Dietary nano iron enhanced intestinal physical barrier, antioxidant capacity and immune function of juvenile Largemouth Bass (Micropterus nigricans)

doi:10.21203/rs.3.rs-4676994/v1

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Dietary nano iron enhanced intestinal physical barrier, antioxidant capacity and immune function of juvenile Largemouth Bass (Micropterus nigricans)

https://doi.org/10.21203/rs.3.rs-4676994/v1

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Nano-additives can improve the absorption and utilization of feed nutrients. In this study, we reported the effects of supplementary nano iron (0, 30, 60, 120, 240 and 480 mg/kg) on intestinal tract function of largemouth bass juveniles. Firstly, 60 mg/kg of supplementary nano iron improved the specific growth rate, and significantly increased the height and width of intestinal villi and intestinal absorption area, and improved the intestinal physical barrier of juvenile largemouth bass. Secondly, the supplementation of low concentration of nano iron can significantly increase the activity of intestinal antioxidant enzymes (CAT and T-SOD), enhance the expression of antioxidant-related genes (SOD1, Nrf-2) and proteins (CAT; P < 0.05), and decrease malondialdehyde (MDA) content. Meanwhile, supplementation with 60 mg/kg of nano iron resulted in the highest survival rate following intraperitoneal injection of Nocardia. However, supplementation of 480 mg/kg would compromise the immune and antioxidant capacity of juvenile largemouth bass. This was evidenced by increased expression of inflammatory and apoptotic genes, significant reduction in the expression of intestinal tight junction genes (ZO-1, Acludin1, Acludin7; P < 0.05), impairment of intestinal physical barriers, and exacerbation of oxidative stress levels in the organism, ultimately resulting in decreased immunity. In conclusion, moderate supplementation of nano iron appears to enhance the intestinal physical barrier, antioxidant capacity, and immune function of juvenile largemouth bass.

Micropterus nigricans

Nocardia

nano iron

intestinal physical barrier

antioxidant capacity

immune function

In recent years, nanoparticles (NPs) or nanoforms of essential minerals have garnered considerable attention in aquaculture. They have been employed to study the growth performance, feed efficiency, and health status of fish ^[1]. Nano-sized particles, owing to their diminutive size and expanded surface area, can easily penetrate most biological membranes and be effectively absorbed by organisms ^[2]. Compared to traditional forms of minerals, nanoscale minerals can pass through cells more quickly, enhancing their assimilation processes ^[3] and facilitating the absorption of nutrients by animal organisms, thereby improving their metabolism and other physiological functions ^[4]. Therefore, nanoparticles have enormous potential for application in aquaculture.

Iron is indispensable as a trace element for living organisms, crucially involved in fundamental biological processes including oxygen transport, cellular respiration, energy metabolism, and DNA synthesis ^[5]. It is essential for the growth performance, antioxidant defense ^[6], and immune function of aquatic animals ^{[7, 8]}. Iron deficiency in fish can lead to oxidative stress ^[9], resulting in reduced growth performance ^[10] and compromised immune function ^[11]. Fish primarily obtain iron through feed intake to meet their requirements ^[5]. However, the current use of inorganic iron salts as iron sources in aquafeed is limited due to their low bioavailability in aquaculture production ^[12]. In contrast to conventional inorganic iron salts, nanoscale iron demonstrates enhanced surface area and superior particle dispersion, thereby leading to heightened catalytic efficiency ^[13]. Studies have shown that nanoscale iron (Fe₂O₃) can effectively enhance the nonspecific immune response and improve iron bioavailability in Labeo rohita compared to FeSO₄·₇H₂O at the same concentration ^[14]. Moreover, integrating nanoscale particles of selenium, zinc, iron, and other elements into fish feed has been shown to significantly enhance the growth performance of fish ^{[15, 16]}. Hence, the incorporation of nanoscale iron into aquafeed shows significant potential for improving the growth and health of aquatic animals ^[17].

