Effects of Dietary Strontium Supplementation on Growth Performance, Strontium Enrichment, Muscle Nutrition, and Hepatic Lipid Metabolism in Juvenile Hybrid Sturgeon (Acipenser baerii ♀ × Acipenser schrenckii ♂)

Abstract

To explore a safe and effective approach for producing strontium-enriched fish, in this study, we modified the feed for juvenile hybrid sturgeon (Acipenser baerii ♀ × Acipenser schrenckii ♂) and set three different levels of strontium chloride content in their diet (0 mg/kg (Sr0, control), 80 mg/kg (Sr80), and 160 mg/kg (Sr160)) for a period of 8 weeks, analyzing their growth performance, strontium enrichment, muscle nutrition, and hepatic physiological, biochemical, and transcriptomic characteristics. The results show that dietary strontium had no significant impact on sturgeon growth or survival rate (p > 0.05). The strontium content in tissues increased with dietary strontium levels, with the highest enrichment in bone plates (p < 0.05). However, muscle crude fat in the strontium-supplemented groups decreased significantly; the Sr160 group had higher glutamic acid, valine, docosahexaenoic acid methyl ester, lower myristic acid, palmitic acid, etc. (p < 0.05). In addition, strontium treatment alleviated hepatic lipid accumulation and mitochondrial swelling. Biochemical analyses revealed reduced plasma levels of Triglyceride (TG), Total Cholesterol (TC), Alanine Aminotransferase (ALT), and Aspartate Aminotransferase (AST), as well as decreased hepatic Malondialdehyde (MDA) content, while hepatic Glutathione (GSH) levels increased (p < 0.05). Transcriptomic data further showed that strontium downregulated the expression of fasn and tfrc and upregulated the expression of cpt1a, apoa1, cyp7a1, and slc3a2 (p < 0.05). In conclusion, dietary supplementation with 80–160 mg/kg strontium enables safe strontium enrichment in hybrid sturgeon, improves muscle nutritional quality, and protects liver function by regulating the genes related to lipid metabolism and antioxidant defense, providing a scientific basis for the development of strontium-enriched fish products.

1. Introduction

Trace elements play important physiological and biochemical roles in living organisms [1]. Strontium is an example of this [2]; as an essential trace element in the body [3], its functions involve repairing nerve damage, enhancing antibacterial and anti-inflammatory effects [4,5,6]. Additionally, a moderate amount of strontium in the water can enhance the growth efficiency of juvenile chum salmon (Oncorhynchus keta) [7]. Most prior research on strontium has focused on bones, and strontium is the only trace element related to bones’ compressive strength [8]. Strontium can inhibit the proliferation of osteoclasts and promote the differentiation of osteoblasts, thereby promoting bone health [9,10]. Strontium also enhances the antioxidant capacity of the body. By increasing the activities of Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione Peroxidase (GPx) in rats and reducing the content of MDA, it can shorten the duration of chronic inflammation [11]. The nutritional status of strontium may influence the physiological state of the liver [12]. Previous studies have shown that strontium improves endoplasmic reticulum stress by synergistically regulating the expression of endoplasmic reticulum stress and lipid metabolism pathways, thereby improving cases of non-alcoholic fatty liver disease [13].

Products rich in strontium are popular among consumers because strontium is beneficial to human health and significantly increases the added value of agricultural products. Agricultural products rich in strontium, including millet (>1.0 mg/kg), hawthorn (>7.5 mg/kg), and drinking water (>0.4 mg/kg), have greatly enhanced the market value of local agricultural products in China [14]. Fish are rich in various minerals and nutrients and are an excellent source of protein [15]. However, strontium-enriched fish remain rare due to limited natural strontium-rich water in aquaculture areas. To break free from the constraints of strontium-rich water bodies, the exploration of safe, simple and effective new ways to enrich strontium is the inevitable path to the large-scale production of strontium-rich fish.

The hybrid sturgeon (Acipenser baerii ♀ × Acipenser schrenckii ♂) is one of the most important cold-water fish species in the aquatic economy [16]. In 2017, approximately 2329 commercial sturgeon farms were recorded worldwide, representing a 7% increase compared to 2016 [17]. Hybrid sturgeon not only has excellent biological characteristics, but its meat is also highly nutritious. Moreover, it has no intermuscular bones, and its convenience and palatability in terms of consumption are significantly superior to those of common freshwater fish [18]. It is therefore a promising candidate for the large-scale production of strontium-enriched fish.

Therefore, in order to clarify whether making additions to feed can enable hybrid sturgeons to safely, effectively and rapidly accumulate strontium, a comprehensive analysis was conducted. This analysis included data on the growth and survival of hybrid sturgeons, their body composition, physiological and biochemical indicators, and on the metabolomics of important tissues and organs. The research results will clarify the accumulation status of strontium in the organism and its potential effects on growth and lipid metabolism. These findings will provide reference materials for studies on lipid metabolism in organisms related to strontium. They also offer important references for the development of fish with high strontium content.

2. Materials and Methods

2.1. Dietary Formula

A basal diet was formulated using fish meal and chicken meal as primary protein sources, and soybean phospholipid oil and fish oil as lipid sources (

Table 1. Experimental feed formulation.

