The Combined Supplementation of AZOMITE and Citric Acid Promoted the Growth, Intestinal Health, Antioxidant, and Resistance against Aeromonas hydrophila for Largemouth Bass, Micropterus salmoides

Citric acid is an organic acid extensively used in feed industry, and AZOMITE is a hydrated aluminosilicate compound rich in rare earth elements and trace mineral elements. This study investigated the supplemental effects of AZOMITE and citric acid individual or in combination on the growth performance, intestinal microbiota, morphology, digestive enzyme activity, serum indexes, and disease resistance of juvenile largemouth bass. Six diets were designed, including the control diet (CON) and the five additive-supplemented diets with the addition of 4 or 8 g/kg citric acid (CA4, CA8), 3 g/kg AZOMITE (A3), and their combined addition as 4 g/kg citric acid + 1.5 g/kg AZOMITE) (C4A1.5) and 8 g/kg citric acid + 3 g/kg AZOMITE (C8A3). Juvenile largemouth bass with initial body weight of 22.01 ± 0.09 g were fed the six diets for 56 days. The results revealed that the combined addition of 4 g/kg citric acid and 1.5 g/kg AZOMITE (C4A1.5) increased weight gain by 7.99% (P < 0.05), and decreased feed conversion ratio by 0.07 (P < 0.05). The protein retention in the C4A1.5 group and the lipid retention in all additive-supplemented groups were significantly higher than those in the control group (P < 0.05). In serum, all additive-supplemented groups showed significantly higher glutathione peroxidase activity than the control group (P < 0.05). The activities of superoxide dismutase and catalase in the CA8, A3, C4A1.5, and C8A3 groups were significantly higher (P < 0.05), while the concentration of malondialdehyde was significantly lower than those in the control group (P < 0.05). Moreover, the total antioxidant capacity in the A3 and C4A1.5 groups, and lysozyme activity in the A3, C4A1.5, and C8A3 groups were significantly increased when compared to the control group (P < 0.05). In digestive enzyme, the protease activity in the A3, C4A1.5 groups, and amylase activity in the CA4, CA8, and C4A1.5 groups were significantly higher than those in the control group (P < 0.05). In intestinal microbiota, Firmicutes abundance was elevated in all additive groups, while the Fusobacteriota and Plesiomonas shigelloides abundance were decreased. In the intestinal histology, the CA8, A3, and C4A1.5 groups showed significantly higher villus height than the control group (P < 0.05). After the infection with Aeromonas hydrophila, the cumulative mortality of all additive-supplemented groups was significantly lower (P < 0.05), and the C4A1.5 group demonstrated the lowest mortality. In conclusion, the combined supplementation of 4 g/kg citric acid + 1.5 g/kg AZOMITE increased the growth, antioxidant, immune capacity, improved the intestinal morphology and microbial flora of juvenile largemouth bass, and promoted the resistance against Aeromonas hydrophila infection.


