Seasonal variations in the skin epidermal structure and mucosal immune parameters of rainbow trout skin (Oncorhynchus mykiss) at different stages of farming.

The aim of this study was to investigate the seasonal changes in the epidermal structure and the innate immunity parameters of skin mucus in rainbow trout. The skin epidermis and mucus samples were collected over three consecutive seasons including winter, spring and late summer from three different weight groups i.e., 2-20 g (W1), 100-200 g (W2) and 400-600 g (W3) fish. The skin mucosal immunity analysis of rainbow trout showed that the haemagglutination activity increased significantly with increasing fish size from W1 to W3 in all three seasons, while no significant seasonal changes occurred in haemagglutination activity. Moreover, the bactericidal activity against fish pathogens increased significantly with increasing water bacterial load in late summer. The SDS-PAGE analysis of mucus showed a high amount of low molecular weight proteins (<35 kDa) in the late summer that was correlated with the increase in bactericidal activity. Histological analysis of the epidermis structure of rainbow trout skin showed that the density and size of goblet cells and consequently the mucus secretion significantly increased in W3 group in all seasons. In all three weight groups of fish, the density of goblet cells significantly increased from winter to spring and late summer along with increasing water temperature. Moreover, the goblet cell density showed a significant positive relationship with the soluble protein concentration and haemagglutination activity (p < 0.01). The results of this study demonstrated the more active immune role of the skin epidermal cells and mucus in rainbow trout during summer to protect fish against the pathogenic microorganisms. Given its potent bactericidal properties and the lack of haemolytic activity, the rainbow trout mucus might be used as a safe and inexpensive source for developing antimicrobial agents to prevent and treat some bacterial diseases in human and fish.

Can Betadine (10% povidone-iodine solution) act on the survival rate and gill tissue structure of Oranda goldfish (Carassius auratus)?

Industrial chemical solutions are widely used as a method to disinfection of aquaculture water and environments. The aim of the present study was to evaluate the toxicity effect of Betadine (10% solution of povidone-iodine) as a disinfectant solution on the survival and gill tissue of Oranda goldfish (Carassius auratus). For these purposes, 225 fingerling Oranda goldfish with an average weight 5 ± 0.67 g were divided into 15 groups with 3 replications. Fish were exposed to series of concentrations (0, 10, 20, 40, 60, 80, 90, 100, 120, 140, 160, 180, 200, 220 and 240 mg/L) of Betadine for 96 h. The mortality of fish and the samples of gill were observed at 6, 12, 18, 24, 48, 72 and 96 h after exposure. The results of the present study showed that the half-life of Betadine was less than 24 h and mortality were not record after 24 h. The highest mortality rate was at 240 mg/L, and LC50 24 h of Betadine was 158.800 mg/L. Histopathological results showed that lethal concentrations of Betadine lead to hyperemia, hypertrophy, hyperplasia and adhesion of secondary lamellar of the gill. Moreover, fish that were exposed to these concentrations displayed clinical signs such as anxiety, darkening of the skin. Overall results showed that Betadine have short half-life in the aquatic environment and are toxic to fish at very high concentrations, therefore it can be considered as practically non-toxic and useful for disinfection of the aquatic environment.

Evaluation of Behavioral Changes and Tissue Damages in Common Carp (Cyprinus carpio) after Exposure to the Herbicide Glyphosate.

Pesticides can induce changes in behavior and reduce the survival chance of aquatic organisms. In this study, the toxic effects of glyphosate suspension (Glyphosate Aria 41% SL, Tehran Iran) on behavior and tissues of common carp (Cyprinus carpio) were assessed. For this purpose, a 96 h LC50 of glyphosate suspension (68.788 mL·L-1) was used in the toxicity test. All individuals were divided into control and treatment groups with four replicates. Exposure operations were performed under two conditions: increasing concentration of suspension from 0 to 68.788 mL·L-1; then, decreasing to the first level. The swimming pattern was recorded by digital cameras during the test and tissue samples were collected at the end of the test. There were significant differences between the swimming pattern of treated individuals and control ones during both steps. The sublethal concentration of glyphosate led to hypertrophy, hyperplasia and hyperemia in the gill of fish. However, changes were obvious only after sampling. The exposed fish also displayed clinical signs such as darkening of the skin and increasing movement of the operculum. Moreover, glyphosate suspension affected swimming patterns of fish suggest that the swimming behavior test can indicate the potential toxicity of environmental pollutants and be used as a noninvasive, useful method for managing environmental changes and assessing fish health conditions by video monitoring.

