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Article

Effects of Carnosine Addition in Low-Fishmeal Feed on the Growth Performance, Muscle Antioxidant Capacity and Flesh Quality of Orange-Spotted Grouper (Epinephelus coioides)

by
Dong Li
1,
Weijun Chen
1,
Yanxia Yin
1,
Lulu Yang
1,
Mingfan Chen
1,
Yunzhang Sun
1,2 and
Jidan Ye
1,2,*
1
Xiamen Key Laboratory for Feed Quality Testing and Safety Evaluation, Fisheries College of Jimei University, Xiamen 361021, China
2
Fujian Engineering Research Center of Aquatic Breeding and Healthy Aquaculture, Xiamen 361021, China
*
Author to whom correspondence should be addressed.
Fishes 2024, 9(11), 467; https://doi.org/10.3390/fishes9110467
Submission received: 21 October 2024 / Revised: 14 November 2024 / Accepted: 15 November 2024 / Published: 18 November 2024
(This article belongs to the Special Issue Growth, Metabolism, and Flesh Quality in Aquaculture Nutrition)
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Abstract

:
Carnosine is a natural dipeptide made up of L-histidine and β-alanine which is rich in muscle tissues and has multiple physiological functions. The current research aimed to investigate the effects of varied carnosine concentrations in low-fishmeal feed on the growth, muscle antioxidant capacity and flesh quality of orange-spotted grouper. Carnosine was supplemented at doses of 0, 10, 20, 40, 80, 160, and 320 mg/kg in low-fishmeal feed. Seven groups with three tanks of fish (11.4 ± 0.1 g/fish) were allotted one of the diets during the 8-week feeding trial. The growth rate, body protein content, muscle activities of superoxide dismutase and catalase, and muscle adhesiveness showed positive linear response and/or an open upward parabola with increasing carnosine concentrations, with a peak at 160 mg/kg of carnosine. Feed utilization, serum total protein content, gut trypsin activity, muscle glutathione peroxidase, total antioxidant capacity, muscle hardness, gumminess, chewiness and resilience followed the same pattern as the growth rate, reaching a peak at 320 mg/kg of carnosine; while the opposite trend was observed, reaching a minimum at 320 mg/kg for muscle malondialdehyde and 160 mg/kg for muscle liquid and water loss. The results indicated that appropriate carnosine addition could improve growth performance, muscle antioxidant capacity and flesh quality of grouper. The suitable inclusion concentration was estimated to be 195.14 mg/kg to achieve the best percent weight gain.
Keywords:
carnosine; Epinephelus coioides; growth performance; muscle antioxidant capacity; flesh quality
Key Contribution: This study contributes to exploring the effect of carnosine supplementation at multiple levels to a low-fishmeal diet on growth, muscle antioxidant capacity and flesh quality of grouper and determined the dosage for best growth promotion.

Graphical Abstract

1. Introduction

Carnosine is made up of L-histidine and beta-alanine and exists primarily in the animal muscle and nervous tissue as a natural dipeptide [1]. In vivo, carnosine has two forms of methylation, β-alanyl-1-methyl L-histidine (anserine) and β-alanyl-3-methyl L-histidine (balenine). Anserine is prevalent in fishes and birds, while the latter is mostly found in reptiles and marine mammals [2]. The three types of dipeptides are referred to as histidine-containing dipeptides [2,3], of which carnosine has received more attention due to its importance [2,4]. Carnosine has multi-functions such as pH buffering, chelating metal ions, antioxidant protection, inflammation regulation, as well as scavenging of lipid peroxides and carbonyl compounds [4,5]. Many studies on animals showed that carnosine can eliminate the harm of some detrimental factors to animals, such as ethanol [6,7], lipopolysaccharide [8], heavy metals [9], drugs [10,11], and nutritional imbalance [12,13]. Thus far, carnosine has been widely used in cosmetics, athletic supplements and functional foods, and is regarded as a potential complementary therapeutic medicine [14,15,16].
Carnosine is rich in animal-origin feedstuffs, but not present in plant-origin ingredients. This makes carnosine application in low-fishmeal feed for grouper worth further investigation. Currently, carnosine has already been used as a feed additive to enhance the antioxidant capacity, immunity, muscle quality, and animal welfare of broilers [17,18], laying hens [19], finishing pigs [20], as well as cats [21]. Research on the potential application of carnosine in aquafeeds as a feed additive is limited. However, these finished studies have given rise to inconsistent conclusions. Several studies indicated that dietary carnosine addition could promote the growth of fish such as tilapia [22] and zebra fish [23], while others showed the effect of carnosine addition was minimal on rainbow trout [24], or even negative on turbot [25]. Further studies are warranted to explore the potential use of carnosine in aquafeeds across different species.
Grouper is a widely farmed marine carnivorous fish in Asian coastal countries including China [26]. The aquaculture yield of grouper in China has been kept growing in recent years and reached 205,800 tons in 2022 [27]. Meanwhile, the merits of good taste and high nutritional value had ensured grouper a good price as well as growing consumption in the market, and thus the grouper aquaculture remained profitable and luring to new farmers. One limitation of grouper’s production is that, like other farmed carnivorous fish species, it relies heavily on fish meal for feed manufacture [28]. Fish meal replacement in the feed usually led to adverse effects on grouper, including: poor growth and feed utilization, oxidative stress, and intestinal damage in previous studies [29,30,31,32,33]. Further, flesh quality was also reported to be affected by fish meal replacement in grouper [34], and other fish species [35,36]. One possible explanation to these drawbacks is that some functional substances are missing in the alternative feedstuffs [37,38]. For this reason, the supplement of certain feed additives in low fish meal feed has been proved to reverse this negative effect caused by the lack of bio-active substances [39,40,41].
In our present study, the growth performance, muscle antioxidant capacity, and flesh quality of orange-spotted grouper (Epinephelus coioides) were investigated with varied carnosine supplementation in a low-fishmeal diet to explore the potential use of carnosine as a functional additive in this certain fish species.