Introduction
Largemouth bass (Micropterus salmoides) is native to North America, and it is a typical carnivorous freshwater fish species with delicious flavor and without intermuscular bones.China is the largest producer in largemouth aquaculture with a production of 702,093 tons in 2021 (China Fishery Statistical Yearbook, 2022) [1].In the past, the high-density farming and the unreasonable use of compound feed [2] increased the possibility and risk of disease occurrence.In the prevention and treatment of disease, antibiotics have been widely used [3].However, the potential hazards posed by antibiotic use, such as drug resistance and residue, increased the potentially threatening to both humans and the environment [4][5][6].In many countries, the use of antibiotics has been strictly restricted FAO [7].Thus, the search for efficient and ecofriendly feed additives to enhance the immune function and disease-resistant ability of aquaculture animals has become a hot topic in the industry [8].AZOMITE, a hydrated aluminosilicate mineral, is formed from volcanic eruption with plenty of natural mineral elements essential for the growth of animals and plants, along with rare earth elements such as lanthanum and actinium.AZOMITE has been used as mineral fertilizer and soil amendment in crop cultivation [9,10].In poultry, it was reported that dietary AZOMITE improved broiler's growth performance [11] and enhanced the mineral utilization such as calcium and phosphorus [12].In aquaculture, AZOMITE supplementation in diets significantly enhanced the growth performance, immune capacity, and digestive function of tilapia (Oreochromis niloticus × O.aureus) [13] and grass carp (Ctenopharyngodon idella) [14], as well as improved the disease resistance of tilapia (Oreochromis mossambicus) [15].
Our previous studies have demonstrated that the addition of 2-3 g/kg AZOMITE in the diet significantly improved the growth performance, feed utilization, nonspecific immunity, Aeromonas hydrophila resistance, and intestinal morphology in juvenile largemouth bass [33].However, the dietary application of citric acid has not been reported in largemouth bass.As citric acid could enhance the utilization of minerals, it is speculated that there may be a synergistic effect between citric acid and AZOMITE rich in minerals and rare earth elements.Therefore, this study investigated the supplemental effects of AZOMITE and citric acid individual or in combination on the growth performance, intestinal microbiota, morphology, digestive enzyme activity, serum antioxidant and immune indexes, and resistance against Aeromonas hydrophila infection of juvenile largemouth bass.Findings from this study will direct the development of efficient and eco-friendly feed additives for carnivorous fish.
The ingredients were ground and passed through a 60 mesh screen, mixed evenly, and then made into sinking pellets with a particle size of 2 mm using a single-screw extruder (LX-75 extruder, Longxiang Food Machinery Factory, Hebei, China).Granulation was performed at 85°C, and feed was dried to an approximate moisture content of 100 g per kilogram, then sealed for storage at room temperature.

Experimental
Procedure.Largemouth bass were purchased from Fengmin Aquaculture Farm in Huzhou, Zhejiang Province (China).Initially, the fish were temporarily reared in indoor concrete pools for 2 weeks.Then, 450 healthy fish (22.01 AE 0.09 g) were selected and allotted in 18 indoor net cages (1.5 × 1.2 × 1.2 m), which were suspended in concrete pools (5 × 3 m).There were six treatment groups with three cages per group and 25 fish per cage.The water source was filtered with lake water (freshwater).
In the rearing period, the fish were fed twice daily (at 8:30 and 16:30) at a rate of 3%-5% of their body weight.All cages received the similar amount of feed.The cages were cleaned, and about 1/10 of the water was renewed once every 3 days to maintain the water quality.During the rearing period, the water quality was as follows: temperature 23-30°C, pH 6.5-7.3,dissolved oxygen >4 mg/L, salinity 0.5%-1.0%,nitrite <0.1 mg/L, and ammonia nitrogen 0.1-0.2mg/L.The trial was carried out at Binhai Base of Shanghai Ocean University with a culture cycle of 56 days.

Samples Collection.
Upon completion of 56 days of feeding, the fish were fasted for 24 hr, then the number of fish in each cage was counted, and the weight was measured.Weight gain (WG), feed conversion ratio (FCR), and survival were calculated from the above data.

2
Aquaculture Nutrition Each cage contained three randomly selected fish for whole-body composition analysis, and blood was drawn from another three fish per cage via caudal vein after measuring body length and weight.The blood samples were centrifuged at 3,000 r min −1 for 10 mins, then the serum was collected for the determination of biochemical indicators.
The viscerosomatic index (VSI) and hepatosomatic index (HSI) of the three fish were calculated after blooding and dissection.Under sterile conditions, the intestinal tract was separated, and 1-2 cm of the anterior intestine was fixed in Bouin's solution for tissue observation, and the remaining parts were used for the measurement of intestinal digestive enzyme activity.Based on the growth performance, the CON, A3, CA8, C4A1.5, and C8A3 groups were selected for the intestinal microbial analysis, and three fish per cage were rapidly dissected in a sterile environment, then 1-2 cm of the intestine was sampled and stored in liquid nitrogen (the intestines of three fish were pooled as one sample) for the detection of intestinal microbial community composition.

