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. 2025 Jan 2;15:451. doi: 10.1038/s41598-024-83494-1

Effect of partial and total replacement of fishmeal by soybean meal in feed on growth and gut performance of Penaeus vannamei

Chanadda Kasamechotchung 1, Natthinee Munkongwongsiri 2,3, Pichet Plaipetch 4, Kanokwan Lertsiri 2, Siripong Thitamadee 3,5,6, Rapeepun Vanichviriyakit 3,7, Siriwan Khidprasert 1, Kallaya Sritunyalucksana 2,3, Felipe Nobre Façanha 8, Thanapong Kruangkum 3,7,✉
PMCID: PMC11697264  PMID: 39747937

Abstract

This study evaluated the growth and gut performance of shrimp fed three isonitrogenous diets (37% crude protein) with varying inclusions of fish meal (FM) and soybean meal (SBM): F1 (27.5% FM), F2 (10% FM + 23.5% SBM), and F3 (38% SBM). Over a 28-day period, feed intake, feed conversion ratio (FCR), and survival rates showed no significant differences among the groups. However, shrimp fed F2 and F3 exhibited significantly higher weight gain and average daily growth (ADG) compared to those fed F1 (P < 0.05). Gut performance analysis revealed that F3 consistently had the highest gut passage time (GPT), while F1 had the lowest. By day 28, shrimp fed F2 displayed elevated gut retention time (GRT). F1-fed shrimp showed a high gut passage rate (GPR), whereas F3-fed shrimp had a low GPR until day 21, with differences becoming negligible by day 28. Histological examination of the hepatopancreas revealed an increased R-cell population in shrimp fed F3. These findings highlight the adaptability of shrimp to different dietary compositions and underscore the importance of considering multiple factors when assessing the impacts of feed on growth and physiology.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-83494-1.

Keywords: Gut performance, Fish meal, Soybean meal, Hepatopancreas

Introduction

Fishmeal (FM) has been used as the key ingredient in shrimp feed due to its high protein content, balanced essential amino acid profile, low content of anti-nutritional factors, and good palatability1. Despite its importance, it was found that global FM production in 2023 decreased by an average of 23% when compared to 20222. This decline in FM production has led to increased prices, impacting shrimp feed costs1. In recent years, attention has been focused on alternative protein sources to replace FM in shrimp feed and to promote the sustainable aquaculture by research of aquaculture industry3. SBM provides high-quality protein and low levels of carbohydrates and fiber, which generally enhance shrimp digestibility4. However, SBM lacks sufficient methionine, an essential amino acid, and contains anti-nutritional factors like phytic acid which can hinder nutrient absorption and growth5. This issue has been addressed by considering the use of SBM to replace 25% of FM6. The imbalance of essential amino acids in SBM can be mitigated by supplementing synthetic amino acids7. Research on protein sources including SBM has been intensively studied and reviewed in terms of its potential impact on feed quality and shrimp growth performance8. Several previous studies have reported a decline in growth performance with increasing levels of FM replacement by SBM8,9. While, there is a study reported that raising up to 75% replacement of soybean protein concentrate could be suggested to use without any harms for shrimp’s growth6.

Most studies have primarily focused on growth performance and survival rates. However, there is limited understanding of the fundamental mechanisms regulating gut passage dynamics in penaeid shrimp, which are closely linked to their digestive physiology. The gut performance parameter, including gut passage time (GPT) is defined as the elapsed time between the first ingestion of the first feed pellet to its earliest or first defecation observed. Gut retention time (GRT) refers to the duration between the initial string of defecation and the moment when the intestine is empty, resulting in the release of fecal matter into the water. The gut length of each individual shrimp was measured to calculate the gut passage rate (GPR), which indicated the rate of gut content movement (Fig. 1). While the GPT and GPR refer to the timing and velocity of ingesta transportation from feeding to defecation along the digestive tract that reflects the ability of digestion and absorption efficiencies10, the GRT could be considered by the duration of all crude ingesta remaining in the tract. Previous studies on the effect of feed quality on these gut performance parameters have provided conflicting results.

Fig. 1.

Fig. 1

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Schematic diagram to measure gut passage time (GPT, min), gut retention time (GRT, min) and gut passage rate (GPR) in this study.