2. Materials and Methods

2.1. Test Diets

Carnosine was obtained from Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China (98%, No. B25565). A low-fishmeal (20%) feed (basal diet) was designed to contain 48.6% protein and 10.3% fat (Table 1), in which carnosine was not detected. An incremental level of carnosine (0, 10, 20, 40, 80, 160, and 320 mg/kg) was supplemented to the basal diets. According to our previous feed production method [42], the seven test diets (diets 1–7) were produced, dried, and then sealed in plastic bags, followed by storage under −20 °C until used in the feeding experiment.

2.2. Feeding Trial

The growth experiment was conducted at the marine aquaculture experimental farm of Jimei University (Xiamen city, Fujian province, China). After arrival at the experimental farm, the juvenile fish were maintained with Diet 1 in a cement pond for 2-week acclimatization before the beginning of growth experiment. Five hundred and twenty-five healthy fish with an initial weight of 11.4 ± 0.1 g/fish were randomly assigned to seven groups. Each group was allotted three 500 L tanks (25 fish/tank) in a water temperature-controlled recirculating culture system and was given two meals (7 a.m. and 3 p.m.) daily, achieving apparent satiety each meal in a natural light cycle across the 8 weeks feeding duration. Collection of excessive feed was carried out 40 min after each meal, followed by the removal of feces via siphoning. The feed consumption was determined after wet feed was dried. Daily measurement of dissolved oxygen (DO) and water temperature (WT) and ammonia nitrogen detection were respectively undertaken at 3 pm and twice per week using a multi-parameter photome (HI83200; Hanna Instruments, Woonsocket, RI, USA). During the growth experiment, WT ranged between 28 and 30 °C, DO ranged from 5.6 to 7.7 mg/L, as well as ammonia-N was below 0.21 mg/L.

2.3. Tissue Sample Collection

After the growth experiment, the fish were caught and anaesthetized with a dose of 100 µL/L solution of eugenol (Nanjing wensenbao International Trade Co., Ltd., Nanjing, China). Fish per tank were counted and batch-weighed for the determination of survival, the percent weight gain (WG), the specific growth rate (SGR), and the feed conversion ratio (FCR). Three fish in each tank were randomly sampled and stored at −20 °C for whole-body composition analysis. Another four fish in each tank were measured and weighed individually for the calculation of condition factor (CF). Subsequently, blood was drawn using 1 mL medical disposable syringes, and placed at 4 °C for 12 h and the serum was separated by centrifugation (1027 g, 10 min) and stored at −80 °C until analysis. After hemospasia, the fish abdomen was aseptically dissected out, followed by the removal of liver and viscera for the purpose of calculating the hepatosomatic index (HSI), visceralsomatic index (VSI), and intraperitoneal fat rate (IPF). The gut and muscle samples were respectively pooled into one tube by tank to eliminate inter-individual variation, followed by quick freezing using liquid nitrogen and then placed at −80 °C for the analysis of gut digestive enzyme activity, muscle components, and muscle antioxidant capacity. Another two fish of average size in each tank were selected for dorsal muscle collection, and testing of muscle texture, pH value and liquid holding capacity (LHC) was implemented immediately on site.

2.4. Nutritional Composition Analysis

The nutritional composition of feeds and fish samples was measured following the guidance of the Association of Official Analytical Chemists [43]. The whole-body fish samples were cooked in autoclave vessel at 121 °C for 20 min and crushed, followed by drying at 65 °C for 24 h, prior to the analysis of proximate composition. The content of dry matter was measured using a ventilation oven at 105 °C to a stable weight. The content of crude protein was measured using the Kjeltec 2300 Analyzer Unit (Foss Tecator AB, Hoganas, Sweden). The content of crude lipid was measured using Soxtec Avanti 2050 Fat Extraction System (Foss Tecator AB). Ash content was measured using a muffle furnace at 550 °C for 8 h.

