The hot water extract of Chlorella vulgaris (CVE) is a nucleotide-peptide complex enriched with amino acids, peptides, vitamins, minerals, nucleic acids, and carbohydrates, which not only strengthens the immune system and improves antioxidant activity but also promotes cell growth and enhances the regenerative capacity of normal cells (Merchant and Andre, 2001; Kang et al., 2013; An et al., 2016; Yamaguchi, 1996; Kumar et al., 2020; Kumar et al., 2020). Chlorella (Chlorella sp.) is a genus of single-cell green algae characterized by relatively easy cultivation, high productivity, and high protein, chlorophyll, lutein, and other essential micronutrients (Buono et al., 2014; Jeon et al., 2012; Choi et al., 2021; Hao et al., 2021). Various Chlorella-based products, including liquid, powdered, and fermented Chlorella and Chlorella extracts, are being introduced as feed additives (An et al., 2016). In recent years, more research has been conducted on powder of Chlorilla as a feed additive (Ma and Hu, 2024) and less on CVE as a feed additive. As a feed additive, CVE has been found to reduce vertebrate plasma peroxidation and increase antioxidant enzyme activity (Hu et al., 2007; Vijayavel et al., 2007), but it has not been extensively studied in aquatic animals. The cell wall of Chlorella vulgaris may limit the entry of digestive enzymes into the cell for appropriate digestion and absorption of components (Nemcova and Kalina, 2000). Therefore, CVE is important as a feed additive in aquatic animals.

Litopenaeus vannamei is one of the most important shrimp species cultured worldwide. Owing to its high nutritional value, fast growth rate, and strong adaptability to the environment, it has high economic value and is widely cultivated worldwide (Aktaş et al., 2014; Eissa et al., 2023). During aquaculture production, with prolonged culture time, the residual feed and waste metabolites of aquaculture animals tend to accumulate, thereby elevating the concentrations of ammonia nitrogen in the water, which, upon reaching or exceeding the tolerance limit of the aquaculture stock, will have the adverse effects of retarding growth and reducing metabolic capacity (Jensen et al., 2013). Shrimp culture is susceptible to various environmental factors, and changes in these factors can cause physiological changes and oxidative stress (Amaya et al., 2007; Wang et al., 2009). Oxidative stress weakens the immune system, making animals susceptible to opportunistic pathogens (Li et al., 2016; Thirugnanasambandam et al., 2019). This leads to a decline in immunity, thereby increasing the likelihood of infection with various pathogens and potentially widespread mortality (Bhoopathy et al., 2021). Therefore, improving the antioxidant and immune systems of shrimp is a practical and urgent problem in the aquaculture industry, especially intensive aquaculture (Huynh et al., 2017). Research has shown that the addition of Caulerpa racemosa (Agardh, 1873) polysaccharides (CRP) to feed can enhance the antioxidant and immune properties of shrimp (Lee et al., 2020). This illustrates that active microalgal substances as feed additives can enhance immunity and stress resistance in aquatic animals (Chen et al., 1988; Pulz and Gross, 2004), and research on active microalgal substances is essential.

In the study of feed additives, amino and fatty acid contents serve as important indicators of the nutritional value and flavor of aquatic products (Peng et al., 2021). The amino acids comprising muscle are not only important nutrients but also make a valuable contribution to determining the color, aroma, and taste of fish meat. During the culture of aquatic organisms, the amino acid content of the muscle can be modified to a certain extent by adding trace elements to the basic feed and optimizing the ratios of proteins, which in turn can enhance the nutritional quality of the muscles of aquatic animals (Long et al., 2020). Inosinic acid is an intermediate metabolite widely distributed in animals, and the freshness of aquatic products is often influenced by the accumulated amount of this acid (Tanimoto et al., 1993). Furthermore, it has been demonstrated that using the alga Chlorella vulgaris instead of fish meal as a protein source can significantly enhance the amino acid and fatty acid content of African catfish (Tikk et al., 2016), and other functional feed additives have been established to enhance the development, immunity, and antioxidant capacity of shrimp.