Hepatopancreas and midgut are the essential organs which play the important role in digestion, reabsorption, including transportation in crustaceans11–13. The histological and cytological organization reflect the putative function of the organ13. However, studies examining the histological or histopathological effects of replacing fishmeal (FM) with soybean meal (SBM) in shrimp diets remain limited, except a recent study by fermented soybean meal (FSBM) in juvenile white shrimp, Litopenaeus vannamei14.

This study evaluates the effects of partial and total FM replacement with SBM on shrimp performance indicators such as survival rate, feed conversion ratio, and growth. Additionally, it investigates gut physiology and histological changes to provide insights into the impacts of SBM-based diets on digestive health. The findings aim to inform sustainable feed formula development to support growth and digestion in shrimp aquaculture.

Results

Proximate composition of the diets

The proximate analysis of three diets was shown in Table 1. Although most parameters measured in the diets were similar among the three feeds, the feed diets with the components of SBM, F2, and F3 showed higher fiber contents than those of F1.

Table 1.

Proximate analysis of the test feeds.

Components g/100 g feed (as dry basis)
F1 (control) F2 F3
Protein 37.88 ± 0.21 37.89 ± 0.15 37.48 ± 0.07
Fat 6.79 ± 0.11 6.54 ± 0.09 6.35 ± 0.05
Carbohydrate 38.42 ± 0.19 37.94 ± 0.90 37.41 ± 0.59
Fiber 0.71 ± 0.04 1.68 ± 0.09 2.50 ± 0.02
Ash 8.62 ± 0.03 9.01 ± 0.04 9.23 ± 0.02
Moisture 7.55 ± 0.19 6.94 ± 0.07 7.03 ± 0.42
Gross Energy (Kcal/kg) 3,663.33 ± 8.21 3,623.27 ± 17.73 3,566.87 ± 21.42
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Note: Values for the nutritional components of the diet are expressed as mean ± S.D

Effect of diets on growth performance

Shrimp were fed with three different diet for 28 days. The initial weight of all feeding groups was in a range of 1.72–1.79 g. After 28 days post feeding, there were no significant differences in feed intake (P = 0.099), FCR (P = 0.247) and percentage of survival (P = 0.985) among three groups, as shown in Table 2. In contrast, there were significant differences on the final weight (P = 0.05), weight gain (P = 0.05) and ADG (P = 0.05). Determination of these growth parameters was performed every 7 days post feeding. There were no significant differences in the growth performance among the three groups on day 7, 14 and 21 post feeding, as shown in Fig. 2. The significant difference in growth performance was clearly demonstrated on day 28 post feeding. Shrimp fed with F2 (FM + SBM) and F3 (SBM) showed higher final weight, weight gain, and ADG than those fed with F1 (FM) (See the Supplement Table S1). The weight gain/ ADG of 7.86 ± 0.32 g/ 0.28 ± 0.01 g/day in the F2 group and 7.46 ± 0.08 g/ 0.27 ± 0.002 g/day in the F3 group were significantly higher than 6.24 ± 0.57 g/ 0.22 ± 0.02 g/day in the F1 group (6.24 ± 0.57 g and 0.22 ± 0.02 g/day).

Table 2.

Comparisons of the growth performance, survival rate, feed intake and FCR of F1, F2 and F3 group on day 28 post feeding.

F1 F2 F3 p-value
Initial weight 1.78 ± 0.04a 1.79 ± 0.11a 1.72 ± 0.16a 0.687
Final weight 8.02 ± 0.54a 9.65 ± 0.33b 9.17 ± 0.17b 0.005
Weight gain 6.24 ± 0.57a 7.86 ± 0.32b 7.46 ± 0.08b 0.005
ADG 0.22 ± 0.02a 0.28 ± 0.01b 0.27 ± 0.002b 0.005
FCR 1.05 ± 0.09a 0.96 ± 0.04a 0.98 ± 0.01a 0.247
Feed intake (g) 216.09 ± 5.56a 240.62 ± 13.36a 231.03 ± 13.61a 0.099
Survival (%) 86.67 ± 12.02a 86.67 ± 6.67a 85.56 ± 7.70a 0.985
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Note: ADG: Average Daily Gain, FCR: Feed Conversion Ratio, all data was presented as mean ± S.D

The different superscript letters in the same row represent statistically significant differences (p < 0.05).

Fig. 2.

