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Article

Glycerol Monolaurate Affects Growth, Amino Acid Profile, Antioxidant Capacity, Nutrient Apparent Digestibility, and Histological Morphology of Hepatopancreas in Juvenile Pacific White Shrimp (Litopenaeus vannamei)

1
Zhejiang University Zhongyuan Institute, Zhengzhou 450001, China
2
College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China
*
Authors to whom correspondence should be addressed.
Fishes 2025, 10(3), 124; https://doi.org/10.3390/fishes10030124
Submission received: 14 January 2025 / Revised: 5 March 2025 / Accepted: 7 March 2025 / Published: 11 March 2025

Abstract

An eight-week feeding trial was conducted to evaluate the effects of dietary supplementation with glycerol monolaurate (GML) on Pacific white shrimp (Litopenaeus vannamei). A basal diet was formulated containing 100 g fish meal, while four additional GML-supplemented diets were prepared: GML1 (0.25 g), GML2 (0.50 g), GML3 (0.75 g), and GML4 (1.00 g). Each diet was given to triplicate tanks containing 50 shrimp, each weighing 1.67 ± 0.25 g. GML2 supplementation enhanced the final body weight, weight gain, condition factor, specific growth rate, and viscerosomatic index of the shrimp compared to the other diets (p < 0.05). The whole-body amino acid profile was significantly high in the GML3 group. The antioxidant and immune indicators in the serum, like total protein, triglyceride, and aspartate aminotransferase, were significantly high in the GML2-supplemented group. The immune and antioxidant indicators in the hepatopancreas of the shrimp, like total protein, triglyceride, total cholesterol, and complement protein 3, were significantly high in the GML2 group. However, the malondialdehyde in their livers and serum were significantly high in the control group. Digestive enzymes were significantly high in the GML2 group. In conclusion, this study confirms that GML may benefit the health of Pacific white shrimp, offering new insights into aquaculture.
Key Contribution: This research focused on glycerol monolaurate, which enhanced the growth, digestive enzyme activity, immune system, and blood antioxidant levels, while reducing oxidative stress in the hepatopancreas of Pacific white shrimp (Litopenaeus vannamei) in the GML-supplemented groups.

1. Introduction

The Pacific white shrimp (Litopenaeus vannamei) belongs to the family Penaeidae, is native to the eastern Pacific Ocean, is globally farmed using intensive and semi-intensive aquaculture methods [1], and is an economically important species [2]. Due to its palatability, tenderness, nutritional value, and high tolerance to salinity and temperature, L. vannamei has been the choice of many aquaculture farmers and consumers [3]. The need to increase production led to an increase in the shrimp culture density; as a result of this, the increased crowding can affect the water quality and the immune system, allowing microorganisms to take advantage of this situation, resulting in oxidative stress, disease outbreaks, and the suppression of immunological and physiological functions in shrimp [4]. The susceptibility of L. vannamei to devastating and prevalent diseases, including white spot syndrome virus and acute hepatopancreatic necrosis disease, is a big challenge and causes enormous losses in the world [5]. The overuse of antibiotics further aggravates these issues, reducing the shrimps’ ability to combat pathogens due to the development of antibiotic resistance. This underscores the urgent need to identify alternative strategies to sustain shrimp health. In aquafeeds, fishmeal serves as an ideal source of protein due to containing plenty of protein and having high palatability, good taste, and a good amino acid profile [6]. As a result, nowadays, researchers are more focused on feed additives that could replace antibiotics and improve growth. Among these alternatives, the medium-chain fatty acid (MCFA) has attracted significant interest. Medium-chain fatty acids are a group of energy-yielding compounds with unique physiological roles, making them promising feed additives and potential alternatives to antibiotics [7]. The potential of antimicrobial agents has attracted attention due to their contribution to good metabolic functions, and these have been found naturally occurring in coconut oil [8]. The swift uptake of MCFAs suggests that these fatty acids have an important physiological function in aquatic animals [9]. Studies indicate that MCFA monoglycerides, particularly GML, exhibit antipathogenic properties [10]. Glycerol monolaurate (GML), a fatty acid glyceride from the medium-chain monoglyceride group, which is readily absorbed, exhibits potent antioxidant properties, and is highly digestible [11]. Glycerol monolaurate is approved by the U.S. Drug Administration and Food; it is a food emulsifier classified as Generally Recognized as Safe (GRAS) and is considered to be a nontoxic and natural compound [12].
Glycerol monolaurate exhibits relative stability, and it goes through the gastrointestinal tract, resulting in an extended residence time [13]. The extended presence enables direct engagement with the gut microflora, which is essential for physiology and host health, especially regarding the immune system and metabolism [14]. Additionally, GML demonstrated health benefits, along with strong antimicrobial and anti-inflammatory properties [15,16]. Glycerol monolaurate has the potential to decrease bacterial and viral infectivity in feed, which may aid in reducing disease transmission [17]. The influence of diverse symbiotic gut bacteria on the host’s metabolism and immune system is now well established, stemming from interactions involving gene products and microbial cell components, including lipopolysaccharides, flagellin, and peptidoglycan [18]. To date, supplementation with GML has demonstrated considerable potential in improving meat quality and productivity in poultry [19] and aquaculture species [20]. Glycerol monolaurate, a metabolite in animals, does not encourage the resistance of pathogens and is used in shrimp [11]. Glycerol monolaurate can enhance the growth performance in shrimp by improving immune function, gut microbiota, and intestinal histomorphology [11]. Research indicates that incorporating GML into the diet of broilers markedly improves the feed intake, average body weight, and carcass yield in male broilers [21]. Nonetheless, a substantial gap persists in the research concerning the GML effects on aquatic animals. Very few studies suggest that GML can notably improve the growth of zebrafish [20], Chinese softshell turtles [22], and Larimichthys croceus [23]. Additionally, glycerol monolaurate enhances the growth, lipid metabolism, and antioxidant capacity in white-leg shrimp, Penaeus vannamei [24]. Although GML has been shown to boost the nonspecific defense mechanisms in shrimp, the optimal amounts (both frequency and quantity) for inclusion in diets remain undefined for some species. This study aimed to evaluate the effects of varying levels of glycerol monolaurate supplementation in aquatic feed on the performance of growth, body composition, digestive enzymes, serum biochemical, and hepatopancreatic and immunological parameters, as well as histological changes in the hepatopancreas (H&E staining) of Pacific white shrimp, with the goal of determining the optimal GML dose for this species.

