Production of Walleye,Stizostedion vitreum,in Earthen Ponds Fertilized with Organic and Inorganic Fertilizers and Stocked at Three Rates

Walleye, Stizostedion vitreum, fry were raised at densities of 20,30 and 40/m³ in ponds initially fertilized with alfalfa and yeast and supplementally fertilized with liquid inorganic fertilizers. Liquid fertilizers were added weekly to maintain N and P concentrations of 600 and 30 ug/L, respectively. Fish growth was not affected by stocking rate, but survival was significantly (P < 0.05) reduced in ponds stocked at the two higher rates. The fertilization procedure we used did not provide a sufficient forage base to support more that 20 walleye/m³.     

Epidermal hyperplasia of walleye, Stizostedion vitreum vitreum (Mitchill), associated with retrovirus-like type-C particles: prevalence, histologic and electron microscopic observations

Epidermal hyperplasia consisting of discrete translucent raised outgrowths of cells were observed on the skin of walleye, Stizostedion vitreum vitreum (Mitchill), during their spawning period in the spring. The cells constituting the hyperplastic growths were limited to the epidermal layer, and were associated with surface budded, 120-nm-diameter, retrovirus-like particles located in the expanded intercellular spaces. These tumour-like growths were distinct from the other virus-associated skin lesions of walleye including dermal sarcoma, lymphocystis disease and herpesvirus-associated hyperplasia. Lesions could be differentiated by careful observation in the field and comparison of portions of each growth by histologic and electron microscopic observations.     

Pond Production of Fingerling Walleye,Stizostedion vitreum,in the Northern Great Plains

The cultural practices used to produce fingerling walleye, Stizostedion vitreum, in drainable earthen ponds are described for a state fish hatchery in Nebraska and two federal hatcheries in North Dakota. The ponds were filled 1 to 7 days before D2-D4 (Dl=the day of hatch) walleye fry were stocked. At one hatchery, ponds were sometimes double-cropped, first for production of northern pike, Esoxlucius. The two federal hatcheries fertilized ponds with ground alfalfa hay or pellets, while the standard practice at the Nebraska hatchery was not to fertilize walleye ponds, because of concern that fertilization would result in weed problems and oxygen depletion. One hatchery seeded the ponds with rye grass in the fall. Two of the hatcheries regularly used herbicides to prevent the stranding of fingerlings during harvest and their mortality caused by entangment with net algae, Hydrodicton. When used, herbicide treatment was applied before ponds were filled (Aquazine^TM) or as needed during the culture interval (Aquazine^TM) or copper sulfate). Harvesting was done after 24 to 58 days; the extreme range represented variation among hatcheries; the variation among ponds at a given hatchery ranged from 4 to 10 days. Harvest occurred when fingerlings were 25 to 50 mm total length and weighed 1,500-5,440 fish/kg. Harvests ranged from 11,933 to 308,537 fingerlings/ha. Survival ranged from 3 to 104% of the estimated number of fry stocked.     

Length reduction and dry weight loss in frozen and formalin-preserved larval walleye, Stizostedion vitreum (Mitchill)

Abstract We examined the relationships between fresh and preserved measurements of length and dry weight for larval walleye, Stizostedion vitreum (Mitchill), subjected to freezing and preservation in formalin. Length reductions for both preservation techniques were <5% and decreased with larval size. Dry weight losses ranged from 32·1 to 54·0% for frozen larvae and from 18·2 to 30·1% for larvae preserved in formalin with larger larvae losing proportionately less weight. Freezing caused significantly greater length shrinkage and dry weight loss than preservation in formalin.     

Establishment and characterization of a new cell line from koi brain (Cyprinus carpio L.)