The intestine serves as a primary interface between fish and its external environment, playing crucial roles in various physiological functions. It functions not only as the primary site for nutrient digestion and absorption but also as a vital immune organ within the fish's body ^[18]. Prior research has demonstrated that supplementation with nano-selenium effectively mitigates intestinal damage in juvenile grass carp by enhancing intestinal barrier function and suppressing intestinal inflammation and oxidative stress ^[19]. Similarly, administering Trichogaster trichopterus with nano iron at a concentration of 40 mg/kg enhances growth performance and increases the activity of intestinal digestive enzymes and antioxidant enzymes ^[20]. However, the effects of adding nano iron to the diet of other species warrants further investigation. Therefore, the purpose of this study was to investigate the impacts of different dietary concentrations of nano iron on intestinal physical barrier, antioxidant capacity and immune function of largemouth bass juvenile, so as to find the appropriate supplemental amount of nano iron.

2.1. Experimental diets design

This experiment formulated six diets with nano iron concentrations of 0 (A), 30 (B), 60 (C), 120 (D), 240 (E), and 480 (F) mg/kg (Table 1). The final approximate composition of feed is shown in Table 2. Nano iron (Fe₂O₃) was sourced from the Chemical Biology Laboratory of Sichuan Agricultural University, and the regular feed ingredients were purchased from Sanwang Co., Ltd. Our previous study ^[21] described the process of diets preparation. After drying, all rations are stored at -20 ℃ until use.

Table 1

The basal diet was used in the feeding
Ingredients	Content (g/kg dry basis)
Casein	250
Fish meal	150
Soybean concentrate	140
Cellulose	120
α-starch	120
Gelatin	80
Ca(H₂PO₄)₂	15
Yeast hydrolysate	10
Soybean oil	100
Vitamin premix ^a	5
Fe- free mineral premix ^b	10
^a Vitamin Premix (mg kg ^− 1 diet): vitamin A, 16000 IU; vitamin D3, 8000 IU; vitamin K3, 14.72; vitamin B1, 17.80; vitamin B2, 48; vitamin B6, 29.52; vitaminB12, 0.24; vitamin E, 160; vitamin C, 800; niacinamide, 79.20; calcium pantothenate, 73.60; folic acid, 6.40; biotin, 0.64; inositol, 320; choline chloride,1500; L -carnitine, 100.
^b Mineral Premix Fe-free (mg kg ^− 1 diet): Cu (CuSO₄), 2.00; Zn (ZnSO₄), 34.4; Mn (MnSO₄), 6.20; I (Ca (IO₃) ₂), 1.63; Se (Na₂SeO₃), 0.18; Co (COCl₂), 0.24; Mg (MgSO₄.H₂O), 52.70.

Table 2

The nano iron addition and proximate composition in all experiment treatments
Treatments	A	B	C	D	E	F
Nano-Fe₂O₃ (mg/kg)	0	43	86	172	344	688
Proximate composition
Moisture	82.4	80.7	85.8	82.6	85.8	85.6
Crude protein	461.8	464.4	466.4	467.0	464.6	465.8
Total lipid	100.4	102.0	108.1	104.8	106.3	104.0
Ash	51.7	51.9	51.6	52.1	51.7	52.4
Fe (mg/kg)	197.56	222.55	253.78	307.80	414.88	629.70

2.2. Fish and feeding trial

Juvenile largemouth bass, initially weighing about 5 g, were supplied by Hongbo Company. Experimental fish were held for two weeks and fed commercial feed to provide an assimilation period prior to beginning the experiments. After an acclimation period, 540 healthy fish of similar size were randomly distributed into six groups (each with 3 replicates of 30 fish per replicate) and fed experimental feed twice daily (at 9:00 and 17:00) until visual satiety for a duration of 10 weeks. The water temperature was maintained at 23 ± 2℃, and natural light conditions were maintained throughout the experiment. Dissolved oxygen levels were kept above 6 mg/L, and pH was between 6.7 and 7.6 throughout the experiment. To maintain water quality, fish excrement and residual feed were removed and one-quarter of the water volume was replaced with fresh water after feeding,

