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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. [ABSTRACT FROM AUTHOR]

Xenogenesis is an innovative tool for hybrid catfish (female Channel Catfish Ictalurus punctatus × male Blue Catfish I. furcatus) seed production, accomplished by transplanting undifferentiated germ cells derived from a donor diploid fish into a sterile recipient, which then enables recipient fish to produce donor‐derived gametes. There is potential to collect donor‐derived germ cells from mature fish during certain times of the year depending upon seasonal temperature and serum sex steroid hormonal fluctuations. The objective of this study was to evaluate seasonal variations in germ cell counts and serum sex steroid hormonal profiles in mature Blue Catfish. Mature fish were collected monthly over the full annual cycle to quantify the number of live germ cells (spermatogonia A, oogonia), viability of germ cells, and levels of serum sex steroid hormones, including testosterone, 11‐ketotestosterone, and 17β estradiol. Extracted spermatogonia A counts were highest from April to June, whereas a significant decline was detected from July to November. Extracted live oogonia counts were highest in April and gradually decreased to zero over the months of May to August. Seasonal variations in serum testosterone, 11‐ketotestosterone, and 17β estradiol followed a similar pattern as the live spermatogonia A and oogonia counts. Even though spermatogonia A counts were relatively lower in mature than in immature Blue Catfish males, extracting spermatogonia A from mature Blue Catfish males during April to June provides an added advantage to the process of artificial fertilization, as it is required to sacrifice these fish to collect sperm. [ABSTRACT FROM AUTHOR]

Exogenous oestrogen 17β-oestradiol (E2) has been shown to effectively induce feminization in teleosts. However, the molecular mechanisms underlying the process remain unclear. Here, we determined global DNA methylation and gene expression profiles of channel catfish (Ictalurus punctatus) during early sex differentiation after E2 treatment. Overall, the levels of global DNA methylation after E2 treatment were not significantly different from those of controls. However, a specific set of genes were differentially methylated, which included many sex differentiation-related pathways, such as MARK signalling, adrenergic signalling, Wnt signalling, GnRH signalling, ErbB signalling, and ECM–receptor interactions. Many genes involved in these pathways were also differentially expressed after E2 treatment. Specifically, E2 treatments resulted in upregulation of female-related genes and downregulation of male-related genes in genetic males during sex reversal. However, E2-induced sex reversal did not cause sex-specific changes in methylation profiles or gene expression within the sex determination region (SDR) on chromosome 4, suggesting that E2-induced sex reversal was a downstream process independent of the sex determination process that was regulated by sex-specific methylation within the SDR. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs), also known as host defence peptides, are evolutionarily ancient defensive weapons that can combat infections caused by pathogens. Fish‐derived AMPs have shown microbicidal properties, host immunomodulatory responses, and are induced by a variety of factors, especially pathogenic infection and abiotic stress, which may activate downstream signalling pathways to initiate the expression of antimicrobial peptide genes. Large‐scale genome sequencing has been applied in many aquatic animals, providing databases for the systematic identification of putative AMPs in these species. Recently, the whole genome and the proteome of channel catfish (Ictalurus punctatus) were released; by taking advantage of the antimicrobial peptide database and combining the proteome of catfish, we established a bioinformatic analysis pipeline to identify catfish AMP repertoire. This review introduces an effective method for the identification and development of putative AMPs based on protein datasets, and summarizes the structural properties, immunomodulation and molecular responses in vivo of catfish AMPs. In addition, the evaluation of the antibacterial activity of synthetic or isolated AMPs in vitro, and the applications of AMPs in fish via genetic engineering and clustered regularly interspaced short palindromic repeats associated proteins system (CRISPR/Cas9) were presented, which supports antimicrobial research and aquaculture therapy. The summary and comparison of these AMPs will enhance our understanding and their cross‐species applications. Combined with current genetic engineering and genome editing techniques, it is possible to promote the development of immune processes to protect valuable aquatic animals from infectious diseases. [ABSTRACT FROM AUTHOR]

