Blue catfish (Ictalurus furcatus) is an important aquaculture species because the F1 hybrid produced from mating female channel catfish with male blue catfish exhibits strong heterosis with a number of important performance and production traits. While the mechanism of sex determination has been well studied in channel catfish, only limited work has been conducted with sex determination in blue catfish. As a first step toward understanding of sex determination in blue catfish, in this study, we conducted whole genome re-sequencing using pooled DNA of 64 male and 64 female blue catfish. Genomic variations were identified between the males and females, with a focus on the sex chromosome. We focused our work on insertions and deletions because the size differences caused by indels are amenable to PCR analysis. Here we report the identification of two Y chromosome-specific insertions. The two insertions are nearby and, therefore, a single PCR allowed amplification of the locus containing both the insertions. Upon PCR amplification, two bands were produced from males whereas only one band was produced from females, making this locus a marker suitable for genetic sex identification at any stage of blue catfish. This marker will be very useful for stock management for hybrid production and will facilitate studies to identify the sex determination gene of blue catfish.

This study evaluated the texture, flavor, and color of commercially available frozen catfish fillets, including channel catfish (Ictalurus punctatus), hybrid catfish, and swai (Pangasianodon hypophthalmus), with and without sodium phosphate treatment. Genetic testing confirmed the species' identity. Untreated fillets were redder, while phosphate-treated fillets were softer and less firm. Control swai exhibited the firmest and toughest texture, whereas phosphate-treated fillets were mushiest, as confirmed by mechanical and sensory analyses. Sensory panels noted phosphate-treated fillets as significantly softer than untreated counterparts. This study highlights texture differences between Ictalurus and Pangasianodon catfish, with swai demonstrating a chewier, firmer texture overall.

Transgenic technologies have been used for genetic improvement of catfish performance with notable success. However, these developments are useless from a commercialization standpoint without extremely efficient confinement. Transgenic sterilization has the potential to accomplish 100

AbstractDespite catfish being the dominant freshwater aquaculture product in the United States, catfish texture and sensory evaluation are understudied compared with other aquaculture species, and very few studies have been conducted to evaluate these traits in catfish. Texture, sensory, carcass yield, flavor, visceral fat deposition, gonadal development, and fillet color analyses were conducted on four size classes, small (<0.68 kg), medium (0.68–0.92 kg), large (0.93–1.75 kg), and extra‐large (>1.75 kg), for channel catfish (n = 456) (Ictalurus punctatus), blue catfish (n = 78) (I. furcatus), and hybrid catfish (n = 195) (channel catfish ♀ × blue catfish ♂). Within genetic type comparisons indicated that the texture traits, hardness, and chewiness and the sensory trait toughness increased with increasing size in hybrid catfish and channel catfish but were the most pronounced in channel catfish. Overall, channel catfish had the firmest fillets based on several attributes. Blue catfish were found to have differences among texture traits between the extra‐large size class and the three remaining size classes, but overall size had less of an effect compared with the channel catfish and hybrid catfish. A trend of paternal predominance was observed as the hybrid catfish was more similar to the blue catfish than the channel catfish. Hybrid catfish had the highest fillet percentage. This study is the first large‐scale analysis of texture and sensory traits within two catfish species and their interspecific hybrid at different sizes and highlights the differences in commercially important texture and sensory traits.