2.5. Assay for Serum Biochemical Indices

The contents for total protein (TP), cholesterol (TC), triglycerides (TG), and immunoglobulin M (IgM), as well as lysozyme activity (LZM) in serum were assayed by means of kits (cat: A045-4-2, A111-1-1, A110-1-1, A050-1-1) provided by Jiancheng Bioengineering Institute (Nanjing, China) according to the manual of the manufacturer.

2.6. Assay for the Digestive Enzyme Activity

The gut activities of digestive enzymes (amylase, trypsin and lipase) were determined using their respective kits (cat: C016-1-1, A80-2-2, A054-2-1) provided by Jiancheng Bioengineering Institute.

2.7. Muscle Antioxidant Capacity Determination

The value of muscle total antioxidant capacity (TAOC), activities of muscle glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT), as well as the content of muscle malondialdehyde (MDA) were measured respectively using kits (cat: A015-3-1, A005-1-2, A001-3-2, A007-1-1, A003-1-2) provided by Jiancheng Bioengineering Institute.

2.8. Muscle Determination of Carnosine and Anserine

The muscle contents of carnosine and anserine were measured using the method of high-performance liquid chromatography (HPLC) [44]. One gram of muscle sample was grinded in liquid nitrogen and homogenized in pure water, followed by smashing in an ultrasonic crusher to release intracellular carnosine and anserine. The protein of samples was removed by adding 10% trichloroacetic acid, followed by centrifugation at 20,000× g at 4 °C for 5 min. The supernatant was purified through ultrafiltration using a 3 kD cutoff. Finally, the injection volume of 10 μL filtrate was used to separate and measure carnosine and anserine using a HPLC UltiMate 3000 (Thermofisher Scientific, Waltham, MA, USA). The contents of carnosine and anserine were defined as mg/g wet muscle weight.

2.9. Flesh Quality Analysis

Approximately 1 g of minced dorsal muscle was crushed in a homogenizer using deionized water (1 g/10 mL). pH was then detected using a pH meter (206 PH1, Testo, Titisee-Neustadt, Germany). The liquid holding capacity (LHC) was assayed by gravimetric method [45]. Three sampling points were selected for each dorsal muscle. Approximately 1 g of dorsal muscle (W0) was wrapped in the filter paper (W1), followed by centrifugation at 500× g for 10 min at 10 °C. The wet paper (W2) was weighed and then dried in the oven (75 °C) to constant weight (W3). The liquid loss, water loss and lipid loss were calculated using the following formulas 100 × (W2 − W1)/W0, 100 × (W2 − W3)/W0, and 100 × (W3 − W1)/W0, respectively.
Dorsal muscle samples were cut into cubes (1.0 cm × 1.0 cm × 0.5 cm). The muscle texture was then detected using a texture analyzer TA. XT2i (Stable Micro System, London, Britain) equipped with a P/36R probe. The experimental conditions comprised two continuous pressures at the fixed speed of 1.0 mm/sec with the compression degree of 30%, and an interval time of 5 s. Subsequently, the parameters of muscle texture were detected [46].

2.10. Calculations

Percent weight gain (WG, %) = 100 × (W2 − W1)/W1,
Specific growth rate (SGR, %/d) = 100 × (ln W2 − ln W1)/56 days,
Daily feed intake (DFI, %/d) = 100 × FI/((W2 + W1/2) × 56 days),
Feed conversion ratio (FCR) = 100 × FI/(W2 − W1),
Survival (%) = 100 × final number of fish/initial number of fish,
Condition factor (CF, g/cm3) = 100 × BW/BL3,
Hepatosomatic index (HSI, %) = 100 × LW/BW,
Viscerasomatic index (VSI, %) = 100 × VW/BW, and
Intraperitoneal fat rate (IPF, %) = 100 × IPFW/BW.
where W1, initial individual weight (g); W2, final individual weight (g); FI, feed intake (g); LW, liver weight (g); BW, body weight (g); BL, body length (cm); VW, visceral wet weight (g); IPFW, intraperitoneal fat weight (g).

2.11. Statistical Analysis

Analysis of variance (one-way ANOVA) was conducted to examine the significance among treatments. A test of normality and homogeneity of variance was performed before multiple comparisons using the Kolmogorov–Smirnov test and Levene’s test in SPSS Statistics 22.0 (SPSS, Michigan Avenue, Chicago, IL, USA). Percentage or ratio data underwent either arcsine or square root transformation prior to statistical analysis. The orthogonal polynomial contrasts were used to test the dose–response relationship of the dependent variable in linear or quadratic response to feed carnosine concentrations. The results were displayed as the mean and standard error of the mean (SEM). p value < 0.05 was set as the significant difference.