Long‐term storage of oogonia and germ‐line stem cells provides an alternative to the limitations associated with cryopreserving eggs of important fish species. These cell types are less vulnerable to the stresses of freezing. Cryopreservation has enormous potential for aquaculture advancement, but protocols must be developed for each species and cell type since its success hinges on various input factors. Blue Catfish Ictalurus furcatus were selected as the test species in this study because of the need to improve fry production of Blue Catfish ♂ × Channel Catfish I. punctatus ♀ hybrids, which can be facilitated by storing oogonia in gene banks. Our objective was to develop a freezing protocol for oogonia of this species. We tested different permeating and nonpermeating cryoprotectants, concentrations of these agents, and freezing rates. We proved that all three factors influenced postthaw recovery of oogonia. Of the permeating cryoprotectants, 1.0 M dimethyl sulfoxide resulted in the most live cells with the highest viability percentages, and adding 0.2 M lactose with 10% egg yolk further improved the results. There were also specific interactions in which the effects of concentration and freezing rate varied among the cryoprotectant treatments. The most effective freezing rate was −1.0°C/min, and cell viability was reduced at −2.5°C/min and −5.0°C/min. From these results, we propose adding 1.0 M dimethyl sulfoxide with 0.2 M lactose and 10% egg yolk to cryomedia and freezing it at a rate of −1.0°C/min. By developing a cryopreservation protocol for a commonly cultured catfish, this work may guide the development of protocols for other species of interest. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9-mediated m ultiplex g enome e diting (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 s ingle- s gRNA-based g enome e diting (ssGE) and m ulti- s gRNA-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 % ∼ 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. [ABSTRACT FROM AUTHOR]

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. • First conduct transgenic-embryo-based genome editing. • Crossbreeding combined with CRISPR/Cas9-mediated systems increases the integration rate of dual transgenes. • Dual-AMG transgenic fish showed enhanced disease resistance compared to WT individuals. • Logistic regression models are feasible to determine the synergistic expression of genes. [ABSTRACT FROM AUTHOR]

Constructs bearing the cecropin B gene from the moth Hyalophora cecropia , driven by the cytomegalovirus (CMV) promoter, or the common carp beta-actin (β-actin) promoter were transferred to channel catfish, Ictalurus punctatus via electroporation. One F 3 channel catfish family transgenic for cecropin transgene driven by the CMV promoter, and one F 1 channel catfish family transgenic for cecropin transgene driven by the common carp β-actin promoter were produced. F 3 and F 1 individuals exhibited enhanced disease resistance when challenged in tanks with Edwardsiella ictaluri , the causative agent of enteric septicemia of catfish (ESC). Inheritance of the transgene by the F 1 and F 3 generation was 15% and 60%, respectively. Growth rates of the cecropin transgenic and non-transgenic full siblings (controls) channel catfish were not different (P > 0.05). All transgenic fish showed significant resistance to infection by ESC at day 3 and day 4 post exposure (P = 0.005). No correlation was detected between body weight and time to death for all genetic groups (P = 0.34). Results of our study confirmed that genetic enhancement of E. ictaluri resistance can be achieved by cecropin transgenesis in channel catfish. In addition to survival rate, improving survival time is essential because the extension of survival time gives a better chance to apply treatments to stop the bacterial infection. • Genetic enhancement of E. ictaluri resistance can be achieved by transgenesis. • Non-transgenic channel catfish survived less time than cecropin-transgenic. • No correlation was detected between weight and time to death for all genotypes. [ABSTRACT FROM AUTHOR]

Sustainability of channel catfish, Ictalurus punctatus ♀ × blue catfish, Ictalurus furcatus ♂ hybrid aquaculture relies on new innovative technologies to maximize fry output. Transplanting spermatogonial stem cells (SSCs) from blue catfish into channel catfish hosts has the potential to greatly increase gamete availability and improve hybrid catfish fry outputs. Cryopreservation would make these cells readily accessible for xenogenesis, but a freezing protocol for blue catfish testicular tissues has not yet been fully developed. Therefore, the objectives of this experiment were to identify the best permeating [dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol, methanol] and non-permeating (lactose or trehalose with egg yolk or BSA) cryoprotectants, their optimal concentrations, and the best freezing rates (−0.5, −1.0, −5.0, −10 ° C/min until −80 ° C) that yield the highest number of viable type A spermatogonia cells. Results showed that all of these factors had significant impacts on post-thaw cell production and viability. DMSO was the most efficient permeating cryoprotectant at a concentration of 1.0 M. The optimal concentration of each cryoprotectant depended on the specific cryoprotectant due to interactions between the two factors. Of the non-permeating cryoprotectants, 0.2 M lactose with egg yolk consistently improved type A spermatogonia production and viability beyond that of the 1.0 M DMSO control. The overall best freezing rate was consistent at −1 °C/min, but similar results were obtained using −0.5 °C/min. Overall, we recommend cryopreserving blue catfish testicular tissues in 1.0 M DMSO with 0.2 M lactose and egg yolk at a rate of either -0.5 or −1 °C/min to achieve the best cryopreservation outcomes. Continued development of cryopreservation protocols for blue catfish and other species will make spermatogonia available for xenogenic applications and genetic improvement programs. • Effects of cryoprotectants and freezing rates were evaluated. • Optimal concentration varied by cryoprotectant. • 1.0 DMSO with 0.2 M lactose and egg yolk was the best treatment combination. • Freezing rate of −1 °C/min was more effective than −5 or −10 °C/min. • Cryopreservation protocol established for blue catfish Type A spermatogonia cells. [ABSTRACT FROM AUTHOR]

