastocysts and cat intracytoplasmic sperm injection blastocysts. In conclusion, ZP-free cloning has improved the quality of cat embryos with respect to the other cloning techniques evaluated and was successfully applied in iSCNT complemented with embryo aggregation.

Isolation of Primary Fibroblast Culture from Wildlife: the Panthera onca Case to Preserve a South American Endangered Species

“…Therefore, the Biobank of Genetic Resources of Buenos Aires Zoo (BRG) acquires, processes, stores, uses and supplies sperm, oocytes, embryos, tissues and cells such as fibroblasts to conserve wildlife that is viable and available for research. The applications developed so far with fibroblasts from the biobank created with the protocols described here are related to cell cloning (Moro et al., ; Moro et al., ,b). These and other applications in artificial insemination, in vitro fertilization, intracytoplasmic sperm injection, embryo transfer, genetic diversity of cellular metabolism (Holt et al., ; Wildt et al., ; Watson and Holt, ; Roldan and Garde, ) allow the exchange of genetic material between wild and captive populations, aiding inbreeding and avoiding loss of genetic variability.…”

The potential and pitfalls of de‐extinction

“…Mitochondria can also be added by using the cell fusion method for importation of the nucleus (Moro et al . ).…”

“…More promising, however, is that although a further possible conflict is between mitochondria and nucleus (Hiendleder et al 2004), working cells have been created using depletion of mitochondria in the oocyte and addition of mitochondria from the same species as the nuclear DNA (Jiang et al 2011). Mitochondria can also be added by using the cell fusion method for importation of the nucleus (Moro et al 2015).…”

ARTs in wild felid conservation programmes in Poland and in the world

“…The fact that cloning requires only a tiny skin section to establish fibroblast lines that provide the source of genetic material is an unquestionable advantage of this technique. When it comes to recipient cells for nuclei, there are two choices: either oocytes of the same species can be used, or if there is a shortage of such cells from valuable species, oocytes obtained from the domestic cat or other species can be applied (cross-species cloning) (11, 27).…”