3. Results

3.1. Growth Performance and Proximate Body Composition

Table 2 shows the results of growth performance of groupers. Dietary carnosine addition significantly (p < 0.05) affected the growth rate and feed efficiency. The FBW, WG and SGR showed a dose–response relationship in a linear and an open upward parabola manner as the carnosine concentration in feed was increased (p < 0.05), reaching a maximum in the diet with 160 mg/kg carnosine, while the FCR showed the opposite trend, reaching a minimum in the diet with 320 mg/kg carnosine. The values of other parameters were not influenced (p > 0.05) by treatments.
The regression analysis of WG against the carnosine concentration in feed is shown in Figure 1. The estimation of optimal carnosine concentration for the growth rate of grouper was 195.14 mg/kg of feed (YWG = −0.0019x2 + 0.7415x + 238.9971, R2 = 0.6284).
As shown in Table 3, the crude protein content of whole-body showed a dose–response relationship in linear and an open upward parabola manner with the increase in carnosine concentration in feed (p < 0.05), reaching a maximum at 160 mg/kg carnosine. Meanwhile, whole-body crude lipid in all diets with carnosine addition was lower than that of the basal diet (p < 0.05). No differences (p > 0.05) were observed in whole-body moisture and ash contents between treatments.

3.2. Serum Components

As shown in Table 4, serum TP content showed a positive linear responses and an open upward parabola to increasing dietary carnosine level (p < 0.05), reaching a maximum at 320 mg/kg carnosine. The LZM activity displayed a dose–response relationship in an open upward parabola manner with the increase in carnosine concentration in feed, reaching a peak at 40 mg/kg carnosine (p < 0.05). No differences (p > 0.05) were observed in serum TG, TC, and IgM contents between treatments.

3.3. Intestinal Digestive Enzyme Activity

Table 5 displays the intestinal activities of digestive enzymes. The activity of trypsin followed the same trend as serum TP in response to the increase in carnosine concentration in feed (p < 0.05), reaching a peak at 320 mg/kg dietary carnosine. The activity of amylase was lower at doses of 10–80 mg/kg carnosine than at the basal diet (p < 0.05). No dose–response relationship was observed between the activities of lipase and dietary carnosine concentrations (p > 0.05).

3.4. Muscle Antioxidant Capacity

The indices of muscle antioxidant capacity are presented in Table 6. The value of TAOC and the activities of CAT, SOD and GSH-Px followed the same pattern as WG (p < 0.05) in response to the increase in carnosine concentration in feed, reaching a maximum at 160 mg/kg or 320 mg/kg carnosine, while MDA value showed the opposite trend, reaching a minimum at 320 mg/kg carnosine.

3.5. Flesh Quality

As shown in Table 7, both liquid and water loss followed a negative linear response and an open downward parabola to increasing dietary carnosine level (p < 0.05), reaching the minimum at 160 mg/kg carnosine. No dose–response relationship was observed between pH-5 min, pH-24 h, and lipid loss and the carnosine concentration (p > 0.05).
Table 8 shows the trends of muscle texture in response to carnosine concentrations in feed. The trends of hardness, adhesiveness, gumminess, chewiness and resilience were found to follow the pattern of WG (p < 0.05), in response to the increase in carnosine concentration in feed, reaching a maximum at 320 mg/kg carnosine except for adhesiveness with a peak at 160 mg/kg carnosine. No alterations were observed in values of cohesiveness and springiness between treatments (p > 0.05).

3.6. Muscle Contents of Histidine-Containing Dipeptides

As shown in Table 9, diets with carnosine addition had marginally higher muscle contents of carnosine and anserine than the basal diet (p > 0.05). Meanwhile, the ratios of carnosine to anserine maintained a constant content among treatments (p > 0.05).