A novel permanently growing brain cell line from koi (Cyprinus carpio L.) (KB cell line) was established, and its suitability for detection of koi herpesvirus (KHV) was demonstrated in this study. The KB cell line was optimally maintained at 27°C in Leibovitz's L‐15 medium supplemented with 10% foetal bovine serum (FBS). It was subcultured more than 100 times, and chromosome analysis revealed that 51.54% of KB cells at passage 80 maintained the abnormal diploid chromosome number 2n = 96 while the modal chromosome number was 2n = 100. The cell line was cryopreserved in liquid nitrogen at ?196°C and was recovered from storage after 1 year with good cell viability and vitality. The results of virus isolation demonstrated that KB cells were susceptible to KHV, which was shown by the presence of an obvious cytopathic effect and abundant virus particles. The viral titres of KHV in KB reached 10^5.73TCID₅₀/0.1 ml within 7 days. Immunofluorescence and Western blot assays confirmed that KB replicated KHV. The newly established KB cell line will serve as a useful tool to elucidate KHV disease (KHVD) pathogenesis.     

Development of a walleye cell line and use to study the effects of temperature on infection by viral haemorrhagic septicaemia virus group IVb

A cell line, WE-cfin11f, with a fibroblast-like morphology was developed from a walleye caudal fin and used to study the intersection of thermobiology of walleye, Sander vitreus (Mitchill), with the thermal requirements for replication of viral haemorrhagic septicaemia virus (VHSV) IVb. WE-cfin11f proliferated from 10 to 32 °C and endured as a monolayer for at least a week at 1–34 °C. WE-cfin11f adopted an epithelial shape and did not proliferate at 4 °C. Adding VHSV IVb to cultures at 4 and 14 °C but not 26 °C led to cytopathic effects (CPE) and virus production. At 4 °C, virus production developed more slowly, but Western blotting showed more N protein accumulation. Infecting monolayer cultures at 4 °C for 7 days and then shifting them to 26 °C resulted in the monolayers being broken in small areas by CPE, but with time at 26 °C, the monolayers were restored. These results suggest that at 26 °C, the VHSV IVb life cycle stages responsible for CPE can be completed, but the production of virus and the initiation of infections cannot be accomplished.     

Differential viral haemorrhagic septicaemia virus genotype IVb infection in fin fibroblast and epithelial cell lines from walleye,Sander vitreus (Mitchill), at cold temperatures

A cell line, WE‐cfin11e, with an epithelial‐like morphology was developed from a caudal fin of walleye, Sander vitreus (Mitchill), characterized as distinct from the established walleye caudal fin fibroblast‐like cell line, WE‐cfin11f, and compared with WE‐cfin11f for susceptibility to VHSV IVb. Immunocytochemistry and confocal microscopy were used to localize the intermediate filament protein, vimentin, the tight junction protein, zonula occludens‐1 (ZO‐1), the extracellular matrix protein, collagen I, and the viral protein, G. Although both cell lines contained vimentin, only WE‐cfin11e stained for ZO‐1 and only WE‐cfin11f stained for collagen I. Ascorbic acid increased the accumulation of collagen I and caused the appearance of collagen fibres only in WE‐cfin11f cultures. At 14 °C, both cell lines produced VHSV IVb, but the infection developed more rapidly in WE‐cfin11f. At 4 °C, both cell lines became infected with VHSV IVb as judged by the expression of viral proteins, N and G, but only WE‐cfin11f produced virus. The results suggest that cold temperatures can modulate viral tropism.     

Development and characterization of a fibroblastic‐like cell line from caudal fin of the red‐line torpedo,Puntius denisonii (Day) (Teleostei: Cyprinidae)

A fibroblastic‐like cell line was established from the ornamental fish, red‐line torpedo (Puntius denisonii). The red‐line torpedo fin (RTF) cell line is being maintained in Leibovitz's L‐15 medium supplemented with 10% fetal bovine serum (FBS) for over 1 year at 28 °C on a continuous basis in normal atmosphere. The growth rate of RTF cells increased as the FBS proportion increased from 5% to 20% at 28 °C with optimum growth at the concentrations of 10% FBS. The morphology of RTF cell was predominantly fibroblastic like. Propagation of these cell lines was serum dependent, with a low plating efficiency (<15%). Karyotyping analysis of RTF cells at the 25th passage indicated that the modal chromosome number was 2n=50. The cell line was cryopreserved in liquid nitrogen at ?196 °C and could be recovered from storage after 6 months with good cell viability. Polymerase chain reaction amplification of a fragment of two mitochondrial genes, 16S rRNA and CO1, confirmed the identity of these cell lines with those reported from this animal species, confirming that the cell lines originated from P. denisonii. The bacterial extracellular products from Vibrio cholerae MTCC3904 and Aeromonas hydrophila were found to be toxic to RTF. The cell lines were not susceptible to viral nervous necrosis virus, a marine fish virus.     

