Performing Department
Marine Biotechnology
Non Technical Summary
Aquaculture is becoming increasingly important to resolve the current and projected shortfalls in aquatic food production. Among aquaculture fish species, salmonids are the most valuable. They are considered one of the most nutritious and healthy foods because of their high omega-3 fatty acids, protein, vitamins, and minerals. As such, salmonids are believed to be the most critical drivers for finfish aquaculture and healthy food consumption. Among salmonids, Atlantic salmon is the most popular and consumed fish in the US.At present, over 99% of Atlantic salmon aquaculture production is from ocean net-pens worldwide. However, the limited US production of Atlantic salmon in coastal pens continues to be overshadowed by federal and state regulations and loss of social acceptance, preventing its domestic growth. Therefore, it is becoming increasingly evident that alternative farming systems must be used for Atlantic salmon aquaculture to expand in the US. Land-based recirculating aquaculture system (RAS) technology is a viable and sustainable means to expand US Atlantic salmon production. It provides the ability to capture and treat waste effectively, enhances biosecurity to prevent fish escapes, minimizes pathogen entry, offers optimal conditions to maximize fish performance, and increases flexibility in site selection. Recently, over $2.5 billion has been announced to invest in RAS Atlantic salmon production throughout the US. However, for the RAS Atlantic salmon industry to be thriving in the US, it must be continuously optimized and overcome biological and technological hurdles.Early sexual maturation is one of the significant challenges in farmed Atlantic salmon. It was estimated at 20-30% in open-water farming practices. Shockingly, precocious male maturation has been reported as high as 80% by harvest time in land-based, closed-containment grow-out systems. Sexual maturation is associated with a substantial decrease in muscle growth due to the diversion of energy into the development of the gonads. It results in deterioration of flesh quality and an increase in susceptibility to stress and disease. Early maturation in RAS-grown salmon can have significant biological and business consequences. Using an all-female population is a promising approach for preventing early maturation in RAS farming as most grilsing has been associated with males. Currently, the only commercial available all-female Atlantic salmon seed production relies on using a male steroid (17α-methyltestosterone ) to reverse females (carrying XX sex chromosomes) to males that produce all-X sperm. Due to regulatory hurdles and the increasing concern of 17α-methyltestosterone releasing into the environment, alternative approaches to produce an all-female population are needed. Hence, we propose an alternative approach that employs fish sterilization and germ cell transplantation technology to generate the all-X milt for all-female salmon production. By transplanting female germ cells (carrying XX sex chromosomes) to sterile male gonads, all-X sperm can be produced in the recipients and used for an all-female production without the need of using steroids.For a commercial application to achieve cost-effective operation, the germ-cell-transplantation technology needs to be further optimized to achieve a higher success rate. As such, we will study and optimize this technology by 1) systematically investigating the candidate factors for improving fish ovarian-germ-cell transplantation; 2) verifying and optimizing the improved conditions in transplantation; 3) producing all-male sterile rainbow trout to carry Atlantic salmon ovarian germ cells and produce all-X sperm successfully. It is known that male Atlantic salmon generally reach sexual maturity at age 2 to 3 years, while male rainbow trout mature at age 1 to 2 years. Thus, the generation time for Atlantic salmon all-X milt production can be shortened through the use of trout recipients.In salmonids, substantial investigation of the factors that influence germ cell transplantation and optimizing the conditions that can enhance transplantation efficiency is very limited due to their seasonal availability of embryos and a relatively long generation time. Therefore, we will use fish species that can spawn year-round and have a short generation time for this objective. Zebrafish and Nile tilapia are considered great models for this purpose since they breed year-round and have relatively short generation time, and well-established methodologies will help the optimization of ovarian germ cell transplantation. The optimized conditions achieved in zebrafish and tilapia transplantation works will be evaluated and modified in rainbow trout and Atlantic salmon germ cell transplantation. We will establish an optimal salmonid germ cell transplantation SOP for all-female Atlantic salmon production in a commercial setting.The valuable outcomes of this research will help overcome the restriction of using 17α-methyltestosterone to reduce or even eliminate the use of sex steroids for all-female production, which will support aquaculture development toward more environmentally responsible and sustainable.
