Performing Department
(N/A)
Non Technical Summary
Our long-term goal is to improve production traits of farmed catfish through transgenesis and to accomplish reversible genetic sterility in fish, specifically catfish, to accomplish absolute reproductive confinement, preventing establishment of transgenic genotypes in the natural environment. Such a technology could be used in a variety of fish including any domestic genotype, interspecific hybrid, transgenic or exotic, to minimize impacts on the natural environment, protect genetic biodiversity and ecosystems, increasing environmental friendliness of aquaculture and transgenic fish. The successful culmination of the project would result in a system that would prevent any gene flow of transgenes or domestic genes into wild populations.We have produced transgenic fish possessing multiple insertions of transgeneswhile mutating other genes. One strategy includes the mutation of one reproductive gene or a second such gene to redundantly guarantee sterility. These fish have improved growth, disease resistance, and n-3 fatty acid levels. The infertility was reversed with hormone therapy for the fish with one mutated reproductive gene. However, the success has been variable and relatively low, and the spawning rate needs to be improved to prove efficacy to government regulatory agencies and for economical commercialization. Two reproductive gene knockouts have not been evaluated for fertility restoration. Thus, the main thrust of this proposal is optimization of hormone therapy and to determine if the redundant system requires the same or different hormone dosage regimen to restore fertility as the editing of a single reproductive gene.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
Specific Objectives-1) To determine the optimum dose of luteinizing hormone releasing hormone analogue (LHRHa) and human chorionic gonadotropin (HCG) as either liquid injections or slow-release cellulose acetate implants to restore fertility to transgenic channel catfish that have had one or two HPG axis related genes knocked out.2) To determine if equine chorionic gonadotropin (eCG) is more effective than HCG for restoring fertility to sterilized transgenic- gene-edited channel catfish.3) To verify that hormone therapy regimens do not adversely affect progeny development and early life history performance.4) Confirm the inheritance of performance level in the resulting generation of offspring of the transgenic-gene-edited parents and examine any potential epigenetic effects on long-term performance due to hormone therapy.
Project Methods
Body weight of males and females will be obtained and those with good secondary sexual characteristics indicative of readiness for spawning will be paired with the appropriate genotype in partitioned spawning tanks. A 2 x 2 x 3 x 4 factorial will be utilized for the hormone treatments with 2 pooled genotypes (1 or 2 HPG axis genes knocked out), 2 methods of delivery (liquid injection or slow-release implant), 3 doses of HCG and 4 doses of LHRHa. Fish that are one reproductive gene or two reproductive genes mutated will have 10 replicate pairings each per hormone treatment. Forty replicate pairings of wild-type pairings will be utilized and will be injected with LHRHa at 100 ug/kg, which is the current industry standard for wild-type domestic catfish. Reproduction by gene-edited channel catfish comparable to that found by wild types that are spawned with industry protocols would be considered a success. The injection/implantation regimens are the same for males and females.The following injection treatments will be administered: (1) 1,200 IU hCG, (2) 1,600 IU hCG, and (3) 2,000 IU hCG coupled with LHRHa [0 (saline (0.85% NaCl), 50, 100 ug/kg] and hCG [0 (saline), 1,200, 1,600, 2,000 IU] and the same dosages for the implants.Fry (n = 10 individuals) will be randomly sampled from each family, at peak hatch, before first feeding (when >50% of fry swim-up to the surface), and at 30 days post-hatch. Total length, notochord length, and body morphology measurements (e.g., eye diameter, jaw length, yolk sac area, and yolk utilization rates) will be obtained from fry using imaging software. Deformed fry will also be identified visually based on pericardial, head, eye, yolk, tail, and spine deformities. Body weight and survival will also be recorded during these "critical" stages in ontogeny.Growth rate, carcass rate, and disease resistance will be determined. Standard bacterial disease challenges will be performed.