4. Discussion

It is worth noting that a beneficial effect and possible mechanisms of carnosine has been evidenced by studies on human and mice, which could give reference to similar studies on fish species, as performed on zebra fish to explore the role of carnosine on mitigating the negative effects of dietary soybean meal in a recent study [23]. The results of our current study displayed that the growth of grouper had a dose–response relationship in response to the carnosine concentration in feed, with a lowest growth occurring for the basal diet without carnosine addition. This finding indicates that the growth of grouper could benefit from appropriate carnosine addition, which was similar to what has been reported in previous studies with broiler chicken [18,47], tilapia [22] and turbot [25]. However, excessive carnosine addition in feed could result in growth retardation in some cases [25,48]. In our current study, the maximum WG was achieved in the diet with 160 mg/kg of carnosine and maximum feed utilization was achieved at a dosage of 320 mg/kg, but in fact, there were no differences in growth and feed utilization between 160 mg/kg and 320 mg/kg of carnosine. This indicated that the dose of 160 mg/kg of carnosine or higher will not produce a significant impact on growth and feed utilization of grouper. The HSI, CF, VSI and IPF were not affected by dietary carnosine addition, reflecting a good nutritional state of fish [24,49], which was coincident with the previous studies on rainbow trout [24], and turbot [25]. The health conditions could also be evidenced by constant survival rates across dietary treatments [50].
In our current study, body protein content had a dose–response relationship in response to the carnosine concentration in feed, while the lipid content declined adversely, indicating that body protein preferentially deposits in fish fed with high carnosine feed. These findings corresponded with a previous study on cats, where dietary carnosine supplement increased the lean body mass while the fat body mass decreased [21].
Serum TP, TG and TC are valuable indicators that reflect the nutritional state of fish body [51]. In the present study, serum TP content had the same ascending trend as body protein content and the growth rate as dietary carnosine concentration was increased. Furthermore, the activities of trypsin also showed a dose-dependent relationship with dietary carnosine concentrations, similar to the changing trend for serum TP content. This indicated an enhanced ability of dietary protein digestion, which improved body protein accumulation, and thereby promoted fish growth [52]. We could, therefore, speculate that the increment of fish body protein accumulation may be partly attributed to the synthesis enhancement of tissue protein as a result of carnosine addition. A previous study observed an alteration in the protein metabolism of skeletal muscle in carnosine synthase knockout mice, as evidenced both by the declination in gene expression and insulin-like growth factor-1 (IGF-1) levels and by the degrading enzyme cathepsin B, in comparison with that in wild-type mice [53].
Lysozyme is a glycanhydrolase that could hydrolyze the bonds in the peptidoglycan of bacterial cell wall, causing the lysis of pathogen, which plays a vital role in the innate immunity of fish [54,55]. IgM mainly originates from the secretion of plasma cells in the spleen and has high specificity and affinity, which exerts bacteriolysis and virus neutralization via activating the complement system [56]. In the current study, serum lysozyme activity showed a dose-dependent relationship with dietary carnosine concentrations, whereas serum IgM content remained stable. This finding indicates that appropriate dietary carnosine addition may activate the non-specific immune response but does not affect the specific immune response of grouper. In previous studies on murine and human, carnosine proved to play a role as a regulator in the activation of macrophage [10] and neutrophil [57]. But in birds, what’s different was that dietary carnosine addition elevated serum Ig A and Ig G contents, indicating the activation of specific immune system [19].
Nowadays, the fish consumers are increasingly concerned about the flesh quality of farmed fish. The flesh quality is closely related to the muscle antioxidant capacity, especially when there exists a stress like dietary fish meal declination [36,58]. Such stresses were reported to disturb the balance of oxidation and antioxidation balance, accompanied by an elevation of reactive oxygen species (ROS), which will induce lipid peroxidation (LPO) and protein carbonylation, causing damage to cell components [59,60]. The MDA is a final product of lipid peroxidation which was itself cytotoxic and also commonly regarded as a reliable biomarker for the judgement of LPO [59]. Moreover, the antioxidant enzymes including SOD, GSH-Px and CAT play a key role in the inhibition of LPO and thus elevate tissue oxidative stress [61]. Carnosine is known for its in vivo antioxidant capacity [1,62] and is proved to effectively prevent lipid and protein oxidation through regulating antioxidant enzyme activity [7,63]. In this study, the TAOC values and activities of SOD, GSH-Px and CAT in the muscle had a close dose–response relationship in a positive linear manner and an open upward parabola in response to carnosine concentration in feed, while muscle MDA values showed the opposite trend. This finding indicated that dietary carnosine inhibited lipid peroxidation through an improvement in muscle antioxidant enzyme activities, thus reducing the oxidative stress. Similar findings were reported in previous studies with broiler chickens [18,47]. Further, the enhanced antioxidant capacity may contribute to the growth promotion observed in the current study in accordance with the previous reports on grouper [61] and other species [58,64].
Muscle glycolysis is initiated to produce a large amount of lactic acid after slaughter, causing a decrease in pH value, which is regarded as a degradation of flesh quality [65,66]. Meanwhile, muscle carnosine immediately acts as a proton buffer to reduce lactic acid accumulation. Although in previous studies dietary carnosine supplementation had led to an elevation of pH value after slaughter [18,20], no significant increase in the pH value of the flesh 15 min or 24 h after slaughter was observed in our current study. But our present results support those of a previous report with broilers [17]. The LHC value is another commonly used index which negatively correlated with the degree of muscle degradation [65,67]. The increased LHC values caused by dietary carnosine addition were observed in pigs [17] and broilers [18,47,68] in previous studies, and now also in grouper in our current study.
As mentioned above, marine fish species have been popular due to their good taste and high fillet quality, of which muscle texture plays a key role [69]. The muscle texture indicators are used for the evaluation of the flesh quality through simulating the chewing sensation of consumers, including hardness, adhesiveness, springiness, cohesiveness, gumminess, chewiness, and resilience [70,71]. Previous studies have proved that muscle texture of fish could be influenced by feed ingredients [35,72], feed additives [73,74,75], and protein levels [76]. Our current study found that muscle texture could be improved by dietary carnosine supplementation. All the muscle texture indicators except cohesiveness showed a dose–response relationship in a linear manner and an open upward parabola with increasing carnosine concentration in feed and achieved a maximum at 160 or 320 mg/kg carnosine, indicating an elevation of muscle texture. Considering the similar changing tendency of muscle texture with muscle antioxidant capacity in the current study and their close association supported by previous researchers [73,74], the elevation of the muscle texture of grouper triggered by carnosine supplementation could be partially attributed to the enhancement of muscle antioxidant capacity.
Carnosine and anserine are commonly present in the muscle of various animal species including marine teleost [2,3]. Further, their content in muscle could be influenced by dietary carnosine supplement, which was observed in fish [24,77], rats [78], broilers [17,68], as well as human beings [79,80]. The increased contents of carnosine and anserine in flesh are generally regarded as an extra benefit for consumers owing to their health promoting functions [4,44,81]. In the present study, fish fed on diets supplemented with carnosine had higher muscle contents of carnosine and anserine than the control group (without significance), while the ratio of carnosine to anserine remained stable. Notably, the content of carnosine was much higher than that of anserine in grouper, which was similar to eels, but different from most other marine teleost according to data published [2,3]. Whether the muscular contents of carnosine and anserine vary with fish species and their relative changes in response to feed carnosine additions needs further exploration.

