Production of reproductively sterile Atlantic salmon for environmentally-responsible aquaculture in Norway

Innovation of Technology

Working together with Professor Yonathan Zohar and Dr. Ten-Tsao Wong at the University of Maryland, Baltimore County (UMBC), ACD Pharma has developed a simple and efficient bath-immersion technology to produce reproductively sterile fish. This non-GMO technology is based on short-term “silencing” of the genes that are responsible for the development of the very early gonads leading to fish without ovaries, testes or gametes.

We discovered that a molecular transporter known as Vivo, can effectively carry a silencing agent (Morpholino oligomer, MO) across the chorion of the eggs, enter the embryos and reach the target cells. Vivo-conjugated MO against deadend (dnd-MO-Vivo), an essential gene for fish primordial germ cell (PGC) development [1, 2], effectively disrupted PGC development, which led to the elimination of germ cells and resulted in the production of reproductively sterile fish without affecting any other physical characteristics of the fish. Since sterility is inducible by bath-immersion, this technology makes it convenient to produce a fertile broodstock population (i.e., skip the treatment) when required.

Figure 1. dnd-MO-Vivo induced sterility in zebrafish. No difference in (A) appearance or (B) body weight was observed between dnd-MO-Vivo treated adult fish and control males. Examination of (C) gonadal tissue showing a fully-developed testis of a control male, (D) a fully-developed ovary of a control female, and (E) the gonads of dnd-MO-Vivo treated fish that developed into a thin filament-like tissue. Photomicrographs (F–H) show (F) advanced spermatogenesis in the testis of a control male, (G) a well-developed ovary of a control female with oocytes at advanced stages of gametogenesis, (H) the gonad of dnd-MO-Vivo treated fish, which appears to be under-developed and surrounded with a large amount of adipocytes, without advanced gonadal structure or germ cells.


Figure 2. dnd-MO-Vivo treatment induced infertility in rainbow trout. A well-developed testis (A) of an untreated male fish; A well-developed ovary (B) of an untreated female fish; The gonads (C) of dnd-MO-Vivo treated fish developed into a thin filament-like tissue. Photomicrograph of dissected gonads (D). Active spermatogenesis in the testis (E) of an untreated male fish; A well-developed ovary (F) of an untreated female with oocytes at different developmental stages; Histological examinations of gonadal tissue (G) showed that the gonad of a treated fish that appears to be under-developed and surrounded by a large amount of adipocytes without advanced gonadal structure or germ cells.


This non-GMO technology was first developed and optimized in zebrafish (Fig. 1) and has been subsequently tested in salmonids. Our initial results indicated that the technology can be used to produce reproductively sterile rainbow trout (Fig. 2) and Atlantic salmon (Fig. 3). Since Dnd is highly conserved in fish and its essential role in PGC development has been demonstrated in multiple species of fish [2-4], it is expected that the technology can be successfully applied to a wide range of aquaculture species, which will also benefit aquaculture in general and globally. In addition to developing Vivo, we also discovered a new molecular transporter that can enhance the uptake of dnd-MO without the need to conjugate to dnd-MO. This discovery is expected to significantly reduce the cost of the technology. Our approach is innovative since it is the first time that a non-GMO gene silencing technology has been shown to have the potential to produce reproductively sterile fish to prevent breeding of escapees and alleviate the problem of early maturation in the industry. Moreover, our approach can be seamlessly integrated into the Atlantic salmon egg production process by simply adding the silencing compounds before or at the time of fertilization. The technology that we developed so far has been filed as two separate patent applications which are now pending approval.

Figure 3. dnd-MO-Vivo treatment blocked gonadal development in 10-month-old Atlantic salmon. (A) A developing testis of an untreated control male; (B) A developing ovary of an untreated control female; (C) The gonads of Salmonidae’s dnd-MO-Vivo treated fish developed into a thin filament-like tissue. (D) Photomicrograph of dissected gonads from each fish. (E) Active spermatogenesis in the testis of an untreated male fish; (F) A developing ovary of an untreated female with oocytes at different developmental stages; (G) Histological examinations of gonadal tissue show the gonad of a Salmonidae’s dnd-MO-Vivo treated fish appears to be under-developed, consisting of connective tissue with no germ cells.


Research ethics

All of the experimental procedures and protocols described in this study were approved by the University of Maryland Animal Care and Use Committee and adhere to the National Research Council’s Guide for Care and Use of Laboratory Animals.

Role and competence of each partner in project

Drs. Zohar and Wong at UMBC are exceptionally well positioned to collaborate and carry out this work. Drs. Zohar’s and Wong’s research has focused on biotechnology development towards the manipulation of fish breeding and reproduction, with extensive basic and applied research on salmonid species. They are also the sole inventors of the inducible sterilization technology described herein. Furthermore, in UMBC’s on-site Aquaculture Research Center (ARC), the Department of Marine Biotechnology (DMB) maintains extensive fish-holding facilities, including large and small tanks, egg incubation trays, quarantine tanks, etc, that are available for this research.

References

1. Weidinger, G., et al., dead end, a novel vertebrate germ plasm component, is required for zebrafish primordial germ cell migration and survival. Curr Biol, 2003. 13(16): p. 1429-34.
2. Slanchev, K., et al., Development without germ cells: the role of the germ line in zebrafish sex differentiation. Proc Natl Acad Sci U S A, 2005. 102(11): p. 4074-9.
3. Fujimoto, T., et al., Sexual dimorphism of gonadal structure and gene expression in germ cell-deficient loach, a teleost fish. Proc Natl Acad Sci U S A, 2010. 107(40): p. 17211-6.
4. Goto, R., et al., Germ cells are not the primary factor for sexual fate determination in goldfish. Dev Biol, 2012. 370(1): p. 98-109.

Publications

Wong, T.T & Zohar, Y. (2015). Production of reproductively sterile fish by a non-transgenic silencing technology. Scientific Reports 5: 15822