Fig. 2

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The bar chart presented values of weight (A), weight gain (B), and ADG (C) which were compared among 3 feeding groups on Day 7, 14, 21, and 28 post-feeding. The different superscript letters on each day of each study represent statistically significant differences (P < 0.05). All data was presented as mean ± S.D.

Effect of first meal diet on gut performance and defecation behaviors

The GPT, GRT, and GPR of shrimp fed with three diets were determined in shrimp fed for the first time with only one meal of the tested diet and the results were shown in Fig. 3. While there were no significant differences found with the gut length of all tested groups, there were significant differences in GPT, GRT, and GPR among the 3 experimental groups. Shrimp of the F3 group revealed the longest GPT (P =  < 0.001) and GRT (P = 0.004) values than those of the F2 and F1 group. In contrast to GPR, shrimp of the F1 group showed the higher rate of gut content movement than those of the F2 and F3 group (P =  < 0.001). The raw data are presented in Supplementary information, Table S2.

Fig. 3.

Fig. 3

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The bar chart presenting gut passage time (GPT), gut retention time (GRT), and gut passage rate (GPR) of shrimp fed with three different diets; F1, F2 and F3 at the first meal of day 1 of the experimental feeding (A) GPT, (B) GRT, and (C) GPR. The table demonstrated the average gut length of each feeding group. The different superscript letters in each study indicate statistically significant differences (p < 0.05). All data was presented as mean ± S.D.

Effect of diet on gut performance and defecation behaviors during 28 day of feeding trial

The pattern of feeding and defecation behaviors was observed during the experiment. While ingestion behavior of shrimp fed with three different diets were similar, there was difference of the feces pattern observed. The defecation process of the shrimps fed with F1 and F2 showed single continuous intact fecal strings, as shown in Fig. 4. The non-continuous and fragmented fecal string of shrimp fed with F3 was observed throughout the experiment.

Fig. 4.

Fig. 4

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Photographs of fecal strings taken from the shrimps fed with three different fed formulae F1, F2, and F3.

The gut performance of shrimp was recorded on day 1, 7, 14, 21 and 28 post feeding (Fig. 5). With the growth of shrimp, the trend of GPT increased during 28 feeding period. Similar results to the previous study (Fig. 3) for the first meal were observed in Fig. 5. The initial GPT and GRT of shrimp (Day 1) of the F3 were significantly higher than those of F1, while the GPR of F2 was higher than those of F3 (Fig. 5A). The pattern of high GPT in the F3 group and low GPT in the F1 group was continuously observed until day 21 and 28 of experiment. Like the pattern of GPT, the pattern of high GRT was found in the F3 group and the pattern of low GRT was found in the F1 group during 28 days of feeding (Fig. 5B). A significantly high GRT was also found in the F2 group on day 28 post feeding. The pattern of low GPR in the F3 group and high GPR in the F1 group was also observed until day 21 post experimental feeding (Fig. 5C). However, there was no significant difference of GPR found on day 28 post feeding.

Fig. 5.

Fig. 5

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The bar chart presented values of GPT (A), GRT (B) and GPR (C) which were compared among 3 feeding groups on Day 1 and 28 post-feeding. The different superscript letters on each day of each study represent statistically significant differences (p < 0.05). The raw data are presented in Supplementary information Table S3. All data was presented as mean ± S.D.

Histological observation

Histological observation of the shrimp midgut proper, fed with three different diets, was investigated by cross-sections passing through the 1st abdominal segment (Fig. 6). The characteristics of the midgut epithelium of shrimp treated with three different feed formulae were observed and classified individually from the randomized shrimps based on the shape of the cell (Fig. 6 and Table 3). There were no significant differences in the shape of epithelium among the three feed formulae which exhibit intact columnar to cuboidal shapes of epithelium in day 7 of culture (Fig. 6A-C, and Table 3). The intact columnar midgut epithelium remains present in F2 and F3 (Fig. 6E, F, Table 3) after being fed for 14 days of culture, while the epithelium of midgut of the F1 group presents low cuboidal to squamous in shape (Fig. 6D, Table 3). On day 21 post feeding, the midgut epithelium of the F1 and F2 group were cuboidal in shape and in the group fed with F3 showed the midgut epithelial cell of columnar cell type (Fig. 6G, H, and I, respectively, Table 3). On day 28 post feeding, the midgut epithelium of the shrimp fed with F1 turned to be columnar in shape (Fig. 6J), while there was thinning of the epithelium as squamous-shape in F2 and F3 groups (Fig. 6K, L). Interestingly, most of the midgut epithelium of shrimp fed with F2 and F3 after performing 28 days of culture, detached from the basement membrane of the midgut (Fig. 6K, L, arrowhead).