Animal Health Component
0%
Research Effort Categories
Basic
40%
Applied
60%
Developmental
(N/A)
Goals / Objectives
In farmed fish, sexual maturation is associated with a substantial decrease in somatic growth due to the diversion of energy into the development of the gonads. It results in deterioration of flesh quality and an increase in susceptibility to stress and disease. Shockingly, much higher (80%) precocious male maturation has been reported in land-based recirculating aquaculture systems (RAS) compared to other farming systems (20-30%). As RAS technology is a viable and sustainable means to expand US Atlantic salmon production, early maturation in RAS-grown salmon can have significant biological and business consequences. Using an all-female population is a promising approach for preventing early maturation in RAS farming as most grilsing has been associated with males. However, due to the increasing concern of 17α-methyltestosterone, the steroid used to indirectly produce all-female population, released into the environment, no permit is issued to US commercial Atlantic salmon egg producers despite the rising demands. US growers will continuously rely on foreign all-female seed suppliers unless alternative approaches to produce an all-female population are developed.This proposed research intends to study and optimize germ-cell-transplantation technology for a commercial application to produce all-female Atlantic salmon without using steroids. To utilize germ-cell-transplantation technology for commercial seed production, enough recipients carrying donor-derived gametes should be obtained repetitively. However, due to unstable transplantation efficiency, research and development are needed to optimize this technology to be cost-effective. The goal of this proposed research will be achieved by three specific objectives: 1) Investigation of candidate factors for improving ovarian germ cell transplantation efficiency in zebrafish and tilapia; 2) Verification and optimization of the improved conditions obtained from Objective 1 in ovarian germ cell xenotransplantation between Atlantic salmon and rainbow trout; 3) Further optimization of germ cell transplantation to produce more successful male rainbow trout recipients for a commercial-scale all-female Atlantic salmon production.By carrying out these three objectives, we expect to improve the ovarian germ cell transplantation technology to double the success rate to 60% or more compared to the current success rates at 15% to 30%. The outcomes of this proposed research can also be applied to a wide variety of aquaculture species to produce monosex populations without using sex steroids.
Project Methods
Objective 1. Investigation of candidate factors for improving ovarian germ cell transplantation efficiency in zebrafish and tilapiaOvaries from 3-moth-old female zebrafish will be removed and cut into small pieces in L-15 medium (L15) containing 0.2% collagenase. The dissociated cells will be subjected to enrichment procedures, including Percoll density gradient centrifugation (PC), differential plating (DP), or the combination of both methods (PC+DP) to obtain enriched ovarian cell populations.FITC-conjugated Dnd-MO and DsRed-nanos3 3'UTR mRNA will be co-injected into zebrafish embryos at 1-2 cell stage to eliminate germ cells by Dnd knockdown and PGC labeling. The hatched larvae that do not carry DsRed-expressing cells in the gonadal region at 3 pdf will be selected as the sterile recipients for the transplantation. Transparent Casper zebrafish strain due to deficient Nacre and Roy will be used as recipients for zebrafish germ cell transplantation. About 200 nL of cell suspension containing 3,000 cells will be transplanted into the peritoneal cavity of the larva using the glass needle. The transplantation efficiency will be examined by incorporation, colonization, and proliferation of donor cells in the gonads at 5 and 30 days post-transplantation (dpt).The effects of various supplements such as growth factors (Fgf2 or Igf1), small molecules, fish serum, embryo extract, and others that have beneficial effects in promoting germ cell survival and proliferation and inhibiting differentiation will be studied. Transplantation assays will be conducted following incubation (16 h) of ovarian cells with those supplements at different concentrations. Subsequently, the mixture of supplements that showed positive effects at the specific concentration will be tested through transplantation assay and optimized to identify the effective growth factors at a defined concentration. In addition, receptors involved in germ cell migration such as Cxcr4b and Igfr1b will be studied by mRNA transfection of the ovarian cells.When the recipients reach sexual maturity, milt will be collected from each male zebrafish recipient and analyzed by PCR using DsRed2 and EGFP specific primers to screen recipients harboring donor-derived sperms. F1 offspring produced by crossing recipients and Casper will be observed under a fluorescence microscope to detect DsRed2 and EGFP expression. Through these steps, the effects of each optimization method will be evaluated, and the most effective three conditions will be selected from zebrafish works and evaluated in the tilapia model.Tilapia ovarian germ cells