Date Published: April 17, 2019
Publisher: Public Library of Science
Author(s): Fariba Moulavi, Sayyed Morteza Hosseini, Peter J. Hansen.
In this study, a modified method of handmade cloning (m-HMC), which had been originally developed in sheep, was used for somatic cell nuclear transfer (SCNT) in the dromedary camel. The unique feature of m-HMC over current SCNT methods lies in the use of a simple device (a finely drawn micropipette made of Pasteur pipette) for chemically-assisted enucleation of oocytes under a stereomicroscope with improved efficiency and ease of operation. Using this system, the throughput of cloned embryo reconstitution was increased over 2-fold compared to the control SCNT method (c-NT). Stepwise measurement of reactive oxygen species (ROS) revealed that method, steps, and duration of SCNT all influenced oxidative activity of oocytes, but their impact were not similar. Specifically, UV-assisted oocyte enucleation was identified as the major source of ROS production, which explained significantly higher total ROS of reconstituted embryos in c-NT compared to m-HMC. Fusion efficiency (95.3±3.3 vs. 75.4±7.6%) and total efficiency of blastocyst development (22.5±3.0 vs. 14.1±4.3%) were significantly higher in m-HMC compared to c-NT, respectively, and blastocysts of transferable quality were obtained in similar rates (41.9±8.2 vs. 48.0±15.2%, respectively). Significance differences were observed in total cell number (155.3±13.6 vs. 123.6±19.5) and trophectoderm (145±9.5 vs. 114.3±15.2), but not inner cell mass (10.3±4.1 vs. 9.3±5.3) counts between blastocysts developed in c-NT compared to m-HMC, respectively. However, expression of key developmental genes (POU5F1, KLF4, SOX2, MYC, and CDX2) was comparable between blastocysts of both groups. The introduced m-HMC method might be a viable approach for efficient production of dromedary camel clones for research and commercial utilization.
Animal cloning by somatic cell nuclear transfer (SCNT) has been the subject of much attention in recent years and as a result, several mammalian species have been cloned . Somatic cell cloning in camelids, however, has proven an especially inefficient technology due to the biological and technical problems and the birth of cloned camels has still only been reported from one laboratory . High genetic merit dromedary camels (Camelus dromedarius) with exceptional capacities in racing and in production of milk and meat are very valuable genetic resources to regenerate particular populations of camels . Hence, it is expected that the demand for large scale production of cloned dromedary offspring for both research and commercial utilizations will increase. To meet this increasing demand, the efficiency and throughput of camel somatic cell cloning should be improved. The SCNT method used in camels is the original method introduced by Willadsen  in spite of its known disadvantages including expensive equipments, high manipulation skills, and low throughput and efficiency [5–8].
Unless otherwise specified, all chemicals and media were obtained from Sigma Chemical Co. (St. Louis, MO, USA) and Gibco (Grand Island, NY, USA), respectively. The full experimental procedures were performed in accordance with the government of United Arab Emirates’ animal care and use guidelines.
This study describes the first application of a modified method of handmade cloning (we called it “m-HMC”) in dromedary camel. Although the ultimate efficiency in production of clone offspring remains to be evaluated, the established technique significantly increased the throughput of camel cloned blastocyst production in a direct comparison to the standard zona-intact method of cloning (c-NT). In particular, the redox status of reconstituted oocytes and the overall efficiency of cloned blastocyst production were both significantly improved using m-HMC method, although the overall percentage of transferable embryos remained unchanged.
Currently, somatic cell cloning in camelids has proven an especially inefficient technique and successful cloning of dromedary camel has still only been reported from one laboratory using in vivo matured oocytes . The search for biological causes underlying low cloning efficiency is confounded by technical aspects of the procedure. Our data suggest that the new version of m-HMC procedure may be a viable alternative to the current available methods for production of cloned blastocyst. Specifically, low costs, simplicity and high throughput and efficiency may be particular advantages this technique in comparison to available methods. Although the ultimate efficiency of this technique has been confirmed by the birth of healthy cloned goat and sheep, our current focus is for a broader application of cloning technology in dromedary camel for both research and commercial utilizations.