Galactooligosaccharide and a Combination Of

Galactooligosaccharide and a combination of yeast and β-glucan supplements enhance growth and improve intestinal condition in striped catfish Pangasianodon hypophthalmus fed soybean meal diets Amalia Sutriana, Roshada Hashim, Mst. Nahid Akter & Siti Azizah Mohd Nor

Fisheries Science

ISSN 0919-9268 Volume 84 Number 3

Fish Sci (2018) 84:523-533 DOI 10.1007/s12562-018-1195-4

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Fisheries Science (2018) 84:523–533 https://doi.org/10.1007/s12562-018-1195-4

ORIGINAL ARTICLE

Aquaculture

Galactooligosaccharide and a combination of yeast and β‑glucan supplements enhance growth and improve intestinal condition in striped catfsh Pangasianodon hypophthalmus fed soybean meal diets

Amalia Sutriana1 · Roshada Hashim2 · Mst. Nahid Akter3 · Siti Azizah Mohd Nor4

Received: 3 August 2017 / Accepted: 14 February 2018 / Published online: 14 March 2018 © Japanese Society of Fisheries Science 2018

Abstract The efects of galactooligosaccharide (GOS) and a combination of yeast and β-glucan (YβG) supplementation of dietary soybean meal (SBM) on the growth and digestive performance of striped catfsh Pangasianodon hypophthalmus were evalu- ated. Four isonitrogenous (30% protein) and isocaloric (19 MJ/kg diet) diets were formulated to contain 100% fsh meal (FM) protein, 55% FM protein/45% SBM protein, FM-SBM supplemented with 1% GOS, and a combination of 1% yeast and 0.1% β-glucan, respectively. Each diet was fed for 12 weeks to three groups of 30 striped catfsh fngerlings (average weight 16.45 ± 0.07 g) maintained in circular fberglass tanks (600 l). Growth, feed utilization, and muscle protein composi- tion of fsh improved signifcantly after supplementation with either GOS or YβG compared to the unsupplemented SBM diet, but were similar to those of fsh fed the FM diet. Nutrient digestibility, digestive enzyme activities, villi and microvilli length were signifcantly increased in fsh fed the supplemented SBM diets. The gut microbiota ranking profle showed that supplementing the SBM diet with YβG and GOS gave a ranking of Verrucomicrobia, Spirochaetes, Bacteriodetes, and Act- inobacteria phyla similar to that of the FM diet. Thus, diet containing 45% protein from soybean supplemented with either GOS or YβG can be recommended to improve the growth and digestive performance of striped catfsh.

Keywords Fish performance · Nutrient digestibility · Digestive enzyme · Villi · Gut microbiota

Introduction competitive price compared to fsh meal (FM) (Biswas et al. 2007). However, the use of high levels of SBM in the diet For decades, soybean meal (SBM) has been used as an can cause a reduction in growth and feed utilization in rain- alternative plant protein source in the fsh farming indus- bow trout (Oliva-Teles et al. 1994), Atlantic salmon (Ref- try because of its high protein content, comparatively well- stie et al. 1998), and striped catfsh (Phumee et al. 2011), balanced amino acid profle, relative ease of availability, and as well as morphological and functional disruption, such as enteritis, increased susceptibility to bacterial infection, * Amalia Sutriana changes in absorptive cells, presence of infammatory cells, [email protected] and shortening of the villi and microvilli in the intestines of Atlantic salmon (van den Ingh et al. 1991; Baeverford and 1 Faculty of Veterinary Medicine, Syiah Kuala University, Krogdahl 1996; Bakke-McKellep et al. 2000), rainbow trout Banda Aceh, Aceh 23111, Indonesia (Rumsey et al. 1994) and common carp (Uran et al. 2008). 2 Faculty of Science and Technology, Universiti Sains Islam There is also evidence of a decrease in digestive enzyme Malaysia, 71800 Bandar Baru Nilai, Negeri Sembilan, Malaysia activities in the distal intestinal epithelial cell brush bor- ders as reported in Atlantic salmon fed a SBM-containing 3 Faculty of Fisheries, Hajee Mohammad Danesh Science and Technology University, Dinajpur 5200, Bangladesh diet, which was attributed to histopathological changes in the intestine (Bakke-McKellep et al. 2000; Krogdahl et al. 4 Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21300 Kuala Terengganu, Terengganu, Malaysia 2003). Additionally, SBM proteins are mainly compact and