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. • Evaluate colonization capability of transplanted blue catfish stem cells • At 50 DPH, significantly highest cell and cluster area detected at 5.4 DPH & 5.6 DPH. • At 90 DPH, significantly highest cell and cluster area detected at 5.8 DPH & 5.7 DPH. • 83.3% xenogens resulted from gonadal PCR analysis when recipients injected 3–6 DPH. • 4 to 6 DPH is a suitable timespan to inject donor-derived stem cells into recipients. [ABSTRACT FROM AUTHOR]

Xenogenesis has been identified as an innovative hatchery technology for hybrid catfish (channel catfish, Ictalurus punctatus ♀ × blue catfish, I. furcatus ♂) embryo production. The xenogeneic process can be accomplished by transplanting primordial germ cells, spermatogonial stem cells (SSC), or oogonial stem cells (OSC), derived from a donor diploid fish into a sterile recipient, which then enables recipient fish to produce donor-derived gametes. Currently, freshly extracted stem cells are used to create xenogens, which is challenging, especially considering germ cell production is donor size and age specific as well as dependent on seasonal cycles. Thus, having frozen stem cells available in germplasm repositories will help to facilitate xenogenesis technology for hybrid catfish production. This study was conducted to assess the effectiveness of fresh and cryopreserved stem cells for germ cell transplantation to support xenogenesis. At 5 days post hatch (DPH), triploid channel catfish were injected with fresh or cryopreserved SSC or OSC labeled with PKH26 dye. At 45 and 90 DPH, body size (total length and weight) and survival of these recipient fish were evaluated. In addition, colonization of fresh and cryopreserved donor cells was evaluated in recipients using PKH26 dye florescence (for percentage of cell area and cluster area) and PCR from gonadal samples. No significant differences in body size characteristics were detected between the fresh and cryopreserved injection treatments at 45 and 90 DPH. Survival was significantly higher in the control treatment than the SSC and OSC injected treatments at both sampling days. Fluorescent imaging revealed that percentage of SSC cell area, SSC cluster area, OSC cell area, and OSC cluster area did not significantly differ between the fresh and cryopreserved treatments at 45 and 90 DPH. There was a significant increase in cell area and cluster area from 45 to 90 DPH for all treatments. According to PCR analyses, a high proportion of xenogens (at 45 and 90 DPH, respectively) were detected in recipient fish injected with fresh SSC (72.2 and 83.3%), cryopreserved SSC (61.1 and 66.6%), fresh OSC (66.7 and 61.1%), or cryopreserved OSC (61.1 and 61.1%). Taken together, our results show that cryopreserved donor stem cells can recover in recipient gonads and perform as well as their freshly extracted counterparts. Hence, cryopreserved stem cells can be used for future germ cell transplantation to support xenogenesis. These findings will enhance the efficiency of germ cell transplantation in xenogenesis for commercial scale hybrid catfish production. • Evaluate colonization capability of fresh & cryopreserved blue catfish SSC & OSC. • Similar cluster & cell areas resulted in fresh & cryopreserved SSC transplantations. • Similar cluster & cell areas resulted in fresh & cryopreserved OSC transplantations. • Over 61% xenogens resulted from gonadal PCR analysis of SSC & OSC transplantations. • Cryopreserved stem cells can be used for future germ cell transplantation. [ABSTRACT FROM AUTHOR]