Ingredients (%)	Sr0	Sr80	Sr160
Fish meal	35.0	35.0	35.0
Chicken meal	5.0	5.0	5.0
Porcine hemoglobulin powder	2.5	2.5	2.5
46% Soybean meal	12.0	12.0	12.0
Fermented soybean meal	10.0	10.0	10.0
Peanut bran	5.0	5.0	5.0
vital wheat gluten	2.5	2.5	2.5
Seaweed powder	2.5	2.5	2.5
Calcium bicarbonate	2.0	2.0	2.0
Vitamin premix a	1.0	1.0	1.0
Mineral premix b	1.0	1.0	1.0
Soybean phospholipid oil	5.0	5.0	5.0
Fish oil	4.5	4.5	4.5
Strong flour	12.0	12.0	12.0
Strontium SrCl2, (mg/kg) c	0	80	160
Proximate composition (analyzed, % dry matter)
Crude protein	≥45.0
Crude fat	≥12.0
Crude ash	≥12.0
Dry matter	≥94.0
Lysine	≥3.0

^a Vitamin premix (per kg of premix): A, 60.00 g/kg; B1, 8.00 g/kg; B2, 12.00 g/kg; B3, 80.00 g/kg; B5, 24.00 g/kg; B6, 12.00 g/kg; H, 0.40 mg/kg; B12, 0.02 mg/kg; C, 600.00 g/kg; D3, 0.04 mg/kg; E, 96.00 g/kg; K3, 8.00 g/kg; M, 4.00 g/kg; α-cellulose, 95.54 g/kg. ^b Mineral premix (per kg of premix): Calcium Lactate, 350.00 g/kg; K₂HPO₄, 250.00 g/kg; Ca(H₂PO₄)₂·H₂O, 175.00 g/kg; NaCl, 75.00 g/kg; KI, 0.13 g/kg; CuSO₄·₅H₂O, 37.50 g/kg; MnSO₄·H₂O, 2.50 g/kg; ZnSO₄·H₂O, 14.05 g/kg; K₂CO₃, 35.00 g/kg; α-cellulose, 60.823 g/kg. ^c The strontium source added to the experimental diet was anhydrous chlorosilicic acid (SrCl₂, analytical grade, purity ≥ 99.0%), which was used to adjust the strontium content in the diet of each group. Main ingredients: The protein sources include fish meal, chicken meal, 46% soybean meal, and high-quality wheat protein powder; carbohydrates come from strong gluten flour and seaweed powder; fat comes from soybean lecithin oil and fish oil.

). We designed the feed formula using Excel. The feed raw materials were thoroughly ground and passed through a 60-mesh sieve. Then, various raw materials were mixed evenly using a stepwise expansion method, and different amounts of strontium chloride (SrCl₂) (Sinopharm Group Co., Ltd., Shanghai, China) were added to the basic feed. The mixture was then processed into sedimentary granular feed with a particle size of 3 mm using a dry power press (model MUZL180; Muyang Group, Jiangsu, China). The feed was air-dried at room temperature until the moisture content was approximately 10%, then packed into sealed bags and stored at −20 °C. Strontium stability was measured once a week using an inductively coupled plasma mass spectrometer(iCAPQ, Thermo Fisher Scientific, Waltham, MA, USA).

2.2. Experimental Animals and Sample Collection

Hybrid sturgeon were obtained from a breeding base located in Sanjian Township, Fengdu County, and subsequently transported to the Center for Big Science of Germplasm Innovation in the Chongqing High-tech Zone, China, where breeding experiments were conducted. Following a 14-day acclimatization period, healthy juvenile fish (312.21 ± 25.31 g) were randomly assigned to three experimental groups, each consisting of three replicate tanks. The fish were placed in a circular aquaculture tank with a diameter of 3 m and a depth of 2 m. The volume of water in each tank was maintained at approximately 10.6 cubic meters, and 30 fish were placed in each tank. The water used for breeding was treated by aeration and chlorine removal, with the water temperature maintained at 19 ± 2 °C. The dissolved oxygen level was 8.7 ± 0.5 mg/L, and the ammonia nitrogen and nitrite contents were both below the safe limit (Non-ionic ammonia ≤ 0.01 mg/L; Nitrite nitrogen ≤ 0.05 mg/L). The water was filtered and disinfected through sedimentation tanks, micro-filters, ultraviolet irradiation and ozone. Full-spectrum LED was used as the indoor light source, with a light cycle of 12L:12D. Feeding was carried out twice a day, at 09:00 and 16:00, with a total daily feeding amount accounting for 3% of the fish’s body weight. The remaining feed and any feces were removed within an hour of each feeding session to prevent water quality deterioration.

Test samples were collected after eight weeks. All fish were fasted for 24 h and anesthetized using MS-222 (Sinopharm Group Co., Ltd., Shanghai, China). Nine fish were selected for each breeding tank, resulting in a total of eighty-one fish being chosen. The body length and body weight of each fish were recorded. Tissue samples, including muscle, skin-on-muscle, bone plate, cartilage, whole blood, liver, intestine, and heart, were isolated and stored at −20 °C for subsequent analysis of strontium content, muscle nutrient composition, amino acid profile, and fatty acid composition. Blood was collected from the caudal vein using a sterile needle pre-rinsed with sodium heparin (Sinopharm Group Co., Ltd., Shanghai, China), and plasma was separated by centrifugation at 4000 rpm for 15 min under 4 °C conditions, followed by storage at −20 °C for subsequent assays. Liver samples were taken from the corresponding anatomical sites and stored in designated sample tubes. These samples were then preserved in a −80 °C refrigerator for biochemical testing. The remaining liver samples were fixed in paraformaldehyde (Sinopharm Group Co., Ltd., Shanghai, China), glutaraldehyde electron microscopy fixative (Sinopharm Group Co., Ltd., Shanghai, China), or RNA preservation solution (Accurate Biology, Changsha, China), and stored at 4 °C or −80 °C, as appropriate. All animal procedures in this research were conducted according to the Guidelines for the Care and Use of Laboratory Animals in China and approved by the Southwest University Animal Ethics Committee (permit No. IACUC-20230928-01).