Xenogenic individuals are sterilized hosts that are colonized by germ cells transplanted from a donor species and can subsequently produce donor-derived gametes. In our case, unsorted gonadal cells from a donor species injected near the genital ridge of a sterile surrogate should colonize only that region, eventually becoming donor gametes. Unsorted gonadal cells from blue catfish (Ictalurus furcatus) injected into triploid common carp (Cyprinus carpio) embryos and fry were detected by PCR assays predominantly in recipient gonads and, in a subset of individuals, occasionally in DNA extracts from pectoral fin, anal fin, or muscle tissue, resulting in mosaicism in these tissues (presence of donor DNA/cells). Recipients retained normal common carp morphology. Donor signal was not detected in other assayed tissues, including eyes, livers, intestines, and hearts. This is the first report of such aberrant, presumably gonadal stem cell migration and colonization outside the gonads in xenogenic fish. Individuals injected later in development had reduced or no mosaicism compared to those injected at early developmental stages. Catfish donor cells were detected in the gonad, muscle, anal fin, and pectoral fin of both diploid and triploid common carp hosts. The incidence of non-target tissues containing blue catfish DNA trended towards being higher in triploids than diploids, especially when mosaicism involved three tissues. These findings provide preliminary insights into donor unsorted gonadal cell migration, tissue-specific persistence, and developmental timing in reproductive biology. These PCR findings indicate tissue mosaicism but do not establish somatic differentiation.

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.

Xenogenesis, an advanced hatchery technology for hybrid catfish (channel catfish, Ictalurus punctatus ♀ × blue catfish, I. furcatus ♂) production, involves transplanting germline stem cells (GSCs) from diploid donors into sterile recipients. Currently, freshly extracted GSCs are used, however cell production depends on age, size, and seasonal cycle of the donor, causing challenges. In vitro propagation could address these issues by providing a year-round GSCs supply. The present study compared effectiveness of fresh vs. cultured oogonial stem cells (OSCs) and spermatogonial stem cells (SSCs) for transplantation. Triploid channel catfish fry were injected at 5 days post-hatch (DPH) with PKH26 labelled fresh or cultured OSCs or SSCs. Growth and survival of recipient fish were assessed at 45 and 90 DPH, while donor cell colonization was quantified using PKH26. PCR and fluorescence images were used to determine percent xenogens. No significant differences in fry growth were observed between fresh and cultured treatments at 45 and 90 DPH. However, fluorescence imaging revealed significantly higher cell, cluster area in cultured treatments compared to fresh treatments. Cell, cluster areas significantly increased from 45 to 90 DPH in both fresh and cultured treatments. Cell area at 45 DPH was significantly higher in cultured treatments than fresh treatments, while no difference was detected at 90 DPH. PCR analyses revealed a higher proportion of xenogens in recipients injected with cultured cells (85.7%) compared to fresh cells (83.3%). Our findings demonstrate that cultured stem cells perform comparably to fresh stem cells, offering a promising approach for future cell transplantation.

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.

As a new, safe, and efficient method, Atmospheric-Pressure Room-Temperature Plasma (ARTP) mutagenesis has been widely applied in the field of microbial breeding and industrial applications, but it is rarely used in fish. In this study, ARTP mutagenesis technology was applied for the first time to a common carp strain, Songpu mirror carp (Cyprinus carpio L.), to increase genetic variation in this species. The appropriate experimental conditions were determined to include a radio frequency output power of 160 W and the processing of fertilized eggs for 360 s. The ARTP treatment group had a lower survival rate than the control group. The CV of morphological characters in the ARTP treatment group was significantly higher than that in the control group, and the CV of body weight was the highest (p < 0.05). In addition, the deformity rate in the ARTP treatment group was significantly higher than in the control group (p < 0.05). Individuals with high weight and no deformities were screened within the selection pressure of 1:15 of ARTP treatment group and fed in the same pool with the control group of the same age. The measurement of serum indices showed that, in the ARTP treatment group, TP, ALP, ALB, T-CHO, LDL levels were significantly higher than those in the control group (p < 0.05). Furthermore, the relative expressions of SOD, growth-related genes GH, IGF-I, protein synthesis-related genes TOR and 4EBP1 were significantly higher in the ARTP treatment group than in the control group (p < 0.05). In summary, Songpu mirror carp subjected to ARTP treatment showed a higher growth potential and antioxidant capacity.