2. Experimental Procedures and Methodologies

2.1. Dietary Preparation and Formulation

Glycerol monolaurate (GML) was provided by Kangyuan Food Technology Limited, based in Hangzhou, Zhejiang. In accordance with the nutritional requirements of Litopenaeus vannamei, five isoenergetic, e.g., 17.89 kJ/g gross energy, and isonitrogenous, e.g., 40.00% crude protein, diets were developed: a control diet (GML0.00), alongside four additional diets with progressively increasing levels of GML (0.25 g/kg, 0.50 g/kg, 0.75 g/kg, and 1.00 g/kg). The main protein sources in these diets comprised fermented shrimp meal, soybean meal, fishmeal, and soybean protein concentrate. To fulfill the isoenergetic and isonitrogenous requirements in all the diets, α-starch was incorporated. The detailed composition of these ingredients is outlined in Table 1.
The amino acid (AA) composition of five experimental diets is detailed in Table 2. Prior to weighing and thorough mixing, all the ingredients were minced to a good consistency using a 178 μm mesh; homogenized and weighed 0.1% yttrium oxide (Y2O3) was used as an exogenous indicator for apparent digestibility [25]. The homogenized mixture was subsequently processed into pellets with a diameter of 1.2 mm utilizing a feed machine (Model HKJ-218; HUARUI). After the formation of the pellets, they were subjected to steaming for a duration of 10 min and then dried for 72 h in a controlled-air environment. The diets were maintained at −20 °C until utilized.

2.2. Experimental Setup

Healthy Litopenaeus vannamei were sourced from the Mariculture Research Institute located in Zhejiang province, China. The feeding trial was carried out at Zhoushan, China, Xixuan Island. Before the experiment commenced, the shrimp were acclimatized to the culture conditions by being provided with a commercial diet for a duration of four weeks. After a 24 h fasting period, 750 healthy shrimp (mean initial body weight: 1.67 ± 0.25 g) were randomly nominated and placed into 15 fiberglass tanks (Φ = 80 cm and a height of 65 cm) with a capacity of 500 L each and 50 shrimp per tank (number of shrimp = 100 shrimp/m3 × 0.5 m3 = 50 shrimp), with each dietary treatment allocated and divided into three tanks. The tanks were continuously aerated with air stones. Over the course of the eight-week feeding trial, the shrimp were farmed in a flow-through system (2 L/min), which provided a continuous supply of filtered seawater. The fish were fed to apparent satiation three times daily at 08:00, 12:00, and 16:00, all under natural light conditions. The feces and useless food were systematically removed on a daily basis. The water parameters were upheld as follows: dissolved oxygen, ≥6.5 mg/L; pH, 8.0–8.3; temperature, 26–28 °C; total ammonia nitrogen, 0.3–0.5 mg/L; and salinity, 27.2–30.0 g/L. The environments were well within the optimal range for the survival and growth of shrimp.

2.3. Digestibility Assessment

Starting from the 7th week, feces were collected daily before 06:00 using the siphon method. The fecal matter was then allowed to settle, filtered, and transferred into sealed bags for storage at −20 °C. Subsequently, the samples were sieved and dried. Following dry digestion, the yttrium (Y) content in the fecal samples was quantified using a coupled plasma atomic emission spectrometer inductively (U.S, Thermo Electron., Waltham, MA, USA).

2.4. Collection of Samples

Upon the completion of the eight-week trial of feeding and subsequent 24 h fasting period, all the surviving shrimp were meticulously counted and weighed. Fifteen shrimp were randomly selected from each tank to assess their total hepatopancreas weight, body weight, and length. Furthermore, 10 shrimp were collected from each tank for the analysis of their proximate chemical composition. The leftover shrimp were used to collect samples of the gut, hepatopancreas, and dorsal muscles. Hemolymph was taken from the pericardial sinus, positioned at the base of the first abdominal segment, employing a 1 mL disposable sterile syringe. Solution was subsequently combined with an equal volume of precooled anticoagulant, which consisted of 0.16 M disodium hydrogen phosphate, 0.34 M sodium chloride, 0.02 M EDTA, and 0.04 M sodium dihydrogen phosphate, and adjusted to a pH of 7.4. Plasma for the biochemical analysis was collected following centrifugation at 3500× g for 10 min at 4 °C and was subsequently frozen immediately in liquid nitrogen. The samples of hepatopancreas were promptly frozen in liquid nitrogen following dissection for further genetic analysis and biochemical analysis. All the samples preserved in liquid nitrogen were maintained at −80 °C. The hepatopancreas samples were prepared for histological examination by being fixed in a 10% (v/v) formaldehyde solution for a duration of 24 h. Subsequently, they were transferred to 70% ethanol for long-term preservation until further analyses could be conducted.

2.5. The Analytical Procedures for Composition Determination

The crude lipid, moisture, crude protein, and ash contents of the five diets, as well as in the dorsal muscles and the whole bodies of the shrimp, were assessed following the standards set by the Association of Official Analytical Chemists [26]. For the moisture analysis, the ground samples were dried at 105 °C in a forced air oven for 24 h. The crude protein content was calculated using the Kjeldahl nitrogen method, with a conversion factor of 6.25. Crude lipids were extracted using the Soxhlet method, with ether, for a duration of 6 h. To determine the total ash content, the samples were incinerated in a muffle furnace for 6 h at 550 °C. Meanwhile, the total phosphorus concentration was determined using the molybdovanadate method [27]. This process involved weighing the ash, subjecting it to wet digestion with HNO3 and HCl, and subsequently analyzing the resulting solution. The amino acid compositions of the whole-body shrimp and the diets were analyzed using an automatic amino acid analyzer (Hitachi LA8080, Tokyo, Japan) after the samples were hydrolyzed in 6 M HCl for 24 h at 110 °C; for the methionine analysis, a 15 min oxidative hydrolysis was performed using performic acid at 55 °C prior to acid hydrolysis. Tryptophan was degraded by HCl during the acid hydrolysis process and, therefore, was not measured independently [28].