Intestinal Morphology, Digestive Enzyme Activity, and
Microflora.The anterior intestine tissues were dehydrated through a series of ethanol solutions with increasing concentrations, followed by transparentizing in xylene and embedding in paraffin.Sections with thickness of 4 μm were prepared using a microtome (Leika RM 2016, Germany).Hematoxylin and eosin (H&E) staining was performed, and the morphological structure of the intestinal tissue was observed and photographed using a microscope (Nikon YS100 Photomicrography System).Additionally, the height and width of villus and the thickness of the muscle layer were recorded (ImageJ).
The anterior gut samples were thawed at 4°C, followed by homogenization using nine times volume of 4°C physiological saline.After centrifuging for 15 min at (1,500 g, 4°C), the supernatant was collected to determine the digestive enzyme activity.The protease activity measurement referred to the method of Su et al. [35] by using 2% casein solution as the substrate, and the enzyme amount that decomposed casein to generate 1 μg tyrosine per minute per milligram of tissue protein at pH 7.2 was defined as one protease activity unit (U).The amylase activity was determined using a reagent kit, and the hydrolysis of 10 mg starch per milligram of tissue protein at 37°C for 30 min was defined as one amylase activity unit (U).The total protein content was determined by a Coomassie brilliant blue method.
The intestine samples were sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. for intestinal microbiota analysis.DNA was extracted, and the PCR amplification was carried out, followed by high-throughput sequencing using the Illumina MiSeq platform.The primers V338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and V806R (5′-GGACTACHVGGGTWTCTAAT-3′) were employed to amplify the 16S rRNA gene V3-V4 region.PCR conditions were as follows: predenaturation at 95°C for 3 min, followed by 29 cycles of 95°C for 30 s, 53°C for 30 s and 72°C for 45 s, then 72°C for 10 min.The obtained data were processed using the Majorbio Cloud Platform (https://www.majorbio.com) for operational taxonomic unit (OTU) clustering, and the composition and species abundance of microbial communities at the phylum and genus levels were calculated.
2.5.Challenge Test.The Aeromonas hydrophila used in this study was provided by the Aquatic Pathogen Collection Center of Shanghai Ocean University.The bacteria were cultured in Luria-Bertani (LB) broth at 30°C for 18 hr, then the required bacteria were obtained by centrifugation at 4°C for 10 min (3,500 g).A pretest was conducted before the challenge test to determine the LD50 by intraperitoneal injection of three concentrations (3 × 10 7 , 3 × 10 8 , and 3 × 10 9 CFU/mL) at a dose of 0.01 mL/g body weight.Phosphate-buffered saline was used as the control, and the LD50 was determined to be 3 × 10 7 CFU/mL (Bliss method).Ten fish per cage were selected after the sample collection and fed with the original diets for 3 days to restore normal metabolism.After 24 hr of fasting, the fish were intraperitoneally injected with a solution of Aeromonas hydrophila at 3 × 10 7 CFU/mL (0.01 mL/g body weight).Then, the fish were observed continuously for 1 week after challenge, and the cumulative mortality rate was calculated.

Statistical Analysis.
The experimental data were expressed as means AE standard deviation (SD).One-way analysis of variance (ANOVA) was performed using SPSS26.0software to test for homoscedasticity and normal distribution of all data.Duncan's multiple range test was used for multiple comparisons between treatments with a significance level of P <0:05.

Whole-Body
Composition and Nutrient Retention.The whole-fish crude lipid content in the CA8, A3, and C4A1.5 groups, the PR in the C4A1.5 group and the LR in all additivesupplemented groups were significantly higher than those in the CON group (P <0:05), but the crude protein and crude ash contents of all groups showed no significant differences (P >0:05) (Table 3).

Serum Biochemical Analysis.
In serum biochemical indices, all additive-supplemented groups showed significantly higher glutathione peroxidase (GSH-PX) activity than the CON group (P <0:05).The activities of superoxide dismutase (SOD) and catalase (CAT) in the CA8, A3, C4A1.5, and C8A3 groups were significantly higher (P <0:05), while the concentration of malondialdehyde (MDA) was significantly lower than those in the CON group (P <0:05).Moreover, the total antioxidant capacity in the A3 and C4A1.5 groups, acid phosphatase activity in the CA4 and CA8 groups, and lysozyme activity in the A3, C4A1.5, and C8A3 groups were significantly increased as compared to the CON group (P <0:05) (Table 4).