2.3. Sample collection

Prior to sampling and weighing, fish were subjected to a 24-hour fasting period, and the fish number and condition in each tank were documented to determine survival rates and growth. All sampling operations were performed after the fish were anesthetized (0.1g/L MS-222). Firstly, we collected blood samples using a 1mL syringe and obtained plasma and serum through centrifugation (3000 rpm, 15 min, 4 ℃), which was then stored at -80 ℃. Secondly, Intestinal samples were collected from 3 fish per group and promptly fixed in a polyformaldehyde solution. Thirdly, intestinal samples from 9 fish per group were collected and stored at -80°C in frozen storage tubes. These samples were intended for subsequent analysis of biochemical parameters (3 fish per tank), quantitative real-time polymerase chain reaction (3 fish per tank), and protein imprinting (3 fish per tank).

2.4. Growth performance and microscopic observations

At the conclusion of the experiment, all fish were measured for body length, body weight, liver weight, and visceral weight to evaluate the growth performance of Largemouth Bass. The intestinal samples were transferred to a gradient of alcohol (70%-100%) and xylene, and subsequently embedded in paraffin. They were then cut into 5µm sections, stained with H&E, and examined under a light microscope (Olympus DP73, Nikon Corporation, Japan). Intestinal samples were immersed in a fixed medium (2.5% glutaraldehyde dissolved in 0.15M phosphate buffer). The intestinal samples were cleaned four times in PBS and embedded in 1% of osmotic oxide. The sample was dried in increasing order of alcoholic concentration and then embedded in argdite. Transverse ultrathin slides were prepared and stained with huranyl acetic acid and pyridine ferric citrate. Images were taken on a Hitachi Jem-1400 Flash electron transmission microscope.

2.5. Enzyme activity determination

The plasma samples were diluted with 9 volumes of normal saline prior to analysis. The intestinal samples were homogenized with 10 volumes of normal saline on ice. The resulting supernatants were then isolated by centrifugation at 4000 g for 10 minutes at 4°C and subsequently stored at − 20°C. The activities of Malondialdehyde (MDA), Catalase (CAT), Superoxide dismutase (SOD), Glutathione peroxidase (GPX), Acid phosphatase (ACP), and Alkaline phosphatase (AKP) were assessed using commercial assay kits purchased from Nanjing Jiancheng Bioengineering Institute, China.

2.6. Quantitative real time PCR

The primers of target genes for qPCR were designed by Primer Premier 5.0 according to the genome sequences previously reported (Table S1). Total RNA was extracted from intestinal samples using the Animal Total RNA Isolation Kit (Foregene, Chengdu, China). cDNA synthesis was carried out using the Master Premix (For qPCR) RT EasyTM I (Fore Gene Biotechnology Co LTD, Chengdu, China). Real-time PCR was conducted using the CFX96TM Real-Time PCR Detection System. The expression levels of target genes were analyzed using the 2^−ΔΔCT method, with β-actin serving as the reference gene for normalization.

2.7. Western blotting

Firstly, Intestinal tissue samples weighing 0.02g were homogenized in 500µl of lysis buffer, followed by a 30-minute digestion period and centrifugation at 13,000g for 10 minutes. After centrifugation, the resultant supernatant was collected for western blot analysis. A commercial BCA kit (Beyotime, Shanghai, China) was used to quantify protein concentration. Then the proteins were separated by SDS-PAGE gel electrophoresis. And the proteins transferred to PVDF membranes were performed on a membrane transfer apparatus. The PVDF membranes were incubated with 5% skimmed milk powder at room temperature for 2 hours on a shaker.

Secondly, primary antibodies (rabbit anti-Bax, abcam, ab3191, 1:1000; rabbit anti-Keap-1, abcam, ab227828, 1:1000; rabbit anti-CAT, abcam, ab3296, 1:1000; mouse anti-β-actin, Zenbio, 200068-8F10, 1:1000) were applied and incubated overnight at 4°C. The membranes were washed three times with TBST buffer (10 minutes each). Subsequently, secondary antibodies (horseradish peroxidase-coupled goat anti-rabbit IgG, Zenbio, 1:10,000) were applied and incubated for 1 hour at 25°C. After three additional washes with TBST buffer (10 minutes each), freshly prepared DAB substrate was added for 1 minute to develop color. The quantification of band intensity of gel images was captured using Image-J software, and β-actin was served as an internal reference.