2.3. Calculation of Growth Indicators

Each growth performance index was calculated as follows:

Survival rate (SR, %) = 100% × (Nt/No);

(1)

Weight gain rate (WGR, %) = 100% × (Wt − Wo)/Wo;

(2)

Specific growth rate (SGR, %/d) = 100% × (Wt − Wo)/t;

(3)

Feed conversion ratio (FCR) = Wf/(Wt − Wo);

(4)

Viscerosomatic index (VSI, %) = 100% × Wv/W;

(5)

Condition factor (CF, g/cm³) = 100% × W/L³;

(6)

where Nt is terminal number of fish, No is initial number of fish, Wt is terminal body mass (g), Wo is initial body mass (g), t is experimental days (d), Wf is feed intake (g), Wv is visceral weight (g), W is body mass (g), and L is body length (cm).

2.4. Detection of Strontium Content in Tissues

Muscle, skin-on-muscle, bone plates, cartilage, whole blood, liver, and heart tissues of the hybrid sturgeon were thawed and homogenized. The strontium content in the aforementioned tissues was analyzed using an inductively coupled plasma mass spectrometer [19].

2.5. Analysis of Muscle Nutritional Components

Muscle samples of the hybrid sturgeon stored at −20 °C were thawed and homogenized. Crude fat, crude protein, moisture, and ash contents were analyzed per GB 5009.6-2016, GB 5009.5-2016, GB 5009.3-2016 (direct drying method), and GB 5009.4-2016 (feed component testing standards) [20], respectively. A total of 17 amino acids were quantified via an amino acid analyzer (LA8080, Hitachi High-Tech Corporation, Tokyo, Japan) [21], and 37 fatty acids via a gas chromatograph-mass spectrometer (Trace1310 ISQ, Thermo Fisher Scientific, Waltham, MA, USA) [22].

2.6. Observation of Tissue Microstructure

The liver tissue of the hybrid sturgeon, which had been fixed in paraformaldehyde and processed with glutaraldehyde electron microscope solution, was retrieved and appropriate sections were selected for the observation of hepatic morphology. Liver samples were fixed in paraformaldehyde and stored at 4 °C. A graded ethanol (Sinopharm Group Co., Ltd., Shanghai, China) series was used for dehydration, followed by clearing in xylene (Sinopharm Group Co., Ltd., Shanghai, China) and embedding in paraffin (Sinopharm Group Co., Ltd., Shanghai, China). The resulting sections were cut to a thickness of 3–5 μm. Subsequently, the sections were stained with hematoxylin and eosin (HE) [23] and Oil Red O [24], and then examined under a light microscope (Nikon Corporation, Tokyo, Japan).

Liver tissues were fixed in a 2.5% glutaraldehyde solution (Sinopharm Group Co., Ltd., Shanghai, China), post-fixed in 1% osmium tetroxide (Sinopharm Group Co., Ltd., Shanghai, China), dehydrated using a graded ethanol series, embedded in Epon812 resin (Electron Microscopy Sciences, Hatfield, PA, USA), and subsequently sectioned into ultrathin slices. The sections were initially stained with uranyl acetate (Sinopharm Group Co., Ltd., Shanghai, China), followed by staining with lead citrate (Sinopharm Group Co., Ltd., Shanghai, China), and then examined under a transmission electron microscope (JEOL JEM-2100Plus, JEOL Ltd., Tokyo, Japan), with images captured for analysis [25].

2.7. Biochemical Analysis

Total cholesterol (TCH/T-CHO), triglycerides (TG), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were quantified in the collected plasma supernatant of the hybrid sturgeon [26]. The levels of superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) were also assessed in liver tissue samples of the hybrid sturgeon. All biochemical parameters were measured using commercially available assay kits (Jiancheng Biotechnology Co., Ltd., Nanjing, China), following the manufacturer’s instructions.

2.8. Total RNA Isolation and Transcriptome Sequencing

Total RNA was extracted from liver tissue of the hybrid sturgeon using Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA quality and concentration were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). mRNA molecules containing poly(A) tails were enriched using oligo(dT) magnetic beads and subsequently fragmented randomly using divalent cations in a Fragmentation Buffer (Accurate Biology, Changsha, China). The fragmented mRNA was used as a template for first-strand cDNA synthesis via reverse transcription, followed by second-strand cDNA synthesis using dNTPs. The synthesized cDNA fragments were subjected to end repair, phosphorylation, and adenine base addition (A-tailing), in accordance with Illumina library preparation protocols. Following adapter ligation and library amplification, the quality and concentration of the constructed libraries were evaluated. The samples were then sequenced on an Illumina HiSeq platform (Illumina, Inc., San Diego, CA, USA) according to the required sequencing depth [27].

2.9. Differential Gene Expression

The expression of single genes was calculated using fragment per million mapping per kilobase transcript (FPKM), thereby identifying differentially expressed genes (DEGs) among the three groups of this hybrid sturgeon. DESeq2 R package (1.20.0) was used for differential expression analysis of the two groups. DESeq2 provides statistical procedures for determining differential expression in digital gene expression data using models based on the negative binomial distribution. GOseq (v1.46.0, Bioconductor Project, Seattle, WA, USA) and KOBAS (v3.0, Peking University, Center for Bioinformatics, Beijing, China) software were used to perform GO functional enrichment analysis and KEGG pathway enrichment analysis for differential gene sets. The resulting p values were adjusted to control the false discovery rate using the Benjamini–Hochberg method. The difference between gene expression levels was determined by DESeq2 with an adjusted p-value < 0.05 [28].