Currently, relatively little information is available regarding the application of hot water extracts of C. vulgaris (CVE) in crustacean cultures. In this study, we assessed the effects of CVE as functional additives in crustacean aquaculture. We examined the effects of different concentrations of CVE as a feed supplement on the nutritional composition and antioxidant system of muscles in the Litopenaeus vannamei. The optimal CVE concentrations screened were also subjected to ammonia nitrogen stress experiments to verify the immune-protective effects of CVE on shrimp. This study aimed to provide nutritional and immunological support for the application of CVE as a feed additive in aquaculture and to provide a scientific basis for the health of Litopenaeus vannamei.

CVE is a compound enrich with amino acids, polysaccharide and other substances, more easily absorbed by the body, so CVE as a feed additive in aquaculture has a positive prospect of application. In previous study, it was found that CVE has significant effects on the growth of Litopenaeus vannamei. Adding 1% CVE to the feed can effectively improve the final weight, gain rate, specific growth rate, and survival rate of Litopenaeus vannamei, and reduce the feed coefficient (Han et al., 2022).

The composition and content of amino acids are important indicators of the nutritional value of proteins, and CVE is rich in amino acids; therefore, its use as a feed additive in the aquatic industry has significant advantages (Chen et al., 2021). In the present study, we found that supplementing feed with different proportions of CVE promoted an increase in amino acid content in the muscle of L. vannamei. Feeding the 1% CVE feed group significantly increased the “fresh sweet” amino acid content and reduced the “bitter” amino acid content of the shrimp. Similarly, replacing fish meal with C. vulgaris in the diet of African catfish has been shown to promote a significant increase the contents of “fresh” amino acids (Arg, Val, Thr, and Lys) in the muscle of these fish (Tikk et al., 2016), and consistently, replacing 20% of fish meal with microalgae in the diet of L. vannamei was found to enhance the content of fresh amino acids (Phe, Arg, Glu) in the muscle of these shrimps (Pês et al., 2016). Collectively, these findings indicate that supplementing the feed of L. vannamei with 1% CVE had the greatest effect on enhancing the flavor and protein quality of shrimp.

Nucleotide degradation-related compounds are important indicators for evaluating the freshness and flavor of aquatic products (Clausen et al., 2018). The degradation of adenosine triphosphate (ATP) in the muscle begins to take place; ATP breaks down into adenosine diphosphate (ADP), ADP breaks down into adenosine monophosphate (AMP) by creatine kinase (CK), AMP generates inosine monophosphate (IMP) by adenosine monophosphate dehydroammonia enzyme, and IMP will be hydrolyzed to produce hypoxanthine (Hx), which has a bitter flavor (Tikk et al., 2016). The content of inosinic acid and its degradation products in aquacultured species is not only determined by breed but is also associated with factors such as feed composition (Bremner et al., 1988; Greene and Bernatt-Byrne, 1990). To date, comparatively few studies have assessed the flavor-enhancing effects of dietary CVE supplementation on aquaculture products. Our findings revealed that supplementing shrimp feed with different concentrations of CVE contributed to increases in the IMP, AMP, and CK contents of shrimp muscle, with levels gradually increasing with an increase in the proportion of supplemented CVE. The hypoxanthine content in shrimp fed the 1% CVE-supplemented diet was significantly lower than that in the other groups (P< 0.05), whereas the levels of IMP, AMP, and CK were significantly higher than those recorded in the control group (P<0.05). Similarly, the addition of glycerol monolaurate (GML) to feed enhances the inosinic acid content of large yellow croaker (Larimichthys crocea) muscle (Zhuang et al., 2022). Glu and Asp could play roles in the nucleotide synthesis and further trigger the generation of inosinic acid (Kurihara, 2015). Therefore, the contents of Glu and Asp were significantly increased after CVE sup plementation which could be the explanation for the increase in the inosinic acid content. Current research on algal additives has revealed that algae contain various active substances, high protein and low fat contents, and multiple vitamins, which, when incorporated into feed, can contribute to enhancing the quality and flavor of animal muscle (Ahmad et al., 2020).

Amino acids and inosine can also be used together as indicators for evaluating the flavor of aquatic animal meat (Yamaguchi, 1998). Research has shown that replacing fishmeal with C. vulgaris in the deit of L. vannamei can effectively improve muscle quality (Li et al., 2022). Application of a dietary lysine to feed can increase the content of amino acids, fatty acids, and inosine in grass carp muscle, thereby improving the muscle quality of grass carp (Ctenopharyngodon Idella) (Tang et al., 2023). CVE as an extract of C. vulgaris enriched with amino acids that has the same positive effect. Subsequent comparative studies will be conducted on aquatic animals by adding C. vulgaris and C. vulgaris extracts.