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524 Fisheries Science (2018) 84:523–533 globular (glycinin and β-conglycinin) making hydrolysis and Table 1 Ingredients and proximate composition (g/kg dry matter) of absorption slower than those of FM protein (Yamamoto et al. the experimental diets 1998); these characteristics of SBM proteins potentially Experimental diets reduce their digestibility in fsh. FM FM-SBM FM-SBM- FM-SBM-YβG In recent years, considerable attention has been paid to GOS the use of non-nutrient dietary components such as probi- Ingredients otics and prebiotics to mitigate the negative efects of the a SBM diet. Several studies have shown that prebiotic supple- FM 382.50 211.00 211.00 211.00 ments can signifcantly improve the nutritive value of SBM. Soybean – 267.00 267.00 267.00 meal For example, they protect the intestinal mucosa and reduce Corn starch 430.00 324.70 324.70 324.70 the risk of induced morphological changes in salmon gut Fish oil 28.70 43.70 43.70 43.70 (Dimitroglou et al. 2011); increase protein, organic matter, Soybean oil 58.80 53.60 53.60 53.60 and energy availability in red drum (Burr et al. 2008); and Cellulose 20.00 20.00 10.00 9.00 improve feed efciency in hybrid striped bass (Li and Gatlin GOS – – 10.00 – 2005) and red drum (Buentello et al. 2010) because of the Yeast – – – 10.00 upregulation of the activities of specifc digestive enzymes β-Glucan – – – 1.00 (Buentello et al. 2010). All these efects are a consequence CMC 30.00 30.00 30.00 30.00 of the benefcial modulation of the populations of good bac- b Vitamins 20.00 20.00 20.00 20.00 teria in the fsh intestine. c Minerals 20.00 20.00 20.00 20.00 Preliminary studies using probiotics and prebiotics, sin- Cr2O3 10.00 10.00 10.00 10.00 gly and in combination, in diets containing FM as the sole Proximate composition (g/kg) protein source, indicate that the prebiotic galactooligosac- Moisture 80.30 82.40 79.60 86.90 charide (GOS) and the combination of yeast and β-glucan Protein 306.20 309.60 313.00 305.80 (YβG) positively infuence growth performance and feed uti- Lipid 129.20 117.10 125.60 121.30 lization of striped catfsh (Sutriana 2017). Based on the fact Ash 80.20 89.40 79.60 81.70 that these supplements are able to improve nutrient digest- Fibre 24.10 42.20 49.60 49.00 ibility and gut microfora, the present study was designed to NFEd 460.30 441.70 432.20 442.20 investigate the infuence of GOS and yeast combined with GEe (MJ/kg) 19.02 18.91 19.68 19.43 β-glucan on growth, digestibility, intestinal morphology, and microbiota in striped catfsh, Pangasianodon hypophthalmus FM Fish meal, SBM soybean meal, GOS galactooligosaccharide, YβG fngerlings fed a diet containing SBM. As far as we know, yeast and β-glucan, CMC carboxymethyl cellulose, NFE nitrogen-free extract, GE gross energy this is the frst efort to evaluate the efect of GOS and YβG a Danish fsh meal supplementation to improve the nutritive value of SBM diet b Vitamin (Vit.) mix/kg (Rovitai, Chonburi, Thailand): Vit. A, 50 mil- in striped catfsh. lion international units (IU); Vit. D3, 10 million IU; Vit. E, 130 g; Vit. B1, 10 g; Vit. B2, 25 g; Vit. B6, 16 g; Vit. B12, 100 mg; biotin, 500 mg; pantothenic acid, 56 g; folic acid, 8 g; niacin, 200 g; anticak- Materials and methods ing agent, 20 g; antioxidant, 200 mg, Vit. K3, 10 g; Vit. C, 35 g c Mineral mix/kg: calcium phosphate (monobasic), 397.65 g; cal- Diet formulation and feeding cium lactate, 327 g; ferrous sulfate, 25 g; magnesium sulfate, 137 g; potassium chloride, 50 g; sodium chloride, 60 g; potassium iodide, 150 mg; copper sulfate, 780 mg; manganese oxide, 800 mg; cobalt Four diets were prepared containing 30% protein and 12% carbonate, 100 mg; zinc oxide, 1.5 g; sodium selenite, 20 mg lipid with a gross energy content of 19 MJ/kg (Table 1). d [100 − (protein + lipid + ash + fber)] The control diet contained 100% protein from FM and the e Measured using a Parr 6200 bomb calorimeter remaining three diets were formulated so that 45% of the protein was provided by SBM and 55% from FM (FM- SBM). Two of the latter diets were supplemented with 1% The ingredients were mixed thoroughly in a feed mixer GOS (FM-SBM-GOS) and a combination of 1% yeast and (model TR 202; Tyrone, UK); 1% chromium oxide was 0.1% β-glucan (FM-SBM-YβG), respectively. The replace- added as an inert marker for protein and dry matter digest- ment of 45% protein with SBM was based on the report by ibility determination. Distilled water was added, and the Phumee et al. (2011), who showed that inclusion of SBM moist dough was passed through a 3-mm die using a meat protein beyond 30% compromised fnal weight, specifc grinder (model MH 237; Miao Hsien, Taiwan). After drying growth rate and protein efciency ratio of juvenile striped in an electric oven at 40 °C for 24 h, the diets were sealed in catfsh. polyethylene bags and stored at − 20 °C until use.