2.8. Challenge test

The experimental equipment such as conical flasks and gun tips were sterilized and dried using an autoclave. We weighed 3.85 g of BHI and dissolved in 100 ml of double-distilled water, and the BHI liquid medium was autoclaved and naturally cooled to room temperature. After UV-sterilization in the fume hood, Nocardia was inoculated and incubated in a constant temperature incubator (28 ℃, 180 r/min), and the growth of Nocardia was observed regularly. Bacteria were collected during the logarithmic growth period, washed with sterile PBS buffer after centrifugation and adjusted to 3×10⁷ CFU/ml by Mackay's turbidimetric method. At the end of the culture experiment, 24 fish in each group were randomly selected to be injected with 0.5 ml of Nocardia through the peritoneal cavity, and another 6 fish were injected with an equal volume of sterile PBS buffer as a negative control. The period of attack experiment was 1 week, water temperature was 25.0 ± 1.0 ℃, and feeding regimen was the same as during the culture experiment. The infection symptoms and mortality of the diseased fish were recorded in detail each day, and they were examined by autopsy.

2.9. Statistical analysis

The results are expressed as mean ± SEM. Significant differences between treatment groups were assessed using one-way analysis of variance (ANOVA) conducted with GraphPad Prism 8.0 software (GraphPad Software Inc., USA). Statistical significance was defined as α = 0.05. Data plots were generated using GraphPad Prism 8.0.

3.1. Growth performance

All groups of juvenile fish exhibited a survival rate exceeding 95% (Fig. 1, Table S2), and there were no significant variations observed among the groups. Growth parameters, including Final Body Weight (FBW), Weight Gain Rate (WGR), and Specific Growth Rate (SGR), initially showed an increase followed by a decline with increasing concentrations of nano iron. Specifically, group C exhibited a significant 8.8% increase in WGR compared to group A (P < 0.05). Additionally, there were no notable differences among the groups in terms of Hepatosomatic Index (HSI), Protein Efficiency Ratio (PER), Viscerosomatic Index (VSI), and Condition Factor (CF) (P > 0.05).

3.2. Intestinal morphology

The histomorphology and morphological statistics of midgut of juvenile largemouth bass were shown in Fig. 2a and b respectively. With the increase of nano iron supplemental level, the midgut villus height, villus width, and absorption area of largemouth bass increased first and then decreased. In C group, villus height, villus width, and absorption area were significant increases compared to groups supplemented with A (0 mg/kg), E (240 mg/kg), and F (480 mg/kg) of nano iron (P < 0.05). Specifically, villus height and absorption area reached their peak values among all experimental groups. The measurement of intestinal muscle layer thickness showed no significant difference (P > 0.05).

3.3. Intestinal tight junction

Electron microscopic observation showed (Fig. 3a) that the tight junctions between adjacent cells in the midgut epithelium of largemouth bass in groups A, B, and C were clear and structurally intact, while the tight junctions of cells in groups E and F lost their integrity and the gaps were blurred. The relative expression levels of intestinal MUC2 in juvenile largemouth bass were not significantly different between different nano iron concentration groups (P > 0.05; Fig. 3b). The relative expression levels of ZO-1 were significantly lower in groups D, E, and F compared to groups B and C (P < 0.05). Furthermore, the relative expression levels of Claudin1 were significantly lower in groups E and F compared to groups A, B, and C (P < 0.05). Conversely, the relative expression levels of Claudin7 were significantly higher in group C compared to group F (P < 0.05).