2.6. Methods for Biochemical Assays

The hepatopancreases were homogenized in PBS at a ratio of 1:9 and subsequently centrifuged at 4000× g for 10 min at a temperature of 4 °C. The supernatants obtained were utilized to assess various biochemical parameters through the use of suitable assay kits. The total protein (TP) content was assessed using the BCA Protein Assay Kit (A045-3, Nanjing, China, Jiancheng Bioengineering Institute). Malondialdehyde (MDA), superoxide dismutase (SOD), and the total antioxidant capacity (T-AOC) levels in the livers were measured using the A003-1, A001-3, and A015-1 kits, respectively (Nanjing, China, Jiancheng Bioengineering Institute). Complement protein 3 (C3) was quantified using a specific kit from the same supplier. Triglycerides (TG), aspartate aminotransferase (AST), total cholesterol (T-CHO), and lysozyme (LZM) levels were measured using the A110-1-1, C010-2-1, A111-1-1, and A050-1-1 kits, respectively. Lipase, amylase, trypsin, and cellulase activities were assessed using the A054-1-1, C016-1-1, A080-2, and A138 kits, also from Jiancheng Bioengineering Institute 3.

2.7. Formulae and Statistical Analysis

The following equations were used to assess growth performance and feed utilization:
Initial average body weight (IBW, g)
Final average body weight (FBW, g)
Weight gain (WG, %) = 100 × (final body weight − initial bodyweight)/initial body weight
Specific growth rate (SGR, %/day) = 100 × (ln final body weight − ln initial body weight)/days
Mean feed intake (MFI, g shrimp−1 d−1) = air dry fed in g/(shrimp in g × day)
Feed conversion ratio (FCR) = dry feed weight (g)/wet weight gain (g)
Condition factor (CF, g cm−3) = 100 × [(final body weight in g)/(final body length in cm)3]
Hepatosomatic index (HSI, %) = 100 × (liver weight in g/body weight in g)
Viscerosomatic index (VSI, %) = 100 × (viscera weight/body weight)
Survival rate (SR, %) = 100 × (final shrimp number/initial shrimp number)
The apparent digestibility coefficient (ADC) of crude protein, crude lipid, ash, dry matter, and gross energy (kJ/g) (ADCG) were calculated as follows: [29]:
ADC of dry matter (%) = 100 × [1 − (dietary Cr2O3)/faecal Cr2O3]
ADC of nutrients or energy (%) = 100 × [1 − (F/D − DY/FY)]
where FBW is the final body weight in g, IBW is the initial body weight in g, t is the experimental duration in days, F is the per cent of nutrients or energy in the feces, D is the per cent of nutrients or energy in the diet, DY is the per cent of Cr2O3 in the diet, and FY is the per cent of Cr2O3 in the feces.
The data were presented as mean ± standard deviation (SD). The statistical analysis was conducted using a one-way ANOVA, followed by a Tukey’s post-hoc test. A significance level of p < 0.05 was considered for determining statistically significant differences. All the statistical analyses were performed using IBM SPSS 20.0 (IBM, Chicago, IL, USA).

3. Results

3.1. Growth Performance and Feed Utilization Parameters

To visually evaluate the effect of GML in Pacific white shrimp, we first analyzed the growth performance and body indexes. As described in Table 3, the GML effects on the growth performance and body indexes of the shrimp were compared with the control diet. FBW was significantly higher in GML2 and GML3, but no differences were observed in the control, GML1, or GML4. In comparison with the control diet, WG and SGR were not significantly different, but the higher value was observed in GML2, while the lower value was observed in GML4. CF was significantly low in the GML1 and GML4 groups, as compared to GML2 group. However, the GML3 group had significantly lower HSI values than all the other diets, but no differences were observed in the control GML1, GML2, or GML4 groups. The GML1, GML2, and GML3 groups had significantly lower VSI values than the control and GLM4 groups, but no differences were observed in the control and GML4 groups. There were no statistically significant differences in IBW, MFI, FCR, and SR.

3.2. Whole Body and Dorsal Muscle Proximate Composition

Table 4 shows the analysis of the proximate composition of whole bodies and dorsal muscles. No significant variation was found in the proximate values of the whole body and dorsal muscle in moisture, lipid, protein, ash, and phosphorus content (p > 0.05). The ash in the whole bodies of GML1, GML2, GML3, and GML3 shrimp was significantly lower compared to the control group (p < 0.05). However, no differences were observed between GML1, GML2, GML3, and GML4.

3.3. Amino Acid Profiles of Whole Bodies of Litopenaeus vannamei

Table 5 presented the impact of the five different diets on the amino acid profiles of Litopenaeus vannamei. In comparison with the control diet, threonine and methionine were not significantly different, but the higher value was observed in the GML2 group, while the lower value was observed in control diet group. Lysine was significantly lower in the control group and the GML4 group, as compared to the GML3 group. However, valine, isoleucine, phenylalanine, leucine, arginine, and histidine were not significantly affected in any of the groups. In NEAA, aspartate was significantly lower in the GML1, GML4, and control groups, as compared to the GML3 group. Glutamic acid was significantly lower in the GML1, GML2, GML4, and control groups, as compared to the GML3 group. Glycine was significantly lower in the GML4 and control groups, as compared to the GML3 group. Proline was significantly lower in the GML1, GML4, and control groups, as compared to the GML3 group. Alanine, cysteine, serine, and tyrosine were not significantly high in any of the groups.

3.4. Nutrients Apparent Digestibility

Table 6 presents the apparent digestibility coefficients (ADCs) for crude protein, crude lipid, ash, gross energy, and dry matter. No significant variation was observed in the apparent digestibility coefficients (ADCs).

3.5. Serum and Hepatopancreas Antioxidant Parameters

Figure 1 and Figure 2 show the immune and antioxidant parameters in the serum and hepatopancreases. The immune and antioxidant indicators in the serum, like TP and TG, were significantly lower in GML1, as compared to GML2 (0.5 g). AST was significantly lower in GML3, as compared to GML2 (0.5 g). However, the MDA in the serum was significantly high in the control group, while T-CHO, LZM, T-AOC, SOD, and C3 showed no significant effects (p > 0.05) in any of the treatment groups. The immune and antioxidant indicators in the hepatopancreas, like TP and TG, were significantly lower in the control group, as compared to GML2. T-CHO was significantly lower in GML4, as compared to GML2. AST was significantly lower in the control, GML1, and GML4 groups, as compared to GML2. C3 was significantly lower in the control and GML4, as compared to GML2. However, LZM, T-AOC, and SOD showed no significant effects (p > 0.05) in any of the supplemented groups. However, the MDA levels in the hepatopancreases were significantly lower in GML3 and GML4, as compared to the control group.