2.10. qRT-PCR Validation

The reliability of the transcriptomic data of this hybrid sturgeon was validated using the qRT-PCR technique. A quantitative real-time PCR (qPCR) experiment was conducted using the StepOnePlus™ real-time fluorescence quantitative PCR instrument (Thermo Fisher Scientific, Waltham, MA, USA) [29]. Total RNA was extracted from the samples using AG RNAex Pro Reagent (Accurate Biotechnology (Hunan) Co., Ltd., Hunan, Changsha, China). Complementary DNA (cDNA) was synthesized employing the Evo M-MLV Mix Kit with gDNA Clean for qPCR (Accurate Biology, AG11728, Changsha, China), according to the manufacturer’s protocol. A 20-μL qPCR reaction containing 0.4 μL of PCR forward/reverse primers, 1 μL of cDNA template, and 10 μL of 2× SYBR^® Green Premix Ex Taq™ II (Tli RNase H Plus) (Takara Bio Inc., Otsu, Japan) was prepared. The thermocycling conditions were as follows: 95 °C for 10 min, followed by 40 cycles consisting of 95 °C for 15 s and 60 °C for 60 s. The expression level of target genes was normalized to housekeeping gene β-actin and calculated using the 2^−ΔΔCt method. Gene-specific primers were designed using Primer5 software (version 5.0) and are listed in Table S1; primers were chemically synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

2.11. Statistical Analysis

Experimental data are expressed as mean ± standard error. All data were subjected to one-way analysis of variance (ANOVA) using SPSS 22.0 (SPSS, IBM Corporation, Armonk, NY, USA), and differences between groups were considered statistically significant at p < 0.05.

3. Results

3.1. Growth Indicators

The strontium concentration of the diet in this experiment did not significantly affect the weight gain rate, specific growth rate, feed ratio, visceral-body ratio, fatness or survival rate (p > 0.05) (

Table 2. Effect of dietary strontium levels on growth indices of juvenile hybrid sturgeon.

Index	The Amount of Strontium Added to the Feed (mg/kg)
	Sr0 (0 mg/kg)	Sr80 (80 mg/kg)	Sr160 (160 mg/kg)
Wo, g	312.36 ± 8.03	313.51 ± 8.06	311.89 ± 7.43
Wt, g	677.95 ± 13.00	683.68 ± 13.61	685.52 ± 11.14
WGR, %	1.17 ± 0.04	1.18 ± 0.04	1.19 ± 0.03
SGR, %/d	1.38 ± 0.03	1.39 ± 0.05	1.40 ± 0.02
FCR	1.37 ± 0.04	1.40 ± 0.05	1.35 ± 0.04
VSI, %	0.13 ± 0.01	0.13 ± 0.00	0.13 ± 0.00
CF, g/cm3	0.99 ± 0.03	0.92 ± 0.03	0.89 ± 0.02
SR, %	100.00	100.00	100.00

Notes: Values in the same row with different superscripts are significantly different (p < 0.05). Wt is terminal body mass (g); Wo is initial body mass (g); WGR is weight gain rate (%); SGR is specific growth rate (%/d); FCR is feed conversion ratio; VSI is Viscerosomatic index (%); CF is Condition factor (g/cm³); SR is survival rate (%).

).

3.2. Enrichment Levels of Strontium in Tissues

The strontium content in various tissues of hybrid sturgeon increased with elevated strontium intake. In particular, a positive correlation was observed between the strontium content in bone plates and intestine and the strontium concentration in the feed, with statistically significant differences among the experimental groups (p < 0.05). Additionally, the strontium levels in the whole blood and heart tissue of the experimental groups were significantly higher than those in the control group (p < 0.05). Furthermore, the Sr160 group exhibited significantly higher strontium concentrations in muscle, skin-on muscle, cartilage, and liver tissues compared to the Sr0 group (p < 0.05) (

Table 3. Effects of dietary strontium levels on strontium enrichment in various tissues of juvenile hybrid sturgeon.

Index	The Amount of Strontium Added to the Feed (mg/kg)
	Sr0 (0 mg/kg)	Sr80 (80 mg/kg)	Sr160 (160 mg/kg)
Bone plates (mg/kg)	55.86 ± 6.05 c	85.31 ± 1.42 b	110.9 ± 4.59 a
Intestine (mg/kg)	0.39 ± 0.00 c	0.57 ± 0.01 b	0.64 ± 0.02 a
Blood (mg/kg)	0.12 ± 0.00 b	0.15 ± 0.00 a	0.15 ± 0.01 a
Heart (mg/kg)	0.12 ± 0.00 b	0.15 ± 0.01 a	0.15 ± 0.01 a
Muscle (mg/kg)	0.13 ± 0.01 b	0.24 ± 0.05 b	0.46 ± 0.08 a
Skin-on muscles (mg/kg)	2.32 ± 0.14 b	2.47 ± 0.25 ab	3.34 ± 0.28 a
Cartilage (mg/kg)	0.64 ± 0.02 b	0.80 ± 0.02 ab	0.99 ± 0.11 a
Liver (mg/kg)	0.19 ± 0.01 b	0.19 ± 0.00 b	0.28 ± 0.02 a

Notes: Values in the same row with different superscripts are significantly different (p < 0.05).

), while there was no significant difference between the Sr80 group and the Sr0 group (p > 0.05).

3.3. Principal Nutritional Components in Muscle

The crude fat content in the experimental groups was significantly lower than that in the control group (p < 0.05); however, no statistically significant differences were observed among the treatment groups (p > 0.05). Additionally, no significant differences were found in the crude protein, crude ash, or moisture content across all groups (p > 0.05) (

Table 4. Effect of dietary strontium levels on the basic nutrient composition in the muscle of juvenile hybrid sturgeon (wet weight, %).

Index	The Amount of Strontium Added to the Feed (mg/kg)
	Sr0 (0 mg/kg)	Sr80 (80 mg/kg)	Sr160 (160 mg/kg)
Crude fat	5.20 ± 0.46 a	3.93 ± 0.18 b	4.03 ± 0.20 b
Crude protein	17.16 ± 0.34	17.03 ± 0.24	17.30 ± 0.35
Crude ash	1.46 ± 0.08	1.40 ± 0.15	1.36 ± 0.14
Moisture content	76.46 ± 1.21	77.26 ± 0.63	77.46 ± 0.39

Notes: Values in the same row with different superscripts are significantly different (p < 0.05).

