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The Effect of Glycerol Monolaurate on Intestinal Health and Disease Resistance in Cage-Farmed Juvenile Pompano <i>Trachinotus ovatus</i>

The Effect of Glycerol Monolaurate on Intestinal Health and Disease Resistance in Cage-Farmed... Hindawi Aquaculture Nutrition Volume 2023, Article ID 8580240, 11 pages https://doi.org/10.1155/2023/8580240 Research Article The Effect of Glycerol Monolaurate on Intestinal Health and Disease Resistance in Cage-Farmed Juvenile Pompano Trachinotus ovatus 1,2,3 1,2,3 1,2,3 Huaxing Lin , Beiping Tan, and Qihui Yang College of Fisheries, Guangdong Ocean University, Zhanjiang, 524088 Guangdong, China Aquatic Animals Precision Nutrition and High Efficiency Feed Engineering Research Center of Guangdong Province, Zhanjiang, 524088 Guangdong, China Guangdong Provincial Key Lab of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Zhanjiang, 524088 Guangdong, China Correspondence should be addressed to Qihui Yang; qihuiyang03@163.com Received 31 October 2022; Revised 28 November 2022; Accepted 1 April 2023; Published 24 April 2023 Academic Editor: Zhen-Yu Du Copyright © 2023 Huaxing Lin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This research studied the effects of glycerol monolaurate (GML) to diets on the digestive capacity, intestinal structure, intestinal microbiota, and disease resistance for juvenile pompano Trachinotus ovatus (mean weight = 14:00 ± 0:70 g). T. ovatus were, respectively, fed six diets containing 0.00, 0.05, 0.10, 0.15, 0.20, and 0.25% GML for 56 days. The highest weight gain rate was observed in the 0.15% GML group. In the intestine, amylase activities in the 0.10, 0.15, 0.20, and 0.25% GML groups were significantly increased, compared with 0.00% GML group (P <0:05). Lipase activities in the 0.10 and 0.15% GML groups were significantly increased (P <0:05). Similar significant elevations in the protease activities were also found in the 0.10, 0.15, and 0.20% GML groups (P <0:05). Amylase activities were significantly higher in the 0.10, 0.15, 0.20, and 0.25% GML groups than that in the 0.00% GML group (P <0:05). Villus lengths (VL) and muscle thicknesses (MT) of the 0.05, 0.10, 0.15, and 0.20% GML groups were significantly enhanced, and the villus widths (VW) in the 0.05, 0.10, and 0.15% groups were significantly increased (P <0:05). Additionally, 0.15% GML significantly improved the intestinal immunity by upregulating interleukin 10 (il-10), increasing beneficial bacteria abundances (e.g., Vibrio, Pseudomonas, and Cetobacterium), downregulating nuclear factor kappa b (nf-κb) and interleukin 8 (il-8), and decreasing harmful bacteria abundances (e.g., Brevinema and Acinetobacter) (P <0:05). After challenge test, GML significantly increased the survival rate (80%–96%) (P <0:05). In addition, ACP and AKP activities in the GML-supplemented groups were significantly higher than those in the 0.00% GML group, and LZM activity was significantly higher in the 0.05, 0.10, 0.15, and 0.20% GML groups than that in the 0.00% GML group (P <0:05). In summary, 0.15% GML significantly promoted the intestinal digestibility, improved the intestinal microflora, regulated intestinal immune-related genes, and increased resistance to V. parahaemolyticus of juvenile pompano T. ovatus. 1. Introduction quently and are becoming increasingly serious [3]. Diseases have caused great economic losses to T. ovatus aquaculture Pompano Trachinotus ovatus, a carnivorous fish, has many production and seriously hindered the development of the advantages such as fast growth, tender meat, tasty taste, aquaculture industry [4]. Improving or maintaining the ani- and moderate price [1]. In 2020, T. ovatus production in mal’s immunity of this fish plays a vital role and is the foun- China exceeded 120,000 tones, making it a promising candi- dation of disease resistance, which is related to nutrition and date [2]. However, as negative impacts arise, such as increas- feeding [5]. ing scale and intensification of aquaculture and the pollution Disease outbreaks are a key limiting factor for the aqua- of the aquaculture environment, T. ovatus diseases occur fre- culture industry and lead to the use of a large number of 2 Aquaculture Nutrition chemicals [6]. Antibiotics play a vital role in improving ani- Table 1: Sequences of primers used for real-time quantitative PCR. mal performance and health, but problems connected with Gene their use, such as antibiotic residues, increased bacterial ′ ′ Reference Primer sequence (5 −3 ) name resistance, reduced immunity due to long-term use, and sec- F-TGCGACAAAGTCCAGAAAGAT ondary infections, have caused great concern [7]. Therefore, nf-κb [51] R-CTGAGGGTGGTAGGTGAAGGG the search for feed additives that can replace antibiotics and F-GAGAAGCCTGGGAATGGA improve the immunity of aquatic animals is the focus of il-8 [51] R-GAGCCTCAGGGTCTAAGCA many researchers. Medium-chain fatty acids (MCFAs) are F-CTCCAGACAGAAGACTCCAGCA a class of energy substances with specific physiological func- il-10 [52] R-GGAATCCCTCCACAAAACGAC tions that can be developed as feed additives as an alternative to antibiotics [8]. National and international researchers F-CGGACTCGAACGTGGTCACATTC il-1β R-AATATGGAAGGCAACCGTGCT [53] have found that MCFA monoglycerides, particularly glycerol CAG monolaurate (GML), have antipathogenic properties [9–11]. GML, easily digested and well absorbed, is a typical fatty F-TACGAGCTGCCTGACGGACA β-actin [54] R-GGCTGTGATCTCCTTCTGC acid glyceride of the MCFA monoglycerides that has strong antibacterial properties [8, 12]. GML is used to accelerate Abbreviations: nf-κb: nuclear factor kappa b; il-8: interleukin 8; il-10: growth, improve feed conversion rates, and enhance disease interleukin 10; il-1β: interleukin 1β. resistance for livestock and poultry [13]. Moreover, GML is directly absorbed by the intestinal epithelium. GML provides natural drying to about 10% moisture, they are stored at rapid energy to the intestinal epithelium, increases the -20 C. height of the intestinal villi, improves digestion and absorp- Fish acclimatization, feed and raising conditions, and tion, and maintains the integrity of the animal’s intestine diet preparation (Supplementary Table S1) are detailed in [14]. In addition, GML can inhibit harmful bacteria and the supplementary material. adjust and stabilize the balance of the intestinal microflora, thereby affecting the performance of animals [15]. A study 2.2. Sample Collection and Analyses. After 8 weeks, the fish showed that mice fed GML were able to significantly for each floating cage were sampled, and relevant growth increase the abundance of beneficial intestinal bacteria and indicators were counted, including weight gain rate improve intestinal metabolism [16]. To date, studies have (WGR), specific growth rate (SGR), feed coefficient rate revealed that GML has growth-promoting and immuno- (FCR), and survival rate (SR). Afterwards, the serum of three modulatory effects on poultry [17, 18]. fish for each floating cage was obtained to analyze the rele- In our previous experiments, it was found that GML can vant enzyme activity indicators, including alkaline phospha- remarkably improve the fat metabolism of pompano T. ovatus, tase (AKP), acid phosphatase (ACP), and lysozyme (LZM) thus affecting the growth performance [2]. Based on the nutri- activities. Detailed descriptions are in the supplementary tional properties of GML, an investigation into the relationship material. between GML on the growth and immune capacity of pom- The intestine samples of five fish for each floating cage pano T. ovatus was further explored. Therefore, we studied were quickly collected, and part of them were stored for the effects of GML on digestibility, intestinal structure, intesti- enzyme activity analysis, whereas the rest were stored in nal microbiota, and disease resistance for pompano T. ovatus. RNA-later [19]. Complete intestines of three fish for each floating cage are obtained and kept at -80 C for gut flora analysis [20]. 