Lipid is an essential nutrient for aquaculture animals and is an important component of tissue cells, it has the function of providing essential fatty acids, storing and supplying energy and saving protein. Fish oil (FO) is widely used in the aquafeed industry because of its high unsaturated fatty acids (HUFAs) content, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are essential fatty acids for mariculture animals. However, HUFAs are susceptible to oxygen, moisture, light and the metal in the feed additives and other factors, very easy to oxidation and decomposition into alcohols, ketones, aldehydes, and other harmful substances in the production, use and storage process (1). Ultimately, it has a serious impact on the health and quality of the animal. The lipid content in the feed of mariculture fish is generally 12–15% (2), and the farming objects are generally concentrated in subtropical or tropical areas such as high temperature and high humidity, which provides suitable environmental conditions for the oxidation of FO in feed. Therefore, the problem of FO oxidation in marine fish feed has seriously affected the sustainable development of the mariculture industry.

With the development of the world economy and the improvement of living standards, human beings are also demanding higher quality seafood, and the problem of deterioration of fish health and quality caused by feed rancidity has become one of the important problems that cannot be ignored in the aquaculture industry today. In recent years, there has been an increasing amount of research on oxidized lipids in feed related to oxidative stress. Huang and Huang (3) showed that ingestion of oxidized fish oil (OFO) feed reduced growth performance and increased malondialdehyde (MDA) content of liver in juvenile hybrid tilapia (Oreochromis niloticus × O. aureus); Zhang et al. (4) showed that OFO feed caused liver lipid deposition in loach (Misgurnus anguillicaudatus). These studies focused on the effects of OFO on growth performance, liver biochemical parameters, and antioxidant capacity (5). In addition, oxidation of FO may not only reduce the nutritional value of the feed itself, but may also adversely affect fish muscle quality (amino acid composition, fatty acid composition, vitamin E content, flavor, shelf life, etc.) (6). Previous studies have also shown that ingestion of OFO feeds causes increased MDA and decreased n−3 polyunsaturated fatty acids (PUFAs) content in the muscles of fish such Japanese sea bass (Lateolabrax japonicus) (7), Japanese flounder (Paralichthys olivaceus) (8), largemouth bass (Micropterus salmoides) (5), Atlantic cod (Gadus morhua) (9), which seriously affects fish muscle quality and nutritional value. However, there is a lack of systematic studies on the effect of OFO on the nutritional value and quality of fish muscle.

Hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ E. lanceolatu) has become one of the most important breeding species along the southeast coast of China due to its fast growth rate, tasty meat, high nutritional value, strong disease resistance, and adaptability to the environment (10). The annual production of grouper can reach about 200,000 tons in China, which is a high-quality source of protein. At present, research on pearl grouper is mainly focused on nutritional requirements and immunity (11), there are few reports about its muscle quality. Muscle is the primary part of human consumption, and its nutritional composition, flavor substance composition and quality are related to human health and consumption preferences. In the era of intensive development of aquaculture, preventing oxidation of FO in feed becomes challenging, and it remains to be explored whether the ingestion of OFO feed will have a negative impact on the muscle quality and nutritional value of grouper. Therefore, the study was conducted to compare the muscle nutrient composition, fatty acid composition, amino acid composition, flavor substance composition and flesh quality-related genes expression by feeding fresh fish oil (FFO) diet and OFO diet in different rearing periods using hybrid grouper as the research object. The results can provide basic data for regulating the decline of muscle quality caused by the consumption of OFO diet.

As the most important part of fish for human consumption, muscle nutritional composition is particularly important. In present investigation, ingestion time and type of FO did not affect grouper muscle crude lipid and crude protein. However, Yu et al. (13) showed that ingestion of OFO significantly decreased muscle crude lipid and crude protein content in Amur sturgeon (A. schrenckii), this is probably due to differences in fatty acid utilization by different fish or different experimental periods, etc. Consumption of OFO has been shown to cause elevated MDA levels in various tissues of fish (14). The MDA level in the muscle of OFO group was higher than that of FFO group in the current study, which was consistent with previous study (15). Although there was no significant difference about the other indicators between OFO group and FFO group, OFO significantly increased the TC, and TG content of grouper muscle compared with the initial value, all of which reflected the toxic impact of OFO. With the growth of grouper, TG were accumulated significantly in the muscle after OFO ingestion, which also implied a downward tendency of muscle quality of fish due to lipid deposition caused by OFO (16).

