Abstract

   Objectives: The salinization of the water environment worldwide is
   increasing, which has brought great challenges to the sustainability of
   fish farming of aquatic animals. Methods: Three NaHCO[3] concentration
   groups (0 mmol/L, 20 mmol/L, and 60 mmol/L) were set up in this study
   to investigate growth and metabolic differences between diploid and
   triploid crucian carp under saline-alkaline stresses. Purpose: This
   study utilized UPLC-QTOF/MS metabolomics to analyze significant
   metabolites and metabolic pathways in the serum of diploid and triploid
   crucian carp, exposing them to different NaHCO[3] concentrations in
   saline-alkaline habitats, elucidating the mechanism of their metabolic
   differences. Results: Results revealed that in the CA20 group, diploid
   and triploid crucian carp shared 69 differential metabolites, primarily
   enriched in pathways such as sphingolipid metabolism,
   glycerophospholipid metabolism, and linoleic acid metabolism. In the
   CA60 group, 46 differentially metabolites (DMs) were identified, mainly
   enriched in pathways such as linoleic acid metabolism, unsaturated
   fatty acid biosynthesis and sphingolipid metabolism. Conclusions: The
   analysis indicated that under different carbonate-saline-alkaline
   concentrations, diploid and triploid crucian carp primarily enriched in
   metabolic pathways such as glycerophospholipid metabolism, sphingolipid
   metabolism, and unsaturated fatty acid biosynthesis. With increasing
   carbonate-alkaline concentrations, hemolytic phospholipids associated
   with cell apoptosis were significantly upregulated and sphingolipid
   metabolism related to inflammation was more significantly enriched in
   triploid crucian carp, indicating that triploid crucian carp exhibited
   significant sensitivity to high carbonate-saline-alkaline stress and
   poorer carbonate-saline-alkaline tolerance. The results of this study
   provided a scientific theoretical basis for the later cultivation and
   aquaculture research of saline-alkaline-tolerant fish species.

   Keywords: diploid crucian carp, triploid crucian carp, saline-alkaline
   stress, metabolites difference, serum metabolomics

1. Introduction

   With the influence of human activities and climate change, among other
   factors, saline waters are becoming more widely distributed globally,
   with saline areas occupying about one third of the total land area.
   However, as the global demand for aquatic products continues to
   increase, freshwater resources are decreasing [[34]1]. Therefore, the
   development and utilization of saline water resources for aquaculture
   has become a global challenge. China, a prominent freshwater
   aquaculture nation, faces formidable challenges in achieving
   sustainable development due to the limited potential for increased
   utilization of existing freshwater resources [[35]2]. Saline-alkaline
   areas constitute a crucial component of the world’s land resources,
   with China alone accounting for approximately 4.6 million hectares of
   low-lying saline-alkaline land [[36]3]. These saline-alkaline waters
   are extensively distributed in China’s inland regions, including the
   northwest, north, and northeast. Presently, saline-alkaline waters are
   classified into various types based on their chemical composition, with
   the carbonate type being the most extensive [[37]4]. Saline-alkaline
   environments are characterized by high salinity, elevated pH, and an
   imbalanced ratio of major ions, resulting in extremely low biological
   productivity [[38]5]. According to CNKI (China National Knowledge
   Infrastructure) and WOS (Web of Science) databases, the alkalinity of
   the majority of China’s saline-alkaline waters falls within the range
   of 8 to 30 mmol/L, and waters with alkalinity exceeding 15 mmol/L
   significantly impede the normal growth, development, and reproduction
   of fish, leading to prolonged states of biological inability to
   reproduce [[39]6]. Consequently, the breeding of saline-alkaline
   tolerant fish varieties and the establishment of suitable aquaculture
   models are essential for transforming the extensive saline-alkaline
   water resources into valuable assets. This approach not only
   facilitates the economic reuse of fisheries but also addresses the
   scarcity of freshwater resources in the aquaculture sector [[40]7]. It
   is also one of the key ways to efficiently utilize saline-alkaline
   waters.

