GENE EDITING OF REPRODUCTIVE HORMONES TO STERILIZE CHANNEL CATFISH Ictalurus punctatus

Sterilization of cultured fish is the most effective strategy to prevent unwanted genetic communication between domestic and wild populations, which is essential for an environmentally-responsible aquaculture industry. In this research, we sought to reversibly produce sterile channel catfish (Ictalurus punctatus) by knocking out of reproduction critical genes, the gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) genes, using the zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/cas9 (CRISPR/Cas9) technologies.

CEL-I assay and sequencing were applied to identify the mutation of P1 and F1 fish which were generated from gene-edited parents. Spawning trials of the fish with outstanding secondary sexual characteristics were utilized to evaluate fertility. Hormone therapy was conducted to restore reproduction.

GnRH, FSH and LH genes were successfully mutated in channel catfish through all three technologies. LH gene-edited channel catfish showed normal gonadal growth, but had difficulty in ejaculation and ovulation. Less than 25% of the males fertilized the eggs, and all females failed to ovulate. Majority of the GnRH and FSH gene edited fish were infertile. The rest were fertile and displayed normal gametogenesis and reproductive performance. However, these fish had lower (p < 0.05) hatching rate of embryos compared to the control fish. The mutations were transmitted to the F1 generation through nature spawning. Hormone regime with luteinizing hormone-releasing hormone analog (LHRHa) and human chorionic gonadotropin (hCG) administration restored the spawnability and increased embryo hatching rate for some of the GnRH, FSH and LH mutated fish (Table1).