1,720,986 research outputs found
Integration of antimicrobial peptide genes via CRISPR/Cas9 for disease resistance enhancement and reversible sterility in catfish
Full text linkThe CRISPR/Cas9 platform holds promise for modifying fish traits of interest as a precise and versatile tool for genome manipulation. To reduce introgression of transgenes and control reproduction, catfish were studied for upscaled disease resistance coupled with intervention of reproduction to lower the potential environmental risks of introgression of transgenic escapees.
To generate disease resistance and sterility in channel catfish (Ictalurus punctatus), CRISPR/Cas9 systems were utilized to integrate the cathelicidin gene from an alligator (Alligator sinensis; As-Cath) into the target luteinizing hormone (lh) locus of channel catfish using two delivery systems assisted by double-stranded DNA (dsDNA) and single-stranded oligodeoxynucleotides (ssODNs), respectively. 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, implantion with HCG and LHRHa restored the fecundity, spawnability and hatchability of the new transgenic fish line.
To establish disease resistance and sterility in blue catfish (I. furcatus), transgenic blue catfish of primarily Rio Grande strain ancestry were generated with site-specific KI of the As-Cath transgene into the lh locus via two CRISPR/Cas9-mediated KI systems, assisted by the linear dsDNA and double-cut plasmid (dcPlasmid), respectively. High integration rates were observed with linear dsDNA (16.67%, [13/78]) and dcPlasmid strategies (24.53%, [26/106]). In addition, the on-target KI efficiency of the dcPlasmid 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), indicating no pleiotropic effects for growth of the As-Cath transgene integration at the lh locus was observed in the P1 founders.
To generate transgenic channel catfish carrying two exogenous antimicrobial peptide genes (AMGs), CRISPR/Cas9-assisted microinjection of cecropin (Cec) and As-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 analysis indicated that individual body weight and gender did not affect survival, while the transgenes Cec and As-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.
To establish transgenic sterile channel catfish lines with elevated disease resistance and fast growth rate, single-sgRNA-based genome editing (ssGE) and multi-sgRNA-based MGE (msMGE) were used to replace the lh and melanocortin-4 receptor (mc4r) genes with the As-Cath transgene and the myostatin (two target sites: mstn1, mstn2) gene with the Cec transgene, respectively. A total of 9,000 embryos were microinjected from three families, and 1,004 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 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.
Overall, the lh gene was replaced with the As-Cath transgene and then hormone therapy was administered to gain complete reproductive control of disease-resistant transgenic catfish in an environmentally sound manner. In addition, potential sterile catfish with enhanced disease resistance carrying two AMGs at multiple loci using transgenic-embryo-based genome editing or msMGE strategy was achieved. This strategy not only effectively improves the consumer-valued traits, but also guards against genetic contamination of wild populations. This is a breakthrough in aquaculture genetics to confine fish reproduction and prevent the establishment of transgenic or domestic genotypes in the natural environment
Isolation, Identification, Culture, Cryopreservation, Genetic Transformation and Transplantation of Catfish Germline Stem Cells
Full text linkThe hybrid of channel catfish (Ictalurus punctatus) ♀ × blue catfish (Ictalurus furcatus) ♂ (C × B) is the best catfish for pond culture. The current technology, artificial fertilization and handstripping is labor intensive, time consuming, and requires sacrifice of mature 5 ~ 6 year old blue catfish males. Xenogenic catfish produced by transplanting blue catfish stem cell into the gonads of triploid channel catfish could reduce the generation interval to produce blue catfish and might result in channel catfish males that are able to produce blue catfish sperm, and increase the efficiency of C × B hybrid catfish embryo production.
Gonadal cell size and type of 90 ~ 100 day - old fry (TL: 5 ~ 6 cm), two - year old juvenile (TL: 25 ~ 30cm) and mature adults (TL: 65 ~ 70 cm) blue catfish was examined. Gonads of all ages of fish contained oogonia (12 ~ 15 μm diameter, distinct nucleus 7 ~ 8 μm) and spermatogonia (12 ~ 15 μm, distinct nucleus 6 ~ 7.5 μm). Male juvenile blue catfish, which had suitable size and higher percentage of germline stem cells, were used for stem cell isolation for further study. After testes were dissected from the peritoneal cavity, germline stem cells were isolated with discontinuous density gradient centrifugation to enrich the percentage of spermatogonia. Four distinct cell bands were generated after centrifugation. It was estimated that 55% of the total cells in top - first bands, cell band I, were type A spermatogonia (diameter 12 ~ 15 µm) and type B spermatogonia (diameter 10 ~ 11 µm), 35% were spermatocytes (diameter 5 ~ 9 µm), 10% were spermatids (diameter < 1 µm). In the second band - cell bands II, most of the cells were spermatocytes (60%, diameter 5 ~ 9 µm), some were type B spermatogonia (30%), a few were spermatids (10%) and cell clusters could also be observed. The third band ~ cell band III contained most of the spermatids (95%) and very few secondary
spermatocytes (5%, diameter 5 ~ 8 µm). Red blood cells (95%) and a few spermatids (5%) were the predominant cells in the fourth cell band - cell band IV.