Xenogenesis has been recognized as a prospective method for producing channel catfish, Ictalurus punctatus ♀ × blue catfish, I. furcatus ♂ hybrids. The xenogenesis procedure can be achieved by transplanting undifferentiated stem cells derived from a donor fish into a sterile recipient. Xenogenesis for hybrid catfish embryo production has been accomplished using triploid channel catfish as a surrogate. However, having a surrogate species with a shorter maturation period, like white catfish (Ameiurus catus), would result in reduced feed costs, labor costs, and smaller body size requirements, making it a more suitable species for commercial applications where space is limited, and as a model species. Hence, the present study was conducted to assess the effectiveness of triploid white catfish as a surrogate species to transplant blue catfish stem cells (BSCs) and channel catfish stem cells (CSCs). Triploid white catfish fry were injected with either BSCs or CSCs labeled with PKH 26 fluorescence dye from 0 to 12 days post hatch (DPH). No significant differences in weight and length of fry were detected among BSCs and CSCs injection times (0 to 12 DPH) when fry were sampled at 45 and 90 DPH (P > 0.05). The highest survival was reported when fry were injected between 4.0 to 5.5 DPH (≥ 81.2%). At 45 and 90 DPH, cell and cluster area increased for recipients injected from 0 to 5.2 DPH, and the highest cluster area values were reported between 4.0 to 5.2 DPH. Thereafter, fluorescent cell and cluster area in the host declined with no further decrease after 10 DPH. At 45 DPH, the highest percentage of xenogens were detected when fry were injected with BSCs between 4.0 to 5.0 and CSCs between 3.0 to 5.0 DPH. At 90 DPH, the highest number of xenogens were detected from 4.0 to 6.0 DPH when injected with either BSCs or CSCs. The current study demonstrated the suitability of white catfish as a surrogate species when BSCs and CSCs were transplanted into triploid white catfish between 4.0 to 6.0 DPH (27.4 ± 0.4°C). Overall, these findings allow enhanced efficiency of commercializing xenogenic catfish carrying gametes of either blue catfish or channel catfish.

CRISPR/Cas9-mediated multiplex genome editing (MGE) conventionally uses multiple single-guide RNAs (sgRNAs) for gene-targeted mutagenesis via the non-homologous end joining (NHEJ) pathway. MGE has been proven to be highly efficient for functional gene disruption/knockout (KO) at multiple loci in mammalian cells or organisms. However, in the absence of a DNA donor, this approach is limited to small indels without transgene integration. Here, we establish the linear double-stranded DNA (dsDNA) and double-cut plasmid (dcPlasmid) combination-assisted MGE in channel catfish (Ictalurus punctatus), allowing combinational deletion mutagenesis and transgene knock-in (KI) at multiple sites through NHEJ/homology-directed repair (HDR) pathway in parallel. In this study, we used single-sgRNA-based genome editing (ssGE) and multi-sgRNA-based MGE (msMGE) to replace the luteinizing hormone (lh) and melanocortin-4 receptor (mc4r) genes with the cathelicidin (As-Cath) transgene and the myostatin (two target sites: mstn1, mstn2) gene with the cecropin (Cec) transgene, respectively. A total of 9000 embryos were microinjected from three families, and 1004 live fingerlings were generated and analyzed. There was no significant difference in hatchability (all P > 0.05) and fry survival (all P > 0.05) between ssGE and msMGE. Compared to ssGE, CRISPR/Cas9-mediated msMGE assisted by the mixture of dsDNA and dcPlasmid donors yielded a higher knock-in (KI) efficiency of As-Cath (19.93 %, [59/296] vs. 12.96 %, [45/ 347]; P = 0.018) and Cec (22.97 %, [68/296] vs. 10.80 %, [39/361]; P = 0.003) transgenes, respectively. The msMGE strategy can be used to generate transgenic fish carrying two transgenes at multiple loci. In addition, double and quadruple mutant individuals can be produced with high efficiency (36.3 % similar to 71.1 %) in one-step microinjection. In conclusion, we demonstrated that the CRISPR/Cas9-mediated msMGE allows the one-step generation of simultaneous insertion of the As-Cath and Cec transgenes at four sites, and the simultaneous disruption of the lh, mc4r, mstn1 and mstn2 alleles. This msMGE system, aided by the mixture donors, promises to pioneer a new dimension in the drive and selection of multiple designated traits in other non-model organisms.