   Crucian carp (Carassius auratus), recognized as a significant species
   in large-scale aquaculture, is a bony fish adaptable to both freshwater
   and saline-alkaline environments [[41]8]. Globally, the scale of
   crucian carp aquaculture has been growing steadily, with the current
   production exceeding 3.5 million tons annually, of which the annual
   production of crucian carp in China exceeds 2.8 million tons [[42]9].
   Studies indicate that as the concentration of saline-alkaline in water
   increases, the diversity of freshwater fish species decreases [[43]10].
   However, crucian carp exhibits relatively strong tolerance in
   high-concentration saline-alkaline waters compared to other
   saline-alkaline-tolerant species such as bighead carp (Luciobarbus
   capito), Darli Lake Waleh Yarrow fish (Leuciscus Waleckii), and naked
   carp (Kessler) from Qinghai Lake [[44]11]. Crucian carp, as a
   vertebrate, has evolved with multiple ploidy levels and the ability for
   both sexual and asexual reproduction [[45]12]. Among them, compared
   with diploid crucian carp, triploid crucian carp has excellent
   characteristics such as wide feeding habits, a fast growth rate, and a
   high body condition coefficient [[46]13]. Numerous publicly available
   reports detail the phenotypic and genetic breeding differences between
   triploid and diploid crucian carp. For instance, Cai [[47]14]
   investigated the differences in muscle volatile components between
   diploid and triploid crucian carp, Liu [[48]15] studied the biological
   traits of diploid and triploid crucian carp, and Li [[49]16] utilized
   transcriptomics to analyze the differences in growth, reproduction and
   immune-related genes between diploid and triploid crucian carp.
   However, research specifically focusing on the differences in their
   saline-alkaline-tolerance traits has not been publicly reported to
   date.

   In recent years, with the advancement of technology, techniques such as
   transcriptomics, proteomics, and metabolomics have found widespread
   application in the study of the stress response, ecotoxicology, and
   growth and development of aquatic organisms, becoming powerful tools
   for researching physiological changes and organism metabolism in
   aquatic life [[50]17,[51]18]. Transcription controls the translation
   expression of proteins, while protein activity reflects the organism’s
   fundamental physiological activities. Ultimately, the metabolic
   behavior at the cellular level can accurately reflect the terminal
   information of the organism [[52]19]. Metabolomics, being capable of
   both qualitative and quantitative analysis of small-molecule
   metabolites within an organism, provides a novel research approach for
   elucidating the metabolic characteristics and mechanisms of fish under
   saline-alkaline stress. It offers a scientific basis for unraveling the
   molecular mechanisms underlying specific biological processes.
   Currently, there is limited research on the metabolic differences of
   diploid and triploid crucian carp under saline-alkaline stress.
   Publicly available scientific reports regarding the differential
   analysis of saline-alkaline tolerance traits at the cellular level are
   lacking, constraining the development of superior traits in
   saline-alkaline-tolerant fish.

   Blood, as one of the crucial tissues in an animal’s body, plays vital
   physiological roles in energy transport, physiological regulation, and
   defense, and is closely associated with the animal’s growth and
   physiological state [[53]20]. When fish face metabolic disturbances due
   to external environmental stressors, the metabolites in their blood
   also undergo changes [[54]21]. In previous research conducted by our
   team using non-targeted metabolomics based on Ultra-performance liquid
   chromatography coupled with quadrupole time-of-flight mass spectrometry
   (UPLC-QTOF/MS), plasma and gills of the squarehead catfish and bighead
   carp were studied as target tissues and organs under NaHCO[3]
   saline-alkaline stress. This research revealed widespread changes in
   the plasma metabolome under saline-alkaline stress, with cluster
   analysis indicating alterations in key metabolic pathways related to
   amino acid synthesis and metabolism, arachidonic acid metabolism, and
   ketone body metabolism [[55]22,[56]23,[57]24]. However, there is
   currently limited research on the metabolic differences in the serum of
   diploid and triploid crucian carp under carbonate-alkaline stress.
   Hence, this study employed non-targeted metabolomics technology based
   on UPLC-QTOF/MS to analyze significant metabolic differences in the
   serum of diploid and triploid crucian carp under different NaHCO[3]
   concentrations (0 mmol/L, 20 mmol/L, 60 mmol/L) during saline-alkaline
   stress. This research aimed to further elucidate the metabolic
   differences at the cellular metabolism level in diploid and triploid
   crucian carp under NaHCO[3] stress, providing scientific theoretical
   support for the cultivation of superior saline-alkaline-tolerant fish
   varieties.