Fig. 6.

Fig. 6

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Histological images of the midgut proper of the shrimp fed with three different feed formulae: F1, F2, and F3 on day 7- (A, B, C, respectively), 14- (D, E, F, respectively), 21- (G, H, I, respectively), and 28-post experimental feeding (J, K, L), respectively. Arrowheads in K and L indicated the thinning of midgut epithelium with detachment from the basal laminar.

Table 3.

Summary of histological observation of the midgut proper and hepatopancreas in shrimp fed with three different diets (F1, F2, F3) on 7, 14, 21, and 28 days of culture.

Days post feeding Shape of midgut epithelial cell Number of hepatopancreatic R-cell
F1 F2 F3 F1 F2 F3
Day 7 High columnar Cuboid High columnar Absence Presence Presence
Day 14 Cuboid –squamous Columnar- cuboidal High columnar + 1 + 1 + 2
Day 21 Low cuboid Low cuboid High columnar + 2 + 3 + 4
Day 28 Low cuboid Low cuboid—squamous (Detachment) Squamous (Detachment) + 2 + 4 + 5
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Note: (+ 1) to (+ 5) refer to the representative data defined by the semiquantitative evaluation in the number of R-cells presented in the hepatopancreatic tissue per field of observation.

The hepatopancreas (HP) tissue passing through the distal part of cephalothorax using the heart position as a landmark of dissection, was collected for histological observation by routine H&E staining (Fig. 7). The appearance and number of existing R-cells in each group at the proximal region of the main pancreatic ducts and branching into each lobe of the HP was mainly focused. Interestingly, the existence of R-cell population was observed in the HP tissue of shrimp fed with F2 and F3 (Fig. 7B, C, arrow and Table 3), whereas most of the HP tissue of shrimp treated with F1 was absent or present small amount of R-cell in the tissue section on day 7 post feeding (Fig. 7A, arrowhead, and Table 3). A low number of R-cell remained present in the HP tissue of shrimp fed with F1 on day 14 (Fig. 7D), day 21 (Fig. 7G), and day 28 post feeding (Fig. 7J) compared with the number of R-cells in the HP of shrimp fed with F2 and F3. The number of R-cells of shrimp fed with F3 was dominantly maximized in the number compared to F2 treatment throughout the experiment (days 14–28) (Fig. 7E-F, H-I, and K-L, respectively, and Table 3).

Fig. 7.

Fig. 7

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Histological images of the hepatopancreas of the shrimps fed with three different feed formulae: F1, F2, and F3 on day 7- (A, B, C, respectively), day 14- (D, E, F, respectively), day 21- (G, H, I, respectively), and day 28- post experimental feeding (J, K, L), respectively. (A) The disappearance of R-cell in the proximal part of the branching of HP intra-duct lobules in the shrimp fed with F1 (arrowhead). (B and C) The existence of an R-cell was observed in the HP tissue (arrow).

Discussion

Fishmeal (FM) has been a key protein source in shrimp feed due to its high nutritional value, balanced amino acid profile, and palatability. However, its rising costs and declining availability have created a pressing need for sustainable alternatives. Soybean meal (SBM) has been identified as a promising substitute. This study explored the effects of replacing FM with SBM, partially (F2) or fully (F3), on the growth and gut performance of Penaeus vannamei. Notably, while no differences were observed in feed intake, feed conversion ratio (FCR), or survival rates, shrimp fed F2 and F3 exhibited significantly higher average daily growth (ADG) and weight gain compared to those fed FM-based diets (F1). These findings contrast with earlier studies in P. vannamei15,16, E. sinensis17, and Macrobrachium rosenbergii18 reporting reduced growth with higher SBM inclusion, which may reflect differences in SBM processing or the shrimp’s adaptation to moderate dietary fiber levels in this study. The lack of significant differences in weight, weight gain, and ADG during the first 21 days, followed by their emergence between days 21 and 28, can be attributed to the considerable within-group variability observed early in the study, particularly in group F1. This variability likely masked feed-specific effects during the initial period. However, as variability gradually resolved over time, the differences in feed performance became more apparent, resulting in statistically significant outcomes by day 28. Despite the delayed emergence of statistical differences, the overall trend in the data remained consistent throughout the study.