Intestinal Morphology.
In Figure 2, it can be observed that the intestinal villus in the AZOMITE and citric acid mixture groups were arranged neatly and compactly with better clarity and integrity than the other groups.In Table 6, there was no significant difference in villus width Aquaculture Nutrition and muscle thickness among all the groups.However, the CA8, A3, and C4A1.5 groups showed significantly higher villus height than the control group (P <0:05).

Discussion
4.1.Growth Performance and Feed Utilization.Li et al. [28] reported that the addition of 8 and 12 g/kg of citric acid to diets significantly increased the WG and decreased the FCR of rainbow trout.In low-phosphorus diet, the supplemental citric acid (10 g/kg) significantly improved the growth performance of rainbow trout [29].In large yellow croaker, the supplementation of 8 and 16 g/kg of citric acid to high-plant protein diets significantly enhanced the growth performance and protein retention [27].Similarly, the improvement of growth performance by dietary citric acid has also been reported in red drum [19], red sea bream (Pagrus major) [22], Carassius auratus gibelio [26], and white shrimp (Litopenaeus vannamei) [35].The promoting-effects of organic acids might be realized through reducing intestinal pH, stimulating digestive enzyme activity, inhibiting the growth of harmful intestinal microorganisms, and increasing the utilization of minerals [36].However, dietary supplemental citric acid (0-30 g/kg) did not significantly affect the growth performance of turbot [25].In the present study, adding 4 or 8 g/kg of citric acid to diet just showed an increasing trend in the growth performance (P >0:05), which may be related to the low-addition level of citric acid, and further study is needed to investigate dietary effects of citric acid on largemouth bass with graded citric acid level.AZOMITE is a natural hydrated aluminosilicate rich in rare earth elements.According to the study of Xu et al. [33], adding 2.0 g/kg of AZOMITE to the diet of juvenile largemouth bass increased WG by 11.2% and reduced FCR by 0.1.The growth-promoting effects of AZOMITE have also been observed in tilapia [13], grass carp [14], and white shrimp [37].The rich rare earth and trace elements in AZOMITE, such as La, Ce [38][39][40], have antibacterial properties and may enhance the digestive and absorption capacity of animal, thus positively affecting growth performance.In this study, the addition of 3.0 g/kg of AZOMITE just numerically improved the growth performance of largemouth bass, which may be connected with the growth stages, diet composition, and aquaculture environments.
Xun et al. [41] once reported that adding rare earth citrate to diet increased the nutrient digestibility of sheep, and the dietary rare earth citrate also improved the growth performance of broilers [42].In this study, the combination of 4 g/kg of citric acid and 1.5 g/kg of AZOMITE significantly increased WG and decreased FCR.This promoting effect may be due to the synergistic effect of citric acid and AZOMITE, which increases the utilization of minerals, stimulates the secretion of digestive juice, enhances the activity of protease and amylase.However, the high inclusion of citric acid + AZOMITE (8+ 3 g/kg) did not significantly improved the growth of largemouth bass, which may be due to the antagonistic effect of the excessive supplementation.4.2.Antioxidant Capacity.Fish's antioxidant capacity plays an important role in their health [43].Excessive reactive oxygen species (ROS) can cause DNA hydroxylation, lipid peroxidation, and other effects leading to cell apoptosis and compromised immunity [44,45].Antioxidant systems can control the ROS level and maintain the balance in body, thus enhancing immunity [46].
Superoxide dismutase (SOD) can clear superoxide radicals by catalyzing superoxide radicals (O 2− ) to form hydrogen peroxide and water, reducing the synthesis of OH radicals [44].During the reduction of oxygen in the body, hydrogen peroxide is also produced, and excessive hydrogen peroxide will threaten the body's homeostasis [47], while catalase (CAT) and glutathione peroxidase (Gpx) can clear excess hydrogen peroxide.Malondialdehyde (MDA) is a product of lipid peroxidation caused by excessive ROS, which may negatively affect the activity of proteins in the  8 Aquaculture Nutrition body [48].Total antioxidant capacity (T-AOC) directly reflects the total antioxidant capacity of the organism [49].Therefore, the activity of SOD, CAT, the concentration of GPX, MDA, and T-AOC are important indicators of reflecting antioxidant capacity [50].Dietary supplementation of citric acid can help to improve the antioxidant capacity of fish.For example, dietary citric acid has been reported to promote the antioxidant capacity and reduce MDA content in rainbow trout [28], turbot [51], and large yellow croaker [27].In this study, the addition of 8 g/kg citric acid, rather than 4 g/kg, significantly increased the activity of SOD, CAT, and GPx and reduced MDA concentration.Thus, the positive effect of citric acid is closely related to the inclusion level.
Rare earth elements also have positive effects on the body's antioxidant capacity.In tilapia, [13], grass carp [14] and white shrimp [37], the supplemental AZOMITE (2, 4 g/ kg) significantly increased serum SOD activity.Xu et al. [33] also reported that dietary AZOMITE (1-6 g/kg) increased the serum antioxidant enzyme activity (SOD/CAT) of largemouth bass at different levels.In this study, the addition of 3 g/kg of AZOMITE significantly increased SOD, CAT, GPx activities, and T-AOC level, and reduced the MDA concentrations in serum.
In the present study, the combined supplementation of citric acid and AZOMITE (C4A1.5,C8A3) significantly increased serum SOD, CAT, GPx activities, and T-AOC levels, and reduced the MDA concentrations.It is noteworthy that the SOD and CAT activities in the two combined groups were significantly higher than those in the two citric acid groups, and numerically higher than those in the AZOMITE group.Maybe a synergistic effect between citric acid and rare earth elements in AZOMITE was produced, further enhancing the body's antioxidant capacity.However, the mechanism is unclear and needs further study.