2. Materials and Methods 2.3. Histological Morphology. Intestines of three fish for each 2.1. Animals and Diet Preparation. For all procedures relat- floating cage were quickly removed and preserved in a 4% ing to live animals, approval has been authorized by the paraformaldehyde solution and analyzed histologically with Institutional Animal Care and Use Committee of the hematoxylin-eosin (H&E) [21]. Intestinal sections were Guangdong Ocean University (ID GDOU-AEWC- viewed under a light microscope and photographed, with 20180063). random measurements of VL, VW, and MT. The experiment was conducted at an experimental site in Zhanjiang, Guangdong, China. Juvenile T. ovatus was pro- cured from a seedling farm in Hainan Province for this 2.4. Challenge Test. After initial sampling, the experimental investigation. The juvenile T. ovatus was acclimated to fish were transported to the Donghai Island Breeding base experimental conditions for two weeks. of Guangdong Ocean University to be stored in tanks Six isonitrogenous and isolipidic diets were randomly (0.5 m ) for temporary rearing. During transport, the “closed prepared: the basal diet with 0 (0.00%), 500 (0.05%), 1000 transport with circulating water” method was adopted, (0.10%), 1500 (0.15%), 2000 (0.20%), and 2500 (0.25%) including fish-holding techniques, low-temperature dor- mg/kg of GML. All raw materials are crushed, mixed, added mancy techniques, fish transport equipment, and environ- with oil and water, and then granulated (2.5 mm in diame- mental control techniques. The fish in each group ter) with a twin screw extruder (F-75; South China Univer- exhibited 0% mortality during transport and were fed with sity of Technology, Guangzhou, Guangdong, China). After each group of feed during transient rearing, respectively. Aquaculture Nutrition 3 Table 2: Effect of GML on amylase, lipase, and protease activities in the intestine for juvenile pompano T. ovatus. Experimental diets Parameters 0.00% 0.05% 0.10% 0.15% 0.20% 0.25% a a b b b b Amylase (mIU/mg.pro) 237:98 ± 32:52 273:50 ± 18:96 375:66 ± 55:75 427:51 ± 44:73 439:01 ± 1:02 374:30 ± 31:14 a a c b a a Lipase (mU/mg.pro) 1028:18 ± 36:93 1080:37 ± 61:58 1358:07 ± 76:50 1200:83 ± 27:53 1077:84 ± 89:37 1086:58 ± 44:19 a a c b b a Protease (U/mg.pro) 3208:49 ± 68:95 3509:33 ± 188:49 5906:64 ± 280:18 4895:82 ± 153:36 4834:59 ± 159:60 3391:23 ± 129:42 Note: data are mean ± SEM (n =3). Values in the same row with different superscripts represent a significant difference (P <0:05). Table 3: Effect of GML on hindgut intestine morphology for juvenile pompano T. ovatus. Experimental diets Parameters 0.00% 0.05% 0.10% 0.15% 0.20% 0.25% a b bc c bc a VL (μm) 860:64 ± 22:34 958:38 ± 12:12 973:78 ± 7:18 1025:84 ± 59:00 996:96 ± 7:82 881:71 ± 22:55 a b b c a a VW (μm) 131:29 ± 3:13 145:19 ± 5:19 147:59 ± 3:60 168:29 ± 1:27 127:82 ± 2:33 125:86 ± 1:69 a b b d c a MT (μm) 205:34 ± 3:63 222:60 ± 5:33 232:42 ± 4:19 271:46 ± 9:01 254:62 ± 5:04 201:98 ± 7:22 Note: data are mean ± SEM (n =3). Values in the same row with different superscripts represent a significant difference (P <0:05). VL: villus length; VW: villus width; MT: muscle thickness. 0.00% 0.05% 0.10% 0.15% 0.20% 0.25% Figure 1: Light microscopy of the hindgut intestine morphology for juvenile pompano T. ovatus fed diets with GML (H&E staining). V. parahaemolyticus was obtained from Guangdong Pro- cation was performed using primers (27F: 5 -AGRGTTY- vincial Key Laboratory of Pathogenic Biology and Epidemiol- ′ ′ GATYMTGGCTCAG-3 ; 1492R: 5 -RGYTACCTTGTTAC ogy for Aquatic Economic Animals and activated twice [22]. A GACTT-3 ) to obtain full-length 16S rDNA. Amplicons pretest determined a semilethal concentration (LD , 7 d) of were assessed with a 2% agarose gel and purified employing 2×10 cfu/ml. A challenge test was conducted in triplicate the Axy-Prep DNA Gel Extraction Kit (Axy-gen Biosciences, with 10 fish per replicate. All fish were injected intraperitone- Union City, CA, USA). The purified PCR products were ally with a bacterial suspension (0.2 ml). No diets were pro- sequenced in high throughput (Illumina Hiseq2500 vided to these animals during the trial. Mortality was sequencing system). After sequencing was completed, the observed in each tank at 7 d, and SR was determined. Serum raw reads were screened as follows [23]: (1) reads filtering, was obtained similarly to “2.2 sample collection and analyses”. (2) reads assembly, (3) raw tag filtering, and (4) clustering Detailed descriptions are in the supplementary material. and chimera removal. Through the above 4 steps of process- ing, a valid label was finally obtained for further analysis. Sequences of the most abundant tags were chosen as repre- 2.5. Intestinal Microbiota Sequencing Analysis. Microbial DNA was obtained with HiPure Soil DNA Kit (or HiPure sentative of each cluster. Detailed descriptions are in the supplementary material. Fecal DNA Kit) (Magen, Guangzhou, China). PCR amplifi- 4 Aquaculture Nutrition 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 20,000 40,000 60,000 80,000 Number of tags sampled 0.00%-1 0.15%-1 0.00%-2 0.15%-2 0.00%-3 0.15%-3 0.05%-1 0.20%-1 0.05%-2 0.20%-2 0.05%-3 0.20%-3 0.10%-1 0.25%-1 0.10%-2 0.25%-2 0.10%-3 0.25%-3 Figure 2: Sequencing depth coverage curve. Table 4: Effect of GML on alpha diversity indices in the intestinal microbiota for juvenile pompano T. ovatus. Experimental diets Parameters 0.00% 0.05% 0.10% 0.15% 0.20% 0.25% 312:75 ± 28:58 331:12 ± 22:52 333:94 ± 17:01 345:96 ± 1:40 347:46 ± 40:06 378:91 ± 31:20 Ace Richness estimators 318:34 ± 40:42 323:99 ± 20:84 330:11 ± 2:64 356:31 ± 17:59 386:18 ± 40:67 381:41 ± 34:87 Chao1 0:82 ± 0:03 0:81 ± 0:02 0:75 ± 0:06 0:76 ± 0:04 0:85 ± 0:07 0:86 ± 0:10 Simpson Diversity estimators 3:11 ± 0:18 3:08 ± 0:12 2:67 ± 0:33 3:56 ± 0:68 3:10 ± 0:77 4:03 ± 0:94 Shannon Note: data are mean ± SEM (n =3). 2.6. Real-Time PCR Analysis. RNA extraction, cDNA syn- groups (P <0:05). The highest WGR was observed in the thesis, and relative mRNA expression calculations were car- 0.15% GML group (P <0:05; Supplementary Table S2). ried out as previously published [2, 24]. Detailed descriptions are in the supplementary material. The PCR 3.2. Effects of GML on Digestive Enzyme Activities. In the primers were shown in Table 1 with nf-κb, il-10, il-8, il-1β, intestine, amylase activities were significantly higher in the and β-actin, respectively. 0.10, 0.15, 0.20, and 0.25% GML groups than that in the 0.00% GML group (P <0:05; Table 2). Lipase activities were 2.7. Statistical Analysis. All data were analyzed by one-way significantly higher in the 0.10 and 0.15% GML groups than analysis of variance (ANOVA) using SPSS 21.0 (SPSS Inc., that in the 0.00% GML group (P <0:05). Protease activities Chicago, IL, USA), followed by Duncan’s multiple range test were significantly higher in the 0.10, 0.15, and 0.20% groups to determine significant differences between the groups than that in the 0.00% group (P <0:05). (P <0:05). 3.3. Effects of GML on Histological Morphology. In Table 3 and Figure 1, VL, VW, and MT were all significantly higher 3. Results in the 0.15% GML group than that in the 0.00% GML group 3.1. Effect of GML on Growth Performance. The juvenile (P <0:05). The VL in the 0.05, 0.10, 0.15, and 0.20% GML pompano T. ovatus was fed 0 (0.00%), 500 (0.05%), 1000 groups were significantly higher than those in the 0.00% (0.10%), 1500 (0.15%), 2000 (0.20%), and 2500 (0.25%) GML group (P <0:05), whereas the difference between the mg/kg of GML for 56 days. WGR were significantly higher 0.25 and 0.00% GML groups was not significant. The VW in the 0.10% and 0.15% GML groups than that in the other in the 0.05, 0.10, 0.15, and 0.20% GML groups were Good’s coverage Aquaculture Nutrition 5 Upset plot 92 96 50 26 27 17 17 17 10 10 11 11 12 12 14 111111 22222222223 3 3 4 4 4 4 5 555556 6 6 6 6 6 777999 329 0.00% 425 0.05% 269 0.10% 399 0.15% 402 0.20% 778 0.25% 0.00% 0.15% 0.05% 0.20% 0.10% 0.25% Upset plot Upset plot Upset plot Upset plot 220 173 210 213 205 189 200 189 200 160 150 140 120 150 124 96 80 100 50 40 50 0 0 0 0 329 0.00% 329 0.00% 329 0.00% 329 0.00% 425 0.05% 269 0.10% 399 0.15% 402 0.20% 0.00% 0.00% 0.00% 0.00% 0.05% 0.10% 0.15% 0.20% Upset plot 329 0.00% 778 0.25% 0.00% 0.25% Figure 3: Upset diagram for unique and shared OTUs. The left bar is the total number of OTUs contained in each original dataset; the lower intersection point indicates the name of the group corresponding to the left-hand side. The intersection between the corresponding groups is indicated by the vertical realization of the connection between the points; the upper bar indicates the number of intersection elements in this intersection case. significantly higher than those in the 0.00% GML group In Figure 3, the numbers of intestinal unique operational (P <0:05). The 0.15% GML group had the widest VW. MT taxonomic units (OTUs) were 220, 96, 210, 213, and 557 in corresponded to the trend in VW. The MT values in the the 0.05, 0.10, 0.15, 0.20, and 0.25% GML groups, respec- 0.05, 0.10, and 0.15% GML groups were significantly higher tively. The numbers of shared OTUs were 205, 173, 189, than those in the 0.00% GML group (P <0:05). 