The improved growth performance observed in F2 and F3 diets was linked to enhanced gut performance. Shrimp fed F3 exhibited consistently higher GPT and GRT throughout the trial, while those fed F1 had the shortest GPT. A similar trend was observed for GRT, with F2 showing a significant increase by day 28. In contrast, GPR was inversely related to GPT and GRT, except for a lack of significant differences by day 28. These results suggest that prolonged retention of feed in the digestive tract (reflected by long GPT and GRT) enhances digestion and nutrient absorption, contributing to improved growth.

The role of dietary fiber in this adaptation warrants special attention. While higher fiber content is often associated with reduced growth in other species, the shrimp in this study appeared to adapt well to the fiber levels in F2 and F3 diets (0.71–2.5%). This suggests a species-specific response and highlights shrimp’s ability to adjust gut function to varying dietary compositions. It is crucial to note that the increase in dietary fiber was primarily due to SBM inclusion, and the results should not be attributed to fiber content alone. Instead, they underscore the complex interplay of dietary components influencing shrimp performance. However, this study found no significant increase in GPT with higher dietary fiber content, even though diets ranged from 0.71% to 2.5% fiber. This aligns with findings from Beseres et al.19,20, where large variations in fiber levels (2.3% to 11.3%) did not lead to substantial differences in GPT across several shrimp species. Similarly, Bonvini et al.21 reported that increasing insoluble dietary fiber in European seabass diets had minimal effects on gastrointestinal evacuation patterns. These observations suggest that moderate fiber levels may not hinder gut passage dynamics due to species-specific digestive adaptations or the relatively low fiber content tested.

The information on the production of high number of R-cells in the groups fed with F2 and F3 diets agrees with improvement of growth and gut performance. R-cells are the most abundant cell type in shrimp hepatopancreas, which have multiple small vesicles containing digested nutrients absorbed from the hepatopancreatic lumen22. The function of the R-cells are nutrients absorption and storage23. Thus, they could be used as monitor cells for the nutritional value and the availability of a diet for shrimp. Our finding was correspondent with a recent report by Abd El-Naby et al.14 which indicated that the R-cell in the hepatopancreas was significantly increased in 30% fermented SBM composition treated group. The high number of R-cells in the F2 and F3 groups implies the appropriate feed quality for shrimp. The intestinal epithelial cell is columnar in shape presenting with the apical brush border responsible for nutrient absorption23 and performing as the gut barrier against microorganisms24. There are changes in the shape of the epithelial cells at the midgut proper observed among three feeding group. Whereas the cells of shrimp fed with F3 revealed the characteristic of columnar shaped during the first 21 of the feeding, those of shrimp fed with F1 and F2 showed cuboid to squamous in shape. Our results corresponded to a study using the substitution of FM with fermented SBM (FSM) in P. vannamei25. Feeding shrimp with 44.44% of FSM diet affected the micromorphology of enterocytes by decreasing the microvilli fold and heights.

The defecation behavior of shrimp fed with different diet was considered. Shrimp fed with F3 (SBM as main protein source), excreted the fragmented fecal strings, unlike those fed with F1 (FM as main protein source) and F2 excreted the continuously intact fecal string. The role of dietary fiber in shrimp feeds warrants careful consideration. While higher fiber levels are often linked to slower gut transit and potential growth depression26,27, this study demonstrates that shrimp can tolerate and adapt to moderate fiber inclusion without adverse effects. The absence of significant differences in GPT despite varying fiber levels suggests that shrimp gut dynamics are not solely dependent on fiber content but may also involve interactions with other dietary components, such as protein and lipid digestibility. Defecation behavior, particularly the fragmented fecal strings in shrimp fed F3, underscores the impact of dietary composition on waste characteristics. While dietary fiber often influences fecal texture and consistency20,28, SBM inclusion may further modulate nutrient utilization and waste formation. Understanding these interactions is critical for optimizing diet formulations to enhance nutrient retention while minimizing environmental impact in aquaculture systems. The supplementation of SBM should be performed with considerations carbohydrate digestion capacity of shrimp or the presence of anti-nutritional substances of the SBM preparation. The inclusion of SBM in the diet formulas has been found to have an impact on the limitation of carbohydrate digestion capability in shrimp, as indicated by Le Chevalier and Van Wormhoudt29. The presence of the anti-nutritional substances, consisting of glycinin and β-conglycinin, demonstrated dramatically adverse effects on the digestive ability and intestinal health of the juvenile Chinese mitten crab, Eriocheir sinensis, especially with respect to suppression of trypsin and amylase function in the intestine30. More recently, a report by Peng et al.16 revealed that the intestinal trypsin and amylase activities were reduced corresponding to increases in the percentage of SBM when they reached the significant level of 35%-50% in the diet of P. vannamei.