Behavior evaluation of rainbow trout (Oncorhynchus mykiss) following temperature and ammonia alterations.

In the study, we assessed how acute changes in water temperature and dissolved ammonia concentration can affect the swimming behavior pattern of rainbow trout (Oncorhynchus mykiss). The behavior was analyzed in three different stages: 1) increase stage, (where temperature and ammonia concentration increase during this step, respectively); 2) unchanged stage, (where levels of both factors do not change during this stage); 3) reduction stage, (where ammonia concentration and temperature during this stage are reduced), respectively. The results showed that both factors significantly changed the swimming pattern of the rainbow trout. There were significant differences in swimming parameters (distance from the center, swimming speed, total movement and the average of angular changes of movement) of treated fish in the comparison between treatments, and with the control group. The changes in the swimming pattern of fish in response to physicochemical parameters of water were confirmed to be a good tool in ecotoxicological studies.

Fish cartilage: A promising source of biomaterial for biological scaffold fabrication in cartilage tissue engineering.

Here, engineered cartilage-like scaffold using an extracellular matrix (ECM) from sturgeon fish cartilage provided a chondroinductive environment to stimulate cartilaginous matrix synthesis in human adipose stem cells (hASCs). Three dimensional porous and degradable fish cartilage ECM-derived scaffold (FCS) was produced using a protocol containing chemical decellularization, enzymatic solubilization, freeze-drying and EDC-crosslinking treatments and the effect of different ECM concentrations (10, 20, 30, and 40 mg/ml) on prepared scaffolds was investigated through physical, mechanical and biological analysis. The histological and scanning electron microscopy analysis revealed the elimination of the cell fragments and a 3-D interconnected porous structure, respectively. Cell viability assay displayed no cytotoxic effects. The prepared porous constructs of fish cartilage ECM were seeded with hASCs for 21 days and compared to collagen (Col) and collagen-10% hyaluronic acid (Col-HA) scaffolds. Cell culture results evidenced that the fabricated scaffolds could provide a proper 3-D structure to support the adhesion, proliferation and chondrogenic differentiation of hASCs considering the synthesis of specific proteins of cartilage, collagen type II (Col II) and aggrecan (ACAN). Based on the results of the present study, it can be concluded that the porous scaffold derived from fish cartilage ECM possesses an excellent potential for cartilage tissue engineering.

Dietary peppermint (Mentha piperita) powder affects growth performance, hematological indices, skin mucosal immune parameters, and expression of growth and stress-related genes in Caspian roach (Rutilus caspicus).

Peppermint is a popular herbal medicine due to its several pharmaceutical applications. In this study, peppermint powder was used as a feed additive to evaluate growth performance, hematological parameters, protein profile of skin mucus, and immune parameters, as well as growth hormone (GH), insulin-like growth factor (IGF), and 70 kDa heat shock protein (HSP70) gene expression in Caspian roach (Rutilus caspicus). The fingerlings (average weight of 2.40 ± 0.12 g) were fed with diet containing 0 (control), 2, 3, and 4 g/kg peppermint for 8 weeks. The addition of peppermint significantly enhanced the growth parameters and decreased the food conversion ratio. Hematological indices of fish fed with peppermint-supplemented diets were significantly different from the control group (P < 0.05). Soluble protein, alkaline phosphatase, and lysozyme enzyme activity in mucus samples showed an incremental trend by increasing the peppermint levels in the diet. Evaluation of mucosal immunity indicated a remarkable difference between the protein profile in treatments fed with peppermint-supplemented diets and the control group. A new protein band (approximately 27 kDa) was also found in the skin mucus of fish fed with the diet containing 4 g/kg peppermint, and the highest band density was observed in this treatment. The highest IGF and GH gene expression were observed in 4 g/kg peppermint treatment. There was a significant difference in HSP70 expression between the fish fed with peppermint powder and the control group (P < 0.05). Overall, the results showed that dietary oral administration of peppermint at 4 g/kg of feed can act as a growth promoter and immunostimulant.