2. Materials and Methods

2.1. Experimental Materials

   Sodium bicarbonate (NaHCO[3], analytical grade, Tianjin Chemical No.3
   Factory Co, Tianjin, China); Tricaine (MS-222, St. Louis, MO, USA);
   liquid nitrogen (Harbin Liming Gas Co., Ltd., Harbin, China); methanol
   (mass spectrometry grade, Merck & Co, Darmstadt, Germany);
   acetonitrile, formic acid (mass spectrometry grade, Merck & Co,
   Darmstadt, Germany); ammonia solution (mass spectrometry grade, Thermo
   Fisher Scientific, Pasadena, CA, USA).

2.2. Experimental Subjects

   This study was conducted in strict accordance with Heilongjiang River
   Fisheries Research Institute of CAFS Application for Laboratory Animal
   Welfare and Ethical review ([58]Figure S4). The crucian carp used in
   this experiment were procured from the Heilongjiang Fisheries Research
   Institute of the Chinese Academy of Fishery Sciences at the Harbin
   Experimental Station. Individuals with robust health, free from
   diseases or injuries, weighing (130.67 ± 7.19) grams, and measuring
   (17.83 ± 1.28) centimeters were selected. Each fish’s ploidy was
   determined using a flow cytometer. Based on the identification results,
   the fish were categorized into diploid crucian carp (2n with a cell
   chromosome count of 100) and triploid crucian carp (3n with a cell
   chromosome count of 150). The fish were separated into eight indoor
   recirculating aquaculture systems (dimensions: 100 cm × 45 cm × 50 cm)
   to start to acclimate. The experimental water was sourced from tap
   water aerated for 72 h. The water temperature was maintained at 24 °C
   to 26 °C. Freshwater control pH was about 7.1, ammonia <1.0 mg/L, and
   dissolved oxygen > 7.5 mg/L. A 20 mmol/L NaHCO[3] stress group with a
   pH of about 8.4, ammonia < 1.0 mg/L, and dissolved oxygen > 7.5 mg/L
   was established. Finally, a 60 mmol/L NaHCO[3] stress group with a pH
   of about 9.6, ammonia < 1.0 mg/L, and dissolved oxygen > 7.5 mg/L was
   established. The photoperiod was set at 12 to 14 h daily. During the
   acclimatization period, the fish were fed in a prescribed manner at
   scheduled intervals for two weeks.

2.3. Experimental Methods and Sample Collection

   For this experiment, a total of 360 healthy individuals, including both
   2n and 3n, were selected. The experiment comprised two freshwater
   control groups (Con-2n, Con-3n) and four NaHCO[3] stress groups: 20
   mmol/L (CA20-2n, CA20-3n), and 60 mmol/L (CA60-2n, CA60-3n). Each group
   had three replicates, with each replicate containing 20 individuals. To
   prevent acute stress reactions in 2n and 3n, the exposure
   concentrations were gradually increased at a rate of 5 mmol/L per day
   to reach the required concentration after four days, and then the
   exposure experiment was started for 30 days. During the experiment,
   water was changed every 10 days, with the water replacement volume
   being one-third of the tank volume. Sodium bicarbonate was
   appropriately replenished to maintain the experimental concentrations.
   Water quality parameters and NaHCO[3] concentrations were monitored
   throughout the exposure experiment to ensure water stability.

   Feeding was halted 24 h before sampling. Fifteen fish were randomly
   selected from each group and immersed in an anesthetic solution
   (MS-222) with a concentration of 40 mg/L. After deep anesthesia, 2 mL
   of blood was collected from the caudal vein. Subsequently, the blood
   samples were left to stand in a refrigerator at 4 °C for 12 h, followed
   by centrifugation (4 °C, 3500 r/min) for 10 min. The obtained serum was
   stored at −80 °C in an ultra-low temperature freezer for further
   analysis.

2.4. Metabolite Extraction

   Frozen serum samples stored at −80 °C (100 μL) were thawed at 4 °C. To
   each sample, 400 μL of prechilled 80% methanol-water solution (4 °C)
   was added. The mixture was vortexed for 60 s, subjected to
   ultrasonication in an ice bath for 10 min, and then left to stand at
   −20 °C for 30 min. Subsequently, the samples were centrifuged at 13,000
   r/min for 10 min at 4 °C, and 300 μL of the supernatant was filtered
   through a 0.22 μm organic membrane into the injection bottle for
   subsequent analysis. Simultaneously, for each serum sample, 50 μL was
   taken and mixed uniformly to prepare quality control (QC) samples using
   the aforementioned method [[59]25]. These QC samples were utilized for
   assessing sample stability and instrument reliability.