Transgenesis has a wide range of applications in fish breeding and generation of fish models. Previously, it was common to produce transgenic fish by transferring plasmid DNA into early embryos, resulting in random integration, but more precision, targeted integration is possible with CRISPR/Cas9 technology. Channel catfish (Ictalurus punctatus) is an economically important farmed fish in the United States. To make channel catfish an even richer source of nutrients, we produced P1 fish carrying masu salmon (Oncorhynchus masou) elovl2 (OmElovl2) transgene to increase the content of omega-3 (n-3) fatty acids with CRISPR/Cas9-mediated knock-in targeting non-coding region of chromosome 1, and random integration methods. Mosaicism, transgene expression and fatty acids contents were determined. Integration rates of seven-month-old channel catfish generated by CRISPR/Cas9 and random integration methods were 19% and 27.3%, respectively. However, when we tested five tissues including barbel, fin, muscle, liver and kidney of three channel catfish, 13 out of 15 total observations were verified to carry the transgene from three positive P1 fish produced by CRISPR/Cas9 technology. Only five of 15 tissues carrying transgene were detected in three positive P1 fish produced by random integration. Genomic quantitative real-time PCR (qRT-PCR) also suggested that CRISPR/Cas9 transgenic fish had extremely higher average transgene copy numbers than randomly integrated transgenic fish. Additionally, reverse transcription PCR (RT-PCR) and fatty acids analysis revealed that CRISPR/Cas9 P1 fish had strong OmElovl2 transgene expression in most tissues and 20.7% higher DHA than their controls, while randomly integrated P1 fish did not have detectable OmElovl2 expression in any of five tissues detected. There were no significant differences for any fatty acids between transgenic fish produced by random integration and their non-transgenic controls. CRISPR/Cas9 mediated knock-in technology efficiently reduced mosaicism, improved transgene expression and the biological effects of the foreign gene in P1 generation compared to the conventional random integration method. Therefore, transgenesis based on CRISPR/Cas9 technology would shorten breeding programs and improve applications of gene function studies. • Transgenic channel catfish carrying masu salmon elovl2 gene (OmElovl2) were produced by CRISPR/Cas9 and random integration. • CRISPR/Cas9 P1 fish had strong OmElovl2 transgene expression in most tissues and 20.7% higher DHA than their controls. • CRISPR/Cas9 might efficiently reduce mosaicism, improve the transgene expression than random integration in P1 generation. [ABSTRACT FROM AUTHOR]

Channel catfish, Ictalurus punctatus , is the most produced aquaculture species in the United States, although production levels have decreased since their peak in 2003. Myostatin (mstn) regulates skeletal muscle growth and has been identified as an important gene for increasing body weight in aquaculture species. In this study, the effects of CRISPR/Cas9 knockout of the mstn gene in channel catfish were investigated. Three sgRNAs targeting exon 1 of the channel catfish myostatin gene were microinjected with Cas9 protein in embryos. A total of 209 fish survived to 6 mph after microinjection of Cas9 and sgRNA targeting exon 1 of the mstn gene over the 3 yr experiment (2017–2019) with an average mutation rate of 58%. Successful generation of myostatin knockout (MSTN KO) F1 heterozygotes was achieved in 2019 by individually mating two pairs of control females with MSTN homozygous KO males. The offspring of both families inherited the mutation at a mean rate of 88%. Growth was generally higher in MSTN mutants than controls at all life stages and environments. P1 MSTN mutants were 88% larger than controls at the stocker stage (100 to 200 g) and 27% larger than controls at market size. Heterozygous F1 mutants were 218% larger than controls at the stocker stage. MSTN mutants had reduced overall expression levels of mstn compared to controls. When challenged with Edwardsiella ictaluri , the causative agent of enteric septicemia of catfish, MSTN mutants performed equally or better than controls. With a high mutation rate and inheritance as well as improved growth and disease resistance, using MSTN gene-edited channel catfish could greatly benefit commercial farms. • The effects of CRISPR/Cas9 knockout of the myostatin (mstn) gene in channel catfish, Ictalurus punctatus , was investigated. • A high rate of mutation was achieved that was inherited by the mutant's offspring. • Growth was generally higher in MSTN mutants than controls at all life stages and environments. • MSTN mutants had reduced overall expression levels of mstn compared to controls. • When challenged with Edwardsiella ictaluri , MSTN mutants performed equally or better than controls. [ABSTRACT FROM AUTHOR]

