Use of soluble sperm extract to improve cloning efficiency in zebrafish
Technical Article | Published: 18 June 2019
Sukumal Prukudom1,2,3, Gloria I. Perez4, Jose B. Cibelli*,5 and Kannika Siripattarapravat*,1,2,3,7
1Center for Agricultural Biotechnology, Kasetsart University, Kamphaengsaen Campus, Nakhonpathom, Thailand, 2Center of Excellence on Agricultural Biotechnology: (AG-BIO/PERDO-CHE), Bangkok, Thailand, 3Center for Advanced Studies for Agriculture and Food, Kasetsart University Institute for Advanced Studies, Kasetsart University, Bangkok, Thailand (CASAF, NRU-KU), 4Institute for Quantitative Health Science and Engineering, Michigan State University, East Lansing, MI, USA, 5Department of Animal Science and 6Large Animal Clinical Sciences Department, Michigan State University, East Lansing, MI, USA and 7Department of Pathology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand
During somatic cell nuclear transfer (SCNT), egg activation is required to initiate embryonic development. In zebrafish cloning, the reconstructed egg is activated by exposing it to hypotonic water. Egg activation using water-only is not capable of activating the same intracellular calcium release as fertilization which is required for proper embryonic development. Here we test whether the use of soluble sperm extract (SSE) can properly modulate the activation of reconstructed eggs during SCNT. We microinjected SSE from genomic-inactivated zebrafish sperm into unfertilized eggs and reconstructed eggs right after somatic cell nuclear transfer. We also evaluated the most effective approach for SSE microinjection. Microinjection of SSE (with 0.68 mg/ml of protein concentration) into non-activated eggs through the micropyle induced parthenogenetic development beyond the blastula stage, whereas all water-only activated eggs failed to enter the cleavage period. Microinjection of SSE at 1 mg/ml of protein concentration into non-activated reconstructed egg improved the developmental rate of cloned embryos in comparison to non-injected control clones. The cumulative survival time of cloned embryos injected with SSE was significantly longer than reconstructed eggs activated following sham injection (P<0.01). No significant difference was found among controls (P=0.32). SSE benefits both parthenogenesis and the survival cloned embryos which have never been reported in zebrafish. Further work is necessary to define the functional component(s) of SSE as well as the physiological pathway, to understand its principle of action and advance the utilization of SSE in cloning.