The activity of immune enzymes in the hepatopancreas influences the metabolism and antioxidant capacity of shrimp and can serve as an important indicator of the immune capacity of aquatic animals. In the present study, ACP, AKP, and NOS enzyme activities were the highest in the CVE 1% group, SOD and GSH-Px increased with increasing CVE addition, and MDA content was the lowest in the 0.5% CVE group. Hot water extracts of Chlorella can stimulate or enhance the immune system of animals (Kotrbacek et al., 2015). Previous studies have shown that the shrimp fed the Panax ginseng polysaccharides (GSP) diet had significantly increased ACP, AKP, T-SOD and GSH-Px activities in L. vannamei (Liu et al., 2011). Increasing of ACP, AKP and NOS by dietary CVE suggested that the CVE might contain some bioactive substances involving in the regulating of fish immune response. Immunostimulants obtained by hot water extraction of Chlorella cells, such as the powerful antioxidants vitamin C, and polysaccharides (Spolaore et al., 2006) have been observed to increase the immune resistance of shrimp and fish (Maliwat et al., 2016). Microalgae in general are considered as potential sources of natural antioxidants (Tsao and Deng, 2004). Chlorella was also reported to have a better activity in inhibiting lipid peroxidation and have antioxidant property (Bengwayan et al., 2010). Research has shown that supplementing Atlantic salmon feed with 10% AquaArom enhances the activities of SOD and other antioxidant enzymes while reducing temperature responsiveness (Kamunde et al., 2019). The activity found that The addition of 10% Chlorella powder to feed promoted a significant increase in SOD content and a reduction in MDA content in rainbow trout (Chen et al., 2021), possibly attributing to the antioxidant activity of polyphenols and flavonoids in the Chlorella (Shibata et al., 2003). Research has shown that adding amino acids to feed can enhance the antioxidant capacity, non-specific immune ability, and ability to resist adverse environments of aquatic animals (Oehme et al., 2010; Cheng et al., 2012). In this study, the addition of CVE significantly increased the amino acid content in L. vannamei. This indicates that CVE as a feed additive can accelerate liver metabolism and reduce lipid peroxidation and enhances antioxidant capacity of L. vannamei.

The most economical and effective CVE additive (1%) was selected for ammonia nitrogen stress experiments on L. vannamei to verify its protective effect of CVE as a feed additive on the organismal immunity of L. vannamei. Among the shrimp exposed to NH₃-N stress, we found that the activities of ACP and AKP in those fed the supplemented diet were invariably higher than those in the control group after 6 h. The MDA content in the control group was characterized by an initial decline and subsequent increase, and when measured at 24 h, MDA levels in the 1% CVE group were found to 3 be significantly lower than those in the control group (P < 0.05), with an overall contrasting trend of an initial increase and subsequent decline. Moreover, when measured at 3 h, we detected a significantly higher level of NOS activity in shrimps fed the supplemented diet compared with those in the control shrimps (P<0.05), with the levels of activity reaching a maximum at 12 h. Whereas the research found that NOS activity in L. vannamei initially increased and subsequently declined in response to exposure to heat stress (Jia et al., 2014). These authors similarly observed initial increases and subsequent declines in NOS activity in shrimps exposed to 0.1 mg/L NH₃-N stress, whereas in response to treatment with 0.5 mg/L NH₃-N, they detected a significant reduction in NOS activity, a reduction in SOD activity, and increases in the content of MDA (Jia et al., 2017). When shrimp are stimulated by environmental factors it leads to disruption of the body’s antioxidant system. Excessive non-ionic ammonia can cause metabolic disorders in aquatic animals, affecting their ionic regulation ability and damaging cellular structure to cause harm to the organism (Chen et al., 1988; Cheng and Chen, 2001). When exposed to oxidative stress, organisms deploy various antioxidative defense mechanisms, including enzymatic (e.g., SOD and glutathione peroxidase) and non-enzymatic (e.g., MDA) (Al-Ghanim et al., 2020). This is similar with previous reports that dietary Chlorella improved survival rate of L. vannamei under hypoxia challenge (Pakravan et al., 2017).The shrimp fed the CVE diet may contribute to these specific enzymes involved in immunological activity to enhance ammonia nitrogen stress resistance. CVE, as a feed additive, enhanced the antioxidant capacity and detoxification activity of L. vannamei in the treated group compared to those in the control group.