Channel catfish (Ictalurus punctatus) and blue catfish (Ictalurus furcatus) are two economically important freshwater aquaculture species in the United States, with channel catfish contributing to nearly half of the country’s aquaculture production. While differences in economic traits such as growth rate and disease resistance have been noted, the extent of transcriptomic variance across various tissues between these species remains largely unexplored. The hybridization of female channel catfish with male blue catfish has led to the development of superior hybrid catfish breeds that exhibit enhanced growth rates and improved disease resistance, which dominate more than half of the total US catfish production. While hybrid catfish have significant growth advantages in earthen ponds, channel catfish were reported to grow faster in tank culture environments. In this study, we confirmed channel fish’s superiority in growth over blue catfish in 60-L tanks at 10.8 months of age (30.3 g and 11.6 g in this study, respectively; p < 0.001). In addition, we conducted RNA sequencing experiments and established transcriptomic resources for the heart, liver, intestine, mucus, and muscle of both species. The number of expressed genes varied across tissues, ranging from 5,036 in the muscle to over 20,000 in the mucus. Gene Ontology analysis has revealed the functional specificity of differentially expressed genes within their respective tissues, with significant pathway enrichment in metabolic pathways, immune activity, and stress responses. Noteworthy tissue-specific marker genes, including lrrc10, fabp2, myog, pth1a, hspa9, cyp21a2, agt, and ngtb, have been identified. This transcriptome resource is poised to support future investigations into the molecular mechanisms underlying environment-dependent heterosis and advance genetic breeding efforts of hybrid catfish.

In F1 hybrids, phenotypic values are expected to be near the parental means under additive effects or close to one parent under dominance. However, F1 traits can fall outside the parental range, and outbreeding depression occurs when inferior fitness is observed in hybrids. Another possible outcome is heterosis, a phenomenon that interspecific hybrids or intraspecific crossbred F1s exhibit improved fitness compared to both parental species or strains. As an application of heterosis, hybrids between channel catfish females and blue catfish males are superior in feed conversion efficiency, carcass yield, and harvestability. Over 20 years of hybrid catfish production in experimental settings and farming practices generated abundant phenotypic data, making it an ideal system to investigate heterosis. In this study, we characterized fitness in terms of growth and survival longitudinally, revealing environment-dependent heterosis. In ponds, hybrids outgrow both parents due to an extra rapid growth phase of 2-4 months in year 2. This bimodal growth pattern is unique to F1 hybrids in pond culture environments only. In sharp contrast, the same genetic types cultured in tanks display outbreeding depression, where hybrids perform poorly, while channel catfish demonstrate superiority in growth throughout development. Our findings represent the first example, known to the authors, of opposite fitness shifts in response to environmental changes in interspecific vertebrate hybrids, suggesting a broader fitness landscape for F1 hybrids. Future genomic studies based on this experiment will help understand genome-environment interaction in shaping the F1 progeny fitness in the scenario of environment-dependent heterosis and outbreeding depression.