from orange Mozambique tilapia will be used for transplantation. The enriched ovarian germ cells will be labeled with PKH26 dye following the manufacture instruction prior to transplantation. FITC-conjugated Dnd-MO and DsRed-nanos3 3'UTR mRNA will be co-injected into tilapia embryos to select sterile recipients. About 400 nL of cell suspension containing 6,000 cells will be transplanted into the peritoneal cavity of the larva. The recipients will be examined by incorporation, colonization, and proliferation of donor cells in the gonads at 5 and 30 dpt.The three optimal conditions obtained from zebrafish works on supplements and receptor activation will be tested in tilapia. Once recipients are mature, milt will be collected from male recipients and analyzed by PCR using species-specific markers to screen recipients harboring donor-derived sperms. Positive male recipients will be bred with non-transplanted orange Mozambique tilapia females to examine the donor-derived functional gamete production.Objective 2. Verification and optimization of the improved conditions obtained from objective 1 in ovarian germ cell xenotransplantation between Atlantic salmon and rainbow troutOvarian germ cells will be harvested from immature female Atlantic salmon by the selected enrichment methods and optimized conditions achieved in zebrafish, and tilapia transplantation works. The optimal condition of including growth factor, small molecules, embryo extract and/or serum treatments, and the Igfr1b and/or Cxcr4b mRNA transfection obtained from zebrafish and tilapia works will be used to treat Atlantic salmon ovarian germ. These cells will then be labeled with PKH26 dye before transplantation.Rainbow trout eggs will be 1) immersed with Dnd-MO-Vivo using our optimal bath-immersion method that reaches 84% sterility induction and then fertilized and injected with DsRed-nanos3 3'UTR mRNA at 1-cell stage; 2) fresh-fertilized rainbow trout eggs at 1-cell stage will be co-injected with FITC-conjugated Dnd-MO and DsRed-nanos3 3'UTR mRNA; or 3) the combination of 1 and 2 procedures to produce sterile recipients. The newly hatched larvae that do not carry DsRed-expressing PGCs will be selected and used as sterile recipients. Although donor-derived gamete can be easily distinguished from recipient endogenously derived gamete (rainbow trout, the failure of sterility induction in the recipients) by PCR screening, the successful and high-efficiency sterility induction in rainbow trout recipients is one of the crucial methods for promoting germ cell transplantation technology.Around 10,000 PKH26-staining ovarian germ cells will be transplanted into the peritoneal cavity of each recipient using a glass needle. At 20 and 50 dpt, the incorporation, colonization, and proliferation of donor cells in the recipient gonads will be investigated. At least 30 male recipients from each transplantation will be grown and examined donor oogonia-derived gamete production in rainbow trout recipients.Objective 3. Further optimization of germ cell transplantation to produce more successful male rainbow trout recipients that can be used for a commercial-scale all-female Atlantic salmon productionIn this objective, we will improve the productivity of successful male rainbow trout carrying Atlantic salmon oogonia-derived all X milt by increasing the number of transplanted germ cells and using all-male rainbow trout larvae as sterile recipients for ovarian germ cell xenotransplantation.In addition to 10,000 cells/recipient transplantation, we will maximize the number of transplanted cells (30,000 or 50,000) per recipient and examine the efficiency of the success rate. To increase the number of transplanted ovarian germ cells, we will spin the glass needles at 250 g for 1 min to pack the cells into the tips of the needles. We found that this approach can reduce the medium volume and enable more cells to be transplanted into the peritoneal cavity.Allotransplantation in rainbow trout will be carried out to prepare the all-male rainbow trout recipients. 50,000 testicular cells harvested from the immature males (less than 30 g) will be transplanted into each sterile rainbow trout larvae prepared as described above. The mature female recipients that carry donor-derived eggs possess either X or Y chromosome. After insemination with the milt from regular male rainbow trout, 25% of F1 offspring are expected to be YY males separated from XY males by an established qPCR-based method using fin genomic DNA. Subsequently, sperms from YY male rainbow trout will be inseminated with regular eggs from female rainbow trout to generate the all-male recipients.Atlantic salmon ovarian germ cells will be transplanted into the all-male sterile rainbow trout recipients. Photoperiod and temperature manipulation will be applied to induce early maturation. The verified milt carrying only Atlantic salmon X/X sperms will be inseminated with regular eggs of Atlantic salmon. After fertilization, the sex ratio of F1 will be examined by genomic DNA PCR analysis using Y chromosome-specific primers and gonadal histology. The successful production of all-female Atlantic salmon via ovarian germ cell xenotransplantation between Atlantic salmon and rainbow trout without steroids will be confirmed and established.