3.4. Antioxidant status

The activity of T-SOD was significantly higher in groups B and C, and significantly lower in groups D and E (P < 0.05; Fig. 4). Moreover, T-SOD activity in groups B and C was significantly higher compared to groups D, E, and F (P < 0.05). However, the addition of different concentrations of nano iron had no significant effect on the activity of GPx, the intestinal antioxidant enzyme of juvenile Largemouth bass (P > 0.05). The CAT activity in group D was the highest, significantly exceeding that in groups A, E and F (P < 0.05). The MDA content exhibited a trend of initially decreasing and then increasing with the incremental addition of dietary nano iron; however, no statistically significant differences were observed among the groups (P > 0.05). Conversely, the supplementation of nano iron significantly influenced the SOD1, Nrf-2, and Keap-1 expression (P < 0.05). The SOD1 and Nrf-2 were expressed significantly higher in group C compared to group F (P < 0.05). No significant differences were observed among the other groups (P > 0.05) for these genes. Conversely, the expression levels of the Keap-1 gene were significantly lower in group C than in group F (P < 0.05). Similar to SOD1 and Nrf-2, there were no significant differences among the remaining groups (P > 0.05) for Keap-1 gene expression. Furthermore, the addition of nano iron did not significantly affect the relative expression levels of intestinal GPX and CAT genes in juvenile largemouth bass (P > 0.05). The expression level of CAT protein exhibited a pattern of initially increasing and then decreasing across the experimental groups. The expression level of Keap-1 protein showed a trend of decreasing and then increasing and then decreasing, with the lowest in group C and the highest in group F, with significant differences (P < 0.05).

3.5. Inflammation and apoptosis indicators

The relative expression levels of intestinal inflammatory genes in juvenile largemouth bass are shown in Fig. 5. The relative expression levels of the pro-inflammatory genes TNF-α and COX-2 were not significantly affected by the feed nano iron content (P > 0.05). Compared with group B, the relative expression levels of anti-inflammatory genes TGF-β1 were significantly lower in groups D, E, and F (P < 0.05), and the relative expression levels of TGF-β1 in group F were significantly lower than those in groups A, B, C, and E (P < 0.05). The relative expression levels of anti-inflammatory genes IL-10 were highest in group B and C (P < 0.05), and the relative expression of IL-10 in group E was significantly lower than other groups (P < 0.05).

The relative expression levels of intestinal pro-apoptotic related genes Caspase3, Caspase9 and Bax in juvenile Largemouth bass did not show significant differences (P > 0.05; Fig. 6) after feeding different concentrations of iron nano feeds. The relative expression level of Caspase8 in group B was significantly higher than that in group F (P < 0.05), and there were no significant differences among the remaining groups (P > 0.05). The relative expression level of the anti-apoptotic gene Bcl-2 was highest in group B, significantly exceeding that in groups D, E, and F (P < 0.05). Conversely, the relative expression level of Bcl-2 in group F was significantly lower compared to groups A, B, C, and D (P < 0.05). The expression level of Bax protein displayed a pattern of initially decreasing and then increasing across the groups. The highest protein expression level was observed in group E, which was significantly higher than that in groups A, B, and C (P < 0.05).

3.6. Challenge test

The survival rate of all negative control groups (injected with sterile PBS) at 7 d after Nocardia amberjack attack was 100%, and no white nodules, hemorrhages, or other diseases were observed in the body cavity at autopsy (Fig. 7A). Autopsy observation of the diseased and dead fish treated with the attack toxin revealed that all diseased and dead fish had a large number of white nodules in the abdominal mucosa (Fig. 7B). The survival rate of juvenile Largemouth bass at 7 d after Nocardia amberjack attack was highest in group C (60 mg/kg) with 66.7%, followed by groups D (55.6%), B (50.0%), E (38.9%), F (33.3%) and A (27.8%), respectively (Fig. 7C).