3.6. Digestive Enzymes Activities Hepatopancreas

The digestive enzymes activities within the hepatopancreas are presented in Figure 3. Lipase levels were significantly lower in the GML4 group, as compared with the GML2 group (p < 0.05). Trypsin and cellulase levels were significantly lower in the control, as compared with the GML2 group (p < 0.05). While amylase was not significantly high in any of the groups (p > 0.05).

4. Discussion

The potential for balancing amino acid composition through the supplementation of synthetic plant-based protein amino acids for aquaculture has been widely emphasized and studied [30]. GML, an MCFA with nutritional properties, has been shown to promote health and growth in shrimp, broilers, and mice when used as a feed supplement. It achieves this by enhancing immunomodulatory functions, enzymatic digestibility, and the gut microbiota composition [16,31]. A study indicated that incorporating dietary GML enhanced immunity and growth parameters in shrimp [11]. In the current investigation, our results confirmed that supplementing with GML significantly enhanced the body weight, specific growth rate, weight gain rate, hepatosomatic index, and condition factor of the shrimp. Additionally, improvements in digestive enzyme activities were observed in the GML2 group (0.50 g) after eight weeks of the feeding experiment. These findings align with earlier research conducted on shrimp [11,24]. This underscores the efficacy of GML as a growth-enhancing additive in aquatic feed [32].
The current investigation demonstrated that dietary supplementation with GML notably increased the body weight (BW) in shrimp. These results align with earlier research, which similarly indicated an increase in weight gain, enhanced feed intake, and a decrease in the feed conversion ratio after GML supplementation [11]. These findings align with previous studies, where Acanthopagrus schlegelii fed diets with lauric acid exhibited significant increases in their feed intake, specific growth rate, weight gain ratio, and body weight [33]. These findings align with earlier research, indicating that broilers receiving diets enhanced with GML showed notable increases in their feed intake and body weight (BW) by the conclusion of the study [21]. Similar outcomes were observed when GML was incorporated into the diet of black sea bream, where it significantly enhanced their FBW, WGR, and SGR. Additionally, their feed intake and body weight were significantly increased [34]. Comparable results were noted when GML was included in the diet of mice, leading to significant increases in BW [16]. Furthermore, studies involving pigs have demonstrated that GML has significant potential as a growth promoter and a viable alternative to antibiotics in animal management [35], a finding that that the results of our current study are consistent with.
This study also observed no significant impacts of the dietary treatment on the proximate composition of the dorsal muscle or the whole body. These findings align with earlier research on black sea bream [33,34], as well as studies conducted on grass carp and Arctic char [36]. In contrast, feeding African catfish and red drum with LCT or CO did not result in significant changes in their body lipid content [37]. In the current study, dietary GML significantly increased the ash content in the whole body of the control group in shrimp, a finding that contrasts with previous studies, where GML supplementation led to a marked reduction in the ash content [33].
The content of essential amino acids (EAAs), which serves as a critical measure of the nutritional value in meat production [38], displayed a significant rise in the groups treated with GML, indicating that GML improved the nutritional quality. The levels of both EAAs (essential amino acids) and NEAAs (non-essential amino acids) were significantly elevated in the groups supplemented with GML, showing increased relative concentrations of EAAs (threonine, methionine, and lysine) and NEAAs (aspartate, glutamic acid, glycine, and proline) when compared to the control group. Furthermore, the relative abundance of desirable amino acids that contribute to umami (Glu and Asp) or sweet (Ser, Gly, Ala, and Thr) flavors, which influence meat taste, was probably enhanced [39]. Amino acids (AAs) not only contribute to basic tastes like sweet and umami but also act as precursors in flavor formation, playing essential roles in the development of flavor. As a result, the amino acid composition in the shrimp muscle of the GML-treated groups inevitably improved their immune function and cellular function. These findings indicate that GML can improve the amino acid profile in meat production, as observed in broilers [19], while simultaneously enhancing the freshness, nutritional value, and overall flavor of the meat [19]. The presence of sufficient amino acids triggers the activation of the pyruvate dehydrogenase system, leading to the stimulation of the mTOR pathway. This process subsequently promotes protein synthesis while simultaneously inhibiting autophagy [40]. Amino acid deficiency triggers the amino acid response (AAR) pathway while simultaneously inhibiting the nutrient-sensing mTOR pathway, resulting in the halt of cellular proliferation [41]. The composition and relative proportions of amino acids are essential for assessing meat quality, given that proteins are made up of amino acids [42]. As reported by Liu et al. [43], adding supplementation with 0.5 g of GML significantly enhanced the content of both EAAs and NEAAs, suggesting that GML supplementation improves the nutritional value of broilers.
In this study, various immune, antioxidant, and biochemical parameters were assessed to evaluate the nutritional and health uptake of Acanthopagrus schlegelii supplemented with GML. The parameters measured included total protein, triglycerides, total cholesterol, aspartate aminotransferase, total antioxidant capacity, lysozyme, superoxide dismutase, malondialdehyde, and complement component 3. The results demonstrated a beneficial effect of 0.5 g GML supplementation on the immune and physiological functions of shrimp in the treated groups. Previous studies have reported analogous findings regarding black sea bream [33], various poultry breeds [44], and in relation to the enhancement of immune system strength [45]. The GML group exhibited higher levels of total protein (TP) and triglycerides (TG). Previous studies have reported analogous findings in black sea bream [33]. The increased TP observed in this study indicates enhanced protein metabolism [46]. Medium-chain fatty acids (MCFAs) have demonstrated an impact on immune function and lipid metabolism in fish [47].
Biochemical parameters, such as total cholesterol (T-CHO) and aspartate aminotransferase (AST), were elevated in both the serum and the hepatopancreas of the treated groups, suggesting that GML supplementation influenced these tissues. Comparable findings have been reported in rats treated with virgin coconut oil [48].
This study examines dietary supplementation with GML and found that it did not produce significant changes in the immunological parameters of serum or the hepatopancreas, including lysozyme (LZM) activity. This lack of effect may be attributed to potential immunosuppression or immune system fatigue resulting from the prolonged use of immunostimulants [49]. The findings align with earlier studies [34]. A notable elevation in complement protein 3 (C3) levels was recorded in the GML-supplemented group in comparison to the control group. The findings align with those presented in previous studies [34].
The results indicated that GML supplementation influenced T-AOC levels, suggesting that lauric acid (LA) can enhance the body’s antioxidant enzyme activity, which in turn helps combat ROS. These findings are aligned with previous studies [33,50,51], which have proposed that certain compounds in coconut oil, including LA and MCFAs, when used as dietary supplements, can boost antioxidant enzyme levels, thereby mitigating diseases associated with oxidative stress [52]. SOD levels were elevated in the treated groups, as earlier observed in broilers [43]. The increase in SOD activity in the GML-supplemented groups suggests that GML enhanced the antioxidant capacity of the shrimp, which likely contributed to the observed improvements in growth. Additionally, the current study found a significant reduction in serum MDA levels. Consistent with our findings [33], studies on black sea bream [53] have also reported that citric acids and malic possess antioxidant properties. The available data on aquatic animals may be limited; however, the existing literature corroborates the current findings, demonstrating that GML supplementation improves immunity, serum biochemistry, and antioxidant parameters in Pacific white shrimp.
A variety of enzymes are essential for the digestion of food materials in the digestive tract, which can subsequently lead to increased weight gain and improved fish health. The measurement of the activities of digestive enzymes is a valuable method for assessing the assimilation of the nutrient capacity of fish when fed a specific diet [54]. The growth of shrimp is dependent on the efficient utilization of nutrients, which is closely associated with digestive enzymes activity, such as trypsin, amylase, lipase, and cellulase in the intestine and the hepatopancreas [55]. These enzymes play a critical role in food digestion within the digestive tract, ultimately contributing to improved overall health and growth in shrimp. Digestive enzyme activity is a valuable method for evaluating the ability of shrimps’ nutrient assimilation from their diet [56]. One possible explanation is that MCFAs may stimulate the release of a peptide hormone and cholecystokinin in the gastrointestinal system, which in turn triggers secretion of digestive enzymes [57]. The dietary interventions in this study led to a notable enhancement in the activities of lipase, trypsin, and cellulase. Comparable results have been documented, where the supplementation of Cuphea seeds, along with MCFA as a source of lipase, enhanced digestive enzyme activity in piglets. However, while non-significant change was showed in amylase activity, other digestive enzymes in the foregut region showed a marked increase [13,33].
The hepatopancreas, which functions similarly to the pancreas and liver in mammals, plays a crucial role in the metabolism of nutrients and overall health in crustaceans [58]. Serving as an integrated organ that combines the roles of the liver, intestine, and pancreas in vertebrates, the hepatopancreas also acts as an antioxidative center in shrimp [59]. Our findings revealed elevated hepatosomatic index levels in shrimp fed with the control diet. An increased HSI is often associated with liver enlargement [60]. In contrast, the current study observed a reduction in HSI following GML supplementation. It has been described that dietary GML enhances the amino acid profile, and it is well established that a balanced nutrient intake plays a vital role in regulating liver size [61]. Additionally, effective amino acid supplementation in feed has been shown to reduce the accumulation of fat in the liver [62]. A deficiency in dietary essential amino acids (EAAs) has been associated with various physiological disturbances, including damage to the liver and the inhibition of intestinal epithelial growth [63]. In our current findings, the 0.50 g GML-supplemented diet significantly increased the number of growths, which indicated that GML enhanced the serum and hepatopancreas antioxidant parameters and digestive enzymes of juvenile Pacific white shrimp (Litopenaeus vannamei).