). The analysis of fatty acid content in muscle tissues demonstrated that the levels of myristic acid, linoleic acid, palmitic acid and γ-linolenic acid in the Sr80 and Sr160 groups were significantly lower than those in the control group (p < 0.05). Furthermore, the concentration of docosahexaenoic acid methyl ester in the Sr160 group was significantly higher than that in the Sr80 group (p < 0.05), although no statistically significant difference was observed when compared to the Sr0 group (p > 0.05). No significant differences were found among the groups with respect to the levels of pentadecanoic acid, heptadecanoic acid, stearic acid, etc. (p > 0.05) (

Table 5. Effect of dietary strontium level on muscle fatty acid composition in juvenile hybrid sturgeon.

Index	The Amount of Strontium Added to the Feed (mg/kg)
	Sr0 (0 mg/kg)	Sr80 (80 mg/kg)	Sr160 (160 mg/kg)
Myristic acid	0.050 ± 0.005 a	0.030 ± 0.003 b	0.031 ± 0.005 b
Linoleic acid	1.158 ± 0.028 a	0.921 ± 0.030 b	0.915 ± 0.107 b
Palmitic acid	1.066 ± 0.075 a	0.708 ± 0.064 b	0.777 ± 0.107 b
γ-linolenic acid	0.064 ± 0.004 a	0.042 ± 0.003 b	0.048 ± 0.006 b
Docosahexaenoic acid (DHA)	0.163 ± 0.024 ab	0.122 ± 0.017 b	0.250 ± 0.055 a
Arachidic acid	0.006 ± 0.001	0.003 ± 0.000	0.005 ± 0.003
Pentadecanoic acid	0.006 ± 0.002	0.005 ± 0.000	0.009 ± 0.004
Behenic acid	0.002 ± 0.001	0.001 ± 0.000	0.002 ± 0.001
Heptadecanoic acid	0.008 ± 0.001	0.007 ± 0.000	0.011 ± 0.004
Lignoceric acid	0.002 ± 0.000	0.001 ± 0.000	0.002 ± 0.001
Palmitoleic acid	0.102 ± 0.015	0.072 ± 0.008	0.076 ± 0.011
Oleic acid	1.376 ± 0.189	1.045 ± 0.127	1.004 ± 0.131
Stearic acid	0.261 ± 0.027	0.162 ± 0.014	0.237 ± 0.104
Eicosenoic acid	0.055 ± 0.005	0.036 ± 0.004	0.061 ± 0.031
Erucic acid	0.005 ± 0.001	0.004 ± 0.000	0.007 ± 0.003
Nervonic acid	0.008 ± 0.001	0.007 ± 0.001	0.007 ± 0.002
Alpha-linolenic acid	0.043 ± 0.002	0.058 ± 0.007	0.090 ± 0.041
Eicosadienoic acid	0.058 ± 0.002	0.036 ± 0.004	0.051 ± 0.021
Eicosatrienoic acid (n3)	0.008 ± 0.002	0.005 ± 0.000	0.008 ± 0.003
Eicosatrienoic acid (n6)	0.032 ± 0.007	0.028 ± 0.002	0.036 ± 0.013
Arachidonic acid (ARA)	0.054 ± 0.005	0.045 ± 0.006	0.051 ± 0.013
Eicosapentaenoic acid (EPA)	0.053 ± 0.005	0.034 ± 0.002	0.054 ± 0.020
Docosadienoic acid	0.002 ± 0.000	0.002 ± 0.000	0.003 ± 0.001

Notes: Values in the same row with different superscripts are significantly different (p < 0.05).

). The analysis of muscle amino acid content revealed that glutamate and valine levels were significantly higher in the Sr160 group compared to the control group (p < 0.05). In contrast, strontium supplementation did not significantly affect the concentrations of methionine, isoleucine, leucine, phenylalanine, lysine, histidine, arginine, aspartic acid, glycine, alanine, proline, serine, cystine, tyrosine, or threonine in the muscle tissue of juvenile hybrid sturgeon (p > 0.05) (

Table 6. Effect of dietary strontium levels on amino acid composition in the muscle of juvenile hybrid sturgeon (dry weight, %).

Index	The Amount of Strontium Added to the Feed (mg/kg)
	Sr0 (0 mg/kg)	Sr80 (80 mg/kg)	Sr160 (160 mg/kg)
Glutamic	1.65 ± 0.05 b	1.86 ± 0.08 ab	1.94 ± 0.01 a
Valine	0.66 ± 0.00 b	0.71 ± 0.04 ab	0.74 ± 0.02 a
Methionine	0.26 ± 0.08	0.33 ± 0.05	0.31 ± 0.05
Isoleucine	0.60 ± 0.01	0.65 ± 0.02	0.68 ± 0.01
Leucine	1.08 ± 0.02	1.16 ± 0.05	1.20 ± 0.01
Phenylalanine	0.56 ± 0.01	0.61 ± 0.02	0.62 ± 0.00
Lysine	1.28 ± 0.02	1.38 ± 0.06	1.43 ± 0.02
Histidine	0.44 ± 0.01	0.45 ± 0.02	0.46 ± 0.00
Arginine	0.80 ± 0.01	0.87 ± 0.04	0.89 ± 0.02
Aspartic acid	1.23 ± 0.02	1.33 ± 0.05	1.38 ± 0.01
Glycine	0.68 ± 0.00	0.76 ± 0.05	0.75 ± 0.03
Alanine	0.78 ± 0.01	0.85 ± 0.04	0.87 ± 0.01
Proline	0.50 ± 0.00	0.54 ± 0.03	0.54 ± 0.02
Serine	0.52 ± 0.01	0.55 ± 0.02	0.57 ± 0.00
Cysteine	0.06 ± 0.00	0.09 ± 0.00	0.08 ± 0.01
Tyrosine	0.37 ± 0.00	0.41 ± 0.02	0.40 ± 0.01
Threonine	0.26 ± 0.08	0.33 ± 0.05	0.31 ± 0.05

Notes: Values in the same row with different superscripts are significantly different (p < 0.05).