Seasonal changes of hydrolytic enzyme activities in the skin mucus of rainbow trout, Oncorhynchus mykiss at different body sizes.

The innate immune factors in the skin mucus of fish are affected by the ecological and physiological conditions such as developmental stage and seasonal cycle. The aim of this study was to investigate the seasonal changes in soluble protein and the hydrolytic enzyme activities of the skin mucus of rainbow trout including lysozyme, alkaline phosphatase (ALP) and proteases at different body sizes. Skin mucus samples were collected over three consecutive season periods including winter, spring and late summer. In each season, sampling was performed separately from three different weight groups including 2-20 g (W1), 100-200 g (W2) and 400-600 g (W3) fish. Our results showed a significant increase of soluble protein in all three weight groups from winter to spring when water temperature elevated from 9 °C to 14 °C. Moreover lysozyme activity was remarkably elevated in W1 fish from winter to late summer. In all three seasons, the activity of lysozyme was significantly decreased along with increasing the fish size. Contrary to lysozyme, the activity of proteases and ALP showed a decreasing trend from winter to late summer. A significant positive correlation was found between the proteases and ALP activity, proposing that both proteases and ALP might have important synergic roles in the mucosal innate immune function of rainbow trout. Moreover, using reverse transcription PCR (RT-PCR) analysis of some proteases genes including cathepsin-L and cathepsin-D, we demonstrated that the proteases are transcribed and likely synthesized in epidermal mucus cells of rainbow trout. The present study confirmed seasonal changes of hydrolytic enzyme activities in the skin mucus of rainbow trout across all three weight groups, with the highest variation in juvenile fish.

Effect of dietary supplementation with apple cider vinegar and propionic acid on hemolymph chemistry, intestinal microbiota and histological structure of hepatopancreas in white shrimp, Litopenaeus vannamei.

This experiment was conducted to evaluate the effects of dietary supplementation of Apple cider vinegar (ACV) and propionic acid (PA) on biochemical parameters of hemolymph, intestinal microbiota and histology of hepatopancreas in white shrimp (Litopenaeus vannamei). Five experimental diets were evaluated in this study including diets supplemented with 1.0, 2.0 and 4.0% of ACV, 0.5% propionic acid, and a control diet with no supplements. Shrimps (initial weight of 10.2 ±â€¯0.04 g) in triplicate groups with the density of 25 shrimps per tank were fed the diets for 60 days. At the end of the feeding trial, shrimps fed with ACV and PA supplemented diets had significantly higher total protein level than those fed the control diet (P < 0.05). The number of Vibrio spp., R-cells (lipid storage cells) of hepatopancreas and cholesterol level in shrimps fed the diets containing ACV and PA were lower compared to the control group (P < 0.05). However, there was no remarkable variations in glucose concentration, B-cell number and tubule diameter among the experimental diets (P > 0.05). In addition, shrimps fed the ACV diets had significantly lower total heterotrophic marine bacteria compared to the control or PA groups, and the lowest bacterial number was observed in shrimp fed 4% ACV supplemented diet (P < 0.05). Supplementation of 2 and 4% ACV as well as 0.5% PA in the diet led to a significantly higher calcium concentration than the control treatment (P < 0.05). The lowest triglyceride concentration was observed in the shrimps fed diets containing 2.0 and 4.0% ACV, which resulted in 15 and 20% reduction, respectively (P < 0.05). Overall, the findings indicates that ACV and PA possess antimicrobial activity and demonstrate beneficial effects on health status, so they can be potentially used as feed additive in the feeding of L. vannamei.

The effects of dietary supplement of galactooligosaccharide on innate immunity, immune related genes expression and growth performance in zebrafish (Danio rerio).