Intestinal Microbiota.
The intestinal microorganism is not only an important defense line in fish immune systems, but also participates in nutrient absorption [52].Various intrinsic or extrinsic factors, such as fish species, development stage, water environment, and food would affect the composition of intestinal microbiota [53].The present study indicated that the dominant intestinal microbiota (at the phylum level) of largemouth bass were Firmicutes, Fusobacteriota, and Proteobacteria, which accounted for more than 98% of total bacteria in all groups.At the genus level, the dominant species were Cetobacterium (69.71%),Mycoplasma (12.78%), and Plesiomonas (7.87%).In other groups except the A3 group, the dominant genus were Mycoplasma, Cetobacterium, and Achromobacter.Such results were consistent with the reports by Yang et al. [54], He et al. [55], and Zhou et al. [56].
At the phylum level, the addition of citric acid and AZOMITE did not affect the dominant species composition in the intestinal microbiota.However, at the genus level, the abundance of Mycoplasma was significantly increased in all additive groups compared to the control group.There are few reports on the role of Mycoplasma in the intestinal microbiota of fish.Although some species of Mycoplasma are pathogenic, previous studies have shown that certain species of this genus are harmless commensals in the natural intestinal microbiota.For example, Mycoplasma is the dominant species in the normal intestinal microbiota of Gillichthys mirabilis [57] and wild salmon (Salmon) [58].
It has been reported that Plesiomonas shigelloides is a conditionally pathogenic bacterium that causes significant loss to aquatic animals [59].In this study, Plesiomonas shigelloides accounted for 7.87% in the CON and 0.58%, 0.11%, 1%, and 2.37% in the CA8, A3, C4A1.5, and C8A3 groups, respectively, which indicated that citric acid and AZOMITE addition inhibited the growth and colonization of this bacterium in the intestine, possibly enhancing the host's disease resistance.

