Highlights
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Non-viable nuclei obtained from an Asiatic cheetah carcass preserved at –20°C were used for iSCNT.
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Cloned cheetah embryos developed in vitro up to the morula stage but then arrested.
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Evidence of embryonic fragmentation and abnormal chromosome segregation was observed in cheetah iSCNT embryos.
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Genomic instability was identified as the main cause of failure to reach the blastocyst stage.
Abstract
Recent advances in somatic cell nuclear transfer (SCNT) have raised great hopes for preserving the genetic resources of endangered animals through ex situ conservation technologies. Most SCNT studies use live donor cells for nuclear transfer into recipient oocytes. However, in certain situations where only frozen tissues of rare animals—without proper cryopreservation—are available, obtaining viable cells is difficult or impossible.
In this study, the feasibility of interspecies somatic cell nuclear transfer (iSCNT) in the Asiatic cheetah (Acinonyx jubatus venaticus) was investigated. Nuclei were isolated from frozen tissue preserved at –20°C without cryoprotectant, and in vitro–matured domestic cat oocytes were used as recipients.
No cell growth was observed from initial cultures of skin and tendon fragments or after enzymatic digestion and single-cell culture. Viability staining revealed that nearly all cells had ruptured membranes and were non-viable. Therefore, nuclei were directly injected into enucleated domestic cat oocytes.
Early signs of nuclear reprogramming were observed 2 hours after iSCNT and significantly increased by 4 hours. The percentages of iSCNT reconstructions that developed to the 4–16 cell and morula stages were 32.3 ± 7.3%, 18.2 ± 9.8%, and 5.9 ± 4.3%, respectively. However, none of the embryos progressed to the blastocyst stage.
In the control group using cat cells and cat oocytes, cleavage rates (51.3 ± 13.9% and 77.3 ± 4.0%) and blastocyst formation (11.3 ± 3.3% and 16.8 ± 3.8%) were comparable to standard results.
Overall, this study demonstrated that domestic cat oocytes could partially reprogram non-viable nuclei from the Asiatic cheetah and return them to early embryonic stages. This is the first report of iSCNT in the Asiatic cheetah using frozen non-viable cells.
Introduction
Earth’s current biodiversity is the product of over 3.5 billion years of evolution. However, in the past century, human activities have caused rapid biodiversity loss, placing many species at the brink of extinction.
While in situ conservation plays a vital role in species survival, new technologies such as SCNT allow scientists to utilize genetic resources to reproduce rare species. To date, SCNT has resulted in successful cloning in more than 20 species.
Cloning an animal species requires appropriate donor cells, recipient oocytes, and surrogate mothers. For extinct or critically endangered species, this is nearly impossible, as live donor cells are unavailable and genomic integrity in frozen tissues is often compromised.
Nevertheless, recent studies have shown that even tissues frozen at very low temperatures (–20°C to –80°C) without cryoprotectant can preserve parts of genomic integrity for years. Moreover, iSCNT has previously been successfully used to produce embryos or offspring in animals such as the wild cat, gray wolf, gaur, mouflon, and Pyrenean ibex.
The Asiatic cheetah (Acinonyx jubatus venaticus), one of the rarest subspecies and the only surviving cheetah in Iran, is currently listed as critically endangered by the IUCN Red List. Its population is estimated to be fewer than 110 individuals, with road accidents accounting for about 40% of deaths.
In this study, tissues were obtained from a cheetah that died in a road accident in Yazd Province on May 27, 2010, and whose carcass had been stored at –20°C for 10 days, in order to assess the feasibility of cloning.
Discussion
This study represents the first attempt to generate interspecies cloned embryos of the Asiatic cheetah in vitro using nuclei extracted from frozen tissue without cryoprotectant. The results showed that although cell division up to the early stages was possible, genomic instability prevented further development to the blastocyst stage.
These findings mark an important step toward understanding the capabilities and limitations of iSCNT in conserving endangered species and provide prospects for future use of this method in preserving the genetic resources of rare animals.
Acknowledgments
The authors dedicate this study to the memory of the late Hosseini Sh., former head of the Yazd Province Department of Environment (who passed away on December 31, 2013), and to all Iranian rangers who sacrifice their lives to protect nature and wildlife.
