鲫肠道上皮细胞原代培养方法的研究

探讨了健康鲫(Carassius auratus)幼鱼肠道上皮细胞的原代培养方法。结果显示,用含有抗生素、胶原蛋白酶Ⅰ、EDTA和胶原蛋白酶Ⅳ组成的D-Hanks液消化无菌分离的鲫肠道可以获得大量细胞团及绒毛隐窝;用含有抗生素、胎牛血清(FBS)、表皮生长因子(EGF)、胰岛素的DMEM培养液进行培养,并根据肠道上皮细胞与成纤维细胞贴壁时间的差异进一步纯化,连续培养12d后可以得到纯化的肠道上皮细胞。     

Cell wall disruption: An effective strategy to improve the nutritive quality of microalgae in African catfish (Clarias gariepinus)

The rigid cell walls of microalgae may hinder their utilization in fish feeds. The current experiment assessed the correlation between the accessibility of microalgae nutrients and their in vivo digestibility in African catfish. Nannochloropsis gaditana biomass was subjected to physical or mechanical treatments to weaken its cell wall; untreated—no disruption treatment (UNT), pasteurization (PAS), freezing (FRO), freeze‐drying (FRD), cold pasteurization (L40) and bead milling (BEM). Six experimental diets formulated from differently treated and untreated microalgae (at 30% diet inclusion level) were tested on growth performance and apparent nutrient digestibility (ADCs) in juvenile African catfish. A basal diet (REF) containing no microalgae was used as reference diet. Results showed that biomass gain and feed conversion ratio of fish fed L40 and BEM diets increased by 13% and 11%, respectively, relative to the UNT diet. Additionally, FRD, FRO, L40 and BEM cell wall disruption treatments improved protein digestibility by 0.5%, 5.9%, 8.4% and 16.3%, respectively, compared to the UNT treatment. There was a positive correlation between accessibility of microalgal nutrients and their digestibility in African catfish. Nutrient digestibility of microalgae was dependent on extent of cell disruption. Also, the impact of cell disruption on nutrient digestibility of microalgae differs between African catfish and Nile tilapia.     

Development of a continuous cell line from larval Atlantic cod (Gadus morhua) and its use in the study of the microsporidian,Loma morhua

In vitro cell culture methods are crucial for the isolation, purification and mass propagation of intracellular pathogens of aquatic organisms. Cell culture infection models can yield insights into infection mechanisms, aid in developing methods for disease mitigation and prevention, and inform commercial‐scale cultivation approaches. This study details the establishment of a larval cell line (GML‐5) from the Atlantic cod (Gadus morhua) and its use in the study of microsporidia. GML‐5 has survived over 100 passages in 8 years of culture. The line remains active and viable between 8 and 21°C in Leibovitz‐15 (L‐15) media with 10% foetal bovine serum and exhibits a myofibroblast phenotype as indicated by immuno‐positive results for vimentin, α‐smooth muscle actin, collagen I and S‐100 proteins, while being desmin‐negative. GML‐5 supports the infection and development of two microsporidian parasites, an opportunistic generalist (Anncaliia algerae) and cod‐specific Loma morhua. Using GML‐5, spore germination and proliferation of L. morhua was found to require exposure to basic pH and cool incubation temperatures (8°C), in contrast to A. algerae, which required no cultural modifications. Loma morhua‐associated xenoma‐like structures were observed 2 weeks postexposure. This in vitro infection model may serve as a valuable tool for cod parasitology and aquaculture research.     

Volume regulation in glutathione-treated brook trout (Salvelinus fontinalis) erythrocytes

Brook trout erythrocytes that were washed with and suspended in Ringer's solution with reduced glutathione (1.0 mM) maintained steady state cell volume for up to 24h, while those without the thiol-protective agent steadily shrank. Changes in cell volume (measured as packed cell volume, PCV) were evoked by acidic media (Ringer's at pH 6.8), hypoosmotic solutions (or both) and intracellular K⁺ and Cl⁻ concentrations were monitored over 4h. Acid-swollen cells failed to volume regulate or release K⁺ but had significantly elevated intracellular Cl⁻ Osmotically-swollen cells at pH 7.8 but not at pH 6.8 underwent regulatory volume decrease (RVD) and returned to initial levels in 2h, accompanied by release of K⁺ and Cl⁻ In contrast, osmotically-shrunken cells did not show regulatory volume increase. The regulatory volume decrease and concomitant K⁺ release were dependent on Cl⁻ implying a direct or indirect coupling of K⁺ to Cl⁻ transport in volume regulation. RVD was partially blocked by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, 0.1 mM), an anion exchange blocker, but was unaffected by amiloride (1.0 mM) which blocks Na⁺/H⁺ exchange. Amiloride and DIDS prevented the swelling response to low pH but had no effect on control cells, suggesting involvement of Na⁺/H⁺ and Cl⁻/HCO₃ ⁻ exchanges in acid-induced cell swelling. Quinine (1.0 mM) a known blocker of K⁺ channels, exacerbated the osmotically-induced swelling but had little effect on the subsequent RVD and release of KCl. The results suggest that low extracellular pH inhibits neutral C⁻-dependent K⁺ release and the resultant regulatory volume decrease in osmotically-swollen cells.     