189, and 221, respectively, compared with the 0.00% GML group. A total of 131 shared OTUs were found in the six experimental sample groups. The unique OTUs in the 0.00, 3.4. Effects of GML on the Intestinal Bacterial Community. In 0.05, 0.10, 0.15, 0.20, and 0.25% GML groups were 61, 92, Figure 2, the coverage of the samples was greater than 99% in 26, 96, 110, and 388, respectively. all 18 samples, indicating that the bacteria were largely identi- fied. No significant differences were observed in Ace, Chao1, At the phylum level, ten dominant phyla were certified, including Proteobacteria, Tenericutes, Cyanobacteria, Simpson, and Shannon among all groups (P >0:05; Table 4). Group Group Group 300 300 200 200 0 0 Group Group Group 0 6 Aquaculture Nutrition Phylum 2.23 Tenericutes Bacteroidetes Proteobacteria Firmicutes Fusobacteria 0.42 Cyanobacteria Planctomycetes Actinobacteria Verrucomicrobia Spirochaetes 0 –1.4 0.00 0.05 0.10 0.15 0.20 0.25 Groups (%) Unclassified Fusobacteria Other Firmicutes Verrucomicrobia Spirochaetes Actinobacteria Cyanobacteria Planctomycetes Tenericutes Bacteroidetes Proteobacteria Figure 4: At phylum level, relative abundance and heat map analysis of intestinal microbiota for juvenile T. ovatus with different dietary. Genus 2.23 Brevinema Acinetobacter 70 -Mycoplasma Vibrio Photobacterium 0.3 Pseudomonas Cetobacterium Epulopiscium Ralstonia Endozoicomonas 0 –1.63 0.00 0.05 0.10 0.15 0.20 0.25 Groups (%) Unclassified Cetobacterium Other Pseudomonas Epulopiscium Acinetobacter Endozoicomonas Brevinema Ralstonia Vibrio Photobacterium Mycoplasma Figure 5: At genus level, relative abundance and heat map analysis of intestinal microbiota for juvenile T. ovatus with different dietary. Spirochaetes, Bacteroidetes, Firmicutes, Fusobacteria, Planc- phylum showed a gradual decrease in abundance as the sup- tomycetes, Actinobacteria, and Verrucomicrobia (Figure 4). plemented GML was increased. At the genus level, the intes- In the intestinal microbiota, the abundance of Proteobacteria tinal bacterial flora of juvenile T. ovatus comprised and Tenericutes was primarily dominant; their sum was Mycoplasma, Vibrio, Brevinema, Acinetobacter, Pseudomo- above 60% in all groups. Proteobacteria abundance was nas, and Cetobacterium, along with some genera that could higher in all experimental groups than that in the control not be identified (Figure 5). Vibrio abundance in the GML- group. In the 0.00% GML group, Spirochaetes abundant in supplemented groups was higher than that in the 0.00% 22.71% of the intestinal microbiota were significantly higher GML group. Brevinema abundance was 22.71% in the than that in the other groups (P <0:05). The Spirochaetes 0.00% GML group, significantly higher than that in the Relative abundance (%) Relative abundance (%) 0.00% 0.00% 0.05% 0.05% 0.10% 0.10% 0.15% 0.15% 0.20% 0.20% 0.25% 0.25% Aquaculture Nutrition 7 GML group revealed an increase in seven taxa and a decrease in nine taxa, compared with the 0.00% GML group. 3.5. Effects of GML on Intestinal Immunity-Related Gene Expression. In Figure 7, the mRNA levels of il-10 decreased first and then significantly increased. Compared with the 0.00% GML group, il-10 expression level was significantly upregulated in the 0.15 and 0.20% GML groups (P <0:05). il-8 expression level was significantly downregulated with GML supplementation (P <0:05), with the lowest il-8 expression that was found in the 0.15% GML group. nf-κb expression levels were significantly lower in the 0.10, 0.15, 0.20, and 0.25% GML groups than that in the 0.00% GML group (P <0:05). No significant differences were found in il-1β expression levels (P >0:05). 0.00% GML H: clostridia 0.15% GML I: novosphingobium 3.6. Effects of GML on Challenge Test with V. A: actinomarinales J: alcanivorax B: rothia K: alcanivoracaceae parahaemolyticus. In Figure 8, after the challenge test of V. C: prevotellaceae_Ga6A1_group L: brevinema parahaemolyticus, dietary supplementation with GML sig- D: sulfurimonas M: brevinemataceae nificantly increased the SR (P <0:05) compared with the E: thiovulaceae N: brevinematales 0.00% GML group. The SR values in the 0.05, 0.10, 0.15, F: subdoligranulum O: spirochaetia 0.20, and 0.25% GML groups were significantly higher than G: clostridiales that in the 0.00% GML (P <0:05). After the challenge test, ACP and AKP activities in the Prevotellaceae_Ga6A1_group GML-supplemented groups were significantly higher than Subdoligranulum those in the 0.00% GML group (P <0:05;Table 5).LZM activ- Novosphingobium ity was significantly higher in the 0.05, 0.10, 0.15, and 0.20% Sulfurimonas Thiovulaceae GML groups than that in the 0.00% GML group (P <0:05). Clostridiales Clostridia Rothia 4. Discussion Alcanivorax Alcanivoracaceae The effect of GML on animal health and growth has caused Actinomarinales Spirochaetes widespread concern [25, 26]. Studies has shown that a diet Brevinemataceae supplemented with 0.075% GML significantly improved Brevinematales Spirochaetia WGR and SGR of Danio rerio [15] and L. croceus [27]. In Brevinema addition, 0.07 and 0.105% GML significantly improved –5 –4 –3 –2 –1 01234 WGR and improved the intestinal microbiota of L. vannamei [28]. These results are in line with those obtained in previous LDA score (log 10) research reported on the growth-promoting potential of GML 0.00% GML [29]. In the present study, a similar conclusion was reached; 0.15% GML 0.15% GML in the diet significantly improved the growth. Animal growth is closely related to feeding utilization, Figure 6: Linear discriminant analysis (LDA) score threshold which is influenced by digestion and absorption capacities greater than 3 was presented to demonstrate the variation in the relative abundance of the intestine microbial communities [30]. Digestive enzymes speed up the breakdown and utiliza- between pompano T. ovatus fed diet 0.00% GML and 0.15% GML. tion of the corresponding nutrients by the intestines, thereby improving feed utilization and promoting animal growth. A study has shown that GML could penetrate deep into the GML-supplemented groups (P <0:05). With increasing intestinal tract and greatly affect digestibility and nutrient GML rate, the abundance of Brevinema showed a gradual utilization [31]. In L. vannamei, GML improved protein decrease, whereas that of Acinetobacter showed a decrease digestibility by increasing lipase and protease activities followed by an increase. The percentages of Pseudomonas [28]. In addition, MCFAs and the corresponding glycerides in the 0.00, 0.05, 0.10, 0.15, 0.20, and 0.25% GML groups increased chymotrypsin activity and protein digestibility, were 3.57, 2.50, 2.09, 6.72, 5.21, and 6.60%, respectively. thereby affecting growth similar to that of Atlantic salmon In Figure 6, the linear discriminant analysis (LDA) effect (Salmo salar L.) [32]. Similar conclusions were reached in size (Lefse) package was applied to identify the relative this study. Thus, GML can significantly enhance the intesti- abundance of microbial taxa that differed between the nal digestibility of T. ovatus. 0.15% GML group and the control group. The LDA score The intestine is the most important organ for digestion threshold was set to >3. The linear discriminant analysis and absorption in animals, and a healthy intestinal structure (LDA) shows that the LDA score of Lefse in the 0.15% is a basis for digestive and absorption functions [33]. The 8 Aquaculture Nutrition 1.6 BC 1.4 C BC 1.2 AB AB 1.0 AB AB A B AB AB AB 0.8 0.6 0.4 0.2 0.0 il-10 il-8 il-1𝛽 nf-𝜅 b 0.00% 0.15% 0.05% 0.20% 0.10% 0.25% Figure 7: Relative expression levels of immune-related gene in intestine for juvenile T. ovatus. Data were expressed as means ± SEM. Different letters above a bar are statistically significant different among treatments (P <0:05). 