5. Conclusions

In conclusion, this study suggests that an appropriate level of dietary GML in an SBM-based diet can improve growth performance, immune response, and digestive enzyme activity. The findings indicate that GML is highly beneficial, with 0.50 g being the optimal dosage identified in our study. Additionally, incorporating GML into the diet resulted in diminished cellular damage in the hepatopancreas. However, further molecular research is needed to better understand the regulatory mechanisms and physiological functions of GML.

Author Contributions

S.U.: Conceptualization. Data curation. Formal analysis. Investigation. Methodology. Writing—original draft. Writing—review and editing. B.L.: Conceptualization and editing. Y.Z.: Methodology. Formal analysis. H.G.: Data curation. Formal analysis. Investigation. Y.Y.: Data curation. Formal analysis. Review. M.I.A.: Formal analysis. Writing—review and editing. S.L.: Data curation. Formal analysis. Review. S.D.: Formal analysis. Investigation. M.Z.: Conceptualization. Formal analysis. Writing—review and editing. F.F.: Conceptualization. Funding acquisition. Methodology. Supervision. Investigation. Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the Project of Zhejiang Provincial Department of Agriculture and Rural Affairs (Grant Number: 2024SNJF055) and the Key Research and Development Program of Ningbo, Grant Number: 2023Z113.

Institutional Review Board Statement

The experimental procedures followed in this study were in accordance with the Guidelines for the Care and Use of Laboratory Animals in China. Ethical approval for the study was granted by the Animal Ethics Committee of Zhejiang University (Ethics code: ZJU20190052), approval date: 11 June 2019. Strict protocols were adhered to in order to ensure the proper handling and welfare of all the shrimp throughout the duration of the experiment.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are included in this article; further inquiries can be directed to the corresponding authors.

Acknowledgments

We acknowledge the China–Norwegian Joint Laboratory of Nutrition and Feed for Marine Fish, Xixuan Island, Zhoushan City, Zhejiang Province, and the Key Laboratory of Mariculture and Breeding of Zhejiang Province for their provision of the experimental rearing system and logistical support during the fish growth trial.

Conflicts of Interest

The authors declare that they have no financial or personal conflicts of interest that could have influenced the outcomes of this research.