).

3.4. Results of Biochemical Analyses

The results of blood biochemical indices demonstrated that, compared with the control group, the plasma levels of triglycerides (TG), total cholesterol (TC), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in the Sr80 and Sr160 groups were significantly decreased (p < 0.05). No significant differences were observed between the Sr80 and Sr160 treatment groups (p > 0.05) (Figure 1). With regard to liver antioxidant indices, no significant differences were found in catalase (CAT) and superoxide dismutase (SOD) levels among the groups (p > 0.05) (Figure 2A,B). Compared with the control group, glutathione (GSH) content was significantly elevated in both the Sr80 and Sr160 groups (p < 0.05) (Figure 2C), with no significant difference between the two strontium treatment groups (p > 0.05). Additionally, malondialdehyde (MDA) content was significantly reduced in both Sr80 and Sr160 groups compared to the control group (p < 0.05) (Figure 2D), and the Sr160 group exhibited significantly lower MDA levels than the Sr80 group (p < 0.05). However, total antioxidant capacity (T-AOC) was significantly enhanced only in the Sr160 group (p < 0.05) (Figure 2E).

3.5. Histological Morphology and Ultrastructure Observation

HE and Oil Red O staining showed that the degree of vacuolization in liver cells was higher in the control group, and lipid deposition in the tissue was obvious. In contrast, the degree of vacuolization in liver cells and lipid deposition in the tissue in the Sr80 group and Sr160 group were reduced compared with the control group (Figure 3A). The lipid area in the unit tissue was measured and statistically analyzed. The results showed that lipid accumulation in the Sr80 group was reduced by 18% compared with the control group, and lipid accumulation in the Sr160 group was reduced by 27%. Overall, lipid accumulation in the liver of the strontium treatment groups was significantly lower than that in the control group (p < 0.05) (Figure 3B).

Ultrastructural examination of the liver revealed that, in the control group, the chromatin distribution within hepatocytes was relatively uniform, although mitochondrial swelling and vacuolation were evident. Within these mitochondria, some cristae appeared fragmented or dissolved, and the matrix exhibited signs of dissolution, presenting a flocculent appearance. Autophagic lysosomes were also observed within the cytoplasm (Figure 4A). In both the Sr80 and Sr160 groups, mitochondrial swelling was ameliorated to a certain extent. In most mitochondria, the cristae were densely and regularly arranged, and the matrix displayed a uniform electron density, appearing as a homogeneous gray color under the electron microscope (Figure 4B,C). Lipid droplets of varying sizes were present in the cytoplasm across all three experimental groups.

3.6. DEG Identification and Analysis

To investigate the effects of different concentrations of strontium in the diet on the liver transcriptome of hybrid sturgeon, nine Illumina HiSeq sequencing libraries were constructed, and the clean reads obtained from sequencing were assembled using Trinity. The quality assessment of the transcriptome sequencing data is presented in

Table 7. The descriptive statistics of the raw RNA-seq datasets for all of the nine individuals sequenced in this study.

Sample	Clean_Reads	Clean Base (bp)	Q20	Q30
Control-1	22,772,149	3,415,822,350	97.99	94.35
Control-2	21,712,526	3,256,878,900	98.13	94.77
Control-3	20,794,346	3,119,151,900	98.06	94.56
S80-1	21,201,051	3,180,157,650	98.07	94.61
S80-2	20,824,146	3,123,621,900	98.13	94.78
S80-3	23,285,119	3,492,767,850	97.82	93.96
S160-1	24,725,401	3,708,810,150	97.88	94.1
S160-2	23,419,993	3,512,998,950	97.63	93.49
S160-3	20,056,676	3,008,501,400	97.67	93.59

. The ranges of Q20 and Q30 for the clean reads were 97.63% to 98.13% and 93.49% to 94.78%, respectively. These results indicate that the sequencing data quality is reliable and can be used for subsequent bioinformatics-related analyses.

Transcriptomic analysis of the liver revealed 2092, 2335, and 1458 differentially expressed genes (DEGs) in the Sr80 vs. Control group, Sr160 vs. Control group, and Sr160 vs. Sr80 group, respectively (|log2fold change| > 2, p < 0.05). Among these, 963 DEGs were upregulated and 1129 were downregulated in the Sr80 group compared to the Control group (Figure 5A). In the Sr160 vs. Control comparison, 909 DEGs were upregulated and 1426 were downregulated (Figure 5B). Additionally, 583 DEGs were upregulated and 875 were downregulated in the Sr160 vs. Sr80 comparison (Figure 5C). The Venn diagram analysis indicated that three DEGs were significantly upregulated (Figure 5D) and one DEG was significantly downregulated (Figure 5E) across all three groups. GO functional enrichment analysis of differentially expressed genes in the liver revealed that, compared with the control group, the Sr80 group showed significant enrichment in Gene Ontology terms related to the regulation of endonuclease activity (GO:0004175), hydrolase activity (GO:0016787), catalytic activity (GO:0003824), monosaccharide transmembrane transporter activity (GO:0015145), rhamnose transmembrane transport activity (GO:0015153), and cell component movement (GO:0051270), among other areas (Figure 6A). In contrast, the Sr160 group exhibited significant enrichment in GO terms including sodium transport (GO:0006814), lipid transport (GO:0006869), mitochondrial outer membrane transport enzyme complex (GO:0005742), transcriptional co-inhibitory activity (GO:0003714), guanine ribonucleotide binding (GO:0032561), and extracellular matrix (GO:0031012), among other biological processes (Figure 6B).