Effects of cortisol and angiotensin II on the number and size of juxtaglomerular cells in masu salmon, Oncorhynchus masou

The authors previously reported that the number and size of juxtaglomerular cells (JGCs) in the kidney increased during smoltification in masu salmon, Oncorhynchus masou. In the present study, the effects of cortisol and/or angiotensin (Ang) II ([Asn¹, Val⁵]-Ang II) on the JGC number and size in masu salmon were examined to elucidate hormonal regulation of the changes in the JGC number and size during smoltification. These hormones were injected intraperitoneally every 2 days for a total of 6 injections. There was a significant increase in the JGC number and size with time following the start of the experiment in cortisol- and cortisol + Ang II-treated groups and no significant change in control and Ang II-treated groups. On both days 5 and 11, the JGC number and size in the cortisol-treated group were significantly large compared to those of control and Ang II-treated groups, respectively. The JGC number and size in the cortisol + Ang II-treated group were significantly large compared to those of control on both days 5 and 11, and those of the Ang II-treated group only on day 11, respectively. On the other hand, there was no significant difference in the JGC number and size between the Ang II-treated and control groups and between the cortisol- and cortisol + Ang II-treated groups during the experiment, respectively. The means of the JGC number and size in cortisol-treated group on day 11 were close to those previously reported in smolt. These results suggest that cortisol induces an increase in JGC number and size during smoltification in masu salmon. This revised version was published online in August 2006 with corrections to the Cover Date.     

Non‐permissive C6/36 cell culture for the Australian isolate of Macrobrachium rosenbergii nodavirus

Macrobrachium rosenbergii nodavirus (MrNV) that causes white tail disease (WTD) is an emerging disease that contributes to serious production losses in Macrobrachium hatcheries worldwide. Mosquito cell lines (C6/36) have been reported to support the growth of MrNV and used to observe the cytopathic effects (CPE) in infected cells. This study determined the susceptibility of C6/36 mosquito cells to the Australian isolate of MrNV in order to use fewer animals in further investigations. Different staining methods were used to observe MrNV viral activity in C6/36 cells. Typical cytopathic effects such as vacuolation and viral inclusion bodies were observed in infected C6/36 cells with H&E and Giemsa staining. With acridine orange, it was easier to detect presumptive MrNV messenger ribonucleic acid in the infected cells. Using neutral red staining to measure mitochondrial activity showed light absorption of infected cells maximized at day 4 (O.D. = 0.6) but was significantly lower (chi‐square = 41.265, df = 1, P < 0.05) than control groups (O.D. = 2) which maximized at day 12. Using trypan blue staining to count the number of cells with disrupted cell membranes, the maximum number of presumptively dead cells at day 8 (4 × 10⁵ cells) in infected treatments was higher than the control treatment at day 10 (1.8 × 10⁵ cells). However, TaqMan real‐time PCR did not confirm the replication of MrNV in the cells over 14 days. The mean viral copies and mean cycle times of positive samples were stable at 2.07 × 10⁴ and 24.12, respectively. Limited evidence of viral replication was observed during four serial passages. This study determined the mortality of the C6/36 cell line to the Australian isolate of MrNV but suggests limited patent replication was occurring. Trying different cell lines or adapting the virus to the C6/36 cells may be necessary to successfully replicate Australian MrNV in cell lines.     

Antioxidant and anti‐inflammatory activities of Pinus radiata bark extract in salmonid cell lines