Different bands generated from Percoll density gradient centrifugation were used for germ cell marker identification studies. Expression of seventeen genes (pfkfb4, urod, Oct4, Plzf, sycp3, SOX2, Integrin6, neurogenin3, ID - 4, integrinV, Thy1, GFRα, CDH1, Smad, Pum2 and Prdm14 and Kit) from cells of different bands were analyzed by qRT - PCR in both channel and blue catfish. pfkfb4, urod, Plzf, Integrin6, ID - 4, integrinV, Thy1 and CDH1 genes, which showed the same expression change pattern in different types of testicular germ cells of both channel and blue catfish, were identified as spermatogonia marker. SOX2 gene, which was up - regulated in spermatocytes and even higher up - regulated in spermatids, was identified as spermatids marker.
Blue catfish testicular and ovarian germ cell in vitro cultures were studied. When testicular tissues were used as the initial culture materials, testicular germ cells migrated outward the edges of tissues and formed monolayers during the first two - week culture. Cultured testicular germ cells were fibroblast - like, growing fast after the third passage. For ovaries, ovarian germ cell isolated after trypsin digestion was used as the initial culture material, and the cells attached to the plate during the first week of culture. Cells from ovaries were also fibroblast - like in culture.
Electroporation was used for both channel and blue catfish germ cell genetic transformation. Both blue catfish ovarian germ cells and channel catfish testicular germ cells expressed green florescence protein (GFP) for a long time period after cells were transformed by the expression construct FRMwg driven by the carp β - actin promoter.
Two transplantation techniques, surgery and catheterization, were studied for transplanting spermatogonian stem cell enrichment and testicular germ cells into triploid channel catfish gonads. Ten months after the surgery transplantation, eight fish were recaptured; all three recipient fish which were transplanted by surgery were proved containing blue catfish cells; four out of five recipient fish which were transplanted by catheterization contained blue catfish cells. Both surgery and catheterization were efficiency transplantation method and had a very high transplantation ratio
Evaluation of Disease Resistance in Selectively Bred Channel Catfish (Ictalurus punctatus) and Hybrid Catfish (Channel Catfish ♀ x Blue Catfish (I. furcatus) ♂)
Full text linkSelectively bred channel catfish (Ictalurus punctatus) and hybrid catfish (channel catfish ♀ x blue catfish (I. furcatus) ♂) were evaluated for resistance to channel catfish virus (CCV) and Flavobacterium covae (FC). In the CCV challenge, survival following CCV exposure varied significantly among families. Overall analysis showed slightly higher resistance for the hybrid catfish. Blue catfish paternal effects were more impactful than channel catfish maternal effects. These results contradicted earlier studies that indicated the resistance of hybrid catfish to CCV was less than or equal to channel catfish.
Two FC challenges were conducted, one high dose and one low dose, resulting in extremely rapid and slower mortality, respectively. In the high dose challenge, overall survival fell below 10 % within 72 h, and channel catfish families had longer survival than the hybrid catfish (mean 19.9 h vs 16.8 h; log rank p = 0.01), Family effects were also observed (p < 0.05). In the low dose trial, median survival reached the 176 h study maximum for all families, and channel catfish still averaged longer survival than hybrid catfish (157.4 h vs 138.3 h; p = 0.056).