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Fish and culture system PER = wet weight gain (grams)/total protein intake (grams), Striped catfsh were obtained from a commercial farm and FCR = total dry feed intake (grams)/wet weight gain acclimatized for 2 weeks prior to starting the experiment (grams), at the Aquatic Research Complex, Universiti Sains Malay- SR (%) = (fnal number of fsh/initial number of fsh × sia. At the start of the experiment, 30 fsh (average weight 100, 16.45 ± 0.07 g) were randomly distributed into 12 circular HSI (%) = {100 × [liver weight (grams)/body weight fberglass tanks (600 l). The experimental diets were fed to (g)]}, triplicate groups of fsh for 12 weeks. Continuous aeration IPF (%) = {100 × [intraperitoneal fat weight (grams)/ was provided to each tank. All groups were fed their respec- body weight (grams)]}, tive diets twice daily at 3% body weight/day and adjusted VSI (%) = {100 × [viscera weight (grams)/body weight biweekly according to fsh body weight. During the experi- (grams)]}. ment, water temperatures were 27–29 °C, pH 6.2–6.5, and dissolved oxygen between 5 and 6 mg/l. Growth and feed Analysis of samples efciency were monitored biweekly by collectively weigh- ing each group of fsh. At the end of 12 weeks, fsh were Proximate analyses of ingredients, experimental diets, and sampled for muscle proximate analysis, digestive enzyme fsh muscle were performed according to the standard pro- activities, histological examination, and microbiota analysis. cedure described by the Association of Ofcial Analytical The digestibility of the experimental diets was determined Chemists (AOAC 1997). Briefy, crude protein was deter- using a diferent set of tanks (300-l rectangular fberglass mined by measuring nitrogen (N × 6.25) using the Kjeldahl tanks) but with a similar experimental design as described procedure with concentrated sulfuric acid for digestion, 40% above, and fecal collection was made 1 month after feeding sodium hydroxide for distillation, and 0.1 N hydrochloric the respective diets. At approximately 4 h post-feeding, the acid for titration. Crude lipid contents were determined walls and bottom of the tanks were thoroughly cleaned of using the chloroform–methanol (2:1 volume/volume; v/v) uneaten food and fecal residue. Feces were collected 5–8 h extraction method (Folch et al. 1957). Crude ash contents thereafter by siphoning immediately after defecation, and were determined after heating dry samples in a mufe fur- samples from each tank were pooled as a single composite nace at 550 °C for 5 h. Crude fber content was determined sample for each treatment (three tanks per diet). The pooled as loss on ignition of dried lipid free residues after digestion fecal material was freeze dried, placed into sterile bags and with 5% sulfuric acid. Nitrogen-free extract was calculated stored at − 20 °C until analysis. as 100 − (crude protein + crude lipid + crude fber + crude ash contents). Apparent protein digestibility (APD) and apparent dry Growth, feed utilization, survival and body indices matter digestibility (ADM) were analyzed by the method of assessment Furukawa and Tsukuhara (1966) and calculated using the following formulae. At the termination of the experiment, the fsh were starved % Cr2O3 in diet (a) ADP(%) = 100 − 100 for 24 h. Fish in all tanks were counted and individually % Cr2O3 in faeces weighed. Three fsh were taken randomly from each tank and % protein in faeces × , fsh muscle from each tank was pooled and stored at − 20 °C  % protein in diet  for subsequent proximate analysis. The fnal weight, weight % Cr O in diet gain (WG), specifc growth rate (SGR), protein efciency ADP(%) = 100 − 100 2 3 . (b) ratio (PER), feed conversion ratio (FCR), survival rate (SR), % Cr2O3 in faeces hepatosomatic index (HSI), intraperitoneal index (IPF), and viscerosomatic index (VSI) were calculated using the fol- Digestive enzyme assays lowing formulae: Final weight/fsh (grams) = total weight of fsh per tank/ For enzyme activity analysis, three fsh from each tank no. of fsh, were sampled 4 h after feeding (Grisdale-Helland et al. WG/fsh (grams) = fnal weight − initial weight, 2008). The whole intestinal tract was isolated and rinsed with cold distilled water at 4 °C, weighed and transferred SGR (%/day) = [(ln Wf − ln Wi)/T] × 100, to a sterile tube. The intestinal samples were homogenized where ln Wf is the natural logarithm of the fsh weight in phosphate bufer saline and the supernatants were kept (grams) at the end of experiment, ln Wi is the natural loga- rithm of the fsh weight (grams) at the start of the experi- at − 80 °C until analysis. Intestinal enzyme extracts were ment, T is experiment duration (days). obtained by homogenizing 1 g intestinal tract in 10 ml