Endocrine and gonadal changes during the annual reproductive cycle of the freshwater teleost,Stizostedion vitreum

The annual reproductive cycle of walleye (Stizostedion vitreum) was characterized by documenting changes in gonadal development and serum levels of estradiol-17β (E₂), testosterone (T), 17α,20β-dihydroxy-4-pregnen-3-one (17,20-P), and 11-ketotestosterone (11-KT) in wild fish captured from upper midwestern lakes and rivers throughout the year. Fish from the populations used in this study spawn annually in early- to mid-April. Walleye showed group synchronous ovarian development with exogenous vitellogenesis beginning in autumn. Oocyte diameters increased rapidly from ∼ 200 μm in October to ∼ 1,000 μm in November, and reached a maximum of 1,500 μm just prior to spawning. Changes in gonadosomatic indices (GSIs) paralleled changes in oocyte diameters. Serum E₂ levels in females increased rapidly from low values in October (< 0.1 ng ml⁻¹) to peak levels of 3.7 ng ml⁻¹ in November, coinciding with the period of the most rapid ovarian growth. Subsequently, E₂ levels decreased from December through spawning. Serum T levels exhibited a bimodal pattern, increasing to 1.6 ng ml⁻¹ in November, and peaking again at 3.3 ng ml⁻¹ just prior to spawning. We detected 11-KT in the serum of some females at concentrations up to 5.6 ng ml⁻¹, but no seasonal pattern was apparent. In this study (unlike our results in a related study) 17,20-P was not detected. In males, differentiation of spermatogonia began in late August, and by January the testes were filled (> 95% of germ cells) with spermatozoa. Mature spermatozoa could be expressed from males from January through April. GSIs ranged from 0.2% (post-spawn) to 3.2% (pre-spawn). Serum T levels rose from undetectable levels in post-spawn males to 1.6 ng ml⁻¹ by November, remained elevated throughout the winter, and peaked at 2.8 ng ml⁻¹ I prior to spawning. Levels of 11-KT in males remained low (< 10 ng ml⁻¹, from post-spawning through January, then increased significantly by March and peaked just prior to spawning at 39.7 ng ml⁻¹. Our results indicate that vitellogenesis and spermatogenesis are complete or nearly so, in walleye by early winter, and suggest that it may be possible to induce spawning in this species several months prior to the normal spawning season by subjecting fish to relatively simple environmental and hormonal treatments.     

Evaluation of Experimental and Practical Diets for Walleye Stizostedion vitreum

Culture of walleye Sfizostedion vitreum is one of the largest components of public sector aquaculture in the eastern U.S. and there is increasing interest in private sector culture. However, the nutritional requirements of walleye are unknown and experimental diets for use in quantifying nutritional requirements have not been identified. We formulated four experimental and four practical diets and fed those to triplicate groups of walleye with an initial weight of 13 g per fish. The experimental diets contained either casein (CAS), casein + gelatin (CG), casein + arginine (CA), or casein + gelatin + crystalline amino acids (CGAA) as sources of amino acids. The practical diets were formulated to mimic salmon grower (SG) and trout grower (TG) diets, a fish meal‐free diet for trout (TFMF), and a walleye grower (WG) diet. Fish were fed twice daily to satiation for 9 wk. Feed consumption, percent weight gain, specific growth rates, feed efficiency, protein efficiency ratio, and protein retention efficiency were not significantly different among fish fed CGAA, SG, and TG, but those values were significantly higher than in fish fed other diets. Weight gain of fish fed CGAA was approximately 80% of that in fish fed SG and 91% of that in fish fed TG. Protein retention efficiency of fish fed CGAA was approximately 69% and 81% of that observed for fish fed SG, and TG, respectively. In general, the carcasses of fish fed diets CGAA, SG and TG had significantly lower moisture and ash concentrations, and higher lipid levels than fish fed other diets. There were no significant differences in carcass protein concentration, muscle proximate composition, or liver lipid concentration among treatments. Livers from fish fed all diets were characterized by microvesicular degeneration and glycogen accumulation in hepatocytes. Results from the study indicate that CGAA can be used as a basal experimental diet in future nutritional research with juvenile walleye and confirms the benefits of trout and salmon grower diets. Fish meal‐free diets formulated around the requirements for rainbow trout were consumed at approximately 80% of the values in fish fed TG and SG, but weight gain was approximately 20% of that in fish fed TG and SG. It appears the nutritional requirements for walleye are different than those of rainbow trout.     