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Figure 1. The effect of GML on serum antioxidant parameters for Pacific white shrimp (Litopenaeus vannamei). Different letters on bars show significant difference (p < 0.05). a, ab, b letters indicate statistical significance between the diet groups. a and ab are not significantly different, while a vs. b show a significant difference. (a) TP, total protein; (b) TG, triglyceride; (c) T-CHO, total cholesterol; (d) AST, aspartate aminotransferase; (e) LZM, lysozyme; (f) T-AOC, total antioxidant capacity; (g) SOD, superoxide dismutase; (h) MDA, malondialdehyde; (i) C3, complement protein.
Figure 1. The effect of GML on serum antioxidant parameters for Pacific white shrimp (Litopenaeus vannamei). Different letters on bars show significant difference (p < 0.05). a, ab, b letters indicate statistical significance between the diet groups. a and ab are not significantly different, while a vs. b show a significant difference. (a) TP, total protein; (b) TG, triglyceride; (c) T-CHO, total cholesterol; (d) AST, aspartate aminotransferase; (e) LZM, lysozyme; (f) T-AOC, total antioxidant capacity; (g) SOD, superoxide dismutase; (h) MDA, malondialdehyde; (i) C3, complement protein.
Fishes 10 00124 g001
Figure 2. Effect of GML on hepatopancreas antioxidant parameters for Pacific white shrimp (Litopenaeus vannamei). Different letters on bars show significant difference (p < 0.05). a, ab, b letters indicate statistical significance between the diet groups. a and ab are not significantly different, while a vs. b show a significant difference. (a) TP, total protein; (b) TG, triglyceride; (c) T-CHO, total cholesterol; (d) AST, aspartate aminotransferase; (e) LZM, lysozyme; (f) T-AOC, total antioxidant capacity; (g) SOD, superoxide dismutase; (h) MDA, malondialdehyde; (i) C3, complement protein 3.
Figure 2. Effect of GML on hepatopancreas antioxidant parameters for Pacific white shrimp (Litopenaeus vannamei). Different letters on bars show significant difference (p < 0.05). a, ab, b letters indicate statistical significance between the diet groups. a and ab are not significantly different, while a vs. b show a significant difference. (a) TP, total protein; (b) TG, triglyceride; (c) T-CHO, total cholesterol; (d) AST, aspartate aminotransferase; (e) LZM, lysozyme; (f) T-AOC, total antioxidant capacity; (g) SOD, superoxide dismutase; (h) MDA, malondialdehyde; (i) C3, complement protein 3.
Fishes 10 00124 g002
Figure 3. (ad) Effect of GML on hepatopancreas digestive enzyme for pacific white shrimp (Litopenaeus vannamei). Different letters on bars show significant difference (p < 0.05). a, ab, b letters indicate statistical significance between the diet groups. a and ab are not significantly different, while a vs. b show a significant difference.
Figure 3. (ad) Effect of GML on hepatopancreas digestive enzyme for pacific white shrimp (Litopenaeus vannamei). Different letters on bars show significant difference (p < 0.05). a, ab, b letters indicate statistical significance between the diet groups. a and ab are not significantly different, while a vs. b show a significant difference.
Fishes 10 00124 g003
Table 1. Analysis and proximate composition of the basal diet (g/kg).
Table 1. Analysis and proximate composition of the basal diet (g/kg).
IngredientsDiets
Control
(0.00 g)
GML1 (0.25 g)GML2
(0.50 g)
GML3
(0.75 g)
GML4
(1.00 g)
Fishmeal a100.0100.0100.0100.0100.0
Soybean meal a100.0100.0100.0100.0100.0
Fermented soybean meal a100.0100.0100.0100.0100.0
Soybean protein concentrate a170.0170.0170.0170.0170.0
Squid liver meal a30.030.030.030.030.0
Shrimp meal a50.050.050.050.050.0
Chicken meal a60.060.060.060.060.0
Fish oil a30.030.030.030.030.0
Beer yeast40.040.040.040.040.0
Wheat flour220.0220.0220.0220.0220.0
L-carnitine2.02.02.02.02.0
Soybean lecithin20.020.020.020.020.0
Carrageenan3.73.73.73.73.7
Ascorbic phosphate ester1.01.01.01.01.0
α-Starch2.02.02.02.02.0
GML b0.000.250.500.751.00
Yarrowia lipolytica2.02.02.02.02.0
Carboxymethyl cellulose5.05.05.05.05.0
Mineral premix c5.05.05.05.05.0
Vitamin premix d3.03.03.03.03.0
Zeolite powder21.021.021.021.021.0
Yttrium oxide (Y2O3)1.01.01.01.01.0
L-Lysine3.03.03.03.03.0
Ca(H2PO4)222.022.022.022.022.0
α-Cellulose8.308.057.807.557.30
Butyrin1.01.01.01.01.0
Total1000.01000.01000.01000.01000.0
Proximate composition
 Moisture (%)10.6411.4412.5113.0315.13
 Crude protein (%)37.7337.31 37.6136.8736.34
 Crude lipid (%)7.577.167.187.288.08
 Crude ash (%)13.58 13.51 13.61 12.87 12.93
 Gross energy (kJ/g) e1603.23 1595.49 1592.45 1596.88 1606.37
 Dry matter98.9998.9498.8498.7098.74
a Obtained from Hangzhou, China, Jin Jia Co., Ltd. b Obtained from Shanghai, China, Sangon Biotech Co., Ltd. c The mineral premix composition (mg/kg) includes CaCO3, 544.9; Na2SiO3, 0.4; KH2PO4, 200; NaH2PO4·H2O, 200; MnSO4·H2O, 4; MgSO4·7H2O, 10; ZnSO4·7H2O, 12; CuCl2·2H2O, 2; NaCl, 12; FeSO4·7H2O; CoCl2·6H2O, 0.1; 12; AlCl3·6H2O, 1; KI, 0.1 Na2MoO4·2H2O, 0.5; and KF, 1. d The vitamin premix composition (mg/kg) includes 80; retinyl acetate, α-tocopherol, 40; 0.1; menadione, cholecalciferol, 15; niacin, 165; riboflavin, 22; HCl, 40; 45; D-Ca pantothenate, pyridoxine thiamine mononitrate, 102; vitamin B12, 0.9; folic acid, 10; ascorbic acid, 150; inositol, 450; thiamine, 5; sodium menadione bisulfate, 15; p-aminobenzoic acid, 50; and choline chloride, 320. 1. e the determination of gross energy was conducted utilizing conversion factors of 23.6 kJ/g for protein, 39.5 kJ/g for lipid, and 17.2 kJ/g for carbohydrate. The carbohydrate content was determined by subtracting the sum of moisture, protein, lipid, and ash from 100.
Table 2. The amino acid composition in the glycerol monolaurate (GML) (g/kg dry matter) of the experimental diets.
Table 2. The amino acid composition in the glycerol monolaurate (GML) (g/kg dry matter) of the experimental diets.
Amino AcidsDiet
Control
(0.00 g)
GML1 (0.25 g)GML2 (0.50 g)GML3 (0.75 g)GML4 (1.00 g)
EAA
 Valine1.733 2.602 2.083 2.589 2.943
 Threonine1.261 1.913 1.660 1.393 1.708
 Isoleucine1.426 2.120 1.714 2.112 2.409
 Methionine0.373 0.526 0.445 0.527 0.580
 Histidine0.844 1.254 1.006 1.238 1.412
 Leucine3.171 3.203 3.221 3.261 3.204
 Lysine2.641 3.352 2.982 3.320 3.522
Phenylalanine1.614 2.409 1.927 2.387 2.721
 Arginine2.237 3.380 2.707 3.323 3.783
NEAA
 Alanine1.845 2.769 2.236 2.729 3.122
 Aspartate1.649 2.262 1.701 3.294 4.776
 Cysteine0.096 0.149 0.107 0.139 0.170
 Glutamic
 acid
0.911 1.082 1.330 0.000 0.000
 Glycine2.058 2.957 2.365 3.037 3.474
