Approaches for Improving Viability of Blue Catfish, Ictalurus furcatus, Stem Cells and Embryo Production of Channel Catfish, I. punctatus, Female × Blue Catfish Male Hybrids through Xenogenesis

Abstract

The hybrid channel catfish, Ictalurus punctatus, female × blue catfish, Ictalurus furcatus, male possesses superior characteristics such as faster growth rate, higher survival rate, better disease resistance and low oxygen tolerance, and higher carcass and fillet yield is the best catfish for pond culture in the United States, accounting for approximately 75% of all catfish cultured. Artificial fertilization is the current technology for commercial production of the hybrid catfish embryos, but this method has the disadvantages of intensive labor, time, and sacrifice of valuable blue catfish males. Xenogenesis has been studied as an appropriate alternative for producing the hybrids by mating normal channel catfish female with xenogenic channel catfish male that produces blue catfish sperm, but this technology also needs improved efficiency. One-hundred % of the channel catfish fry and white catfish, Ameiurus catus, embryos that had been pressurized at 7,000 psi 5 min after fertilization were triploid while 85.7 and 82.9% of white catfish and channel catfish, respectively, were triploid when pressurized at 6,500 psi. To achieve 100% triploidy, hatch rate was about 20% of control diploid eggs. The triploid channel catfish in this experiment were transplanted with 4,000 type A spermatogonial stem cells (SSCs) and had a 0-16.7% xenogenesis rate. The triploid white catfish were injected with 9,000-10,000 SSCs, and had 3-4X higher xenogenesis rate. In the white catfish experiment, the donor cells were also treated with Rho protein kinase inhibitor (ROCK I). Injection of 8-14 primordial germ cells per fry resulted in almost zero transplantation, and injection of blastulae resulted in almost zero hatch rate. Experiments were conducted to determine survival rates of spermatogonial stem cells (SSCs) or oogonial stem cells (OSCs) cultured in vitro for 6 days including SSCs/OSCs dissociated from gonad tissues without enzymatic digestion (unsorted cells without enzymatic digestion), SSCS/OSCs dissociated with trypsin-EDTA 0.25% (unsorted cells with trypsin), and SSCs/OSCs dissociated with trypsin-EDTA 0.25% and enriched with discontinuous gradient density Percoll (sorted cells) and supplemented with ROCK I at 0, 10, and 50 µM. Methods to dissociate the SSCs/OSCs did not affect survival rates of the SSCs/OSCs during the 6-day culture period. Application of ROCK I at 10 or 50 µM yielded higher viability of SSCs compared to suspension without using ROCK I. Similarly, the survival rates were highest for the OSCs incubated with ROCK I 50 µM. The SSCs or OSCs had the highest viability in the first day right after being dissociated from the gonads. Polymerase chain reaction (PCR) results from progeny of putative xenogenic channel catfish indicated that only 1 channel catfish male pressurized at 7,000 psi and transplanted with 4,000 SSCs was xenogenic. The fertilization and hatch rates of eggs fertilized by this xenogenic male were 7.3% and 12.3%, respectively. Although the results from the spawning of this xenogenic catfish were low, this is the first report on the success of producing 100% hybrids by mating a xenogenic channel catfish male with a normal channel catfish female via aquaria spawning. Future research should address introduction of larger numbers of donor cells, fine tuning of the best time to inject these cells and examining ROCK I to increase cell viability and colonization rate.