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526 Fisheries Science (2018) 84:523–533 phosphate bufered saline (pH 7.5) using IKA homogenizer pyrosequencing with barcoded primers that target the V2 (T18 Basic Ultra-Turrax; Germany), and centrifuging the region of the 16S rRNA gene. Paired-end 16S community suspension at 15,000 g for 15 min at 4 °C. Supernatants were sequencing on the Illumina MiSeq platform used the bac- transferred to new tubes and stored at − 80 °C until analysis. terial/archaeal primers 515F/806R (Caporaso et al. 2012). The total protein content of the intestinal enzyme extract was determined by the method of Bradford (1976) using the Statistical analysis Bio-Rad protein assay kit. Bradford reagent was prepared by diluting Bio-Rad dye reagent concentrate (#500-0006) Means and SDs were calculated for each parameter meas- with double-distilled water in a ratio of 1:4 (v/v). A 10-µl ured. Data were subjected to ANOVA using SPSS software aliquot of the respective enzyme extract was pipetted into the (version 22). Statistical signifcance was set at P < 0.05 and assigned wells of a 96-well microtiter plate in triplicate and Tukey’s multiple range test was used for comparison of treat- then immediately mixed with 200 µl of the prepared Bio-Rad ments mean. The microbial community data were analyzed dye reagent. The microtiter plate containing the mixture was descriptively. incubated at room temperature for 15 min. Thereafter, the absorbance of the solution was measured by using Bio-Rad micro-plate reader model 680 at 595 nm. The protein content Results in the intestinal enzyme extract was determined using bovine serum albumin (no. 500-0007) as a standard with various Growth performance and feed utilization concentrations (0.0–0.5 mg/ml at 0.05 mg/ml steps). Protease activity was measured as described by Walter Growth parameters, body indices, and survival rates of (1984) using casein hydrolysis at pH 8. Amylase activity was striped catfsh fed diferent experimental diets after 12 weeks quantifed as described in the Worthington Enzyme Manual of feeding are summarized in Table 2. Striped catfsh receiv- (1988) using starch as the substrate at 540 nm; lipase activ- ing either a diet supplemented with GOS or YβG showed ity was determined by measurement of fatty acids released similar weight gain and SGR to fsh receiving the FM diet, following enzymatic hydrolysis of triglycerides in a stabi- but had signifcantly higher weight gain and SGR compared lized emulsion of olive oil (Bier 1955). Digestive enzyme to fsh fed FM-SBM diets. Similarly, feed utilization also activities (i.e., protease, amylase, and lipase) are presented improved with the intake of GOS and YβG, respectively, in as specifc activities (units per milligram protein). which fsh fed the supplemented SBM diets had signifcant higher protein efciency ratio and signifcantly lower food Histological studies conversion ratio compared to fsh fed unsupplemented FM- SBM (P < 0.05). The lowest IPF and highest VSI values Nine fsh from each treatment (three fsh from each tank) were observed in fsh fed the unsupplemented SBM diet. were sacrifced and intestinal samples were used for histo- Cumulative survival after 12 weeks was generally high logical examination by light microscopy (LM) and transmis- with only one case of mortality in the SBM treatment group sion electron microscopy (TEM). Intestinal sections from (Table 2). the anterior (1 cm after the stomach) and posterior regions (1-cm segment before the anus) (Anguiano et al. 2013) Muscle proximate composition were cut for both LM and TEM analysis. Tissue samples for LM and TEM were processed and analyzed following Analysis of fsh body composition showed that protein con- the method described by Akter et al. (2015) and Lewis and tent remained unafected in striped catfsh fed all experimen- Knight (1977), respectively. tal diets (Fig. 1). Lipid content was unafected by the intake of GOS and YβG and did not difer signifcantly from that Gut sample collection for metagenomic DNA of fsh fed unsupplemented SBM diets and FM diet. Gener- extraction ally, no trend was observed in muscle ash content except that levels in fsh fed the FM-SBM-YβG were signifcantly lower Nine fsh were killed by immersion in ice water (≤ 4 °C) than in fsh fed the corresponding unsupplemented diet. at 7 days post-feeding. They were decontaminated in 70% ethanol and transferred to a laminar fow hood. Entire intes- Nutrient digestibility tines were removed from each fsh after dissection under sterile conditions and washed with sterile saline solution. The APD and ADM of the diets tested in this study ranged The intestines from the nine fsh were pooled and preserved from 77.93 to 84.38% and 73.13 to 75.62%, respectively in dry ice and sent to Mycrsynth (Balgach, Switzerland) (Fig. 2). Signifcant highest and lowest APD were obtained where the microbial community profle was determined by for the FM and FM-SBM diet, respectively. Among the

