Gene editing for growth enhancement in channel catfish, Ictalurus punctatus, and xenogenesis in common carp, Cyprinus carpio to produce blue catfish, I. furcatus, sperm

Abstract

Xenogenesis is a method of reproduction where successive generations differ from each other and no genetic material is transmitted from the parent to the offspring. Xenogenesis can be accomplished by transplanting spermatogonial stem cells (SSCs), oogonial stem cells (OSCs) or primordial germ cells (PGCs) from the desired diploid donor species into a sterilized host. Many beneficial ecological and aquaculture applications are possible from this process. Specifically, the production of embryos from hybridizing channel catfish (Ictalurus punctatus) females with blue catfish (I. furcatus) males could be refined to a simpler and more cost-effective practice of xenogenesis. Xenogenesis could alleviate the reliance on growing blue catfish males to maturity and sacrificing them for gonad extraction and subsequent artificial spawning. Male blue catfish cannot be stripped of milt for artificial fertilization and must be sacrificed for sperm collection. Thus, one objective was to produce xenogenic common carp, Cyprinus carpio, to become biological blue catfish milt factories. Common carp embryos, and larvae were injected with blue catfish oogonial stem cells and spermatagonial stem cells at various points in relation to their development. Fish were injected beginning at 0-degree days, every 23-degree days until 621-degree days. In total, 152 potential common carp xenogens were sampled and 57 of them were confirmed to be hosting blue catfish stem cells through polymerase chain reaction (PCR), giving a total 37.5% success rate of common carp hosting blue catfish cells in the gonads. The highest percentage of positive samples were observed at 0 to 46-degree days and 483-575 degree days, averaging 62.3% and 56.7% success respectively. Rate of xenogenesis was similar for male and female common carp hosts. The mean survival among all the treated groups through the first 34 days was 47%, however, among groups injected during embryonic development, mean survival was 8.6%. During the grow out stage (34-545 dpf), mean survival for all treated groups was 70%. At one and half years of age, xenogenic common carp males produced blue catfish sperm based upon DNA analysis of the expressed sperm. In addition to stronger reproductive control, some targeted genetic alterations were performed to aid in higher growth rates and increase yield within catfish aquaculture. Two induced mutations were investigated, melanocortin-4 receptor (mc4r) and myostatin (mstn) gene in channel catfish. In a earthen pond environment stocked at 18,508 fish per hectare, the mean weight for channel catfish mc4r P1, mc4r F1 X control, control X mc4r F1, and mc4r F1 X mc4r F1 mutants was 361, 266, 521, and 426 grams, respectively, was larger (P>0.05,P>0.05, P<0.01, P<0.01) than control (C) channel catfish, 299 grams. In a recirculating system environment, during the first year of growth (354 days post fertilization), channel catfish mc4r F1 mutants had a mean weight of 22.6 grams and channel catfish controls were 10.5 grams, a 73.3% increase in body weight in the mutant fish (P<0.0001). During the next 161 days, mc4r mutants grew 29.2% faster (P<0.0001) than controls, reaching mean body weights of 66.6 grams and 49.7 grams respectively. Additionally, the effects of a CRISPR/Cas9 mediated mc4r and myostatin (mstn) gene knockouts on disease resistance to Flavobacterium covae, in channel catfish and blue catfish (I.furcatus) were examined. Both mc4r and mstn channel catfish mutants had better survival (P=0.0061, P=0.0150) when challenged with F. covae compared to the control channel catfish. Observed survival of blue catfish mc4r mutants was higher than blue catfish controls but was not significantly different (P=0.21).