0.00 0.05 0.10 0.15 0.20 0.25 Experimental diets (%) Figure 8: Effects of GML on the survival rate after V. parahaemolyticus (2×10 cfu/ml) infection of juvenile T. ovatus at 7th day. Data were expressed as the mean ± SEM. Values not sharing a common superscript were significantly different (P <0:05). Table 5: Effect of GML on serum immune parameters after challenge for juvenile pompano T. ovatus. Experimental diets Parameters 0.00% 0.05% 0.10% 0.15% 0.20% 0.25% a b d c bc b AKP (U/mL) 2:49 ± 0:51 3:37 ± 0:42 5:57 ± 0:62 4:34 ± 0:20 4:11 ± 0:09 3:46 ± 0:39 a b d c b b ACP (U/mL) 2:59 ± 0:56 5:18 ± 0:14 8:03 ± 0:74 6:59 ± 0:25 5:66 ± 0:54 5:11 ± 0:58 a b b b c ab LZM (U/mL) 0:99 ± 0:23 1:36 ± 0:13 1:39 ± 0:08 1:49 ± 0:17 2:19 ± 0:17 1:28 ± 0:20 Note: data are mean ± SEM (n =3). Values in the same row with different superscripts represent significant difference (P <0:05). AKP: alkaline phosphatase; ACP: acid phosphatase; LZM: lysozyme. VL, VW, and MT of the intestine are important indicators of [14]. Therefore, GML improves intestinal villi growth in juve- the digestive and absorptive function and the health of the nile T. ovatus, contributing to the intestinal morphological intestinal mucosal tissue structure. The height and length of integrity and absorption of nutrients. the villus are significantly correlated with the number of In addition, as an essential immune organ in animals, the mature cells. Only mature villi can absorb nutrients. The lon- intestine protects against external pathogens. GML has good ger the villus length is, the larger the nutrient absorption area anti-inflammatory effects, alleviates the body’sinflammatory will be. In the present study, GML significantly increased the response through multiple pathways, and participates in the VL, VW, and MT, and these results were direct responses to body’s immune regulation [34]. One of the main pathways the ability of the GML to improve intestinal digestion. GML affecting immunity is the activation of nf-κb,aninevitable cen- improved the morphological structure of the intestine, because tral regulator of the inflammatory response involved in the sig- it was used directly by the intestinal epithelial cells when naling pathways of most intrinsic immune receptors [35]. In absorbed by the villous epithelium as an energy supply sub- the present study, GML significantly decreased the nf-κb stance. GML promoted the growth of the villous epithelial cells expression level, which was in agreement with Kong et al. Relative mRNA expression Survival rate (%) Aquaculture Nutrition 9 [36]. The nf-κb is involved in il-8 transcription and influences GML group. Therefore, 0.15% GML can significantly pro- its regulation. The il-8 is one of the proinflammatory cellular mote intestinal health. factors with a widespread role in promoting inflammation Available methods are considerably limited for conducting [37]. In addition, GML could regulate il-10 expression level, a comprehensive study on fish immunity and disease resis- an inflammatory anticellular factor [15]. The il-10 is the main tance. Thus, finding effective biomarkers of disease resistance anti-inflammatory cytokine in fish and inhibits the overactiva- in fish is difficult. Bacterial challenge experiments facilitate tion of the immune response [38]. In the present study, il-10 the assessment of the effectiveness of feeds in protecting gene upregulation and il-8 gene downregulation were the most against pathogens and are often employed as the final indica- significant in the 0.15% GML group. Therefore, this result pos- tor of the fish health status following nutritional experiments sibly showed that GML supplementation might trigger specific [49]. V. parahaemolyticus, a Gram-negative bacterium, is immunological networks of juvenile pompano T. ovatus,and one of the most serious pathogens in mariculture systems further research is needed. [50]. In the present study, GML could significantly enhance The fish intestinal microbiota is a dynamic community SR (80%-96%) in the V. parahaemolyticus challenge test. of aerobic, partly anaerobic, and anaerobic bacteria. This Moreover, after the challenge test, GML significantly community is a special dynamic environment known as improved serum immune enzyme activities (i.e., AKP, ACP, and LZM). These results may be due to the fact that GML the intestinal “island” microbiota. A balanced intestinal microecology is essential for healthy fish growth, and the can easily cross the cell wall and bind to the biofilm to exert balance of the microecology needs to be maintained by a its inhibitory effect on the pathogen [39]. Therefore, feed sup- wide range of beneficial intestinal bacteria. GML has a good plementation with GML significantly improved the disease antibacterial effect and helps stabilize the balance of the ani- resistance of juvenile T. ovatus. mals’ intestinal microbiota [39]. In the present study, an alpha diversity analysis revealed no significant differences, 5. Conclusion indicating that the microbial diversity of juvenile pompano In conclusion, the research indicated that GML significantly T. ovatus fed with GML was not separated. improved growth and intestinal health for juvenile pompano At the phylum level, the dominant intestinal microbiota T. ovatus. In addition, 0.15% GML significantly increased in juvenile T. ovatus included Proteobacteria and Teneri- serum immune enzyme activity, promoted the intestinal cutes, in agreement with existing research [40, 41]. The pre- dominant beneficial microflora, such as Proteobacteria, digestibility, improved the intestinal microflora, regulated Firmicutes, and Bacteroidetes, provided exogenous digestive intestinal immune-related genes, and increased resistance enzymes that can dissolve food into small molecules [42], to V. parahaemolyticus of juvenile pompano T. ovatus. thereby enhancing absorption and utilization in the fish gut [43]. In the present study, GML increased intestinal Data Availability digestive enzyme activity, possibly associated with an The data that support the findings of this study are available increase in beneficial intestinal bacterial populations. In on request from the corresponding author. The data are not addition, Tenericutes have a beneficial role in fish growth publicly available due to privacy or ethical restrictions. and the suppression of pathogenic bacteria [44]. However, an increase in the proportion of Helicobacter pylori leads to Conflicts of Interest an imbalance in the intestinal microbiota and a decrease in animal immunity. At the genus level, Vibrio is reportedly a The authors declare that they have no conflicts of interest. probiotic for aquatic animals, and some bacteriostatic and growth-promoting species exist, such as Vibrio alginolyticus Authors’ Contributions [45]. Pseudomonas produces a range of compounds with a wide range of antifouling biological activities and enables All authors provided contributions to the article and the production of low-temperature proteases, which remain endorsed the submitted version. active in certain extreme environments [46]. However, in the present study, harmful bacteria, such as Brevinema [47] and Acknowledgments Acinetobacter [48], existed in the intestine. Brevinema rich- ness gradually decreased as GML increased, with the lowest This study was financially supported by the National Key level in the 0.15% GML group. A study indicated that in Research and Development Program (2022YFD2401200), the Sparus aurata, GML could increase the relatively abundance Natural Science Foundation of Guangdong Province of positive bacteria, namely, Lactobacillus [29]. Therefore, (2023A1515011095), and the Correspondent for Science and GML is effective in inhibiting harmful bacteria in the juve- Technology of Guangdong Province (GDKTP2021048400). nile T. ovatus gut. In addition, the 0.00 and 0.15% GML groups were also analyzed using LDA according to their Supplementary Materials growth. 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The Effect of Glycerol Monolaurate on Intestinal Health and Disease Resistance in Cage-Farmed Juvenile Pompano <i>Trachinotus ovatus</i>

Aquaculture Nutrition , Volume 2023 – Apr 24, 2023

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10.1155/2023/8580240
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Abstract