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Table 2 Growth performance, Parameters Experimental diets feed utilization, and body indices of striped catfsh fed FM FM-SBM FM-SBM-GOS FM-SBM-YβG experimental diets for 12 weeks Initial fsh weight (g) 16.52 ± 0.06 16.48 ± 0.03 16.51 ± 0.08 16.54 ± 0.08 Final weight (g) 46.42 ± 1.19 a 34.48 ± 1.25 b 42.68 ± 2.43 a 44.43 ± 2.14 a Weight gain (g) 29.89 ± 1.26 a 18.00 ± 1.27 b 26.18 ± 2.48 a 28.90 ± 3.07 a SGR (%/day) 1.23 ± 0.03 a 0.88 ± 0.04 b 1.13 ± 0.07 a 1.20 ± 0.08 a PER 1.92 ± 0.02 a 1.23 ± 0.17 b 1.76 ± 0.13 a 1.90 ± 0.11 a FCR 1.70 ± 0.02 a 2.67 ± 0.38 b 1.82 ± 0.14 a 1.73 ± 0.10 a HSI (%) 1.55 ± 0.38 1.69 ± 0.30 1.56 ± 0.29 1.47 ± 0.26 IPF (%) 3.21 ± 0.54 a 2.49 ± 0.80 b 2.73 ± 0.65 a 3.03 ± 0.83 a VSI (%) 3.40 ± 0.78 ab 4.36 ± 0.54 c 3.51 ± 0.76 b 3.69 ± 0.68 bc Survival (%) 100.00 96.67 100.00 100.00

Data are expressed as mean ± SD. Mean values in the same row with diferent lowercase letters are signif- cantly diferent (P < 0.05). For abbreviations, see Table 1

Fig. 1 Proximate composition (protein, lipid, ash) of striped catfsh muscle fed experimental diets for 12 weeks on dry weight basis. Diferent lowercase letters indicate signifcantly diferent results (P<0.05)

Fig. 2 Apparent protein digest- ibility (APD) and dry matter digestibility (ADM) for striped catfsh fed experimental diets for 12 weeks. Diferent low- ercase letters indicate signif- cantly diferent results (P<0.05)

diets containing SBM, adding GOS (82.69 ± 0.36) and Although the intake of GOS and YβG improved the ADM YβG (82.20 ± 0.27) improved APD signifcantly (P<0.05) of SBM diets, signifcant improvement was only observed compared to the diet containing SBM alone (77.93 ± 1.15). when GOS was added to the FM-SBM diet.