 Serine0.536 0.807 0.702 0.898 1.785
 Proline0.199 0.195 0.231 0.228 0.350
 Tyrosine0.763 1.125 0.879 1.093 1.245
Abbreviations: GML, glycerol monolaurate. (i) Essential amino acids (EAAs): valine, threonine, isoleucine, methionine, histidine, leucine, lysine, phenylalanine, and arginine. (ii) Non-essential amino acids (NEAAs): alanine, aspartate, cysteine, glutamic acid, glycine, serine, proline, and tyrosine.
Table 3. Impact of GML on growth performance and morphological development of juvenile pacific white shrimp (Litopenaeus vannamei) (n = 3).
Table 3. Impact of GML on growth performance and morphological development of juvenile pacific white shrimp (Litopenaeus vannamei) (n = 3).
ParametersDiets
Control (0.00)GML 1 (0.25 g)GML2 (0.50 g)GML3 (0.75 g)GML4 (1 g)
IBW1.67 ± 0.241.67 ± 0.141.62 ± 0.201.83 ± 0.091.81 ± 0.05
FBW7.12 ± 0.21 b7.27 ± 1.16 b8.87 ± 0.30 a8.80 ± 0.01 a7.13 ± 0.50 b
WG (%)331.23 ± 44.99 ab339.87 ± 94.26 ab452.48 ± 56.92 a381.44 ± 16.22 ab294.25 ± 38.97 b
SGR (%/d)5.91 ± 0.80 ab6.07 ±1.68 ab8.08 ± 1.02 a6.81 ± 0.29 ab5.25 ± 0.70 b
MFI (g shrimp−1 day−1)0.33 ± 0.010.32 ± 0.020.32 ± 0.020.34 ± 0.010.33 ± 0.02
FCR3.35 ± 0.093.34 ± 1.062.48 ± 0.082.73 ± 0.133.44 ± 0.39
CF (%)1.18 ± 0.08 ab1.15 ± 0.04 b1.17 ± 0.27 ab1.52 ± 0.14 a1.12 ± 0.01 b
HSI (%)3.88 ± 0.44 a3.20 ± 0.32 a3.18 ± 0.15 a2.34 ± 0.43 b3.35 ± 0.23 a
VSI (%)5.14 ± 0.45 b4.39 ± 0.36 a4.38 ± 0.14 a3.62 ± 0.41 a4.70 ± 0.13 b
SR (%)97.33 ± 1.1599.33 ± 1.1598.00 ± 2.0098.00 ± 0.0098.00 ± 2.83
Values are presented as mean ± SD of triplicate aquaria (n = 3). Values with different superscript letters within the same row are significantly different (p < 0.05). Abbreviations: FBW, final body weight; WG, weight gain; IBW, initial body weight; MFI, mean feed intake; SGR, specific growth rate; CF, condition factor; FCR, feed conversion ratio, VSI, viscerosomatic index; HSI, hepatosomatic index; SR, survival rate.
Table 4. Effects of different dietary levels of glycerol monolaurate (GML) on proximate compositions (%) of whole bodies and dorsal muscles of juvenile Pacific white shrimp (Litopenaeus vannamei).
Table 4. Effects of different dietary levels of glycerol monolaurate (GML) on proximate compositions (%) of whole bodies and dorsal muscles of juvenile Pacific white shrimp (Litopenaeus vannamei).
ParametersDiets
Control (0.00 g)GML1 (0.25 g)GML2 (0.50 g)GML3 (0.75 g)GML4 (1.00 g)
Whole body
 Crude
protein
72.70 ± 1.9173.53 ± 3.1574.83 ± 0.5574.26 ± 1.5372.90 ± 1.054
 Crude lipid1.60 ± 1.241.44 ± 0.391.68 ± 0.571.30 ± 0.430.94 ± 0.57
 Moisture78.58 ± 1.5877.75 ± 0.9377.03 ± 1.7577.16 ± 1.0677.49 ± 0.91
 Ash15.30 ± 0.36 a11.80 ± 0.53 b12.53 ± 0.47 b12.33 ± 0.50 b12.76 ± 0.65 b
 Phosphorus1.25 ± 0.191.29 ± 0.051.29 ± 0.081.30 ± 0.031.33 ± 0.02
Dorsal Muscle
 Crude
 protein
89.10 ± 0.9589.13 ± 0.4989.16 ± 0.8389.10 ± 0.3688.76 ± 0.95
 Crude lipid0.39 ± 0.360.50 ± 0.390.85 ± 0.690.91 ± 0.520.28 ± 0.28
 Moisture76.25 ± 0.5177.74 ± 1.4976.03 ± 0.3777.36 ± 2.2371.43 ± 8.34
 Ash5.71 ± 0.765.98 ± 0.606.42 ± 0.416.28 ± 0.585.82 ± 0.71
 Phosphorus76.25 ± 0.5177.74 ± 1.4976.03 ± 0.3877.37 ± 2.2371.43 ± 8.34
Values are presented as mean ± SD of triplicate aquaria (n = 3). Values with different superscript letters within the same row are significantly different (p < 0.05).
Table 5. The amino acid composition of the whole bodies of the L. vannamei fed the experimental diets (g/kg dry matter).
Table 5. The amino acid composition of the whole bodies of the L. vannamei fed the experimental diets (g/kg dry matter).
Amino AcidsDiet
Control (0.00 g)GML1 (0.25 g)GML2 (0.50 g)GML3 (0.75 g)GML4 (1.00 g)
EAA
 Valine12.01 ± 1.6911.54 ± 0.5911.03 ± 0.2612.53 ± 1.2610.85 ± 0.77
 Threonine0.15 ± 0.09 b0.8 ± 0.43 ab0.71 ± 0.26 ab0.92 ± 0.03 a0.45 ± 0.39 ab
 Isoleucine5.88 ± 0.495.90 ± 0.506.99 ± 0.456.53 ± 0.256.10 ± 0.36
 Methionine3.85 ± 0.19 b4.32 ± 0.28 ab4.43 ± 0.27 ab5.11 ± 0.67 a4.35 ± 0.34 ab
 Histidine0.00 ± 0.000.09 ± 0.160.00 ± 0.000.00 ± 0.000.00 ± 0.00
 Leucine13.99 ± 1.7315.77 ± 2.0015.92 ± 2.1017.97 ± 1.6713.94 ± 0.45
 Lysine18.69 ± 1.06 b20.59 ± 1.14 ab20.72 ± 0.19 ab21.42 ± 0.22 a19.14 ± 0.67 b
Phenylalanine6.43 ± 0.506.72 ± 0.897.26 ± 1.237.94 ± 1.016.53 ± 2.82
 Arginine6.39 ± 2.615.79 ± 0.754.84 ± 0.395.46 ± 0.495.01 ± 1.47
NEAA
 Alanine11.81 ± 0.7911.16 ± 0.3311.34 ± 0.9812.27 ± 1.5210.69 ± 0.22
 Aspartate19.70 ± 0.88 c20.79 ± 0.69 bc21.69 ± 0.41 ab22.99 ± 0.75 a19.92 ± 0.26 c
 Cysteine0.28 ± 0.220.16 ± 0.080.03 ± 0.010.12 ± 0.140.03 ± 0.02
 Glutamic acid30.04 ± 0.84 c26.83 ± 0.36 d32.13 ± 0.12 b33.87 ± 0.38 a27.09 ± 0.34 d
 Glycine18.94 ± 0.34 b22.28 ± 0.33 a23.08 ± 0.60 a24.90 ± 1.52 a18.68 ± 2.05 b
 Proline11.43 ± 0.24 c12.90 ± 0.25 b14.65 ± 0.29 a15.32 ± 0.21 a13.15 ± 0.76 b
 Serine6.89 ± 1.456.42 ± 0.496.27 ± 0.477.67 ± 0.886.47 ± 2.36
 Tyrosine12.43 ± 0.2412.57 ± 0.4513.32 ± 0.3713.65 ± 0.3613.15 ± 0.76
Values are presented as the mean ± SD of triplicate aquaria (n = 3). Values with different superscript letters within the same row are significantly different (p < 0.05). Abbreviations: (i) essential amino acids (EAAs), (ii) non-essential amino acids (NEAAs).
Table 6. The impact of dietary glycerol monolaurate (GML) concentrations on the apparent digestibility coefficients (ADCs) of nutrients (%) in the feces of juvenile Pacific white shrimp (Litopenaeus vannamei) over an 8 week duration.
Table 6. The impact of dietary glycerol monolaurate (GML) concentrations on the apparent digestibility coefficients (ADCs) of nutrients (%) in the feces of juvenile Pacific white shrimp (Litopenaeus vannamei) over an 8 week duration.
Parameters (%)Diets
Control (0.00 g)GML1 (0.25 g)GML2 (0.50 g)GML3 (0.75 g)GML4 (1.00 g)
Crude protein85.53 ± 0.8286.58 ± 2.7185.41 ± 0.5187.62 ± 3.7383.53 ± 0.86
Crude lipid81.68 ± 5.7283.78 ± 2.3681.42 ± 3.9188.33 ± 7.2683.02 ± 2.14
Ash3.32 ± 0.113.19 ± 0.123.1 ± 0.043.20 ± 0.003.25 ± 0.23
Gross energy (kJ/g)98.22 ± 1.5298.94 ± 2.3699.66 ± 4.5799.73 ± 1.41101.84 ± 0.91
Dry matter81.03 ± 1.0680.98 ± 3.5280.98 ± 0.6883.62 ± 5.1878.16 ± 0.96
Values are presented as mean ± SD of triplicate aquaria (n = 3).
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Ullah, S.; Liu, B.; Zheng, Y.; Guo, H.; Yang, Y.; Ahmad, M.I.; Lv, S.; Deng, S.; Zhao, M.; Feng, F. Glycerol Monolaurate Affects Growth, Amino Acid Profile, Antioxidant Capacity, Nutrient Apparent Digestibility, and Histological Morphology of Hepatopancreas in Juvenile Pacific White Shrimp (Litopenaeus vannamei). Fishes 2025, 10, 124. https://doi.org/10.3390/fishes10030124