The present study investigates the effect of different levels of galactooligosaccharide (GOS) on innate immune parameters, immune related genes expression as well as growth performance in zebrafish (Danio rerio). Four hundred and twenty fish (mean weight 45 ±â€¯0.1 mg) were supplied, randomly stocked in twelve aquaria assigned to four treatments. Zebrafish were fed with either control diet or control diet enriched with different levels (0.5, 1 and 2%) of GOS for eight weeks. At the end of feeding trial innate immune parameters (total immunoglobulin, total protein and alkaline phosphatase activity), immune related genes expression (interleukin 1 beta [il1b], Lysozyme [lyz], tumor necrosis factor alpha [tnf-alpha]) as well as growth performance were measured. Evaluation of immune parameters revealed significant (P < .05) increase of total protein and total Ig in zebrafish fed 1 or 2% GOS compared other treatments. However, in case of lysozyme activity no significant (P > .05) differences were noticed between GOS fed fish and control group. Also, in case of ALP activity, significant increase (P < .05) was observed in 2% GOS treatment. Gene expression studies revealed significant upregulation (P < .05) of tnf-alpha and lyz genes in GOS fed fish. While no significant (P > .05) difference was observed in case of il1b gene expression. Evaluation of growth performance at the end of feeding trial revealed no significant (P < .05) improvement in GOS fed groups. The present results revealed positive effects of GOS on innate immune parameters and related gene expression in zebrafish.

Dietary Myrtle (Myrtus communis L.) improved non-specific immune parameters and bactericidal activity of skin mucus in rainbow trout (Oncorhynchus mykiss) fingerlings.

The present study examined the effects of dietary Myrtle (Myrtus communis L.) on non-specific immune parameters and bactericidal activity of skin mucus in rainbow trout (Oncorhynchus mykiss) fingerlings. Three hundred and sixty fingerlings (6.50 ± 0.55 g (were distributed in twelve cages (65 × 65 × 65 cm) with a metal framework. The study included four treatments repeated in triplicates. The treatments were feeding trouts with experimental diets containing different levels (0, 0.5, 1 and 1.5%) of Myrtle powder. The fingerlings were fed on experimental diet for sixty days and then skin mucus non-specific immune parameters as well as bactericidal activity were measured. At the end of the trial, the highest skin mucus soluble protein level was observed in group fed with 1.5% Myrtle (P < 0.05). The alkaline phosphatase (ALP) activity was significantly increased in fish groups fed 1 and 1.5% Myrtle compared with the control group (P < 0.05). However, evaluation of skin mucus lysozyme activity showed no significant difference between treatments and control group (P > 0.05). Also, no antibacterial activity was detected against Escherichia coli, Staphylococcus aureus and Salmonella enterica in all treatments and control group. Whereas skin mucus of rainbow trout showed antimicrobial activity against fish pathogens (Aeromonas hydrophila and Yersinia ruckeri) in 1 and 1.5% Myrtle treatments. These results indicated beneficial effects of dietary Myrtle on mucosal immune parameters of fingerling rainbow trout.

Dietary effect of apple cider vinegar and propionic acid on immune related transcriptional responses and growth performance in white shrimp, Litopenaeus vannamei.

This experiment was conducted to study the effect of various levels of ACV® and Propionic acid (PA) on expression of immune related genes and growth performance in white shrimp (Litopenaeus vannamei). Three hundred and seventy-five shrimps with an average initial weight of 10.2 ± 0.04 g were collected and acclimatized for two weeks. Five experimental diets including control diet, 0.5% PA diet and 1%, 2% and 4% ACV® diets were applied to feed the shrimps. They were fed 4 times a day with 2.5% of body weight. After 60 days of culture, shrimps fed with ACV® and PA diets showed no significant difference in growth performance. Expression of prophenoloxidase (proPo), lysozyme (Lys), penaeidin-3a (Pen-3a) and Crustin (Cru) genes were determined from hepatopancreas, using the real-time PCR after 15, 30 and 60 days. Expression of Lys and proPo genes was significantly up regulated in shrimps fed with ACV® and PA diets compared to the control group after 30 and 60 days of treatment. After 15 days, Pen-3a gene expression was significantly higher in PA group compared to the control group. Also, shrimps fed with 1% and 4% ACV® and PA diets showed significantly increased expression of Pen-3a after 30 days. In contrast, expression of Cru was significantly down regulated in response to ACV® diets, but, Cru expression in treated shrimps with PA diet was greater than the control group after 30 and 60 days. Overall, the results provided evidence that ACV® could be used as a natural immunostimulant for shrimps in order to adjust and enhance expression of the immune related genes.