This study provides valuable insights into the use of SBM as a partial or total replacement for FM in shrimp diets; however, certain limitations should be noted. The 28-day trial duration may not fully capture long-term growth trends, and the focus on juvenile shrimp (2g) limits the applicability of findings to other life stages. Additionally, only three specific SBM and FM formulations were tested, without accounting for variations in ingredient processing or quality. While gut performance metrics (GPT, GRT, and GPR) were assessed, more comprehensive analyses, such as enzymatic activity and microbial profiling, could provide deeper insights. The study also lacked quantitative histological measurements and did not explore environmental impacts, such as nutrient excretion or water quality effects. Future research should extend trial durations, evaluate SBM across multiple shrimp life stages, incorporate additional gut health parameters, and explore interactions between SBM and other sustainable protein sources in practical feed formulations. Addressing these gaps will help refine SBM-based diets for broader application in shrimp aquaculture.

In conclusion, the study demonstrated the advantageous use of partial or total replacement of FM by SBM in feed for 2g shrimp. Shrimp fed with SBM (F2 and F3) showed good growth performance and gut performance. The findings also challenge the assumption that moderate dietary fiber inherently influences gut passage, emphasizing the complexity of dietary interactions in shrimp nutrition. A nuanced understanding of how different dietary components, including fiber, can impact gut function and growth in shrimp is essential. Future research should explore the synergistic effects of SBM and fiber, including the role of anti-nutritional factors and species-specific digestive adaptations, to refine sustainable feed formulations for aquaculture. Moreover, the dynamics of feed with different fiber concentrations on the digestibility and absorption of nutrient for shrimp growth and gut performance should be propose for further investigation.

Methods

Feed formulation and production

Three isonitrogenous feeds (37% total crude protein) were formulated to determine effect of dietary partial and total replacement of FM by SBM:

  • F1: 27.5% FM / 0% SBM

  • F2: 10% FM / 23.5% SBM (approximately FM used in commercial feed sold in Thailand)

  • F3: 0% FM / 38.0% SBM

The ingredient compositions of the 3 diet formulae, as shown in Table 4, were produced by the Chonburi Aquatic Animal Feed Research and Development Center, Department of Fisheries, Thailand. The stability of feed pellets (size of 1 mm) was tested to be stable in the water up to 4 h. After production, all formulae were subjected to perform proximate analysis.

Table 4.

Composition (g/100 g feed) of three diets used in this study.

Ingredients g/100 g feed (as-is-basis)
F1 (control) F2 F3
Fish meal 27.5 10.0 0
Soybean meal 0 23.5 38.0
Chicken meal 11.0 11.0 11.0
Wheat gluten 7.0 7.0 7.0
Wheat flour 21.0 21.0 21.0
Rice meal 26.3 15.5 8.9
Squid-liver meal 3.0 3.0 3.0
Methionine 0.06 0.22 0.32
Lysine 0.23 0.34 0.42
Vitamins 1.0 1.0 1.0
Minerals 1.0 1.0 1.0
STAY C vitamin C 35% 0.1 0.1 0.1
Marine fish oil 0 1.8 2.8
Soybean oil 0.46 0.22 0.09
Lecithin 1.0 1.0 1.0
Butylated hydroxytoluene 0.02 0.02 0.02
Propionic acid 0.3 0.3 0.3
Monocalcium phosphate 0 2.5 4.0
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Animal ethics

The experimental animals used in this study were handled according to the Thai national guidelines on the care and use of animals for scientific purposes under permits BT- Animal 05/2565 and MUSC64‒035‒584 from the Institutional Care and Use Committee, BIOTEC, NSTDA, and Faculty of Science, Mahidol University.