Hindawi Aquaculture Nutrition Volume 2023, Article ID 8580240, 11 pages https://doi.org/10.1155/2023/8580240 Research Article The Effect of Glycerol Monolaurate on Intestinal Health and Disease Resistance in Cage-Farmed Juvenile Pompano Trachinotus ovatus 1,2,3 1,2,3 1,2,3 Huaxing Lin , Beiping Tan, and Qihui Yang College of Fisheries, Guangdong Ocean University, Zhanjiang, 524088 Guangdong, China Aquatic Animals Precision Nutrition and High Efficiency Feed Engineering Research Center of Guangdong Province, Zhanjiang, 524088 Guangdong, China Guangdong Provincial Key Lab of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Zhanjiang, 524088 Guangdong, China Correspondence should be addressed to Qihui Yang; qihuiyang03@163.com Received 31 October 2022; Revised 28 November 2022; Accepted 1 April 2023; Published 24 April 2023 Academic Editor: Zhen-Yu Du Copyright © 2023 Huaxing Lin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This research studied the effects of glycerol monolaurate (GML) to diets on the digestive capacity, intestinal structure, intestinal microbiota, and disease resistance for juvenile pompano Trachinotus ovatus (mean weight = 14:00 ± 0:70 g). T. ovatus were, respectively, fed six diets containing 0.00, 0.05, 0.10, 0.15, 0.20, and 0.25% GML for 56 days. The highest weight gain rate was observed in the 0.15% GML group. In the intestine, amylase activities in the 0.10, 0.15, 0.20, and 0.25% GML groups were significantly increased, compared with 0.00% GML group (P <0:05). Lipase activities in the 0.10 and 0.15% GML groups were significantly increased (P <0:05). Similar significant elevations in the protease activities were also found in the 0.10, 0.15, and 0.20% GML groups (P <0:05). Amylase activities were significantly higher in the 0.10, 0.15, 0.20, and 0.25% GML groups than that in the 0.00% GML group (P <0:05). Villus lengths (VL) and muscle thicknesses (MT) of the 0.05, 0.10, 0.15, and 0.20% GML groups were significantly enhanced, and the villus widths (VW) in the 0.05, 0.10, and 0.15% groups were significantly increased (P <0:05). Additionally, 0.15% GML significantly improved the intestinal immunity by upregulating interleukin 10 (il-10), increasing beneficial bacteria abundances (e.g., Vibrio, Pseudomonas, and Cetobacterium), downregulating nuclear factor kappa b (nf-κb) and interleukin 8 (il-8), and decreasing harmful bacteria abundances (e.g., Brevinema and Acinetobacter) (P <0:05). After challenge test, GML significantly increased the survival rate (80%–96%) (P <0:05). In addition, ACP and AKP activities in the GML-supplemented groups were significantly higher than those in the 0.00% GML group, and LZM activity was significantly higher in the 0.05, 0.10, 0.15, and 0.20% GML groups than that in the 0.00% GML group (P <0:05). In summary, 0.15% GML significantly promoted the intestinal digestibility, improved the intestinal microflora, regulated intestinal immune-related genes, and increased resistance to V. parahaemolyticus of juvenile pompano T. ovatus. 1. Introduction quently and are becoming increasingly serious [3]. Diseases have caused great economic losses to T. ovatus aquaculture Pompano Trachinotus ovatus, a carnivorous fish, has many production and seriously hindered the development of the advantages such as fast growth, tender meat, tasty taste, aquaculture industry [4]. Improving or maintaining the ani- and moderate price [1]. In 2020, T. ovatus production in mal’s immunity of this fish plays a vital role and is the foun- China exceeded 120,000 tones, making it a promising candi- dation of disease resistance, which is related to nutrition and date [2]. However, as negative impacts arise, such as increas- feeding [5]. ing scale and intensification of aquaculture and the pollution Disease outbreaks are a key limiting factor for the aqua- of the aquaculture environment, T. ovatus diseases occur fre- culture industry and lead to the use of a large number of 2 Aquaculture Nutrition chemicals [6]. Antibiotics play a vital role in improving ani- Table 1: Sequences of primers used for real-time quantitative PCR. mal performance and health, but problems connected with Gene their use, such as antibiotic residues, increased bacterial ′ ′ Reference Primer sequence (5 −3 ) name resistance, reduced immunity due to long-term use, and sec- F-TGCGACAAAGTCCAGAAAGAT ondary infections, have caused great concern [7]. Therefore, nf-κb [51] R-CTGAGGGTGGTAGGTGAAGGG the search for feed additives that can replace antibiotics and F-GAGAAGCCTGGGAATGGA improve the immunity of aquatic animals is the focus of il-8 [51] R-GAGCCTCAGGGTCTAAGCA many researchers. Medium-chain fatty acids (MCFAs) are F-CTCCAGACAGAAGACTCCAGCA a class of energy substances with specific physiological func- il-10 [52] R-GGAATCCCTCCACAAAACGAC tions that can be developed as feed additives as an alternative to antibiotics [8]. National and international researchers F-CGGACTCGAACGTGGTCACATTC il-1β R-AATATGGAAGGCAACCGTGCT [53] have found that MCFA monoglycerides, particularly glycerol CAG monolaurate (GML), have antipathogenic properties [9–11]. GML, easily digested and well absorbed, is a typical fatty F-TACGAGCTGCCTGACGGACA β-actin [54] R-GGCTGTGATCTCCTTCTGC acid glyceride of the MCFA monoglycerides that has strong antibacterial properties [8, 12]. GML is used to accelerate Abbreviations: nf-κb: nuclear factor kappa b; il-8: interleukin 8; il-10: growth, improve feed conversion rates, and enhance disease interleukin 10; il-1β: interleukin 1β. resistance for livestock and poultry [13]. Moreover, GML is directly absorbed by the intestinal epithelium. GML provides natural drying to about 10% moisture, they are stored at rapid energy to the intestinal epithelium, increases the -20 C. height of the intestinal villi, improves digestion and absorp- Fish acclimatization, feed and raising conditions, and tion, and maintains the integrity of the animal’s intestine diet preparation (Supplementary Table S1) are detailed in [14]. In addition, GML can inhibit harmful bacteria and the supplementary material. adjust and stabilize the balance of the intestinal microflora, thereby affecting the performance of animals [15]. A study 2.2. Sample Collection and Analyses. After 8 weeks, the fish showed that mice fed GML were able to significantly for each floating cage were sampled, and relevant growth increase the abundance of beneficial intestinal bacteria and indicators were counted, including weight gain rate improve intestinal metabolism [16]. To date, studies have (WGR), specific growth rate (SGR), feed coefficient rate revealed that GML has growth-promoting and immuno- (FCR), and survival rate (SR). Afterwards, the serum of three modulatory effects on poultry [17, 18]. fish for each floating cage was obtained to analyze the rele- In our previous experiments, it was found that GML can vant enzyme activity indicators, including alkaline phospha- remarkably improve the fat metabolism of pompano T. ovatus, tase (AKP), acid phosphatase (ACP), and lysozyme (LZM) thus affecting the growth performance [2]. Based on the nutri- activities. Detailed descriptions are in the supplementary tional properties of GML, an investigation into the relationship material. between GML on the growth and immune capacity of pom- The intestine samples of five fish for each floating cage pano T. ovatus was further explored. Therefore, we studied were quickly collected, and part of them were stored for the effects of GML on digestibility, intestinal structure, intesti- enzyme activity analysis, whereas the rest were stored in nal microbiota, and disease resistance for pompano T. ovatus. RNA-later [19]. Complete intestines of three fish for each floating cage are obtained and kept at -80 C for gut flora analysis [20]. 