Agaricus bisporus powder improved cutaneous mucosal and serum immune parameters and up-regulated intestinal cytokines gene expression in common carp (Cyprinus carpio) fingerlings.

The aim of the present study was to investigate immunomodulatory effects of Agaricus bisporus, white bottom mushroom powder (WBMP) on common carp (Cyprinus carpio) fingerlings. Carps were fed on different levels of WBMP (0, 0.5, 1 and 2%) for 8 weeks and at the end of feeding trial, skin mucus immune parameters (total Ig, lysozyme and protease activity), cytokines gene expression (TNF-alpha, IL1b, IL8) in intestine as well as serum non-specific immune parameters (total Ig, lysozyme and ACH50) were measured. The results showed significant dose dependent increase of skin mucus immune parameters in carps fed WBMP (P < 0.05). While, no significant difference was observed between 0.5% WBMP and control group (P > 0.05). In case of serum non-specific immune parameters, except lysozyme activity, other parameters (Ig total and ACH50) were significantly affected by dietary inclusion of WBMP (P < 0.05). Also, evaluation of cytokines gene expression in the intestine of carps revealed remarkable up-regulation of TNF-alpha in fish fed 2% WBMP supplemented diet compared other treatment (P < 0.05). Likewise, IL1b gene expression was significantly increased in 1 and 2% WBMP treatments compared to the 0.5% WBMP and control groups (P < 0.05). IL8 gene expression was not affected by inclusion of WBMP in carp diet (P > 0.05). Furthermore, feeding on WBMP supplemented diet significantly improved growth performance (P < 0.05). These results indicated that WBMP can be considered as a promising immunostimulants in early stage of common carp culture.

The effects of garlic-supplemented diets on skin mucosal immune responses, stress resistance and growth performance of the Caspian roach (Rutilus rutilus) fry.

This study was conducted to evaluate the effects of garlic supplementation on some skin mucus immune parameters, mucus antimicrobial activity and growth performance of the Caspian roach (Rutilus rutilus caspicus) fry. Fish (1 ± 0.07 g) were divided into four groups fed diets containing 0 (control), 5, 10 and 15 g kg(-1) garlic for 8 weeks. The results showed that there was a significant increase in weight gain and specific growth rate in those fish fed garlic diets compared with the control (P < 0.05). Condition factor was not significantly affected by garlic dosage. At the end of trial, the epidermal mucus protein level, alkaline phosphatase and antimicrobial activity against 2 g-negative bacteria (Escherichia coli and Serratia marcescens) and gram-positive bacteria (Streptococcus faecium and Micrococcus luteus) were measured. Skin mucus alkaline phosphatase, protein levels and antimicrobial activity were increased following garlic administration, and the bacterial growth inhibition zones were significantly elevated in garlic-fed fish (P < 0.05). In salinity stress experiment, no differences were observed for survival rate among the experimental diets. No mortality was recorded during the feeding trial. These results indicated that dietary garlic beneficially affects the skin mucus immune parameters and growth performance of the Caspian roach fry.

The effects of Lactobacillus acidophilus as feed supplement on skin mucosal immune parameters, intestinal microbiota, stress resistance and growth performance of black swordtail (Xiphophorus helleri).