Development and characterization of five novel cell lines from snubnose pompano,Trachinotus blochii (Lacepede, 1801), and their application in gene expression and virological studies

Five novel permanent cell lines have been established from gill, heart, kidney, eye and fin of snubnose pompano, Trachinotus blochii. They were designated as snubnose pompano gill (SPG), snubnose pompano heart (SPH), snubnose pompano kidney (SPK), snubnose pompano eye (SPE) and snubnose pompano fin (SPF), respectively. All these cell lines were characterized and cryopreserved successfully at different passage levels. Cell lines were passaged every alternate day; SPG, SPH, SPK, SPE and SPF cell lines attained passage levels of 68, 74, 82, 79 and 106, respectively, since the initiation of their development in 2019. The cell lines grew well in Leibovitz's 15 medium containing 15% foetal bovine serum at 28°C. Immunophenotyping of the cell lines revealed the presence of fibronectin and pancytokeratin. No mycoplasma contamination was found. The transfection study revealed the gene expression efficiency of these cell lines by expressing the green fluorescent protein (GFP). The authentication on origin of cell lines from T. blochii was confirmed by amplification of species-specific mitochondrial cytochrome oxidase I gene. The results showed the susceptibility of these cell lines to fish nodavirus (FNV) and tilapia lake virus (TiLV) and resistance to cyprinid herpesvirus 2 (CyHV-2). The FNV infection in the cell lines was confirmed by RT-PCR, Western blot, ELISA and immunocytochemistry, while TiLV infection was confirmed by RT-PCR assay. These results revealed that these cell lines are suitable for virological and foreign gene expression studies.     

Cultural characteristics of salmonid alphaviruses – influence of cell line and temperature

Laboratory studies were carried out to investigate the cultural characteristics of salmonid alphaviruses (SAV) from Atlantic salmon (AS, Salmo salar) and rainbow trout (RT, Oncorhynchus mykiss), particularly in relation to cell line and temperature. In an initial study, SAV was isolated from 12 viraemic sera and passaged in Chinook salmon embryo (CHSE‐214) cells at 15 °C. Geometric mean titres (GMT) after initial isolation were found to be significantly higher (P < 0.05) relative to those after two or four passages. Primary isolation of SAV was conducted from 12 viraemic sera (six AS and six RT) in seven different cell lines at 15 °C: CHSE‐214, rainbow trout gonad (RTG‐2), TO (derived from Atlantic salmon head kidney leucocytes), salmon head kidney (SHK‐1), blue fin‐2 (BF‐2), fat head minnow (FHM) and Epithelioma papulosum cyprini (EPC). Overall, significant differences were found between cell lines in both the numbers of strains where growth was detected and in the GMT obtained. For both AS and RT strains, GMT values were significantly (P < 0.01) higher in both TO and BF‐2 cells relative to the others, including CHSE‐214 and RTG‐2, the cell lines conventionally used for SAV. The effects of temperature of incubation (4, 10, 15 and 20 °C) on growth in TO, CHSE‐214 and RTG‐2 were investigated. In TO and RTG‐2 growth was optimal at 15 °C, whereas in CHSE‐214 results at 10 and 15 °C were more similar. Little or no growth was detected at 4 or 20 °C.     