2.5. UPLC-QTOF-MS Analysis

   Column: BEH C18 column (50 mm × 2.1 mm, 1.7 μm; Waters, Milford, MA,
   USA); Column Temperature: 40 °C; Sample Injector Temperature: 4 °C;
   Injection Volume: 10 μL; Flow Rate: 0.30 mL/min; Positive Ion Scan:
   Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic
   acid in acetonitrile; Negative Ion Scan: Mobile phase A: 0.1% ammonia
   solution in water; Mobile phase B: 0.1% ammonia solution in
   acetonitrile; Gradient Elution Program: 0 to 4 min, 5% to 70% B; 4 to
   11 min, 70% to 85% B; 11 to 12 min, 85% to 100% B; 12 to 13 min, 100%
   B; 13 to 13.2 min, 100% to 5% B; 13.2 to 15 min, 5% B.

   Ion Source: Electrospray ionization source (ESI); Ion Source Voltage:
   Positive mode: 5500 V, Negative mode: −4500 V; Ion Source Temperature:
   550 °C; Collision Voltage: Positive mode: 80 V, Negative mode: −80 V;
   Collision Energy: Positive mode: 35 eV, Negative mode: −35 eV;
   Collision Energy Spread: Positive mode: 15 eV, Negative mode: −15 eV;
   Nebulizing Gas (Gas1) Pressure: 55 PSI, Auxiliary Gas (Gas2) Pressure:
   55 PSI; Curtain Gas (Cur) Pressure: 35 PSI; Dynamic Background Subtract
   (DBS) function enabled; Information-Dependent Acquisition (IDA)
   triggered, with the top 8 peaks exceeding 100 cps subjected to
   second-level mass spectrometry scan; First-level mass spectrometry ion
   scan range: 100–1200 Da; Second-level mass spectrometry ion scan range:
   50–1000 Da.

2.6. Data Processing

   The raw chromatographic peaks obtained from UPLC-QTOF/MS were imported
   into Progenesis QI 2.1 software (Waters Corporation, Milford, MA, USA)
   software for data preprocessing, resulting in a two-dimensional data
   matrix containing information such as mass-to-charge ratio, retention
   time, sample names, and peak areas. Subsequently, the generated
   two-dimensional data matrix was imported into SIMCA 14.0 software for
   Principal Component Analysis (PCA) and Orthogonal Partial Least Squares
   Discrimination Analysis (OPLS-DA) on each group’s data. This analysis
   revealed the discrete trends of metabolites between the control and
   treatment groups. Differential metabolites (DMs) were selected based on
   criteria such as Variable Importance in Projection (VIP > 1) and T-test
   analysis (p < 0.05). Further identification and classification of all
   DMs were conducted using the HMDB database. Finally, the selected DMs
   were imported into MetaboAnalyst 5.0 for metabolic pathway enrichment
   analysis.

3. Results

3.1. Metabolic Profiles

   The obtained data underwent PCA and OPLS-DA model analysis. PCA, an
   unsupervised classification model, is used to continuously reduce
   multidimensional data to several principal components that describe the
   characteristics of the original data. It is primarily employed to
   observe the overall distribution of samples. As shown in [60]Figure 1,
   positive ([61]Figure 1A) and negative ([62]Figure 1B) ion modes
   exhibited a certain separation trend between 2n and 3n under different
   NaHCO[3] concentration stress. Additionally, the distribution of QC
   samples showed good consistency, indicating that the instrument is in a
   stable state, and the obtained data are reliable. OPLS-DA, a supervised
   classification model, emphasizes differences between groups by
   maximizing the contrast between groups. The OPLS-DA score plots in
   [63]Figure 2 for positive ions ([64]Figure 2A,C,E) and negative ions
   ([65]Figure 2B,D,F) showed that samples of 2n and 3n are completely
   distributed in different regions, indicating that different NaHCO[3]
   concentrations have distinct effects on the serum of 2n and 3n.

Figure 1.

   [66]Figure 1
   [67]Open in a new tab

   The PCA scores plots of 2n and 3n in different NaHCO[3] concentration
   groups in positive ion modes (A); The PCA scores plots of 2n and 3n in
   different NaHCO[3] concentration groups in negative ion modes (B); Con
   is freshwater control group, CA20 is the 20 mmol/L NaHCO[3] exposure
   group, CA60 is the 60 mmol/L NaHCO[3] exposure group, 2n is diploid
   crucian carp, 3n is triploid crucian carp.