A fish meal supply shortage is limiting aquaculture development. Currently, plant‐based proteins, such as soya bean meal, are being used as an alternative protein source, despite that such a diet can adversely affect fish, such as by inducing an inflammatory response. A possible solution is to include dietary additives in farm diets to counteract negative effects. One such solution originates from pine bark extracts, which present bioactive properties. In this study, the antioxidant and anti‐inflammatory properties of Pinus radiata bark extracts were evaluated for the first time in a salmonid cell line. This extract chemically demonstrated antioxidant activity through 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH = 58.4 ± 1.1%) and ferric ion reducing antioxidant power (FRAP = 575 ± 17 mgEqFe(II)·g extract^?1) assays. Additionally, the extract showed high flavonoid and phenolic compound contents. Up to 100 mg mL^?1, the P. radiata extract showed no cytotoxicity in the CHSE‐214 salmonid embryo cell line. Moreover, the antioxidant activity of the extract (50 μg mL^?1) was evaluated by a dichlorofluorescein (DCFH) assay in the SHK‐1 salmon cell line challenged with an oxidant stimulus (H₂O₂), showing 58.9% activity. The extract also protected DNA from oxidative damage, as observed through a comet assay. When assessing anti‐inflammatory properties in an in vitro inflammation model, the extract significantly reduced the relative expression of the pro‐inflammatory cytokines interleukin‐1β (IL‐1β), tumour necrosis factor‐α (TNF‐α) and interleukin‐8 (IL‐8) and of the inducible cyclooxygenase‐2 (COX‐2) enzyme. These results suggest a potential application of P. radiata bark extract in functional foods in aquaculture.     

Evaluations of Hilyses™, fermented Saccharomyces cerevisiae,on rainbow trout (Oncorhynchus mykiss) growth performance,enzymatic activities and gastrointestinal structure

This study examined the effects of Hilyses?, fermented Saccharomyces cerevisiae (S. cerevisiae), on rainbow trout growth performance, haematological parameters, digestive enzyme activities and gastrointestinal structure. Rainbow trout (mean weight 100–110 g) were fed dietary Hilyses? (5 g kg^?1) and control diet without Hilyses? for 50 days. Results of this study demonstrated that yeast supplementation in treatment group resulted in increased feed intake, followed by improved feed conversion ratio (FCR) and growth performance. Significant increases were also observed in trypsin and amylase activities in juvenile fish fed treatment diet. Light microscopy demonstrated that both groups of fish displayed normal morphology of proximal intestine and pyloric caeca. In yeast‐treated group, higher density of the goblet cells per villus in the proximal intestine was shown. No effects on haematological parameters and carcass chemical composition were noted. It is therefore possible to use fermented S. cerevisiae supplementation to significantly improve the gastrointestinal structure and growth performance in rainbow trout.     

Intensification of redclaw crayfish Cherax quadricarinatus culture: II. Growout in a separate cell system

In the process of exploring ways to intensify crayfish culture, a growout system of individual cages (cells) was designed to determine the effects of gender and cell size on the growth of the red claw crayfish Cherax quadricarinatus. Cells of three different diameters—large (25 cm), medium (20 cm) and small (16 cm)—were used. When crayfish were stocked at a mean weight of approximately 10 g, growth rate of males was significantly higher than that of females. The growth rate of the males in the large cells was 0.31±0.14 g/day, while that of the females was 0.18±0.09 g/day. The size of the cell had significant influence on the weight of males. Male crayfish in the large and medium cells grew better than those in the small cells. When males were stocked at a higher mean weight (about 23 g), their mean weight after 206 days was higher in the large cells (69.28±15.72 g) than in the small cells (58.11±12.66 g), suggesting that the growth of large males was also affected by cell size. Regardless of cell size, male animals of this species grew faster than females under conditions of individual cells. This intensive culture method appears to present a powerful improvement in yields, by as much as two orders of magnitude, in comparison with communal cultures.     

Effects of adrenaline on ionic equilibria in red blood cells of rainbow trout (Salmo gairdneri)

Effects of adrenaline on the equilibrium distributions of Na⁺ , K⁺ , H⁺ , Cl^– , and H₂O across the cell membrane of rainbow trout (Salmo gairdneri) erythrocytes were determinedin vitro, as a function of P CO₂ (1.76–7.77 torr). CO₂-carrying capacity of the blood was also examined. Plasma catecholamine concentrations inunanaesthetized, unrestrained trout were 3.1 nM adrenaline and 1.2 nM noradrenaline. Elevation of the plasma adrenaline concentrationin vitro to 4.6 × 10³ nM resulted in net gains of Na⁺ , Cl^– and H₂O by red cells, a net loss of H⁺ from red cells, and a pronounced red cell swelling. Adrenaline also reduced the CO₂-carrying capacity of trout bloodin vitro. The magnitudes of these effects increased with P_CO2 and, thus, were sensitive to blood HCO₃ ^– concentrations. The distribution of K⁺ between red cells and plasma was unaffected by adrenaline. Adrenergic-mediated ion movements and red cell swelling were sensitive to both propranolol and SITS. These results are consistent with the symport NaCl uptake model for adrenergic-mediated swelling of Baroinet al. (1984). The adrenergic response of fish erythrocytes may function to ameliorate the effects of blood acidoses on O₂-carrying capacity by maintaining red cell pH in the face of a decrease in plasma pH.