Research has shown that the addition of calcium pyruvate to feed impairs glucose utilization in L. crocea, as evidenced by the elevated plasma glucose levels and reduced activities of enzymes associated with glucose metabolism (HK, PFK, PK, and PDH) in the liver and muscles (Zhang et al., 2023). In the present study, we found that the muscle activities of HK, LDH, and SDH showed similar trends in the control and treatment groups, whereas the KH activity increased to different extents during the early stage of treatment. In contrast, the activity of PK in the muscle was characterized by a declining trend in the control group shrimp and an initial decline and subsequent increase in shrimp fed the supplemented diet, thereby indicating the enhanced blood glucose regulatory capacity of these shrimp. In response to the imposition of nonionic ammonia stress, we detected a decline in LDH activity in the muscle of L. vannamei when measured at 6 h, which implies an initial gradual attenuation of anaerobic respiration in these shrimp, although we detected a subsequent recovery of activity at the 12-h time point. The vigor of shrimp fed a supplemented diet was found to be significantly higher than that of the control group shrimp, which is presumed to be associated with the effects of CVE in regulating metabolic levels in L. vannamei, thus contributing to the enhancement of the anaerobic respiratory capacity of these shrimp when exposed to stress conditions. A similar pattern of response was observed for SDH following exposure to non-ionic ammonia stress, with activities in the muscle of the control group shrimp characterized by a declining trend over time, whereas an initial decline in activity followed by a subsequent increase was detected in shrimp fed the supplemented feed. These findings indicate the higher aerobic respiratory capacity of shrimp in the 1% CVE group and an inhibition of aerobic respiration in the control group, which could be ascribed to tissue hypoxia in these control shrimp. Consumption of a diet supplemented with 1% CVE was found to promote the immune capacity of these shrimps, which, compared with those in the control group, incurred less damage when exposed to non-ionic ammonia stress conditions.

The indicator for determining shrimp stress and health status is the immunological enzyme activity. In terms of immune response, we We added different proportions of CVE and selected the most economical and effective 1% CVE as the feed additive and to verify its promoting effect on the immunity of L. vannamei under ammonia nitrogen stress. CVE can enhance the activity of immune enzymes and antioxidant enzymes in L. vannamei.The study has found that adding C. vulgaris to feed can enhance the immunity of L. vannamei and immune biomarker responses, leading to disease resistance in cultured shrimp (Eissa et al., 2024);. CVE as the extract of C. vulgaris has a positive role in enhancing the immunity of L. vannamei.

Our findings indicate that supplementing the feed of the shrimp L. vannamei with CVE may contribute to improving muscle nutrient composition, antioxidant capacity, and non-specific immunity and that under conditions of NH₃-Nstress, a diet supplemented with 1% CVE may be beneficial in protecting tissues from the adverse effects of oxidative stress and, to a certain extent, reduce lipid peroxidation. However, supplementation of feed with higher levels of CVE did not appear to be conducive to conferring further beneficial effects. In this study, we obtained evidence indicating that supplementary CVE have a significant influence on the nutrient composition and immune response capacity of L. vannamei. In terms of shrimp health, our findings provide a valuable scientific basis for enhancing the quality of aquaculture environments and important insights into the mechanisms underlying the beneficial effects of CVE on the physiology and ecology of aquatic invertebrates. Moreover, these findings will contribute to the further development of microalgal resources and aquaculture applications of CVE.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The Animal Ethics Committee’s scope of approval is limited to studies involving vertebrates (Laboratory Animal Act, Chapter 5Article 25). Our study is on invertebrate species, and does not need ethical approval in China. The study was conducted in accordance with the local legislation and institutional requirements.

DZ: Formal analysis, Visualization, Writing – review & editing. SS: Investigation, Supervision, Writing – original draft. XJ: Investigation, Supervision, Validation, Writing – review & editing. WZ: Conceptualization, Investigation, Writing – review & editing. XS: Formal analysis, Writing – original draft. CH: Formal analysis, Methodology, Writing – original draft. YL: Investigation, Methodology, Writing – original draft.