2. Materials and Methods 2.3. Histological Morphology. Intestines of three fish for each 2.1. Animals and Diet Preparation. For all procedures relat- floating cage were quickly removed and preserved in a 4% ing to live animals, approval has been authorized by the paraformaldehyde solution and analyzed histologically with Institutional Animal Care and Use Committee of the hematoxylin-eosin (H&E) [21]. Intestinal sections were Guangdong Ocean University (ID GDOU-AEWC- viewed under a light microscope and photographed, with 20180063). random measurements of VL, VW, and MT. The experiment was conducted at an experimental site in Zhanjiang, Guangdong, China. Juvenile T. ovatus was pro- cured from a seedling farm in Hainan Province for this 2.4. Challenge Test. After initial sampling, the experimental investigation. The juvenile T. ovatus was acclimated to fish were transported to the Donghai Island Breeding base experimental conditions for two weeks. of Guangdong Ocean University to be stored in tanks Six isonitrogenous and isolipidic diets were randomly (0.5 m ) for temporary rearing. During transport, the “closed prepared: the basal diet with 0 (0.00%), 500 (0.05%), 1000 transport with circulating water” method was adopted, (0.10%), 1500 (0.15%), 2000 (0.20%), and 2500 (0.25%) including fish-holding techniques, low-temperature dor- mg/kg of GML. All raw materials are crushed, mixed, added mancy techniques, fish transport equipment, and environ- with oil and water, and then granulated (2.5 mm in diame- mental control techniques. The fish in each group ter) with a twin screw extruder (F-75; South China Univer- exhibited 0% mortality during transport and were fed with sity of Technology, Guangzhou, Guangdong, China). After each group of feed during transient rearing, respectively. Aquaculture Nutrition 3 Table 2: Effect of GML on amylase, lipase, and protease activities in the intestine for juvenile pompano T. ovatus. Experimental diets Parameters 0.00% 0.05% 0.10% 0.15% 0.20% 0.25% a a b b b b Amylase (mIU/mg.pro) 237:98 ± 32:52 273:50 ± 18:96 375:66 ± 55:75 427:51 ± 44:73 439:01 ± 1:02 374:30 ± 31:14 a a c b a a Lipase (mU/mg.pro) 1028:18 ± 36:93 1080:37 ± 61:58 1358:07 ± 76:50 1200:83 ± 27:53 1077:84 ± 89:37 1086:58 ± 44:19 a a c b b a Protease (U/mg.pro) 3208:49 ± 68:95 3509:33 ± 188:49 5906:64 ± 280:18 4895:82 ± 153:36 4834:59 ± 159:60 3391:23 ± 129:42 Note: data are mean ± SEM (n =3). Values in the same row with different superscripts represent a significant difference (P <0:05). Table 3: Effect of GML on hindgut intestine morphology for juvenile pompano T. ovatus. Experimental diets Parameters 0.00% 0.05% 0.10% 0.15% 0.20% 0.25% a b bc c bc a VL (μm) 860:64 ± 22:34 958:38 ± 12:12 973:78 ± 7:18 1025:84 ± 59:00 996:96 ± 7:82 881:71 ± 22:55 a b b c a a VW (μm) 131:29 ± 3:13 145:19 ± 5:19 147:59 ± 3:60 168:29 ± 1:27 127:82 ± 2:33 125:86 ± 1:69 a b b d c a MT (μm) 205:34 ± 3:63 222:60 ± 5:33 232:42 ± 4:19 271:46 ± 9:01 254:62 ± 5:04 201:98 ± 7:22 Note: data are mean ± SEM (n =3). Values in the same row with different superscripts represent a significant difference (P <0:05). VL: villus length; VW: villus width; MT: muscle thickness. 0.00% 0.05% 0.10% 0.15% 0.20% 0.25% Figure 1: Light microscopy of the hindgut intestine morphology for juvenile pompano T. ovatus fed diets with GML (H&E staining). V. parahaemolyticus was obtained from Guangdong Pro- cation was performed using primers (27F: 5 -AGRGTTY- vincial Key Laboratory of Pathogenic Biology and Epidemiol- ′ ′ GATYMTGGCTCAG-3 ; 1492R: 5 -RGYTACCTTGTTAC ogy for Aquatic Economic Animals and activated twice [22]. A GACTT-3 ) to obtain full-length 16S rDNA. Amplicons pretest determined a semilethal concentration (LD , 7 d) of were assessed with a 2% agarose gel and purified employing 2×10 cfu/ml. A challenge test was conducted in triplicate the Axy-Prep DNA Gel Extraction Kit (Axy-gen Biosciences, with 10 fish per replicate. All fish were injected intraperitone- Union City, CA, USA). The purified PCR products were ally with a bacterial suspension (0.2 ml). No diets were pro- sequenced in high throughput (Illumina Hiseq2500 vided to these animals during the trial. Mortality was sequencing system). After sequencing was completed, the observed in each tank at 7 d, and SR was determined. Serum raw reads were screened as follows [23]: (1) reads filtering, was obtained similarly to “2.2 sample collection and analyses”. (2) reads assembly, (3) raw tag filtering, and (4) clustering Detailed descriptions are in the supplementary material. and chimera removal. Through the above 4 steps of process- ing, a valid label was finally obtained for further analysis. Sequences of the most abundant tags were chosen as repre- 2.5. Intestinal Microbiota Sequencing Analysis. Microbial DNA was obtained with HiPure Soil DNA Kit (or HiPure sentative of each cluster. Detailed descriptions are in the supplementary material. Fecal DNA Kit) (Magen, Guangzhou, China). PCR amplifi- 4 Aquaculture Nutrition 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 20,000 40,000 60,000 80,000 Number of tags sampled 0.00%-1 0.15%-1 0.00%-2 0.15%-2 0.00%-3 0.15%-3 0.05%-1 0.20%-1 0.05%-2 0.20%-2 0.05%-3 0.20%-3 0.10%-1 0.25%-1 0.10%-2 0.25%-2 0.10%-3 0.25%-3 Figure 2: Sequencing depth coverage curve. Table 4: Effect of GML on alpha diversity indices in the intestinal microbiota for juvenile pompano T. ovatus. Experimental diets Parameters 0.00% 0.05% 0.10% 0.15% 0.20% 0.25% 312:75 ± 28:58 331:12 ± 22:52 333:94 ± 17:01 345:96 ± 1:40 347:46 ± 40:06 378:91 ± 31:20 Ace Richness estimators 318:34 ± 40:42 323:99 ± 20:84 330:11 ± 2:64 356:31 ± 17:59 386:18 ± 40:67 381:41 ± 34:87 Chao1 0:82 ± 0:03 0:81 ± 0:02 0:75 ± 0:06 0:76 ± 0:04 0:85 ± 0:07 0:86 ± 0:10 Simpson Diversity estimators 3:11 ± 0:18 3:08 ± 0:12 2:67 ± 0:33 3:56 ± 0:68 3:10 ± 0:77 4:03 ± 0:94 Shannon Note: data are mean ± SEM (n =3). 2.6. Real-Time PCR Analysis. RNA extraction, cDNA syn- groups (P <0:05). The highest WGR was observed in the thesis, and relative mRNA expression calculations were car- 0.15% GML group (P <0:05; Supplementary Table S2). ried out as previously published [2, 24]. Detailed descriptions are in the supplementary material. The PCR 3.2. Effects of GML on Digestive Enzyme Activities. In the primers were shown in Table 1 with nf-κb, il-10, il-8, il-1β, intestine, amylase activities were significantly higher in the and β-actin, respectively. 0.10, 0.15, 0.20, and 0.25% GML groups than that in the 0.00% GML group (P <0:05; Table 2). Lipase activities were 2.7. Statistical Analysis. All data were analyzed by one-way significantly higher in the 0.10 and 0.15% GML groups than analysis of variance (ANOVA) using SPSS 21.0 (SPSS Inc., that in the 0.00% GML group (P <0:05). Protease activities Chicago, IL, USA), followed by Duncan’s multiple range test were significantly higher in the 0.10, 0.15, and 0.20% groups to determine significant differences between the groups than that in the 0.00% group (P <0:05). (P <0:05). 3.3. Effects of GML on Histological Morphology. In Table 3 and Figure 1, VL, VW, and MT were all significantly higher 3. Results in the 0.15% GML group than that in the 0.00% GML group 3.1. Effect of GML on Growth Performance. The juvenile (P <0:05). The VL in the 0.05, 0.10, 0.15, and 0.20% GML pompano T. ovatus was fed 0 (0.00%), 500 (0.05%), 1000 groups were significantly higher than those in the 0.00% (0.10%), 1500 (0.15%), 2000 (0.20%), and 2500 (0.25%) GML group (P <0:05), whereas the difference between the mg/kg of GML for 56 days. WGR were significantly higher 0.25 and 0.00% GML groups was not significant. The VW in the 0.10% and 0.15% GML groups than that in the other in the 0.05, 0.10, 0.15, and 0.20% GML groups were Good’s coverage Aquaculture Nutrition 5 Upset plot 92 96 50 26 27 17 17 17 10 10 11 11 12 12 14 111111 22222222223 3 3 4 4 4 4 5 555556 6 6 6 6 6 777999 329 0.00% 425 0.05% 269 0.10% 399 0.15% 402 0.20% 778 0.25% 0.00% 0.15% 0.05% 0.20% 0.10% 0.25% Upset plot Upset plot Upset plot Upset plot 220 173 210 213 205 189 200 189 200 160 150 140 120 150 124 96 80 100 50 40 50 0 0 0 0 329 0.00% 329 0.00% 329 0.00% 329 0.00% 425 0.05% 269 0.10% 399 0.15% 402 0.20% 0.00% 0.00% 0.00% 0.00% 0.05% 0.10% 0.15% 0.20% Upset plot 329 0.00% 778 0.25% 0.00% 0.25% Figure 3: Upset diagram for unique and shared OTUs. The left bar is the total number of OTUs contained in each original dataset; the lower intersection point indicates the name of the group corresponding to the left-hand side. The intersection between the corresponding groups is indicated by the vertical realization of the connection between the points; the upper bar indicates the number of intersection elements in this intersection case. significantly higher than those in the 0.00% GML group In Figure 3, the numbers of intestinal unique operational (P <0:05). The 0.15% GML group had the widest VW. MT taxonomic units (OTUs) were 220, 96, 210, 213, and 557 in corresponded to the trend in VW. The MT values in the the 0.05, 0.10, 0.15, 0.20, and 0.25% GML groups, respec- 0.05, 0.10, and 0.15% GML groups were significantly higher tively. The numbers of shared OTUs were 205, 173, 189, than those in the 0.00% GML group (P <0:05). 