AMA Style

Ullah S, Liu B, Zheng Y, Guo H, Yang Y, Ahmad MI, Lv S, Deng S, Zhao M, Feng F. Glycerol Monolaurate Affects Growth, Amino Acid Profile, Antioxidant Capacity, Nutrient Apparent Digestibility, and Histological Morphology of Hepatopancreas in Juvenile Pacific White Shrimp (Litopenaeus vannamei). Fishes. 2025; 10(3):124. https://doi.org/10.3390/fishes10030124

Chicago/Turabian Style

Ullah, Sami, Bingge Liu, Yunyun Zheng, Hongbo Guo, Yarui Yang, Muhammad Ijaz Ahmad, Siyu Lv, Shijie Deng, Minjie Zhao, and Fengqin Feng. 2025. "Glycerol Monolaurate Affects Growth, Amino Acid Profile, Antioxidant Capacity, Nutrient Apparent Digestibility, and Histological Morphology of Hepatopancreas in Juvenile Pacific White Shrimp (Litopenaeus vannamei)" Fishes 10, no. 3: 124. https://doi.org/10.3390/fishes10030124

APA Style

Ullah, S., Liu, B., Zheng, Y., Guo, H., Yang, Y., Ahmad, M. I., Lv, S., Deng, S., Zhao, M., & Feng, F. (2025). Glycerol Monolaurate Affects Growth, Amino Acid Profile, Antioxidant Capacity, Nutrient Apparent Digestibility, and Histological Morphology of Hepatopancreas in Juvenile Pacific White Shrimp (Litopenaeus vannamei). Fishes, 10(3), 124. https://doi.org/10.3390/fishes10030124

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