4.1. Moderate supplement nano iron improved the growth performance and enhanced its intestinal physical barrier.

Iron (Fe) has been recognized as an essential mineral element crucial for fish health and growth ^[5]. This experiment demonstrated that supplementing juvenile Largemouth Bass with 60 mg/kg nano iron resulted in improved growth performance, as evidenced by significant increases in their Weight Gain Ratio (WGR) and Specific Growth Rate (SGR). Consistently, previous studies have demonstrated that supplementing fish diets with nano iron significantly enhances growth performance. This effect has been observed in various species including Hybrid Tilapia ^[22], Yellow Catfish ^[23], and juvenile Cobia ^[24]. However, excessive supplementation of nano iron did not yield further improvement in the Specific Growth Rate (SGR). Indeed, the group supplemented with 480 mg/kg of nano iron had a negative impact on the fish growth, in contrast to the group supplemented with 60 mg/kg. Similar declines in growth performance have been noted in other fish species. For example, hybrid tilapia demonstrated substantial reductions in feed efficiency and growth when their diet was supplemented with 400 mg/kg of iron citrate ^[22]. Moreover, juvenile Grass Carp exhibited reduced feed intake and Specific Growth Rate (SGR) when fed a diet containing 120 mg/kg of iron, in contrast to those fed a diet containing 52.8 mg/kg ^[25]. Research has demonstrated that heightened concentrations of nano iron can diminish embryonic hatching rates and induce apoptotic cell death in zebrafish ^[26]. Therefore, the adverse effects of excessive supplementation of nano iron on largemouth bass may stem from iron toxicity, which disrupts the balance of physiological functions, thereby impairing their ability to achieve optimal growth potential.

Intestinal villi are one of the important structures in the fish intestine, and their ability to increase the intestinal absorption area and absorption efficiency has an important impact on the growth and immunity of fish^[27]. The addition of iron in the feed in appropriate amounts can improve the absorption efficiency of iron in fish, thus promoting the growth and development of intestinal villi and improving the growth and immunity of fish^[28]. The findings of this experiment revealed that the length, width, density and absorption area of intestinal villi could be increased by proper addition of nano-iron. However, both insufficient and excessive iron supplementation had adverse effects on these parameters. Excessive iron levels can elevate free radical production in the fish intestine, potentially damaging the structure and function of intestinal villi and impeding fish growth and immunity. Conversely, inadequate iron levels can result in iron deficiency in fish, hindering the growth and development of intestinal villi. Thus, maintaining optimal iron levels is crucial for promoting healthy intestinal morphology and overall fish health ^[29].

The intestinal epithelial barrier primarily comprises tight junctions between adjacent cells, which are composed of several tight junctional proteins such as Claudins, Occludin, and ZOs. These proteins modulate intestinal permeability, facilitate selective nutrient absorption, and act as barriers against the entry of harmful substances into the human body through the intestine ^[30]. The current study demonstrated that supplementing with nano iron did not lead to a significant increase in the relative expression levels of intestinal tight junction-related genes MUC2, Claudin1, Claudin7, and ZO-1. However, higher levels of nano iron supplementation suppressed the relative expression levels of MUC2, ZO-1, and Claudin1. Electron microscopic observations further revealed that the tight junctions of cells in groups E and F exhibited compromised integrity, with blurred gaps, revealing that excessive supplementation of nano-iron adversely affect intestinal tight junctions. Excessive supplementation of nano iron may result in an accumulation of iron ions within intestinal cells, triggering a significant production of free radicals via the Fenton reaction. This oxidative stress can induce an inflammatory response within the intestine, ultimately damaging the physical barrier in juvenile largemouth bass ^[31].