Shrimp specimens and culture conditions

Specific pathogen-free (SPF) shrimp were reared in the hatchery of the Faculty of Agriculture and Natural Resources, Rajamangala University of Technology Tawan-ok (RMUTTO), Chonburi, Thailand until the sizes reached fresh weights of approximately 2g (approximately 4 weeks old). A total of 600 shrimp were acclimatized in 1,000 L-tank containing 800 L of 20 ppt saline water (density = 0.75 shrimp/L) with sufficient aeration and fed daily to satiation body from day 1–30. Shrimp were fed 5 meals per day. During the culture period, water quality was maintained at pH 7.8 -8.0, dissolved oxygen > 5 mg/L, alkalinity > 100 mg/L, total ammonia < 1 mg/L, nitrite < 0.4 mg/L, the water temperature at 28- 30 °C.

Evaluation of shrimp growth performance

Shrimp (n = 270) were divided into 3 feeding groups (F1-F3). Each feeding group contained three replicates of 30 shrimp. Each replicate was cultured in a tank containing 300 L of 20 ppt saline water. The shrimp were fed daily to satiation for 28 days. The growth rate was assessed through the utilization of the bulk weight method on day 0 and day 28 post feeding and by individual samplings (n = 5 each replicate) on days 7,14 and 21 post feeding. Measurements of feed consumption ratio (FCR) and survival rates were on day 0 and day 28 post feeding. The formulae used to calculate the growth performance were,

  • Weight gain (g) = Final weight—initial weight

  • ADG (Average Daily Gain) (g/day) = Weight gain/ 28 (number of days post experimental feeding)

  • FCR (Feed Conversion Ratio) = Total amount of feed consumed (g, dry matter) / total weight gain (g, as-is)

  • Survival rate (%) = (Number of shrimp on 28 day-post feeding /initial number) × 100

Evaluation of gut performance

On days 1, 7, 14, and 28 of culture, ten intermolt shrimp from each group were sampled to investigate GPT, GRT, and GPR. Shrimp were reared in individual acrylic tanks and were starved for one day to clear their gastrointestinal contents. The experimental shrimp were then fed with their corresponding diet at 1.5% BW (10 shrimp/ feeding group). The GPT and GRT were measured as shown in Fig. 1.

Histological procedures and observation

Sample preparation for histological analysis followed the procedures of Bell and Lightner31. Briefly, the specimens (the midgut passing through the 1st abdominal segments and the distal part of the cephalothorax through the hepatopancreas, proximal to heart location, as a landmark of six randomized shrimps from each feeding condition of day 7, 14, 21, and 28 post feeding were fixed with Davidson’s fixative, embedded in paraffin, and stained with hematoxylin and eosin (H&E) for examination with a light microscope (Leica DM750) and photographing with a digital camera (Leica ICC50 HD).

Calculations and statistical analysis

The value of GPT, GRT, and GPR as well as growth performance parameters in this study were analyzed by IBM SPSS Statistics version 22. One-way ANOVA and Tukey HSD method were used to compare the data among groups. All data was presented as mean ± S.D. Differences were statistically significant when p < 0.05.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (308.6KB, docx)

Acknowledgements

The authors would like to thank Prof. T.W. Flegel for assistance in editing the draft manuscript, and Miss Kornchanok Jaiboon for her kind assistance in tissue preparation for histology.

Author contributions

CK, NM, ST, RV, SK, KS, TK Conceptualization; CK, NM, PP, KL, SK, TK Methodology and Investigation; CK, NM, KS, TK Writing the manuscript; CK, NM, ST, ST, KS, FNF, TK Research discussion and validation; RV, TK Funding acquisition; FNF Providing ideas and suggestions; KS, FNF, TK Writing-review and editing the manuscript. CK, TK, FNF Final revision.

Funding

This research has received funding from the NSRF via the Program Management Unit for Human Resources & Institutional Development, Research and Innovation (Grant No: B05F640137) and National Science, Research and Innovation Fund, Thailand Science, Research and Innovation (TSRI) (P2351473).

Data availability

The raw data used for analyses in this study is provided in the supplement (available online).

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Supplementary Materials

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Data Availability Statement

The raw data used for analyses in this study is provided in the supplement (available online).

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