5. Conclusions

In summary, the findings of our current study indicated that dietary carnosine addition could improve the digestion and utilization of feed, and thus promoted the growth rate and reduced FCR. Further, it enhanced the muscle antioxidant capacity evidenced by the elevations of antioxidant enzymes and the declination of muscle MDA content. Furthermore, the flesh quality was elevated and showed better liquid holding capacity and texture. A suitable carnosine addition to low-fishmeal diets for grouper is 195.14 mg/kg according to the regression analysis based on the percent weight gain.

Author Contributions

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

Funding

This research was funded by the Natural Science Foundation of Xiamen City, grant number 3502Z202373027, and the National Natural Science Foundation of China, grant number 32072990.

Institutional Review Board Statement

The animal study protocol was approved by the Animal Ethics Committee of Jimei University, Xiamen, China, Approval number: 2011-58.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data for this research article were available from the corresponding authors by reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Fishes 09 00467 g001
Figure 1. The relationship between the percent weight gain of orange-spotted grouper and dietary carnosine inclusion concentrations.
Figure 1. The relationship between the percent weight gain of orange-spotted grouper and dietary carnosine inclusion concentrations.
Fishes 09 00467 g001
Table 1. Feed formulations and the proximate composition of the control diet (on an as-fed basis, %).
Table 1. Feed formulations and the proximate composition of the control diet (on an as-fed basis, %).
Ingredients
Fish meal (Thailand)20
Soy protein concentrate10
Casein24
Gelatin6
Corn starch25.7
Wheat gluten3.5
Fish oil3
Soybean oil3
Soybean lecithin2
Ca(H2PO4)21.5
Choline chloride0.3
Vitamin premix *0.5
Mineral premix #0.5
Proximate composition
Moisture6.06
Crude protein48.64
Crude lipid10.28
Ash6.63
* Vitamin premix (per kilogram diet): retinol acetate, 10 mg; 1,25-dihydroxycholecalciferol, 10 mg; DL-α-tocopherol acetate, 100 mg; menadione sodium bisulfate, 10 mg; thiamin nitrate, 10 mg; riboflavin, 20 mg; pyridoxine hydrochloride, 20 mg; cyanocobalamin, 0.05 mg; nicotinic acid, 50 mg; calcium-D-pantothenate, 100 mg; D-biotin, 1 mg; meso-inositol, 500 mg; folic acid, 4 mg. # Mineral premix (per kilogram diet): ferric citrate, 497 mg; CuSO4·5H2O, 24 mg; ZnSO4·7H2O, 176 mg; MnSO4·4H2O, 122 mg; CoCl2·6H2O, 0.18 mg; KIO3, 0.51 mg; Na2SeO3, 0.33 mg.
Table 2. The growth performance of orange-spotted grouper fed diets with carnosine supplementation.
Table 2. The growth performance of orange-spotted grouper fed diets with carnosine supplementation.
ItemsDiets with Carnosine Doses (mg/kg)Pooled SEMp-Value
010204080160320ANOVALinearQuadratic
IBW (g)11.4211.3811.4511.4111.411.411.380.010.5330.1870.425
FBW (g) *37.71 d39.88 cd41.86 bc43.69 ab43.10 bc47.04 a44.03 ab0.740.0030.0150.000
WG (%) *230.15 c247.38 bc260.28 bc273.01 b278.23 ab309.89 a281.9 ab6.520.0080.0150.000
SGR (%/d) *2.13 d2.24 cd2.31 bc2.40 ab2.37 bc2.53 a2.41 ab0.030.0020.0120.000
DFI (%/d) *2.262.142.162.152.232.262.140.030.7380.7580.600
FCR *1.18 a1.08 b1.07 b1.04 b1.07 b1.05 b1.01 b0.020.0270.0150.033
HSI (%) #1.722.252.211.961.922.192.010.060.0700.8210.782
VSI (%) #8.008.128.448.048.048.668.210.090.4150.450.396
IPF (%) #2.722.382.482.402.602.212.680.060.1150.7150.132
CF (%) #2.952.973.143.013.052.952.910.030.2330.1390.301
Survival (%)100.0098.6798.6797.33100.00100.0097.330.490.5790.4580.441
* Values are the means of three triplicates per dietary treatment. # Values are the means of 12 fish per dietary treatment. IBW, initial body weight; FBW, final body weight; WG, weight gain; SGR, specific growth rate; DFI, daily feed intake; FCR, feed conversion ratio; HSI, hepatosomatic index; CF, condition factor; VSI viscerasomatic index; IPF, intraperitoneal fat rate. Values in the same row with different superscripts are significantly different (p < 0.05).
Table 3. Whole-body proximate composition (%) of orange-spotted grouper fed diets with carnosine supplementation.
Table 3. Whole-body proximate composition (%) of orange-spotted grouper fed diets with carnosine supplementation.
ItemsDiets with Carnosine Doses (mg/kg)Pooled SEMp-Value
010204080160320ANOVALinearQuadratic
Moisture69.0769.4669.3969.4369.7869.3769.800.100.5930.1600.367
Crude protein17.22 b17.95 a18.19 a18.19 a17.97 a18.42 a18.32 a0.100.0150.0430.033
Crude lipid8.43 a7.47 b7.48 b7.19 b6.90 b7.13 b7.24 b0.140.0400.1610.036
Ash4.214.324.534.514.454.364.250.040.2990.3660.314