189, and 221, respectively, compared with the 0.00% GML group. A total of 131 shared OTUs were found in the six experimental sample groups. The unique OTUs in the 0.00, 3.4. Effects of GML on the Intestinal Bacterial Community. In 0.05, 0.10, 0.15, 0.20, and 0.25% GML groups were 61, 92, Figure 2, the coverage of the samples was greater than 99% in 26, 96, 110, and 388, respectively. all 18 samples, indicating that the bacteria were largely identi- fied. No significant differences were observed in Ace, Chao1, At the phylum level, ten dominant phyla were certified, including Proteobacteria, Tenericutes, Cyanobacteria, Simpson, and Shannon among all groups (P >0:05; Table 4). Group Group Group 300 300 200 200 0 0 Group Group Group 0 6 Aquaculture Nutrition Phylum 2.23 Tenericutes Bacteroidetes Proteobacteria Firmicutes Fusobacteria 0.42 Cyanobacteria Planctomycetes Actinobacteria Verrucomicrobia Spirochaetes 0 –1.4 0.00 0.05 0.10 0.15 0.20 0.25 Groups (%) Unclassified Fusobacteria Other Firmicutes Verrucomicrobia Spirochaetes Actinobacteria Cyanobacteria Planctomycetes Tenericutes Bacteroidetes Proteobacteria Figure 4: At phylum level, relative abundance and heat map analysis of intestinal microbiota for juvenile T. ovatus with different dietary. Genus 2.23 Brevinema Acinetobacter 70 -Mycoplasma Vibrio Photobacterium 0.3 Pseudomonas Cetobacterium Epulopiscium Ralstonia Endozoicomonas 0 –1.63 0.00 0.05 0.10 0.15 0.20 0.25 Groups (%) Unclassified Cetobacterium Other Pseudomonas Epulopiscium Acinetobacter Endozoicomonas Brevinema Ralstonia Vibrio Photobacterium Mycoplasma Figure 5: At genus level, relative abundance and heat map analysis of intestinal microbiota for juvenile T. ovatus with different dietary. Spirochaetes, Bacteroidetes, Firmicutes, Fusobacteria, Planc- phylum showed a gradual decrease in abundance as the sup- tomycetes, Actinobacteria, and Verrucomicrobia (Figure 4). plemented GML was increased. At the genus level, the intes- In the intestinal microbiota, the abundance of Proteobacteria tinal bacterial flora of juvenile T. ovatus comprised and Tenericutes was primarily dominant; their sum was Mycoplasma, Vibrio, Brevinema, Acinetobacter, Pseudomo- above 60% in all groups. Proteobacteria abundance was nas, and Cetobacterium, along with some genera that could higher in all experimental groups than that in the control not be identified (Figure 5). Vibrio abundance in the GML- group. In the 0.00% GML group, Spirochaetes abundant in supplemented groups was higher than that in the 0.00% 22.71% of the intestinal microbiota were significantly higher GML group. Brevinema abundance was 22.71% in the than that in the other groups (P <0:05). The Spirochaetes 0.00% GML group, significantly higher than that in the Relative abundance (%) Relative abundance (%) 0.00% 0.00% 0.05% 0.05% 0.10% 0.10% 0.15% 0.15% 0.20% 0.20% 0.25% 0.25% Aquaculture Nutrition 7 GML group revealed an increase in seven taxa and a decrease in nine taxa, compared with the 0.00% GML group. 3.5. Effects of GML on Intestinal Immunity-Related Gene Expression. In Figure 7, the mRNA levels of il-10 decreased first and then significantly increased. Compared with the 0.00% GML group, il-10 expression level was significantly upregulated in the 0.15 and 0.20% GML groups (P <0:05). il-8 expression level was significantly downregulated with GML supplementation (P <0:05), with the lowest il-8 expression that was found in the 0.15% GML group. nf-κb expression levels were significantly lower in the 0.10, 0.15, 0.20, and 0.25% GML groups than that in the 0.00% GML group (P <0:05). No significant differences were found in il-1β expression levels (P >0:05). 0.00% GML H: clostridia 0.15% GML I: novosphingobium 3.6. Effects of GML on Challenge Test with V. A: actinomarinales J: alcanivorax B: rothia K: alcanivoracaceae parahaemolyticus. In Figure 8, after the challenge test of V. C: prevotellaceae_Ga6A1_group L: brevinema parahaemolyticus, dietary supplementation with GML sig- D: sulfurimonas M: brevinemataceae nificantly increased the SR (P <0:05) compared with the E: thiovulaceae N: brevinematales 0.00% GML group. The SR values in the 0.05, 0.10, 0.15, F: subdoligranulum O: spirochaetia 0.20, and 0.25% GML groups were significantly higher than G: clostridiales that in the 0.00% GML (P <0:05). After the challenge test, ACP and AKP activities in the Prevotellaceae_Ga6A1_group GML-supplemented groups were significantly higher than Subdoligranulum those in the 0.00% GML group (P <0:05;Table 5).LZM activ- Novosphingobium ity was significantly higher in the 0.05, 0.10, 0.15, and 0.20% Sulfurimonas Thiovulaceae GML groups than that in the 0.00% GML group (P <0:05). Clostridiales Clostridia Rothia 4. Discussion Alcanivorax Alcanivoracaceae The effect of GML on animal health and growth has caused Actinomarinales Spirochaetes widespread concern [25, 26]. Studies has shown that a diet Brevinemataceae supplemented with 0.075% GML significantly improved Brevinematales Spirochaetia WGR and SGR of Danio rerio [15] and L. croceus [27]. In Brevinema addition, 0.07 and 0.105% GML significantly improved –5 –4 –3 –2 –1 01234 WGR and improved the intestinal microbiota of L. vannamei [28]. These results are in line with those obtained in previous LDA score (log 10) research reported on the growth-promoting potential of GML 0.00% GML [29]. In the present study, a similar conclusion was reached; 0.15% GML 0.15% GML in the diet significantly improved the growth. Animal growth is closely related to feeding utilization, Figure 6: Linear discriminant analysis (LDA) score threshold which is influenced by digestion and absorption capacities greater than 3 was presented to demonstrate the variation in the relative abundance of the intestine microbial communities [30]. Digestive enzymes speed up the breakdown and utiliza- between pompano T. ovatus fed diet 0.00% GML and 0.15% GML. tion of the corresponding nutrients by the intestines, thereby improving feed utilization and promoting animal growth. A study has shown that GML could penetrate deep into the GML-supplemented groups (P <0:05). With increasing intestinal tract and greatly affect digestibility and nutrient GML rate, the abundance of Brevinema showed a gradual utilization [31]. In L. vannamei, GML improved protein decrease, whereas that of Acinetobacter showed a decrease digestibility by increasing lipase and protease activities followed by an increase. The percentages of Pseudomonas [28]. In addition, MCFAs and the corresponding glycerides in the 0.00, 0.05, 0.10, 0.15, 0.20, and 0.25% GML groups increased chymotrypsin activity and protein digestibility, were 3.57, 2.50, 2.09, 6.72, 5.21, and 6.60%, respectively. thereby affecting growth similar to that of Atlantic salmon In Figure 6, the linear discriminant analysis (LDA) effect (Salmo salar L.) [32]. Similar conclusions were reached in size (Lefse) package was applied to identify the relative this study. Thus, GML can significantly enhance the intesti- abundance of microbial taxa that differed between the nal digestibility of T. ovatus. 0.15% GML group and the control group. The LDA score The intestine is the most important organ for digestion threshold was set to >3. The linear discriminant analysis and absorption in animals, and a healthy intestinal structure (LDA) shows that the LDA score of Lefse in the 0.15% is a basis for digestive and absorption functions [33]. The 8 Aquaculture Nutrition 1.6 BC 1.4 C BC 1.2 AB AB 1.0 AB AB A B AB AB AB 0.8 0.6 0.4 0.2 0.0 il-10 il-8 il-1𝛽 nf-𝜅 b 0.00% 0.15% 0.05% 0.20% 0.10% 0.25% Figure 7: Relative expression levels of immune-related gene in intestine for juvenile T. ovatus. Data were expressed as means ± SEM. Different letters above a bar are statistically significant different among treatments (P <0:05). 