Figure 2.

   [68]Figure 2
   [69]Open in a new tab

   The OPLS-DA score plots of 2n and 3n in Con in positive ion modes (A),
   The OPLS-DA score plots of 2n and 3n in Con in negative ion modes (B);
   The OPLS-DA score plots of 2n and 3n in CA20 concentration in positive
   ion modes (C), The OPLS-DA score plots of 2n and 3n in CA20
   concentration in negative ion modes (D); The OPLS-DA score plots of 2n
   and 3n in CA60 concentration in positive ion modes (E), The OPLS-DA
   score plots of 2n and 3n in CA60 concentration in negative ion modes
   (F).

3.2. Selection of Differentially Expressed Metabolites and Metabolic Pathway
Analysis

   Differentially metabolites (DMs) were screened based on criteria of VIP
   > 1 and p < 0.05, resulting in the identification of 119 DMs.
   Subsequently, cluster analysis was performed, as shown in [70]Figure 3.
   The identified DMs were further validated using the HMDB database. In
   the Con-2n and Con-3n groups, 113 DMs were identified, while 69 DMs
   were common to the CA20-2n and CA20-3n groups, and 46 DMs were common
   to the CA60-2n and CA60-3n groups. These DMs belonged to various
   metabolite types, including glycerophospholipids, sphingolipids, purine
   nucleotides and derivatives, fatty acids, fatty acid amides, bile
   acids, inorganic phosphates, amino acids, and aromatic compounds, among
   others. To further elucidate the metabolic behavior differences between
   2n and 3n under saline-alkaline stress, the identified DMs were
   subjected to metabolic pathway enrichment analysis using MetaboAnalyst
   5.0 software, in which the most affected pathways were highlighted in
   red. The results indicated that DMs were mainly enriched in 15
   metabolic pathways, including linoleic acid metabolism,
   glycerophospholipid metabolism, purine metabolism, sphingolipid
   metabolism, unsaturated fatty acid biosynthesis, ether lipid
   metabolism, GPI-anchor biosynthesis, α-linolenic acid metabolism,
   glycerolipid metabolism, inositol phosphate metabolism, arachidonic
   acid metabolism, fatty acid degradation, primary bile acid
   biosynthesis, fatty acid elongation, and fatty acid biosynthesis.
   Specifically, in both CA20 and CA60 groups, 2n and 3n exhibited
   significant correlations with three metabolic pathways:
   glycerophospholipid metabolism, unsaturated fatty acid biosynthesis,
   and sphingolipid metabolism ([71]Figure 4).

Figure 3.

   [72]Figure 3
   [73]Open in a new tab

   Cluster heatmap of DMs.

Figure 4.

   [74]Figure 4
   [75]Open in a new tab

   The bubble diagram of metabolic pathways.

4. Discussion

   Saline-alkaline water resources are widely distributed in the land
   water system, and the rational development and utilization of saline
   water resources can not only effectively alleviate the problem of the
   serious shortage of freshwater resources but also promote the
   sustainable development of saline-alkaline aquaculture [[76]26].
   Previous studies [[77]27] have indicated that in freshwater aquaculture
   environments, triploid (3n) individuals exhibit superior traits such as
   faster growth and stronger stress resistance compared to diploid (2n)
   individuals. However, the differences in stress resistance and growth
   between 2n and 3n in saline-alkaline water environments are not well
   understood. In this context, unraveling the metabolic differences at
   the cellular level between 2n and 3n in saline-alkaline environment was
   crucial. It held important implications for furthering precise targeted
   metabolic regulation, enhancing the efficiency of aquaculture in
   saline-alkaline water areas, and was considered an effective approach
   for the efficient utilization of saline-alkaline water resources. To
   elucidate the metabolic behavioral differences of 2n and 3n under
   saline-alkaline stress, this study conducted exposure experiments in an
   indoor NaHCO[3] environment, focusing on metabolic profiling of the
   serum from 2n and 3n individuals. The results demonstrated that
   exposure to NaHCO[3] at the same concentration significantly disrupted
   metabolic pathways in the serum, including glycerophospholipid
   metabolism, sphingolipid metabolism, and the biosynthesis of
   unsaturated fatty acids. This finding suggested that the stress
   response in terms of metabolic changes was conserved across both 2n and
   3n individuals under saline-alkaline exposure. Understanding these
   metabolic alterations provides valuable insights for targeted metabolic
   regulation, contributing to improved aquaculture efficiency in
   saline-alkaline water environments. This study serves as a foundational
   step towards the efficient utilization of saline-alkaline water
   resources.