Antimicrobial peptides (AMPs) show promise in enhancing resistance against pathogens. Previously, we integrated two AMP genes, cathelicidin (Cath) from alligator (Alligator mississippiensis or A. sinensis) and cecropin (Cec) from moth (Hyalophora cecropia), into the channel catfish (Ictalurus punctatus) genome. This study examines the efficacy of exogenous AMP gene integration in improving bacterial resistance in transgenic channel catfish and assesses the direct and pleiotropic effects of gene replacement/knockout on survival and growth based on insertion site. Transgenic Cath- and Cec-expressing fish exhibited similar or higher survival rates (P > 0.05) compared to controls during the initial culture. Integration of the Cec transgene doubled the survival rate when challenged with Edwardsiella ictaluri, with knock-in (KI) of Cath further increasing bacterial resistance. Coupling Cec KI with mstn knockout (KO) increased survival 3-fold after E. ictaluri infection and growth by 50% at 4 months post-fertilization (mpf). However, random integration of Cec had a minimal effect on disease resistance and did not enhance growth. Random integration of Cath increased survival 2.5-fold and 4-fold against E. ictaluri and Flavobacterium covae, respectively, without affecting growth. Cath KI at the lh locus increased survival 4-fold when challenged with F. covae and reduced growth by 10% (P > 0.05) at 24 mpf, whereas Cath KI coupled with mc4r KO resulted in a 2.5-fold increase in survival following F. covae infection compared with controls, and increased growth by 80% at 3 mpf. Simultaneous KI of Cath and Cec, along with KO of mc4r and mstn, increased survival 4-fold against E. ictaluri, while increasing growth by 50% at 3 mpf. Dual insertion of AMP genes yielded the greatest resistance to disease. These direct and pleiotropic effects may increase comprehension and societal acceptance of genetic engineering in aquaculture.

As a precise and versatile tool for genome manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) platform holds promise for modifying fish traits of interest. With the aim of reducing transgene introgression and controlling reproduction, upscaled disease resistance and reproductive intervention in catfish species have been studied to lower the potential environmental risks of the introgression of escapees as transgenic animals. Taking advantage of the CRISPR/Cas9-mediated system, we succeeded in integrating the cathelicidin gene (As-Cath) from an alligator (Alligator sinensis) into the target luteinizing hormone (lh) locus of channel catfish (Ictalurus punctatus) using two delivery systems assisted by double-stranded DNA (dsDNA) and single-stranded oligodeoxynucleotides (ssODNs), respectively. In this study, high knock in (KI) efficiency (22.38%, 64/286) but low on-target events was achieved using the ssODN strategy, whereas adopting a dsDNA as the donor template led to an efficient on-target KI (10.80%, 23/213). The on-target KI of As-Cath was instrumental in establishing the lh knockout (LH–_As-Cath+) catfish line, which displayed heightened disease resistance and reduced fecundity compared with the wild-type (WT) sibling fish. Furthermore, administration of human chorionic gonadotropin (HCG) and luteinizing hormone-releasing hormone analogue (LHRHa) can restore the reproduction of the transgenic fish line. Overall, we replaced the lh gene with an alligator cathelicidin transgene and then administered hormone therapy to gain complete reproductive control of disease-resistant transgenic catfish in an environmentally sound manner. This strategy not only effectively improves consumer-valued traits but also guards against unwanted introgression, providing a breakthrough in aquaculture genetics to confine fish reproduction and prevent the establishment of transgenic or domestic genotypes in the natural environment.

The CRISPR/Cas9 platform holds promise for modifying fish traits of interest as a precise and versatile tool for genome manipulation. To reduce introgression of transgene and control reproduction, catfish species have been studied for upscaled disease resistance and intervening of reproduction to lower the potential environmental risks of introgression of escapees’ as transgenic animals. Taking advantage of the CRISPR/Cas9-mediated system, we succeeded in integrating the cathelicidin gene from an alligator ( Alligator sinensis ; As-Cath ) into the target luteinizing hormone ( LH ) locus of channel catfish ( Ictalurus punctatus ) using two delivery systems assisted by double-stranded DNA (dsDNA) and single-stranded oligodeoxynucleotides (ssODNs), respectively. In this study, high knock-in (KI) efficiency (22.38%, 64/286) but low on-target was achieved using the ssODN strategy, whereas adopting a dsDNA as the donor template led to an efficient on-target KI (10.80%, 23/213). On-target KI of As-Cath was instrumental in establishing the LH knockout (LH − _As-Cath + ) catfish line, which displayed heightened disease resistance and reduced fecundity compared to the wild-type sibling fish. Furthermore, implanting with HCG and LHRHa can restore the fecundity, spawnability and hatchability of the new transgenic fish line. Overall, we replaced the LH gene with an alligator cathelicidin transgene and then administered hormone therapy to gain complete reproductive control of disease-resistant transgenic catfish in an environmentally sound manner. This strategy not only effectively improves the consumer-valued traits, but also guards against genetic contamination. This is a breakthrough in aquaculture genetics to confine fish reproduction and prevent the establishment of transgenic or domestic genotypes in the natural environment.