These findings contradict earlier reports that hybrid catfish are more resistant to columnaris than channel catfish (Arias et al., 2012). Genotype-environment interactions may occur due to strain differences in the pathogen, F. covae and/or the confined conditions of the experiments potentially causing more stress on the hybrid catfish leading to greater disease susceptibility in this specific challenge environment
An Evaluation of Natural and Artificial Dietary Lipid Sources on Egg Quality and Fry Production in Channel Catfish (male) X Blue Catfish (female) Hybridization
No full textHybrid catfish (channel catfish Ictalurus punctatus? x blue catfish I. furcatus?) display many characteristics that are favorable to aquaculture production such as increased growth, resistance to disease, higher dress out and ease of seining. The implementation of this hybrid variety into full scale aquaculture production is limited mainly by the difficulties involved with spawning, incubation and larval rearing. One of the confounding stages that has high mortality rates is the complex developmental steps that take place after fertilization. In this phase, the developing embryo must obtain its nutrients and structural components for development from the egg reserves. Therefore, to maximize the survival of these hybrid embryos through hatch, a high quality egg should be supplied by the female. This study was conducted in an attempt to isolate nutritional lipids that lead to high quality egg production in female channel catfish. A 10 week feed trial was conducted in ponds in Auburn, Alabama. In March, 219 female channel catfish brood stock were stocked into nine ponds 0.04 ha in size each for an approximate stocking rate of 1,332 kg/ha. Three dietary treatments were randomly allocated to the fish. Diet-1 was a standard 32% crude protein, 6% lipid floating catfish feed. Diet-2 was the same feed supplemented with forage fish at approximately 28 kg/ha. The third diet was the aforementioned catfish feed top-coated with 2% lipid (1% Menhaden fish oil, 0.5% high DHA oil and 0.5% high ARA oil). The fish were fed the prepared feeds three times a week at 1.5% body weight per feeding. Dissolved oxygen and temperatures were measured twice daily at dawn and dusk, and low DO events were mitigated by nighttime aeration. Ammonia, nitrite and pH parameters were measured twice a week. In May, the females were harvested, administered injections of luteinizing hormone releasing hormone analog to induce ovulation and strip spawned. The eggs were fertilized with blue catfish sperm and incubated in paddle wheel troughs. Percent viable fry was estimated by egg mass assessments 24 hours prior to hatch and resultant fry counts. The spawning data indicates brood fish fed the high lipid diet spawned larger egg masses (+17.6 g eggs/kg brood fish, p=0.003) and had larger eggs both in weight and diameter, when compared to either the control or forage fish treatment (+2.5mg and +0.3mm, p=0.001). These eggs, in turn, had increased complements of high quality lipids such as DHA, EPA and total n-3 fatty acids (p=0.001)
Disruption of Embryonic Development in Channel Catfish, Ictalurus Punctatus, Using 'Sterile-Feral' Gene Constructs
No full textChannel catfish, Ictalurus punctatus, embryos were electroporated with a sterile feral 3 (SF3), sterile feral 4 (SF4), glutamate decarboxylase (GAD) or CAB constructs and a blank control. Doxycycline was applied to some of the SF3 and SF4 replicates, at 50, 100 and 150 ppm, from 4 hours post fertilization to first hatch. The developing embryos were observed from fertilization up through hatch. Dead and deformed embryos were removed during this period. Mortality rates were computed for different time intervals during development as well as for the entire development period. Deformity rates were also analyzed. Objectives were to determine the efficacy of the sterile feral constructs to disrupt embryonic development, the optimal concentration of doxycycline to prevent embryonic disruption and the time interval most crucial for application of doxycycline to prevent the expression of the developmental disrupter.
Embryos electroporated with the SF3 construct consistently demonstrated a higher mean mortality than that of the control groups. The overall mean mortality of the SF3 groups for all three experiments was 35.9% higher than the overall mean mortality of the three blank control groups. Similar results were obtained for the SF4 construct, whose overall mean mortality for the three experiments was 27.2% higher than that of the overall mean mortality for the three experiments with the blank control groups. The GAD construct produced an overall mean mortality that was 34.5% lower than that of the mean mortality of SF3 and 25.6% lower than that of SF4. The single mortality rate produce by the CAB construct was 82.3% lower than that of the mean mortality of SF3 and 79.9% lower than SF4. These consistently lower mortality rates produced by both CAB and GAD demonstrated that the introduction of exogenous DNA was not responsible for the larger mortality rates observed in the SF4 group and SF3 groups, but the action of the SF3 and SF4 constructs. The concentration of doxycycline most optimal for blocking the expression of the blocker in the SF3 construct was 100 ppm and 150 ppm for blocking the developmental disrupter of the SF4 construct. Some portion of the 18-61 hour post fertilization time period appears to be the time interval in which it is most crucial to apply doxycycline to prevent embryonic disruption
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Growth and Morphology of a Synthetic Channel Catfish (Ictalurus punctatus)-Blue Catfish (Ictalurus furcatus) Backcross Breed and the Hybrid Between the Backcross Female and Blue Catfish Male
Full text linkThe F1 hybrid between a channel catfish female (Ictalurus punctatus) × a blue catfish (Ictalurus furcatus) outperforms both parental species in commercial environments for numerous traits. However, reproductive isolation mechanisms between the species make mass production of the F1 hybrid labor intensive and the hybrid is not 100% disease resistant. A synthetic breed be-tween channel catfish and blue catfish was produced by 3 generations of backcrossing with channel catfish followed by one generation of closed breeding. This synthetic channel-blue breed was hybridized with blue catfish males to determine if there could be benefits from both multiple generations of backcrossing followed by heterosis from hybridization. The growth of this hybrid was approximately 30% faster than both the parental synthetic backcross breed and that of chan-nel catfish. The growth rate of synthetic backcross catfish was similar to channel catfish which is not surprising as selection for growth rate was not part of the backcrossing program. The hybrid between the backcross female and blue catfish male had the highest relative body area than back-cross catfish and channel catfish, which predicts that it would also have the higher dressout and fillet percentage. The correlation among morphometric traits was variable among genotypes. Skewness for body weight tended to be low to moderate for all genetic types, which is important considering the oversized fish problem in the commercial industry. Skewness for relative body area was highly negative for the backcross × hybrid, negative for the backcross and positive, but near zero for the channel catfish
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