Long-term storage of spermatogonial stem cells (SSCs) represents the next step for advancing aquaculture research by providing valuable genetic resources for genetic enhancement programs and xenogenesis technologies. Hybrid catfish, the cross between channel catfish, Ictalurus punctatus ♀ and blue catfish, I. furcatus ♂, are in high demand by the US aquaculture industry, but production can be limited by the availability of blue catfish gametes. Therefore, xenogeneic stem cell transplantation is a powerful method for generating blue catfish gametes within faster growing and maturing channel catfish hosts. These technologies could be facilitated by having cryopreserved SSCs from testicular tissue on-hand in gene banks. Cryopreservation protocols for blue catfish testicular tissues and for other fish species are still being optimized, and currently, data is lacking on the effects antioxidants and antifreeze proteins (AFPs) have on cellular post-thaw viability of SSCs. The objective of this study was to analyze the individual and combined effects of antioxidants (catalase, hypotaurine, and ascorbic acid) and AFPs (I and III) on post-thaw type A spermatogonia cell production and viability from testicular tissue. In this study, there were no improvements with individual antioxidant or AFP treatments. However, when the antioxidants hypotaurine and catalase were tested in combination with different AFPs, three treatments had higher type A spermatogonia cell production than the control, including hypotaurine 7 mM + AFPI 1 μg/mL, hypotaurine 3.5 mM + AFPI 0.1 μg/mL, and hypotaurine 7 mM + AFPIII 0.1 μg/mL. These treatments along with hypotaurine 7 mM + AFPI 0.1 μg/mL and catalase 100 IU + AFPI 0.1 μg/mL had higher viability than the control. From these results, we recommend adding these antioxidant-AFP combinations to future cryopreservation protocols. Overall, this is the first study showing that combining specific antioxidants and AFPs can improve spermatogonia cryopreservation for a fish species. • Antioxidants and antifreeze proteins (AFPs) evaluated for cryopreservation. • Individual antioxidants and AFPs did not improve live SSC count or viability. • Some combinations of antioxidants and AFPs yielded positive effects. • Best antioxidant + AFP treatments contained hypotaurine or catalase. • New recommendations added to current blue catfish SSC cryopreservation protocol. [ABSTRACT FROM AUTHOR]

Xenogenesis is emerging as an innovative technology for hybrid catfish (channel catfish, Ictalurus punctatus ♀ × blue catfish, I.furcatus ♂) production, where primordial germ cells (PGC), spermatogonial stem cells (SSCs) or oogonial stem cells (OSCs) are transplanted into infertile host species such as triploid fish to produce xenogens. Currently, donor cells are collected from randomly selected immature fish without having prior knowledge regarding the best donor stage, which is critical to increase the efficiency and success of germ cell transplantation in catfish breeding. Therefore, the current study was carried out with the objective of determining the relationships between quantity of stem cells, total length (T L), total weight (T W), and the level of androgens [testosterone (T) and 11-ketotestosterone (11-KT)] in blue catfish (donor fish). Positive quadratic relationships were observed between T L and the number of extracted live SSCs (r2 = 0.51; P <.0001) as well as T W and the number of live SSCs (r2 = 0.37; P <.0001), revealing that a T L of 25 to 39.9 cm and T W of 100 to 499.9 g, yield the highest number of extracted live SSCs. Similar to the findings from males, T L (r2 = 0.76; P <.0001) and T W (r2 = 0.67; P <.0001), also revealed positive quadratic relationships to the number of extracted live OSCs, where 25 to 39.9 cm and 200 to 600 g females yielded the greatest number of extracted live cells. Positive quadratic relationships were also detected between levels of T (r2 = 0.60; P <.0001), 11-KT (r2 = 0.60; P <.0001) and number of extracted live SSCs in male catfish, where 49 to 51.9 pg/mL of serum T and 37 to 48.9 pg/mL of 11-KT yielded the maximum number of live SSCs. Overall, these relationships between the quantity of stem cells and body size as well as the profile of sex steroid hormones reveal promising results as reliable biomarkers to enhance the efficiency of germ cell transplantation. These results can also be used to make precise predictions on the number of stem cells at a certain body size without sacrificing precious fish housed in living genetic repositories. • Objective was to link quantity of blue catfish SSCs and OSCs to total length (T L), weight (T W), and androgens (T, 11-KT). • Relationships between T L , T W , and number of SSCs revealed that a T L of 25-39.9 cm and T W of 100-499.9 g yielded most stem cells. • T L and T W revealed positive relationships to number of live OSCs, where 25-39.9 cm and 200-600 g females yielded most stem cells. • Relationships detected between T, 11-KT, and SSCs, where 49-51.9 pg/mL of T and 37-48.9 pg/mL of 11-KT yielded most stem cells. [ABSTRACT FROM AUTHOR]