0.00 0.05 0.10 0.15 0.20 0.25 Experimental diets (%) Figure 8: Effects of GML on the survival rate after V. parahaemolyticus (2×10 cfu/ml) infection of juvenile T. ovatus at 7th day. Data were expressed as the mean ± SEM. Values not sharing a common superscript were significantly different (P <0:05). Table 5: Effect of GML on serum immune parameters after challenge for juvenile pompano T. ovatus. Experimental diets Parameters 0.00% 0.05% 0.10% 0.15% 0.20% 0.25% a b d c bc b AKP (U/mL) 2:49 ± 0:51 3:37 ± 0:42 5:57 ± 0:62 4:34 ± 0:20 4:11 ± 0:09 3:46 ± 0:39 a b d c b b ACP (U/mL) 2:59 ± 0:56 5:18 ± 0:14 8:03 ± 0:74 6:59 ± 0:25 5:66 ± 0:54 5:11 ± 0:58 a b b b c ab LZM (U/mL) 0:99 ± 0:23 1:36 ± 0:13 1:39 ± 0:08 1:49 ± 0:17 2:19 ± 0:17 1:28 ± 0:20 Note: data are mean ± SEM (n =3). Values in the same row with different superscripts represent significant difference (P <0:05). AKP: alkaline phosphatase; ACP: acid phosphatase; LZM: lysozyme. VL, VW, and MT of the intestine are important indicators of [14]. Therefore, GML improves intestinal villi growth in juve- the digestive and absorptive function and the health of the nile T. ovatus, contributing to the intestinal morphological intestinal mucosal tissue structure. The height and length of integrity and absorption of nutrients. the villus are significantly correlated with the number of In addition, as an essential immune organ in animals, the mature cells. Only mature villi can absorb nutrients. The lon- intestine protects against external pathogens. GML has good ger the villus length is, the larger the nutrient absorption area anti-inflammatory effects, alleviates the body’sinflammatory will be. In the present study, GML significantly increased the response through multiple pathways, and participates in the VL, VW, and MT, and these results were direct responses to body’s immune regulation [34]. One of the main pathways the ability of the GML to improve intestinal digestion. GML affecting immunity is the activation of nf-κb,aninevitable cen- improved the morphological structure of the intestine, because tral regulator of the inflammatory response involved in the sig- it was used directly by the intestinal epithelial cells when naling pathways of most intrinsic immune receptors [35]. In absorbed by the villous epithelium as an energy supply sub- the present study, GML significantly decreased the nf-κb stance. GML promoted the growth of the villous epithelial cells expression level, which was in agreement with Kong et al. Relative mRNA expression Survival rate (%) Aquaculture Nutrition 9 [36]. The nf-κb is involved in il-8 transcription and influences GML group. Therefore, 0.15% GML can significantly pro- its regulation. The il-8 is one of the proinflammatory cellular mote intestinal health. factors with a widespread role in promoting inflammation Available methods are considerably limited for conducting [37]. In addition, GML could regulate il-10 expression level, a comprehensive study on fish immunity and disease resis- an inflammatory anticellular factor [15]. The il-10 is the main tance. Thus, finding effective biomarkers of disease resistance anti-inflammatory cytokine in fish and inhibits the overactiva- in fish is difficult. Bacterial challenge experiments facilitate tion of the immune response [38]. In the present study, il-10 the assessment of the effectiveness of feeds in protecting gene upregulation and il-8 gene downregulation were the most against pathogens and are often employed as the final indica- significant in the 0.15% GML group. Therefore, this result pos- tor of the fish health status following nutritional experiments sibly showed that GML supplementation might trigger specific [49]. V. parahaemolyticus, a Gram-negative bacterium, is immunological networks of juvenile pompano T. ovatus,and one of the most serious pathogens in mariculture systems further research is needed. [50]. In the present study, GML could significantly enhance The fish intestinal microbiota is a dynamic community SR (80%-96%) in the V. parahaemolyticus challenge test. of aerobic, partly anaerobic, and anaerobic bacteria. This Moreover, after the challenge test, GML significantly community is a special dynamic environment known as improved serum immune enzyme activities (i.e., AKP, ACP, and LZM). These results may be due to the fact that GML the intestinal “island” microbiota. A balanced intestinal microecology is essential for healthy fish growth, and the can easily cross the cell wall and bind to the biofilm to exert balance of the microecology needs to be maintained by a its inhibitory effect on the pathogen [39]. Therefore, feed sup- wide range of beneficial intestinal bacteria. GML has a good plementation with GML significantly improved the disease antibacterial effect and helps stabilize the balance of the ani- resistance of juvenile T. ovatus. mals’ intestinal microbiota [39]. In the present study, an alpha diversity analysis revealed no significant differences, 5. Conclusion indicating that the microbial diversity of juvenile pompano In conclusion, the research indicated that GML significantly T. ovatus fed with GML was not separated. improved growth and intestinal health for juvenile pompano At the phylum level, the dominant intestinal microbiota T. ovatus. In addition, 0.15% GML significantly increased in juvenile T. ovatus included Proteobacteria and Teneri- serum immune enzyme activity, promoted the intestinal cutes, in agreement with existing research [40, 41]. The pre- dominant beneficial microflora, such as Proteobacteria, digestibility, improved the intestinal microflora, regulated Firmicutes, and Bacteroidetes, provided exogenous digestive intestinal immune-related genes, and increased resistance enzymes that can dissolve food into small molecules [42], to V. parahaemolyticus of juvenile pompano T. ovatus. thereby enhancing absorption and utilization in the fish gut [43]. In the present study, GML increased intestinal Data Availability digestive enzyme activity, possibly associated with an The data that support the findings of this study are available increase in beneficial intestinal bacterial populations. In on request from the corresponding author. The data are not addition, Tenericutes have a beneficial role in fish growth publicly available due to privacy or ethical restrictions. and the suppression of pathogenic bacteria [44]. However, an increase in the proportion of Helicobacter pylori leads to Conflicts of Interest an imbalance in the intestinal microbiota and a decrease in animal immunity. At the genus level, Vibrio is reportedly a The authors declare that they have no conflicts of interest. probiotic for aquatic animals, and some bacteriostatic and growth-promoting species exist, such as Vibrio alginolyticus Authors’ Contributions [45]. Pseudomonas produces a range of compounds with a wide range of antifouling biological activities and enables All authors provided contributions to the article and the production of low-temperature proteases, which remain endorsed the submitted version. active in certain extreme environments [46]. However, in the present study, harmful bacteria, such as Brevinema [47] and Acknowledgments Acinetobacter [48], existed in the intestine. Brevinema rich- ness gradually decreased as GML increased, with the lowest This study was financially supported by the National Key level in the 0.15% GML group. A study indicated that in Research and Development Program (2022YFD2401200), the Sparus aurata, GML could increase the relatively abundance Natural Science Foundation of Guangdong Province of positive bacteria, namely, Lactobacillus [29]. Therefore, (2023A1515011095), and the Correspondent for Science and GML is effective in inhibiting harmful bacteria in the juve- Technology of Guangdong Province (GDKTP2021048400). nile T. ovatus gut. In addition, the 0.00 and 0.15% GML groups were also analyzed using LDA according to their Supplementary Materials growth. Compared with the 0.00% GML group, beneficial bacteria, such as Sulfurimonas, Novosphingobium, and Sub- Supplementary material to this article is as follows: 2.1 Animals and diet preparation. 2.2 Sample collection and analyses. 2.5 doligranulum, increased in the 0.15% GML group. By con- trast, harmful bacteria, such as Brevinema, Brevinematales, Intestinal microbiota sequencing analysis. 2.6 Real-time PCR analysis. Table S1: composition and nutrient levels of the Brevinemaceae, and Acinetobacter, decreased in the 0.00% 10 Aquaculture Nutrition experimental diets. Table S2: effects of GML on growth and feed [15] Y. Wang, H. Zhong, J. Wang, and F. Feng, “Dietary glycerol monolaurate improved the growth, activity of digestive utilization for juvenile T. ovatus. (Supplementary Materials) enzymes and gut microbiota in zebrafish (Danio rerio),” Aqua- culture Reports, vol. 20, article 100670, 2021. References [16] Q. Mo, A. Fu, L. 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