Values in the same row with different superscripts are significantly different (p < 0.05).
Table 4. Serum biochemical indices of orange-spotted grouper fed diets with carnosine supplementation.
Table 4. Serum biochemical indices of orange-spotted grouper fed diets with carnosine supplementation.
ItemsDiets with Carnosine Doses (mg/kg)Pooled SEMp-Value
010204080160320ANOVALinearQuadratic
TP (g/L)29.24 c39.24 b41.98 ab41.77 ab41.29 b43.31 ab46.66 a0.640.0020.0060.013
TG (mmol/L)7.336.505.465.706.556.796.740.200.1710.4680.750
TC (mmol/L)1.711.511.361.631.861.461.690.060.3580.6380.896
LZM (µg/mL)13.79 d20.37 c54.93 b69.70 a59.42 b59.08 b24.29 c4.790.0000.7730.001
IgM (µg/mL)291.79305.61279.24283.19336.10293.02364.5412.340.5230.0710.183
Values in the same row with different superscripts are significantly different (p < 0.05). TP, total protein; TG, triglycerides; TC, total cholesterol; LZM, lysozyme.
Table 5. Digestive enzyme activities of orange-spotted grouper fed diets with carnosine supplementation.
Table 5. Digestive enzyme activities of orange-spotted grouper fed diets with carnosine supplementation.
ItemsDiets with Carnosine Doses (mg/kg)Pooled SEMp-Value
010204080160320ANOVALinearQuadratic
Amylase (U/mg protein)0.24 a0.18 ab0.09 c0.13 bc0.13 bc0.21 ab0.20 ab0.010.0130.2980.478
Trypsin (U/mg protein)57.08 e144.11 d185.33 d209.03 cd277.37 bc295.19 b379.14 a27.970.0000.0000.000
Lipase (U/g protein)3.092.902.203.263.513.923.680.200.3240.0790.098
Values in the same row with different superscripts are significantly different (p < 0.05).
Table 6. Muscle antioxidant capacity of orange-spotted grouper fed diets with carnosine supplementation.
Table 6. Muscle antioxidant capacity of orange-spotted grouper fed diets with carnosine supplementation.
ItemsDiets with Carnosine Doses (mg/kg)Pooled SEMp-Value
010204080160320ANOVALinearQuadratic
TAOC (mmol/g)0.002 c0.005 c0.010 bc0.012 b0.011 b0.016 ab0.021 a0.000.0010.0000.000
CAT (U/mg protein)0.14 b0.16 b0.37 b0.46 ab0.53 ab0.68 a0.46 ab0.050.0050.0640.000
GSH-Px (U/mg protein)6.17 d11.80 c12.42 bc14.07 bc14.97 bc15.57 b19.39 a1.070.0000.0000.001
SOD (U/mg protein)2.26 b3.79 a3.75 a3.74 a3.86 a4.38 a4.35 a0.180.0010.0100.005
MDA (nmol/mg protein)1.18 a1.00 b0.90 bc0.93 bc0.86 bc0.76 cd0.73 d0.040.0020.0010.000
TAOC, total antioxidant capacity; CAT, catalase; GSH-Px, glutathione peroxidase; SOD, superoxide dismutase; MDA, malondialdehyde. Values in the same row with different superscripts are significantly different (p < 0.05).
Table 7. Liquid holding capacity and pH in the muscle of orange-spotted grouper fed diets with carnosine supplementation.
Table 7. Liquid holding capacity and pH in the muscle of orange-spotted grouper fed diets with carnosine supplementation.
ItemsDiets with Carnosine Doses (mg/kg)Pooled SEMp-Value
010204080160320ANOVALinearQuadratic
pH-5 min6.766.746.936.886.686.956.780.040.4880.9510.779
pH-24 h6.656.366.636.476.466.526.570.030.1940.7700.673
Liquid loss (%)9.839.529.418.758.647.888.210.220.1270.0130.005
Water loss (%)8.778.858.468.097.517.257.430.190.0850.0100.003
Lipid loss (%)1.070.670.950.661.130.630.790.080.5690.5920.835
Table 8. Muscle texture of orange-spotted grouper fed diets with carnosine supplementation.
Table 8. Muscle texture of orange-spotted grouper fed diets with carnosine supplementation.
ItemsDiets with Carnosine Doses (mg/kg)Pooled SEMp-Value
010204080160320ANOVALinearQuadratic
Hardness (g)109.82 c130.88 bc132.95 bc156.7 ab153.05 ab158.88 ab163.96 a4.730.0150.0000.000
Adhesiveness (g.s)15.43 d18.18 cd20.66 bcd20.66 bcd25.26 ab29.96 a24.38 abc1.210.0010.0000.001
Springiness0.930.940.940.920.920.910.920.010.3120.0470.142
Cohesiveness0.640.610.620.620.650.650.650.010.3620.0780.135
Gumminess (g)70.66 c83.58 bc81.60 bc94.59 abc99.02 ab102.49 ab107.00 a3.450.0370.0000.001
Chewiness (g)58.1579.4276.1986.4790.4892.3198.213.850.1220.0020.007
Resilience0.260.310.330.330.320.330.340.010.1270.0140.020
Values in the same row with different superscripts are significantly different (p < 0.05).
Table 9. Muscle contents of histidine-containing dipeptides of orange-spotted grouper fed diets with carnosine supplementation.
Table 9. Muscle contents of histidine-containing dipeptides of orange-spotted grouper fed diets with carnosine supplementation.
ItemsDiets with Carnosine Doses (mg/kg)Pooled SEMp-Value
010204080160320ANOVALinearQuadratic
Carnosine (mg/g)5.465.95.716.266.016.186.570.160.6500.0770.203
Anserine (mg/g)0.540.580.660.600.640.610.680.020.3190.0910.247
Carnosine/Anserine ratio10.189.589.469.9210.009.979.720.200.9810.9690.942
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MDPI and ACS Style