4.2. Nano iron improved the intestinal antioxidant status and enhanced the immune ability.

Antioxidant capacity refers to an organism's ability to combat free radicals, which are major contributors to numerous diseases and significant agents of oxidative damage to cells ^[32]. In fish, the intestine plays pivotal roles in the digestion and absorption of nutrients. Furthermore, it serves as a critical defense barrier against potentially harmful external substances for the organism. ^[33]. Therefore, investigating the impact of nano iron on the intestinal antioxidant capacity of largemouth bass is crucial for understanding its effects on fish health. The study results revealed that increasing dietary nano iron supplementation led to a pattern where activities of antioxidant enzymes T-SOD and CAT initially increased and then decreased, whereas MDA content exhibited the opposite trend. Additionally, nano iron supplementation significantly influenced the expression of antioxidant-related genes in Largemouth bass. Specifically, the expressions of SOD1, CAT and Nrf-2 genes were increased in the 60 mg/kg group and decreased in the 480 mg/kg group. These findings align with previous research showing that dietary supplementation with a certain level of nano-iron enhanced activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) in the intestinal tissues of snowy salmon, thereby improving their ability to scavenge free radicals ^[12]. This suggested that supplementing nano iron about 60 mg/kg enhanced the fish's ability to scavenge free radicals and protects the intestine from oxidative damage. However, excessive nano iron can disrupt the antioxidant system of the fish gut, resulting in oxidative damage and potentially contributing to the development of intestinal diseases ^[34].

Compared to the 0 mg/kg group, the relative expression levels of intestinal pro-inflammatory genes TNF-α and COX-2 showed a tendency to decrease in the 60 mg/kg and 120 mg/kg groups. These findings suggest that moderate amounts of nano iron have the potential to attenuate the inflammatory response of the intestine, thereby offering protection against inflammatory damage. At moderate concentrations of nano iron, modulating oxidative stress levels through activation of the intracellular antioxidant defense system may be advantageous for mitigating inflammatory responses ^[35]. In contrast, Conversely, supplementation with excessive nano iron (480 mg/kg) significantly reduced the expression of anti-inflammatory genes such as TGF-β1 and IL-10. Excessive iron may influence the Fenton reaction, leading to heightened production of free radicals in the body and increased oxidative stress. Consequently, this oxidative stress may be responsible for the low expression of anti-inflammatory genes, which further increases inflammation in the body ^[29].

In the present study, the intestine of largemouth bass in the 60 mg/kg supplemented group exhibited relatively low expression levels of Caspase3 and Bax (pro-apoptosis-related genes). Concurrently, the relative expression levels of the anti-apoptotic gene Bcl-2 initially increased to the highest level with nano iron supplementation, but then reduced gradually with higher concentrations of nano iron. These findings suggested that moderate levels of nano iron may mitigate intestinal cell apoptosis. This effect could be attributed to the enhancement of the body's antioxidant capacity by moderate nano iron, which helps scavenge excessive free radicals within cells, thereby reducing oxidative damage. Ultimately, this protective mechanism may contribute to maintaining the integrity and stability of intestinal cells and decreasing apoptosis levels. However, excessive supplementation of nano iron may lead to an elevated rate of apoptosis in intestinal cells. This is likely due to the toxic effects of high nano iron levels, which intensify intracellular oxidative damage and subsequently induce apoptosis in intestinal cells. Overall, supplementing with nano iron at appropriate concentrations has the potential to bolster the immunity of the intestinal tract in juvenile largemouth bass and reduce intestinal apoptosis, thereby promoting and maintaining intestinal health.

4.3 Nano iron can improve the disease resistance, but high concentration of nano iron had negative effects.

With the rapid development of aquaculture, the scale of intensive farming is also increasing. However, the increase in culture density has led to an increase in the instability of environmental factors in the culture water, which can easily lead to a decrease in the immunity of the fish and is highly susceptible to large-scale diseases and mortality. Nocardia amberjack is a pathogenic bacterium frequently suffered by largemouth bass, and the main symptom is the appearance of white nodules on the body surface and internal organs, which is highly pathogenic to largemouth bass^[36]. In order to reduce fish mortality and economic losses due to diseases and to improve the survival rate and economic efficiency of culture, it is now crucial to enhance the immunity of cultured fish through the addition of nutrients in feeds under the strict restriction of the use of antibiotics^[37]. Iron plays a dual role in fish physiology: it not only promotes fish growth but also plays a crucial role in enhancing their immune function.