The main indispensable amino acids in grouper muscle are lysine, leucine and arginine, and the main non-essential amino acids are aspartic acid and glutamic acid, in a recent report published from our team, these amino acid levels were similar to the results of the present study (17). Indispensable amino acid/total amino acid (IAA/TAA) ratio is an important reference indicator for evaluating the nutritional value of aquatic protein, and it is generally considered that the ideal IAA/TAA ratio for high quality protein is about 0.4 (18), the ratio of IAA/TAA in grouper muscle in the present study was ~0.5, indicating that grouper muscle is a high-quality protein source. Methionine, phenylalanine, total indispensable amino acid, aspartic acid, serine, glutamic acid, total dispensable amino acid, and total amino acids of muscle in FFO group first decreased significantly and then increased to no significant difference from the initial, suggesting a self-regulatory ability of fish muscles. Proline content increased significantly with ingestion time, regardless of the type of feed consumed. In biological organisms, proline is not only an ideal osmoregulatory substance, but also serves as a protective substance for membranes and enzymes and a free radical scavenger, and provides the material basis for growth promotion, normal metabolism and life maintenance, therefore, proline may play an important role as a functional amino acid in regulating muscle growth and metabolism in grouper. Phenylalanine, as a sweet amino acid recognized by the Joint Committee of Experts of the World Health Organization (WHO) and the Food and Agriculture Organization (FAO), was significantly higher in the OFO group than in the FFO group after 60 days of ingestion, which to a certain extent indicates that the OFO changed the amino acid profile of muscle.

Fatty acid composition of fish tissues was influenced by fatty acid composition of ingested diets (19), the significant decrease in muscle SAFA and MUFA and the significant accumulation of PUFA with prolonged FFO ingestion in this study, further illustrates the importance of FFO as a recognized source of high-quality commercial lipids (20). The PUFA of OFO group was significantly lower than that of FFO group at 60 d, which may be attributed to the fact that part of the LC-PUFA in FO was oxidized to alcohols, aldehydes, acids, ketones, etc. (21), reduced the content of PUFA in FO and thus affected the accumulation of PUFA (especially EPA and DHA) in muscle, this is consistent with the results that OFO adversely affects the fillet quality of Amur sturgeon (A. schrenckii) (13).

In addition to muscle nutritional quality, sensory characteristics also determine the degree of consumer consumption of aquatic products, of which flavor is one of the important factors influencing the determination of sensory characteristics (22). The diversity of muscle volatile compounds decreased from 88 to 57 at 60 days on OFO diet, which reflects the destructive effect of OFO on muscle flavor diversity to a certain extent. In present study, aldehydes, esters, and aromatics were the main differential volatile compounds identified. Aldehydes with low threshold can be formed rapidly in lipid oxidation, so they contribute greatly to the formation of flavor substances in aquatic animals (23). Studies demonstrate that aldehydes are related to the grassy taste (23), fishy taste, and greasy taste of aquatic products (24) and account for a large proportion of the volatile substances. Esters compounds are formed by esterification of carboxylic acids and alcohols, and esters mostly give a fruity or floral flavor to foods (25). The aldehydes enriched in the present project were mainly non-anal, which as a saturated straight chain aldehyde mainly produced fishy and irritating odor and gave an unpleasant smell (24), and its content decreased significantly after ingestion of FFO diet and increased significantly after ingestion of OFO diets, and it was significantly higher in OFO group than that in FFO group at 60 days. While the ester content accumulated significantly in FFO group and was significantly higher than that in OFO group at 60 days. These differential substances may be derived from the interaction between the Maillard reaction (26) and lipid oxidation. In addition, compared with the FFO group, the level of Aromatics such as 2,4-Di-tert-butylphenol was significantly increased by the consumption of OFO diet. Some studies have suggested that the substances may be transferred to the fish by environmental pollutants and may produce negative effects on fish muscle (27), but in the present study, all conditions remained the same except for the difference in feed consumption, which, in our opinion, on the one hand, it may be the result of the toxic effect of MDA and peroxide in OFO; on the other hand, may be due to the low utilization of nutrients, especially lipid, caused by the oxidation of lipids in the body caused by the consumption of OFO, resulting in the excretion of nutrients that further polluted the aquaculture environment. Of course, only our speculation, the specific reasons still need further verification. These indicated that FO itself was a good source of lipids to enhance flavor, but being oxidized would make a considerable negative consequence on the flavor and quality of aquatic products, and even affect the sales of aquatic products.

