Interspecies nuclear transfer using fibroblasts from leopard, tiger, and lion ear piece collected postmortem as donor cells and rabbit oocytes as recipients

Abstract

Skin fibroblast cells were obtained from a small piece of an ear of leopard, lion, and tiger collected postmortem and attempts were made to synchronize the skin fibroblasts at G0/G1 of cell cycle using three different approaches. Efficiency of the approaches was tested following interspecies nuclear transfer with rabbit oocytes as recipient cytoplasm. Fluorescence-activated cell sorting revealed that the proportion of G0/G1 cells increased significantly (P < 0.05) when cells subjected to serum starvation, contact inhibition, and 3 mM sodium butyrate (NaBu) treatment when compared with cycling cells. However, 3 mM NaBu treatment caused alterations in cell morphology and increase in dead cells. Thus, interspecies nuclear transfer was carried out using fibroblast cells subjected to contact inhibition for 72 h, serum starvation for 48 h, and cells treated with 1.0 mM NaBu for 48 h. The fusion rates, the proportion of fused couplets that cleaved to two-cell and developed to blastocyst, were highest in all three species when the donor cells were treated with 1.0 mM NaBu for 48 h. But, the blastocyst percentage of interspecies nuclear embryos (5-6%) was significantly lower when compared with rabbit-rabbit nuclear transfer embryos (22.9%). In conclusion, fibroblast cells of leopard, lion, and tiger were successfully synchronized and used for the development of blastocysts using rabbit oocytes as recipient cytoplasm.

Applied Biotechnologies in the Conservation of Wild Felids: In Vitro Embryo Production and Cellular Regenerative Therapies

“…This study described that the rabbit oocytes were capable to reprogram big felid somatic cells. However, the blastocyst rate after in vitro culture was 5-6% in the three species [62].…”

Potential role of intraspecific and interspecific cloning in the conservation of wild mammals

“…Finally, the third step in the preparation of the karyoplasts is cell synchronization in the G0/G1 stage (Gómez et al , 2003; Yelisetti et al , 2016). In general, nuclear reprogramming is controlled by epigenetic modification.…”

Use of somatic cell banks in the conservation of wild felids

“…Additionally, the temperature during transport is maintained at 4 °C to avoid dehydration and to preserve the viability of the tissue (Mestre‐Citrinovitz et al, ). Another important parameter is the duration of transport, understanding the time between sample collection and processing in the laboratory is imperative; as such, there are reports of transport duration ranging from 1 (Yelisetti, Komjeti, Katari, Sisinthy, & Brahmasani, ) to 24 hr (León‐Quinto et al, ).…”

“…In another study conducted by Guan et al () using Bengal tiger fibroblasts, the PDT was determined to be 28 hr. Yelisetti et al () used fibroblasts derived from ear fragments of leopard ( Panthera pardus ), lion ( Panthera leo ), and tiger ( Panthera tigres ) collected post‐mortem and measured the PDT to be 26.7, 27.2, and 34.7 hr, respectively, attributing the differences to variations in culture conditions, passage number, and age of the animal.…”

“…The establishment and use of biobanks of wildlife has been central to the development of basic scientific research and is indispensable to the long‐term storage of somatic cells (Comizzoli, ). Many studies that aim to conserve somatic tissues of wild felids are carried out in cooperation with zoos (Hashem et al, ; Thongphakdee et al, ; Verma et al, ; Wittayarat et al, ; Yelisetti et al, ). Studies are being developed to preserve somatic cells of endangered wild felids from various continents (Table ).…”