After Nocardia amberjack attack, the survival rate of largemouth bass was highest in the group supplemented with 60 mg/kg iron nanoparticles, lowest in the group without iron nanoparticles (0 mg/kg), and second lowest in the group supplemented with 480 mg/kg iron nanoparticles. This deficiency could potentially reduce the activity of iron-containing enzymes in the body, thereby impacting the function of immune cells and the bactericidal ability of leukocytes, ultimately compromising the overall immunity of the fish ^[38]. The resistance of fish to Nocardia amberjack was diminished, as evidenced by the lower survival rate of juvenile Largemouth bass in the 480 mg/kg group following the attack. This outcome is likely attributed to an excess of iron triggering the Fenton reaction, resulting in the generation of excessive free radicals. Consequently, oxidative stress ensued, causing damage to tissues and organs through oxidative mechanisms ^[39], further compromising the organism's immune capacity ^[40]. In addition, high concentrations of iron may promote bacterial growth and reproduction, causing more widespread infections and accelerating morbidity and mortality in the animal body^[41]. The findings of this experiment align with Sealey et al.'s study, which observed that iron deficiency in feed increased the susceptibility of spotted forked catfish to Edwardsiella infection. Additionally, supplementing with appropriate amounts of iron reversed the decreased immunity resulting from iron deficiency in the fish. However, high iron levels (180 mg/kg) in Sealey et al.'s study also suppressed the immune response and elevated mortality among spotted forked catfish following E. edwardsi attack ^[42]. Therefore, supplementing the feed with nano iron can enhance the antibacterial capacity of juvenile Largemouth Bass by bolstering immune system function. However, excessive doses could potentially induce oxidative damage and compromise the organism's immunity ^[43].

The findings of this study demonstrate that dietary supplementation with nano iron markedly enhanced antioxidant capacity, immune function, and intestinal barrier function in juvenile largemouth bass. However, over supplement of nano iron was found to inhibit antioxidant and immune functions in fish and adversely impact intestinal barrier function. Therefore, meticulous consideration of the dosage is essential when using nano iron as an additive in aquafeed to optimize fish health and productivity.

Ethics approval and consent to participate

All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the College of Animal Science and Technology of Sichuan Agricultural University, Sichuan, China, under permit No. DKY-2022102016. All experimental methods were performed according to relevant guidelines and regulations and the study was carried out in compliance with the ARRIVE guidelines.

CRediT authorship contribution statement

Kuo He: Data curation, Methodology, Writing - review & editing, Conceptualization. Rui Huang: Writing - original draft, Data curation, Sample collection. Hangyu Yang: Sample collection. Qiao Liu: Methodology. Yaoyi Zhang: Sample collection. Haoxiao Yan: Sample collection. Yifan Hu: Sample collection. Rabia Tahir: Writing - review & editing. Liulan Zhao: Resources, Supervision. Song Yang: Project administration, Supervision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

Acknowledgments

This research was supported by the 2022 double branch of discipline construction with grant number [035/2022993226]. The ingredients of experimental feed were supported by Mr. Lei Chi, SAN WANG Co. Ltd (Chengdu, Sichuan, China).

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No competing interests reported.

Supplementaryfile.docx
Table S1 Primer sequences of target gene for real-time PCR. Table S2 Effects of various nano iron levels on growth and physiological parameters in juvenile Largemouth Bass.

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Dietary nano iron enhanced intestinal physical barrier, antioxidant capacity and immune function of juvenile Largemouth Bass (Micropterus nigricans)

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Experimental diets design

2.2. Fish and feeding trial

2.3. Sample collection

2.4. Growth performance and microscopic observations

2.5. Enzyme activity determination

2.6. Quantitative real time PCR

2.7. Western blotting

2.8. Challenge test

2.9. Statistical analysis

3. Results

3.1. Growth performance

3.2. Intestinal morphology

3.3. Intestinal tight junction

3.4. Antioxidant status

3.5. Inflammation and apoptosis indicators

3.6. Challenge test

4. Discussion

4.1. Moderate supplement nano iron improved the growth performance and enhanced its intestinal physical barrier.

4.2. Nano iron improved the intestinal antioxidant status and enhanced the immune ability.

5. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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