4.1. Glycerophospholipid Metabolism Pathway

   The metabolomic results indicated that exposure to
   carbonate-saline-alkaline environments disrupts glycerophospholipid
   metabolism in the serum of both 2n and 3n individuals, leading to a
   destabilization of the serum. Lipids play a crucial role as a source of
   energy for organisms and contribute significantly to various
   physiological functions, including metabolism [[78]28], protein
   recognition on cell membranes, and signal transduction [[79]29].
   Glycerophospholipids, such as phosphatidylcholine (PC),
   phosphatidylethanolamine (PE), and phosphatidic acid (PA), are
   essential components of cell membranes. Changes in their concentrations
   directly affect the composition and permeability of cell membranes,
   thereby influencing cellular processes like apoptosis [[80]30], immune
   response [[81]31], and inflammation [[82]32]. PA [[83]33], in
   particular, serves as a vital mediator in various lipid metabolic
   pathways. PC [[84]34] and PE [[85]35], major constituents of biological
   membranes, account for approximately 75% of the total phospholipid
   content in cells. They are essential to maintaining cell integrity and
   storing energy [[86]36]. Additionally, they serve as substrates for
   generating various lysophospholipids, including lysophosphatidylcholine
   (LysoPC), lysophosphatidylethanolamine (LysoPE), and lysophosphatidic
   acid (LysoPA). The production of Lysophospholipids can lead to cell
   membrane damage or cell death [[87]37].

   Under external environmental stress, excess reactive oxygen species
   (ROS) bind to unsaturated fatty acids and cholesterol on cell
   membranes, causing lipid peroxidation, leading to reduced cell membrane
   fluidity and altered cellular function, which can cause irreversible
   damage to the organism [[88]38]. The identified glycerophosphocholine
   (GPC) in this study, upregulated significantly in 3n but not
   significantly in 2n at CA20 concentration, indicated that 2n exhibits a
   favorable adaptive response to low carbonate
   saline-alkaline-concentration stress compared to 3n. However, both
   showed a downregulation trend in CA60, with 3n exhibiting a
   significantly higher degree of downregulation than 2n. This suggested
   that with increasing saline-alkaline concentration, the organism’s
   stress response surpassed its tolerance, exacerbating damage and
   compromising its ability to maintain GPC balance.

   Furthermore, in CA20 and CA60 concentrations, there was a significant
   downregulation trend of Lysophospholipids (LysoPC(22:6(4Z, 7Z, 10Z,
   13Z, 16Z, 19Z)/0:0), LysoPC (16:0/0:0), LysoPC (18:1(9Z)/0:0), LysoPA
   (18:1(9Z)/0:0), and LysoPE (18:0/0:0)) in 3n, while no significant
   changes were observed in 2n. These results collectively suggested that
   under high carbonate saline-alkaline stress, the rate of lipid
   peroxidation was faster in 3n than in 2n. The increase in phospholipids
   in 2n played a crucial role in maintaining cell integrity, indicating
   that 2n had the capability to ensure the normal physiological function
   of PC and PE substances. Simultaneously, at CA20 and CA60
   concentrations, Lysophospholipids were significantly upregulated in 3n,
   indicating more severe destruction of the double-layer structure of the
   lipid membrane, higher levels of cell death, and subsequent
   inflammation. This reflected 3n’s poorer performance in saline-alkaline
   stress tolerance compared to 2n.

4.2. Sphingolipid Metabolism Pathway

   Sphingolipids, as structural molecules in biological membranes
   [[89]39], play a crucial role in maintaining the barrier function of
   cells and the fluidity of cell membranes. They are involved in
   regulating essential signal transduction processes, influencing cell
   growth, aging, and programmed cell death [[90]40]. Sphingomyelins (SM),
   the most abundant type of sphingolipids, are essential lipids in cell
   membranes and plasma lipoproteins. They play a vital role in regulating
   various cell processes’ biological activities, such as proliferation,
   differentiation, apoptosis induction, and inflammation. SM is
   hydrolyzed by sphingomyelinase to produce a crucial product, ceramide
   (Cer). Cer, acting as a second messenger, can activate downstream
   effector molecules and further metabolize into sphingosine (Sph) and
   free fatty acids through ceramidase [[91]41]. Studies have shown that
   Cer and Sph can effectively inhibit cell growth and induce apoptosis
   [[92]42]. Palmitic acid (PA), the most abundant free fatty acid in the
   blood, is a major inducer of lipotoxicity. Research has indicated that
   PA can significantly induce cell damage, and the severity of cell
   apoptosis increases with its concentration [[93]43].