A variety of molecular mechanisms regulate the physiological processes, degeneration, and quality of muscle growth (28). With specific aspects of muscle growth and catabolism dependent on nutritional condition, the expression of genes related to myogenic signaling pathways was frequently analyzed to determine the molecular response to various diets (29). MyoG, MyoD, Myf5, MRF4, MSTN 2, IGF1, IGF2, COL1A-1, and COL1A-2 are all myogenic regulatory factors (MRFs) growth-related factors, which are internal factors that control specification, activation, and differentiation of myogenic cells (30). MyoG plays an essential part in myoblast differentiation, MyoD and Myf5 are primarily myogenic determinants (31). The expressions of MyoG, MyoD, and Myf5 were up-regulated after ingesting two diets, the results indicated that OFO has no adverse effect on muscle differentiation and formation in grouper. However, the expression of MyoG and MyoD in the muscle of grouper consuming OFO diet for 60 days was significantly lower than that of the FFO group, this also illustrated the negative effect of OFO on grouper muscle differentiation. It also confirms that MyoG and MyoD levels always seem to correlate with nutritional status (32). Also, muscle is regulated by MSTN, which normally inhibits the growth of skeletal muscle (33). IGF1 is a key positive regulatory growth factor with abundant receptors in fish muscle that promotes cell activation, proliferation and differentiation (34); whereas, IGF2 is a negative regulator (35). In turn, COL1A-1 and COL1A-2 regulate the diameter and stiffness of muscle fibers (36). Although muscle IGF1, COL1A1, and COL1A2 were significantly upregulated after feed intake, IGF1, COL1A1 were significantly higher in OFO group than those in FFO group, and correspondingly, the expression of MSTN and IGF2 was downregulated. This might be due to the increase of MDA and TG content in the muscles of grouper feeding on OFO feed, and the deposition of lipid and harmful substances play a certain stimulating effect on the growth of muscles, showing negative feedback regulation. We speculate that the ingestion of OFO may lead to the inhibition of myotubular value-added and the reduction of fusion number in grouper muscles, and inhibit the proliferation and differentiation of myoblasts, but the by-products produced may play a certain negative feedback regulation on the growth of muscles, which may also be the reason why the ingestion of OFO fish can grow normally but with poor quality, but these speculations need to be further verified.

GPX, CAT, Hsp70, and Hsp90 are all antioxidant-related factors in the body. CAT could make a dynamic balance between the production and elimination of free radicals and reduce oxidative damage (37). GPX catalyzes the breakdown of peroxides, thereby protecting the structural and functional integrity of cell membranes (38). Hsp factors can modulate stress tolerance by preventing protein denaturation, refolding damaged proteins, or ensuring irreversible degradation of damaged proteins (39). OFO intake down-regulated the expression of muscle GPX, CAT, Hsp70, and Hsp90 compared with the initial values or those of FFO group at the same time points. The results implied that OFO caused damage to the antioxidant system of grouper muscles, this corresponded to the above results of elevated MDA and TC levels in grouper muscle caused by OFO.

To overview, ingestion time and diet lipid type both could affect the muscle quality of grouper, while the effect of diet lipid type is the main factor. OFO may increase the deposition of harmful substances and lipid in muscle through oxidative damage, thus affecting nutrient metabolism and reducing muscle nutritional quality, while oxidative decay of PUFA in FO is an important factor in muscle flavor deterioration, and the mechanism needs further investigation. The project is expected to provide reference data for the feed industrial use of FO.

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author/s.

The animal study was reviewed and approved by the Ethics Review Board of the Institutional Animal Care and Use Committee at Guangdong Ocean University in Zhanjiang, China.

XY: data analysis and manuscript writing. ZL: experimental implementation. XD: experimental design and manuscript revision. BT: experimental design. SP, TL, SL, WH, and XS: sample analysis. YY: data management. All authors contributed to the article and approved the submitted version.

This study was supported financially by the National Natural Science Foundation of China (NSFC31972808), the General Program of Natural Science Foundation of Guangdong Province (2021A1515011165), Department of Education of Guangdong Province (2021ZDZX4005), Postgraduate Education Innovation Project of Guangdong Ocean University (202139), Science and Technology Bureau of Zhanjiang (Grant No. 2020A03010 and 2020A05003), supported by China Agriculture Research System of MOF and MARA (CARS-47), and Research and Demonstration of Precision Functional Compound Feed Technology of Major Cultured Fishes and Shrimps in South China (2021B0202050002).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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