The KEGG enrichment analysis of differentially expressed genes in the liver demonstrated that the Sr80 group was significantly enriched in pathways related to protein digestion and absorption (ko04974), DNA replication (ko03030), and the insulin signaling pathway (ko04910) when compared to the control group (Figure 6C). In the Sr160 group, metabolic pathways such as bile secretion (ko04976), phenylalanine metabolism (ko00360), calcium signaling pathway (ko04020), hematopoietic cell lineage (ko04640), and PPAR signaling pathway (ko03320) were significantly enriched (Figure 6D).

3.7. Analysis of Functional Gene Expression and qPCR Validation

The key genes involved in fatty acid metabolism were identified in the liver tissues of juvenile hybrid sturgeon using transcriptomic analysis. These included fatty acid transport genes (mttp, fabp1, slc27a1, slc27a4), lipid oxidation genes (cpt1a, acox1, atgl), lipid synthesis genes (acly, fasn, srebp1, dgat, scd1, acc), cholesterol metabolism genes (cyp7a1, apoa1, abcb11, hmgcr), and lipid peroxidation genes (gpx4, trfc, slc3a2) (

Table 8. Genes involved in lipid metabolism and lipid peroxidation in the liver.

Categories	Gene Name	log2FoldChange (Sr80 vs. Control)	p Value	log2FoldChange (Sr160 vs. Control)	p Value
Fatty acid transport	mttp	0.31193	0.21063	0.0047234	0.98712
	fabp1	0.038456	0.93093	−0.76649	0.21357
	slc27a1	0.31291	0.37459	0.23485	0.48278
	slc27a4	−0.2594	0.32448	−0.29672	0.3061
Lipid oxidation	cpt1a	1.5804	0.016786	0.72888	0.37006
	acox1	−0.20454	0.51732	−0.1773	0.5405
	atgl	0.038456	0.93093	0.48495	0.31546
Lipid synthesis	acly	0.1931	0.87024	0.8137	0.4841
	fasn	−1.6731	0.036733	−1.2299	0.10991
	srebp1	−0.20086	0.57096	0.080901	0.79149
	dgat	−0.04927	0.89429	0.31723	0.34817
	scd1	−0.012212	0.97168	−0.26931	0.43831
	acc	−0.76335	0.063297	−0.042024	0.90245
Cholesterol metabolism	cyp7a1	0.15241	0.78853	2.1688	0.018518
	apoa1	1.0466	0.0047229	0.77567	0.035034
	abcb11	0.67599	0.17399	−1.5662	0.0075855
	hmgcr	−0.11967	0.67545	−0.19356	0.52885
Lipid peroxidation	gpx4	0.095012	0.74914	0.29769	0.3401
	trfc	−1.0039	0.0046288	−0.98614	0.0058384
	slc3a2	0.42772	0.35209	1.1903	0.0088911

). Compared with the control group, the Sr80 group exhibited significantly downregulated expression of fasn, along with significantly upregulated expression of cpt1a and apoa1 (p < 0.05). In the Sr160 group, a significant upregulation of cyp7a1 and apoa1 expression was observed, accompanied by a significant downregulation of abcb11 expression (p < 0.05). All strontium-treated groups showed significantly increased expression of slc3a2 and significantly decreased expression of trfc (p < 0.05).

To validate the reliability of the transcriptome sequencing data, seven key genes were selected from the set of DEGs identified in the liver transcriptome for qPCR analysis. These expression values were then converted to log₂FoldChange values for correlation analysis with the corresponding RNA-Seq data. The results demonstrated a strong agreement between the qPCR and RNA-Seq datasets (p < 0.0001) (Figure 7), thereby confirming the accuracy and reliability of the RNA-Seq results.

4. Discussion

(a)

Growth Performance

In this study, adding 80 mg/kg and 160 mg/kg of strontium to the feed of juvenile hybrid sturgeon for eight weeks did not significantly affect their growth performance and survival rate. Studies have indicated that soaking Larimichthys crocea fry in water containing 18 mg/L of strontium for seven days did not significantly influence their average body length [30]. In contrast, the young chum salmon raised in water containing 10 mg/L of strontium for 30 days showed a significant increase in weight gain and specific growth rate [7]. This might be due to the relatively low concentration of strontium used and the short experimental period.

(b)

Physiological Performance

In the current study, the extent of strontium enrichment in major strontium-rich tissues of juvenile hybrid sturgeon was ranked as bone plate > skin muscle > cartilage > intestinal tract > muscle > liver, with particularly pronounced strontium concentration observed in the bone plate. Strontium is similar to calcium in terms of its physicochemical properties and is mainly deposited in the skeletal part of animals [31,32]. In a study involving white rabbits, the experimental subjects were continuously given deionized water containing 2 milligrams per liter of strontium for 30 days. The results showed that as the intake increased, the strontium content in the blood and bones gradually rose [33]. When zebrafish were exposed to strontium in water at concentrations of 0, 10, 50 and 250 mg/L for 14 days, strontium accumulation across tissues followed the order bone > liver > whole fish [34]. In this study, we investigated strontium accumulation in hybrid sturgeon through oral administration. It was hypothesized that strontium is primarily absorbed in the intestine after ingestion, subsequently entering the circulatory system and distributing throughout the body, which constitutes a key pathway for strontium enrichment in various tissues and organs.

The present study found that 80/160 mg/kg dietary strontium had no effect on hybrid sturgeon muscle crude ash, protein, or moisture, but reduced crude fat; it also increased muscle glutamic acid (a glutathione component aiding oxidative stress reduction) [35] and valine (a branched-chain amino acid protecting mitochondria) [36] while lowering myristic acid (a pro-obesity/inflammation saturated fatty acid) [37], palmitic acid (a lipid homeostasis disruptor) [38], and linoleic acid (n-6 PUFA) [39] and increasing docosahexaenoic acid methyl ester (n-3 PUFA) promoting fatty acid oxidation [40]. Previous studies showed that the daily administration of 5 mL strontium-containing (6 mmol/kg) drinking water to mice for 3 weeks reduced adipogenesis in mesenchymal stem cells [41], and the administration of 625 mg/kg strontium plus carboxymethylcellulose (Sr + CMC) (5 days/week) to mice post-ovariectomy and exercise for 8 weeks significantly decreased bone fat content [42]. This suggest that strontium improves sturgeon muscle nutrition and lipid homeostasis, and alleviates oxidative stress and inflammation via amino acid/fatty acid regulation.