   In this study, at CA20 and CA60 concentrations, the content of SM
   [d18:1/18:1 (9Z)] was downregulated in both 2n and 3n, while SM
   [d18:1/24:1 (15Z)], PA, and Sph content were upregulated in both 2n and
   3n. However, the changes in these DEMs were significantly higher in 3n
   than in 2n. This indicated that carbonate saline-alkaline stress
   disrupts sphingolipid metabolism in the crucian carp organism,
   specifically leading to the disturbance of sphingolipid metabolism
   related to inflammatory responses. The degree of inflammation response
   in 3n is significantly higher than in 2n, resulting in severe lipid
   metabolism disruption and inducing cell apoptosis.

   Simultaneously, crucian carp produced a large amount of sphingomyelin
   through sphingolipid metabolism to resist damage caused by
   saline-alkaline environments. This finding is consistent with the
   metabolic study of crucian carp gills under saline-alkaline stress
   conditions [[94]44].

4.3. Biosynthesis Metabolic Pathway of Polyunsaturated Fatty Acids

   Unsaturated fatty acids can be classified into monounsaturated fatty
   acids and polyunsaturated fatty acids (PUFAs) based on the number of
   double bonds. Studies have shown that monounsaturated fatty acids such
   as oleic acid (OA) have a preventive and protective effect on lipotoxic
   metabolic abnormalities caused by saturated fatty acids [[95]45].
   PUFAs, as unique bioactive substances, play a crucial role in
   antioxidant [[96]46], anti-inflammatory [[97]47], and immune regulation
   [[98]48]. According to the position and function of double bonds, PUFAs
   can be further divided into ω-3, ω-6, ω-9, etc. [[99]49]. In PUFA
   molecules, the first unsaturation appears at the third position from
   the methyl end of the carbon chain, called ω-3 PUFA, mainly including
   alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and
   docosahexaenoic acid (DHA). Research has shown that ω-3 PUFAs can
   improve inflammatory responses. When the repair function of the body is
   stronger than the occurrence of inflammation, the body can maintain
   normal physiological activities. Conversely, when the repair function
   cannot compensate for inflammation damage, the body tends to
   physiological imbalance, and prolonged imbalance can lead to
   irreversible physical damage or even death. DHA plays an important role
   in enhancing the body’s immune system [[100]50].

   In the PUFA molecule, ω-6 PUFA is typically characterized by its
   farthest double bond from the carboxyl group on the penultimate sixth
   carbon atom, mainly including linoleic acid, gamma-linolenic acid, and
   arachidonic acid (AA), among others [[101]51]. Linoleic acid, as an
   essential fatty acid in the body, is converted into gamma-linolenic
   acid under the action of the rate-limiting enzyme Δ6 desaturase, which
   is finally upgraded to dihomo-γ-linolenic acid, and then generates
   arachidonic acid by the successive action of an elongase and a D5
   desaturase [[102]52]. Arachidonic acid, as an inflammatory signaling
   factor in the biosynthesis metabolic pathway of unsaturated fatty
   acids, can improve oxidative stress reactions and regulate lipid
   metabolism [[103]53]. Some studies have shown that ω-3 PUFAs and ω-6
   PUFAs have a competitive inhibitory relationship. Therefore, an
   imbalance in the ω-6/ω-3 PUFAs ratio in the body can cause confusion in
   various physiological functions and aggravate lipid metabolism
   disorders [[104]54]. Moreover, an excessively high content of ω-3 PUFAs
   can inhibit immune function and increase lipid oxidation [[105]55].

