Int. J. Dev. Biol. 63: 287 - 293 (2019)

https://doi.org/10.1387/ijdb.180367ks

Vol 63, Issue 6-7

Use of soluble sperm extract to improve cloning efficiency in zebrafish

Technical Article | Published: 18 June 2019

Sukumal Prukudom^1,2,3, 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

Abstract

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.

Keywords

zebrafish, soluble sperm extract, somatic cell nuclear transfer, cloning