Establishment and characterization of a heart-derived cell line from goldfish (<Emphasis Type="Italic">Carassius auratus</Emphasis>)

The goldfish Carassius auratus, a freshwater fish in the family Cyprinidae, was one of the earliest fish to be domesticated for ornamental purposes. A cell line was established from goldfish heart (GH) tissue to create a biological monitoring tool for viral diseases. The GH cell line was optimally maintained at 25 °C in M199 medium supplemented with 10–20% fetal bovine serum. A chromosomal analysis indicated that the cell line remained diploid, with a mean chromosomal count of 100. In viral inoculation assays, significant cytopathic effects (CPEs) were caused by epizootic hematopoietic necrosis virus (EHNV), Andrias davidianus iridovirus (ADIV), and Bohle iridovirus (BIV) infections in the fish cells and the viral titers (average value) of EHNV, ADIV, and BIV in GH cells reached 10^5.0, 10^4.5, and 10^5.0 TCID₅₀/0.1 mL, respectively, within 7 days. However, no CPE was observed in the cells infected with viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), spring viremia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), channel catfish virus (CCV), or grass carp reovirus (GCRV). These results suggest that the GH cell line is a valuable tool for studying viral pathogenesis.     

Effects of Fertilization and of Fry Stocking Density on Pond Production of Fingerling Walleye,Stizostedion vitreum

The influence of fertilization and of fry stocking density on production of fingering walleye, Stizostedion vitreum, was evaluated in earthen ponds at North Platte State Fish Hatchery, North Platte, Nebraska. In 1990, five 0.4-ha ponds were fertilized with alfalfa pellets, and five were fertilized with soybean meal; four unfertilized ponds served as controls. All ponds were stocked with D2 (Dl = the day at hatch) walleye fry at 250.000ha. Differences in yield, number of fingerlings harvested, mean length, and mean weight amone treatments were not statistically significant (P> 0.05). In 691, two fertilization schedules (no fertilizer and fertilization with alfalfa pellets) and two fry stocking rates (250.000 and 375,000 fry/ha) were evaluated. Four ponds were used for each treatment. Statistically significant treatment differences were found in yield, number of fingerlings harvested/ha, average length, and average weight. Yield was higher in fertilized ponds compared with yield from unfertilized ponds at both stocking densities, but yield did not differ significantly between stocking density treatments given the same fertilizer treatment. Survival did not differ between density treatments, but total number of fish harvested was significantly greater from ponds stocked at the higher density. Fingerlings with the largest average weight were raised in fertilized ponds that were stocked at 250,00O/ha, while the smallest fingerlings were from unfertilized ponds that were stocked at 375,000ka. Days in culture interval, which varied among ponds by 9 days in 1990 and 10 days in 1991, was significantly correlated with most production variables in 1990 and with all production variables in 1991. Means of water quality variables were not significantly different between fertilized and unfertilized ponds in either year, but significant differences were found in means of three water quality variables between 1990 and 1991. Yield in both fertilized and unfertilized ponds in 1991 was less than in 1990.     

Critical thermal maxima and minima for curimbatá, Prochilodus scrofa Steindachner, of two different sizes

Critical thermal maxima (CTMax) and minima (CTMin) were determined for Prochilodus scrofa Steindachner of two sizes (19.5 ±7.2 g and 249 ± 42.4 g). acclimated at 15. 20, 25, 30 and 35 ± 1°C. The CTMax and the CTMin for the smaller fish were 33.9. 36.7. 38.7, 40.3. 42.0°C and 5.0, 7.2. 9.2. 10.3. 13.4^oC and for the larger (Ish 33.3, 35.7, 38.2. 40.6. 42.6°C and 6.5, 8.2. 10.8. 12.4, 14.6°C. respectively, at each acclimation temperature. The CTMin from smaller fish were significantly lower than those from larger ones but the CTMax did not show any such difference. These results indicate that P. scrofa is suitable for culture in south-eastern and even in southern Brazil where winter temperatures may drop to very low levels, mainly at night. The zone of thermal tolerance calculated by CTMax and CTMin was equivalent to 1046°C² and 964.2°C², respectively, for smaller and larger fish, showing a high degree of eurythermicity.