Channel catfish, Ictalurus punctatus , have limited ability to synthesize Ω-3 fatty acids. The ccβA-msElovl2 transgene containing masu salmon, Oncorhynchus masou , elongase gene driven by the common carp, Cyprinus carpio, β-actin promoter was inserted into the channel catfish melanocortin-4 receptor ( mc4r ) gene site using the two-hit two-oligo with plasmid (2H2OP) method. The best performing sgRNA resulted in a knockout mutation rate of 92%, a knock-in rate of 54% and a simultaneous knockout/knock-in rate of 49%. Fish containing both the ccβA-msElovl2 transgene knock-in and mc4r knockout (Elovl2) were 41.8% larger than controls at 6 months post-hatch ( p = 0.005). Mean eicosapentaenoic acid (EPA, C20:5n-3) levels in Elov2 mutants and mc4r knockout mutants (MC4R) were 121.6% and 94.1% higher than in controls, respectively ( p = 0.045; p = 0.025). Observed mean docosahexaenoic acid (DHA, C22:6n-3) and total EPA + DHA content was 32.8% and 45.1% higher, respectively, in Elovl2 transgenic channel catfish than controls ( p = 0.368; p = 0.025). To our knowledge this is the first example of genome engineering to simultaneously target transgenesis and knock-out a gene in a commercially important aquaculture species for multiple improved performance traits. With a high transgene integration rate, improved growth, and higher omega-3 fatty acid content, the use of Elovl2 transgenic channel catfish appears beneficial for application on commercial farms.

CRISPR/Cas9-mediated genome editing has paved new avenues for improving production-valued traits in aquaculture by knocking out or disrupting functional genes. However, utilizing CRISPR/Cas9-based knock-in (KI) of exogenous genes can expedite genetic improvement of traits such as disease resistance, which remains problematic in farmed fish. In this study, we successfully generated transgenic blue catfish (Ictalurus furcatus) of primarily Rio Grande strain ancestry with site-specific KI of the alligator (Alligator sinensis) cathelicidin (As-Cath) gene into the luteinizing hormone (lh) locus via two CRISPR/Cas9-mediated KI systems, assisted by the linear double-stranded DNA (dsDNA) and double-cut plasmid, respectively. High integration rates were observed with linear dsDNA (16.67%, [13/78]) and double-cut plasmid strategies (24.53%, [26/106]). In addition, the on-target KI efficiency of the double-cut plasmid strategy (16.04%, [17/106]) was 1.67 times higher than that of the linear dsDNA strategy (10.26%, [8/78]) based on the odds ratio. The relative expression of the As-Cath transgene of P1 founders was detected in nine tissues, dominated by the kidney, skin, and muscle (14.30-, 7.71- and 6.92-fold change, P < 0.05). Moreover, the As-Cath transgenic blue catfish showed a higher cumulative survival rate than that of wild-type controls (80% vs. 30%, P < 0.05) following Flavobacterium covae infection. Survival during culture supports the challenge data as survival of As-Cath transgenic individuals was 97.1% while that of pooled non-transgenic individuals was observed to be less 87.0% (P = 0.15). The growth rates and external morphology of the transgenic and wild-type siblings were not different (P > 0.05), indicating no pleiotropic effects of the As-Cath transgene integration at the lh locus in the P1 founders for this trait. Taken together, our findings demonstrate that CRISPR/Cas9-assisted KI of an antimicrobial peptide gene can be achieved in blue catfish with high integration efficiency, and As-Cath transgenic blue catfish have improved disease resistance, which is a promising strategy for disease reduction in aquaculture.