In this study, we observed that the plasma levels of ALT, AST, TC, and TG in hybrid sturgeons fed with strontium-supplemented feed were significantly lower than those in the control group. Meanwhile, in this study, we also found that the strontium treatment group significantly increased liver GSH levels and reduced MDA levels. Biochemical blood indicators are important parameters that assess the physiological state of organisms [43]. As reported by McGill [44], the markers ALT and AST are released from the hepatocytes with increasing severity of the liver injury. TG is produced in the liver and is often used to assess the health status of bony fish. High levels of triglycerides can harm liver function [45]. TC levels reflect the equilibrium of cholesterol metabolism in the liver; increased TC levels are indicative of disturbances in hepatic cholesterol metabolism [46]. Therefore, ALT, AST, TG, and TC are considered essential biomarkers for evaluating liver function [47]. Lin et al. [48] reported that both 4 mg/L and 6 mg/L high-strontium mineral water-based culture media could promote the proliferation of LO2 human hepatocyte cell lines and reduce transaminase activity. Jiang et al. [7] further demonstrated that the presence of 1–3 mM strontium in the culture medium could significantly decrease TC and TG levels in human hepatocytes in a dose-dependent manner. Animal studies have also shown that providing mice with drinking water containing 5–20 mg/kg strontium can effectively reduce the TC level [49]. This suggests that strontium may reduce the body’s oxidative stress levels by inhibiting lipid peroxidation. These results indicate that dietary supplementation with lower concentrations of strontium not only regulates lipid metabolism but also maintains liver homeostasis and enhances liver health.

(c)

Hepatic Tissue Performance

Our findings indicate that dietary strontium supplementation significantly alleviates mitochondrial swelling and reduces hepatic lipid accumulation in hybrid sturgeon hepatocytes. The liver serves as a critical organ for lipid metabolism in fish. Previous studies have demonstrated that strontium can lower blood lipid levels, inhibit fat synthesis, and enhance antioxidant capacity in animals [50,51,52]. Studies have shown that lipid accumulation in hepatocytes can disrupt the normal structure of mitochondria [53]. Excessive lipid accumulation in the liver can lead to a significant increase in reactive oxygen species (ROS) [54], and excessive ROS can induce the opening of the mitochondrial permeability transition pore (mPTP), thereby causing mitochondrial swelling [55]. Mitochondria are key sites for lipid metabolism, not only participating in fatty acid β-oxidation and ketone body production, but also providing energy and raw materials for lipid synthesis and regulating cellular lipid metabolism signaling pathways [56,57]. Therefore, the occurrence of mitochondrial swelling in the liver will further lead to an imbalance in liver lipid metabolism. Thus, it is hypothesized that strontium may, through some mechanism, improve the swelling of liver cell mitochondria and thereby modulate liver lipid metabolism.

(d)

Functional Gene Expression

In this study, the expression of cyp7a1 (the gene encoding CYP7A1, the rate-limiting enzyme in the classical pathway of bile acid biosynthesis) in the liver of the Sr160 group was significantly upregulated. The liver, as the core organ regulating lipid metabolism, has the bile acid secretion pathway as the main route for cholesterol decomposition [58]. It is known that dietary calcium supplementation can affect bile acid secretion and thereby regulate the accumulation of fat in the liver [59], and since strontium can exert its functions through the calcium transport system, it may have a similar effect on this pathway. CYP7A1 is of great significance for maintaining bile acid homeostasis and regulating liver lipid accumulation [60], and previous studies have shown that upregulating cyp7a1 can promote cholesterol degradation and reduce lipid levels under high-fat-diet-induced metabolic disorders [61,62].

Meanwhile, supplementing strontium in the diet can downregulate the expression of fasn (the gene encoding fatty acid synthase, a key enzyme in fatty acid synthesis that promotes liver lipid accumulation) [63] in the liver, and upregulate the expressions of cpt1a (the gene encoding a subtype of carnitine palmitoyltransferase 1, a key enzyme in fatty acid oxidation that inhibits liver lipid accumulation) [64] and apoa1 (the gene encoding apolipoprotein A1) in relation to lipid transport, as its overexpression can reduce liver lipid levels [65]. This is consistent with the research results of culturing bovine liver cells with strontium solution [66], indicating that strontium can affect lipid metabolism and transport by regulating the expression of these genes. In addition, in this study, we found that strontium could also upregulate the expression of slc3a2 (the gene encoding a subunit of the transport protein that promotes glutathione synthesis) [67] and downregulate the expression of tfrc (the gene encoding a marker of lipid peroxidation-induced apoptosis and an iron transport receptor) [68]. Therefore, it is speculated that strontium may inhibit lipid peroxidation by promoting the synthesis of glutathione in the liver and reducing the accumulation of intracellular iron ions. The above results suggest that strontium plays an important role in the regulation of fish lipid metabolism and that its regulatory mechanism is relatively complex.

5. Conclusions

In this study, we applied 80 and 160 mg/kg dietary strontium to hybrid sturgeon for strontium enrichment, confirming oral administration as safe and effective. The addition of strontium to the feed has no effect on the growth of sturgeon, but it improves the fatty acid and amino acid composition in the muscle and reduces lipid accumulation in the liver by regulating related genes, enhancing antioxidant capacity. This provides support for the production of strontium-rich fish and helps to clarify our understanding of the physiological role of strontium.