The confinement of transgenic fish is essential to prevent their escape and reproduction in natural ecosystems. Reversible transgenic sterilization is a promising approach to control the reproduction of transgenic fish. Therefore, the present study was conducted to develop a reversibly sterile channel catfish (Ictalurus punctatus) via the transgenic overexpression of the goldfish (Carassius auratus) glutamic acid decarboxylase (GAD) gene driven by the common carp (Cyprinus carpio) β-actin promoter to disrupt normal gamma-aminobutyric acid (GABA) regulation. Three generations of GAD-transgenic fish were produced. All studied generations showed repressed reproductive performance; however, this was not always statistically significant. In F₁, 5.4% of the transgenic fish showed a sexual maturity score ≥ 4 (maximum = 5) at five years of age, which was lower (p = 0.07) than that of the control group (16.8%). In the spawning experiments conducted on F₁ transgenic fish at six and nine years of age, 45.5% and 20.0% of fish spawned naturally, representing lower values (p = 0.09 and 0.12, respectively) than the percentages in the sibling control fish of the same age (83.3% and 66.7%, respectively). Four of six pairs of the putative infertile six-year-old fish spawned successfully after luteinizing hormone-releasing hormone analog (LHRHa) therapy. Similar outcomes were noted in the three-year-old F₂ fish, with a lower spawning percentage in transgenic fish (20.0%) than in the control (66.7%). In one-year-old F₂-generation transgenic fish, the observed mean serum gonadotropin-releasing hormone (GnRH) levels were 9.23 ± 2.49 and 8.14 ± 2.21 ng/mL for the females and males, respectively. In the control fish, the mean levels of GnRH were 11.04 ± 4.06 and 9.03 ± 2.36 ng/mL for the females and males, respectively, which did not differ significantly from the control (p = 0.15 and 0.27 for females and males, respectively). There was no significant difference in the estradiol levels of the female transgenic and non-transgenic fish in the one- and four-year-old F₂-generation fish. The four-year-old F₂-generation male transgenic fish exhibited significantly (p < 0.05) lower levels of GnRH and testosterone than the control fish. In conclusion, while overexpressing GAD repressed the reproductive abilities of channel catfish, it did not completely sterilize transgenic fish. The sterilization rate might be improved through selection in future generations.

Optimization of xenogenesis for hybrid catfish (♀ channel catfish, Ictalurus punctatus × ♂ blue catfish, I. furcatus) embryo production was the goal. The effect of density of unsorted gonadal cells (80,000, 100,000, or 120,000 cells/fry) from blue catfish (BGCs) injected into triploid channel catfish surrogates, and BGCs or channel catfish (CGCs) into triploid white catfish (Ameiurus catus) surrogates on proliferation and colonization rates in surrogates injected at 4-, 5-, or 6-days post-hatch (DPH) was evaluated. At 45 and 90 DPH, survival and size of surrogates, and colonization/proliferation of donor cells (cell area < 150 μm² and cluster area > 150 μm²) were evaluated. Survival and size of all surrogate species were not impacted by cell density or donor. All surrogate species injected with 100,000 cells/fry had larger cluster cell areas than those injected with 80,000 cells/fry. White catfish surrogates with BGCs and CGCs had larger cell areas when injected with 100,000 cells/fry than those injected with 80,000 cells/fry. Both cell and cluster area increased by 90 DPH for all surrogates. PCR and PKH26 red fluorescence analysis confirmed that > 89% and > 86% of surrogates were positive xenogens at 45 and 90 DPH, respectively. No surrogate type or donor was superior to the others regarding colonization and proliferation, survival or growth, thus, channel catfish or white catfish were equally effective surrogates. Potential advantages of white catfish are small size, early sexual maturity, and spawning early in the season. These findings enhance the efficiency of germ cell transplantation for commercial hybrid catfish production.