Li, D.; Chen, W.; Yin, Y.; Yang, L.; Chen, M.; Sun, Y.; Ye, J. Effects of Carnosine Addition in Low-Fishmeal Feed on the Growth Performance, Muscle Antioxidant Capacity and Flesh Quality of Orange-Spotted Grouper (Epinephelus coioides). Fishes 2024, 9, 467. https://doi.org/10.3390/fishes9110467

AMA Style

Li D, Chen W, Yin Y, Yang L, Chen M, Sun Y, Ye J. Effects of Carnosine Addition in Low-Fishmeal Feed on the Growth Performance, Muscle Antioxidant Capacity and Flesh Quality of Orange-Spotted Grouper (Epinephelus coioides). Fishes. 2024; 9(11):467. https://doi.org/10.3390/fishes9110467

Chicago/Turabian Style

Li, Dong, Weijun Chen, Yanxia Yin, Lulu Yang, Mingfan Chen, Yunzhang Sun, and Jidan Ye. 2024. "Effects of Carnosine Addition in Low-Fishmeal Feed on the Growth Performance, Muscle Antioxidant Capacity and Flesh Quality of Orange-Spotted Grouper (Epinephelus coioides)" Fishes 9, no. 11: 467. https://doi.org/10.3390/fishes9110467

APA Style

Li, D., Chen, W., Yin, Y., Yang, L., Chen, M., Sun, Y., & Ye, J. (2024). Effects of Carnosine Addition in Low-Fishmeal Feed on the Growth Performance, Muscle Antioxidant Capacity and Flesh Quality of Orange-Spotted Grouper (Epinephelus coioides). Fishes, 9(11), 467. https://doi.org/10.3390/fishes9110467

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