The present study evaluates the effects of different levels of dietary Lactobacillus acidophilus as feed supplement on intestinal microbiota, skin mucus immune parameters and salinity stress resistance as well as growth performance of black swordtail (Xiphophorus helleri). One-thousand and eight hundred healthy black swordtail larvae (0.03 ± 0.001 g) were randomly distributed in 12 tanks (100 L) at a density of 150 fish per aquaria and fed different levels of dietary L. acidophilus (0, 1.5 × 10(8), 3 × 10(8) and 6 × 10(8) CFU g(-1)) for 10 weeks. At the end of trial, there were significant differences among antibacterial activity of skin mucus in probiotic fed fish and control group (P < 0.05). Furthermore, the skin mucus protein level and alkaline phosphatase activity in control group were significantly lower than those of L. acidophilus fed fish (P < 0.05). Microbiological assessments revealed that feeding with probiotic supplemented diet remarkably increased total autochthonous bacteria and autochthonous lactic acid bacteria levels (P < 0.05). The results showed that dietary administration of L. acidophilus significantly elevated black swordtail resistance against salinity stress (i.e survival %) (P < 0.05). Also, dietary administration of different levels of L. acidophilus improved weight gain, SGR, FCR compared to fish fed unsupplemented diet (P < 0.05). These results demonstrate beneficial effects of dietary L. acidophilus on mucosal immune parameters, intestinal microbiota, stress resistance and growth parameters of black swordtail and the appropriate inclusion is 6 × 10(8) CFU g(-1).

The effects of dietary vitamin C on mucosal immune responses and growth performance in Caspian roach (Rutilus rutilus caspicus) fry.

This study was conducted to investigate the effects of different levels of dietary vitamin C on some skin mucus immune parameters, mucus antimicrobial activity and growth performance of Caspian roach (Rutilus rutilus caspicus) fry. Six hundred sixty Caspian roach (1.4 ± 0.02 g) fry were allocated to 12 tanks (55 fish per tank), and triplicate groups were fed diets containing 0, 1,000, 1,500 and 2,000 mg kg(-1) vitamin C for 60 days. At the end of the trial, the epidermal mucus protein level, alkaline phosphatase and antimicrobial activity against two gram-positive bacteria (Streptococcus faecium and Micrococcus luteus) and gram-negative bacteria (Escherichia coli and Serratia marcescens) as well as growth performance were measured. The results demonstrated that feeding on vitamin C significantly elevated skin mucus alkaline phosphatase and protein levels compared to the control group (P < 0.05). However, lysozyme activity was undetectable in both the vitamin C-fed roach fry and the control group. Skin mucus antimicrobial activity was increased following vitamin C administration, and the bacterial growth inhibition zones were significantly elevated in vitamin C-fed roach (P < 0.05). Similar results were obtained in case of the minimum inhibitory concentration of skin mucus. Also fish fed the control diet had a significantly lower weight gain, specific growth rate and condition factor compared to the other treatments (P < 0.05). These results revealed that dietary vitamin C beneficially affects the skin mucus immune parameters and growth performance of Caspian roach fry.

Effect of starvation and refeeding on digestive enzyme activities in juvenile roach, Rutilus rutilus caspicus.

We evaluated the effects of starvation and refeeding on digestive enzyme activities in juvenile roach, Rutilus rutilus caspicus. Fish were divided into four feeding groups (mean mass 1.68 ± 0.12 g). The control group was fed to satiation twice a day throughout the experiment with formulated diet (SFK). The other three groups were deprived of feed for 1(S1), 2(S2), and 3(S3) weeks, respectively, and then fed to satiation during the refeeding period. The results showed that trypsin specific activity was not affected significantly either by starvation or refeeding, in all experimental groups. Chymotrypsin specific activity did not change significantly in S1 fish during the experimental period. In S2 and S3 fish no significant changes were observed during the starvation period. Upon refeeding, the activity increased in S2 fish, while it decreased in S3 fish. Amylase specific activity decreased significantly during the starvation period in all experimental groups. Upon refeeding, the activity increased. Alkaline phosphatase specific activity did not change significantly during the experiment period in S3 fish, while it showed significant changes during the starvation and refeeding period in the S1 and S2 fish. Starvation also had a significant effect on the structure of the intestine.