   The results of the screened DEMs in this study showed that, in the CA20
   and CA60 groups, the content of dihomo-γ-linolenic acid (DGLA) in 3n
   serum was upregulated, while it was downregulated in 2n. This suggested
   that oxidative stress reactions were more severe in 3n than in 2n under
   saline-alkaline stress, leading to the disturbance of biosynthesis
   metabolic pathways of unsaturated fatty acids involving DHA and
   triggering inflammation and cell apoptosis in crucian carp cells. At
   the same time, in the CA20 and CA60 groups, the significantly altered
   metabolites dihydroxyacetone phosphate (DHAP) and oleic acid (OA) in 3n
   were significantly upregulated, while they were significantly
   downregulated in 2n. This indicated that under saline-alkaline stress,
   the increase in the content of DHA and OA in 3n was excessive,
   inhibiting the body’s immune function and causing an imbalance in the
   body’s immune and antioxidant systems, resulting in aggravated lipid
   metabolism disorders. Therefore, under carbonate saline-alkaline
   stress, the inflammatory symptoms of 3n were significantly more severe
   than those of 2n.

5. Conclusions

   By employing non-targeted metabolomics using UPLC-QTOF/MS, we conducted
   a comprehensive analysis of the serum metabolome of 2n and 3n crucian
   carp under saline-alkaline stress. The results revealed significant
   alterations in various compound classes, including
   glycerophospholipids, sphingolipids, purine nucleotides and
   derivatives, and amino acids, under stress conditions. Further
   statistical analysis indicated that the differentially expressed
   metabolites were primarily associated with glycerophospholipid
   metabolism, sphingolipid metabolism, and the biosynthesis of
   unsaturated fatty acids. Specifically, the disturbance in serum
   sphingolipid metabolism suggested that both 2n and 3n experienced
   toxicity under NaHCO[3] stress, with 3n displaying greater sensitivity
   to saline-alkaline stress, evidenced by its weaker capacity to regulate
   oxidative stress and inflammatory responses, resulting in reduced
   tolerance to external saline-alkaline environments. Conversely,
   pathways related to glycerophospholipid metabolism and the biosynthesis
   of unsaturated fatty acids appeared to mitigate the organismal damage
   caused by saline-alkaline stress. Consequently, in saline-alkaline
   environments, 2n was deemed more suitable for aquaculture activities
   compared to 3n.

   This study provides a preliminary insight into the metabolic
   differences between 2n and 3n crucian carp at the cellular level under
   saline-alkaline stress, offering a theoretical foundation for
   understanding the regulatory mechanisms of 2n and 3n crucian carp in
   adapting to saline-alkaline environments.

Supplementary Materials

   The following supporting information can be downloaded at:
   [106]https://www.mdpi.com/article/10.3390/metabo14120721/s1, Table S1:
   The data matrix of positive ion model for PCA and OPLS-DA analysis.
   Table S2: The identified significantly different metabolites between
   diploid crucian carp and triploid crucian carp. Figure S1:
   Cross-validation plot of 2n-20 VS 2n-60 blood samples. Figure S2:
   Cross-validation plot of 3n-20 VS 3n-60 blood samples. Figure S3: Map
   of saline-alkaline areas in China. Figure S4: Heilongjiang River
   Fisheries Research Institute of CAFS Application for Laboratory Animal
   Welfare and Ethical review.
   [107]metabolites-14-00721-s001.zip^ (6.2MB, zip)

Author Contributions

   Conceptualization, Y.S.; methodology, X.W. and F.Y.; software, F.Y.;
   validation, F.Y., X.W. and J.W.; formal analysis, J.W.; investigation,
   X.J. and D.L.; resources, Y.S.; data curation, X.W.; writing—original
   draft preparation, F.Y. and X.W.; writing—review and editing, Y.S.;
   visualization, F.Y.; supervision, J.W.; project administration, Y.S.;
   funding acquisition, Y.S. All authors have read and agreed to the
   published version of the manuscript.

Institutional Review Board Statement

   This study was conducted strictly according to the ethical principles
   of laboratory animal welfare in China and approved by the committee of
   Heilongjiang Fisheries Research Institute.

Informed Consent Statement

   Not applicable.

Data Availability Statement

   The data presented in this study are available on request from the
   corresponding author due to privacy.

Conflicts of Interest

   The authors declare no conflict of interest.

Funding Statement

   This work was financially supported by the Natural Science Foundation
   of Heilongjiang Province for Distinguished Young Scholars (JQ2023C007),
   Central Public-interest Scientific Institution Basal Research Fund.
   HRFRI (HSY202304M), Sub project of National Agricultural Biological
   Breeding Major Project (2023ZD04065), and China Postdoctoral Science
   Foundation funded project (2023T160720, 2020M681124), Postdoctoral
   Science Foundation of Heilongjiang Province of China (LBH-Z20099).

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References