Intestinal Morphology and Digestive Enzyme Activity.
The intestinal morphology directly reflects the health of the intestine, and the digestive enzyme activity is an important indicator reflecting the ability to digest nutrients.Dai et al. [25] found that the intestinal absorption area of turbot was increased by the supplementation of 15 g/kg citric acid.Huang et al. [60] reported that dietary calcium sulfate increased the villus height and width of largemouth bass.Dietary citric acid (10 g/kg) has also been reported to significantly improve the digestive enzyme activity in intestine of tilapia [13].Similar results were obtained in white shrimp, rainbow trout and red drum [19,28,35].In this study, the villus height in the CA8 group and the amylase activity in the CA4 and CA8 groups increased, which may be related to citric acid's ability to reduce the pH and stimulate the secretion of intestine.
Rare earth elements also have the function of improving intestinal structure.Xu et al. [33] found that dietary AZOMITE increased the intestinal villus height and digestive enzyme activity of largemouth bass [33], and Liu et al. [13] reported the similar results in tilapia.In this study, the villus height and protease activity in the A3 and C4A1.5 groups were also significantly increased, the improvement of intestinal structure and digestive enzyme activity may be related to the improvement of the intestinal microbial community and the synergistic effect of acid and rare earth elements.However, the C8A3 group did not present the promoting effects on villus height and protease activity, which may be due to the antagonistic effect of the excessive supplementation.4.5.Immunological Indices and Disease Resistance.Acid phosphatase (ACP) and lysozyme (LZM) are important indicators for the immune function [61].Su et al. [35] reported that dietary supplementation of citric acid significantly increased the lymph lysozyme activity and the survival after Vibrio parahaemolyticus infection.Zhang et al. [26] found that citric acid and malic acid enhanced the alkaline phosphatase activity of Carassius auratus gibelio and increased the gene expression of IL-1β.Adding 2-5 g/kg AZOMITE to the diet of largemouth bass also significantly increased lysozyme activity and reduced cumulative mortality after Aeromonas hydrophila challenge [33].Similar findings in AZOMITE have been reported in tilapia [15] and white shrimp [37].In this study, the ACP Aquaculture Nutrition activity in the CA4, CA8 groups, the LZM activity in the A3, C4A1.5, and C8A3 groups were significantly increased, and the cumulative mortality after Aeromonas hydrophila infection in all citric acid and (or) AZOMITE groups was decreased, when compared to the CON group.Such results indicated that the immunity and disease resistance were promoted by the supplementation.Specially, the lowest cumulative mortality was observed in the C4A1.5 group, indicating a synergistic effect of citric acid (4 g/kg) and AZOMITE (1.5 g/kg) in enhancing disease resistance.This may be due to the anions of citric acid chelating the active components in AZOMITE in the intestine, stimulating the immune function, and enhancing the resistance to bacterial infection.

Conclusion
In conclusion, the combined supplementation of citric acid and AZOMITE increased the growth, antioxidant, immune capacity, improved the intestinal morphology and microbial flora, and promoted the disease resistance against Aeromonas hydrophila infection.A dietary dosage of 4 g/kg citric acid + 1.5 g/kg AZOMITE are recommended for juvenile largemouth bass.
, A3, C4A1.5, and C8A3 groups, respectively.As compared to the CON group, the proportion of Firmicutes increased and the proportion of Fusobacteriota decreased in the CA8, A3, C4A1.5, and C8A3 groups.At the genus level (Figure1(b))

FIGURE 3 :
FIGURE 3: The cumulative mortality of largemouth bass after challenging against A. hydrophila during 7 days.Values are means AE SD (n = 10).Bars bearing with different letters are significantly different among treatments (P <0:05).

TABLE 1 :
Ingredients and proximate composition of experimental diets (air-dry basis, g/kg).

TABLE 2 :
Effects of dietary AZOMITE and citric acid on growth performance and physical indices of largemouth bass.Values in the same row with different superscripts alphabets indicate significant differences (P <0:05), the same as below.IBW, initial body weight; FBW, final body weight; WG, weight gain; FCR, feed conversion ratio; HSI, hepatosomatic index; VSI, viscerosomatic index; and CF, condition factor.

TABLE 3 :
Effects of dietary AZOMITE and citric acid on whole-body composition and nutrient retention of largemouth bass.

TABLE 4 :
Effects of dietary AZOMITE and citric acid on hematology and biochemical parameters of largemouth bass.

TABLE 5 :
Effects of dietary AZOMITE and citric acid on digestive enzyme activities of largemouth bass.

TABLE 6 :
Effects of dietary AZOMITE and citric acid on intestinal morphology of largemouth bass.