Integrating a vector-engineered antimicrobial peptide gene (AMG) into the fish genome effectively modulated the innate immune system and increased resistance to infectious disease in channel catfish (Ictalurus punctatus). CRISPR/Cas9-assisted microinjection of cecropin (Cec) and cathelicidin (Cath) was employed to create dual-AMG integrated (*_Cec+/*_Cath+) transgenic embryos with high integration rates. Additionally, a univariate-multiple logit regression model was fitted to determine the synergistic expression of transgenes and endogenous AMGs in the head kidney post-bacterial infection. Transgenic-embryo-based genome editing significantly increased the efficiency of dual-AMG integration from 17.6% to 37.3%. The survival rate of single-AMG (50% vs. 20%, P = 0.023) and dual-AMG (70% vs. 20%, P = 0.005) integrated fish was dramatically higher than that of wild-type fish (20%) following Edwardsiella ictaluri challenge. More dual-AMG fry survived than expected based on integration and inheritance rates of single-AMG transgenics compared to other genotypes. Logistic regression (LR) analysis indicated that individual body weight and gender did not affect survival, while the transgenes Cec and Cath contributed directly to the survival during the bacterial infection. Furthermore, transgenes enhanced fish disease resistance by regulating the expression of TCP and NK-lysin genes. This study demonstrates that it is promising to generate dual-gene integrated genetic lines with a high integration efficiency by adopting transgenic-embryo-based CRISPR/Cas9-mediated genome editing, and an LR model is feasible for assessing the synergistic effects of gene expression.

Xenogenesis is an innovative technology for hybrid catfish (♀ channel catfish, Ictalurus punctatus, × ♂ blue catfish, I. furcatus) embryo production. The xenogeneic process can be accomplished by transplanting undifferentiated diploid germline stem cells derived from donor fish into sterile recipients. This methodology enables recipients to produce donor-derived gametes. Until recently, the timing of transplantation of donor cells into hosts was done with limited knowledge of the best age to inject cells. The age of the host could critically affect the success of germ cell transplantation. The present study aimed to identify the best age of the triploid channel catfish to transplant blue catfish stem cells for production of xenogeneic catfish. Triploid channel catfish fry were injected with blue catfish stem cells labeled with PKH26 dye from 0 to 18 days post-hatch (DPH). Then at 50 DPH (1st time interval) and 90 DPH (2nd time interval), total length (TL), weight (BW), and survival of recipients were evaluated. Colonization of donor cells was evaluated in recipients using PKH26 dye fluorescence to calculate percent cell (<150 μm2) and cluster areas (>150 μm2). PCR determined the percentage of xenogens from gonads. Day of stem cell injection had no impact on TL and BW of recipient fish when evaluated at both sampling intervals. Survival of recipients injected with blue catfish stem cells increased from 0 to 5.4 DPH. After 5.4 DPH, survival remained high (≥82%) for fry injected until 18 DPH. At the 1st time interval, cell and cluster area increased as recipients fish injected from 0 to 5.4 DPH and 0 to 5.6 DPH, respectively. Thereafter, fluorescent cell and cluster area in the host declined with no further decrease after 11.3 and 10.4 DPH, respectively. At the 2nd time interval, cell and cluster area increased as recipients were injected from 0 to 5.8 DPH and 0 to 5.7 DPH and significantly declined with no further decrease after 10.2 and 11.3 DPH, respectively. At the 1st time interval, the highest percentage of xenogens were detected when recipients were injected from 3 to 5 DPH (83.3%), while at the 2nd time interval, the highest percentage of xenogens was detected from 4 to 6 DPH (83.3%). Our results show that 4 to 6 DPH is a suitable timespan to inject donor-derived stem cells into recipients. These findings will enhance the efficiency of germ cell transplantation for commercial-scale hybrid catfish production.