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Fig. 3 Specifc activity of amylase, protease, and lipase (unit/mg protein) in the intestine of striped catfsh fed experimental diets for 12 weeks. Diferent lowercase letters indicate signifcantly diferent results (P < 0.05)

Digestive enzyme activity were signifcantly higher for the FM-SBM diet compared to the FM-SBM-YβG treatment but not signifcantly diferent The supplementation of GOS and YβG to the FM-SBM from those in the FM and FM-SBM-GOS treatments. TEM diet signifcantly improved (P < 0.05) amylase, protease and micrographs of the posterior intestine region are shown in lipase activities in striped catfsh (Fig. 3). A comparison Fig. 4. Fish maintained on GOS and YβG supplemented between the supplemented diets and the FM diet showed diets showed a signifcant increase in microvilli length com- highest signifcant amylase activity (P< 0.05) in fsh fed pared to the FM-SBM fed fsh (Table 3). the FM-SBM-GOS diet, while fsh fed the FM-SBM-YβG diet had signifcantly higher protease activity. No signifcant Gut microbiota composition diferences were observed in any of the digestive enzymes tested between the FM-SBM-GOS and FM-SBM-YβG diets. A total of ten bacterial phyla were identifed from the gut of striped catfsh; the most abundant were Firmicutes, Ver- rucomicrobia, Bacteriodetes, Proteobacteria, and Fusobac- Gut histomorphology teria (Fig. 5). The abundance of the diferent microbiota varied among diets. However, regardless of the diet, Fir- The results of the histological examination of intestinal mor- micutes was the most dominant phylum in all fsh. Fish fed phology are presented in Table 3. LM analysis of the ante- the unsupplemented SBM diet showed an increased relative rior intestine indicated that the GOS and YβG supplements abundance of Firmicutes (51.70%) in contrast to fsh fed signifcantly increased villi length compared to the unsup- the GOS and YβG supplemented diets (42.40 and 48.10%, plemented SBM-fed fsh, but was similar to those fed the respectively). FM diet. However, mean villi length signifcantly increased Verrucomicrobia was the next most abundant phylum in the posterior intestine of fsh fed the supplemented diets (range 12.20–33.10%) but had notably lower abundance in compared to the unsupplemented SBM diet and FM diet. fsh fed the unsupplemented SBM (12.20%) diet over all the Villi width in the anterior gut was not altered by any of other diets. Likewise, the percentages of Proteobacteria and the dietary treatments, but in the posterior gut the values Fusobacteria were lowest in fsh fed the unsupplemented

Table 3 Intestinal villi lengths Diets Anterior gut Posterior gut Posterior gut and widths (µm) in the anterior and posterior gut of striped Villi length Villi width Villi length Villi width Microvilli length catfsh fed experimental diets for 12 weeks FM 465.57 ± 18.13 a 95.50 ± 5.80 369.97 ± 14.16 b 81.75 ± 3.99 ab 1.34 ± 0.10 a FM-SBM 329.53 ± 13.68 b 95.25 ± 6.57 308.04 ± 9.74 c 89.12 ± 2.35 b 0.92 ± 0.07 b FM-SBM-GOS 486.52 ± 14.91 a 88.69 ± 7.15 404.77 ± 15.99 a 81.14 ± 4.56 ab 1.23 ± 0.01 a FM-SBM-YβG 462.53 ± 6.88 a 82.31 ± 9.38 395.50 ± 10.47 a 77.80 ± 3.28 a 1.33 ± 0.12 a

Data are expressed as mean ± SD. Mean values in the same column with diferent lowercase letters are sig- nifcantly diferent (P < 0.05). For abbreviations, see Table 1

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Fig. 4 Transmission electron microscopic micrograph of microvilli in the posterior gut region of striped catfsh fed experimental diets for 12 weeks. Yellow line indicates microvillus length. Scale bar equals 2 µm

Bacteria Phyla Actinobacteria (0.10–0.30%). A percentage of other bacteria was also found

100% Bacterioidetes ranging from 1.00 to 1.90%. 90% Firmicutes The gut microbiota profle after the 12-week feeding ) 80% Fusobacteria period was determined by ranking the dominance of each 70% phyla within each treatment. With the exception of Firmi- Lentisphaerae 60% cutes, which ranked frst for all treatments tested, the posi- Planctomycetes 50% tion of other phyla varied among dietary treatment. The gut

Abundance (% Proteobateria 40% microbiota ranking profle of fsh fed FM-SBM was diferent 30% Spirochaetes from that of the FM treatment for Verrucomicrobia, Syner- 20% Synergistetes Relative gistetes Bacteriodetes Fusobacteria Spirochaetes Lenti- 10% , , , , Verrucomicrobia 0% sphaerae and Actinobacteria. Supplementing the SBM diet other with YβG and GOS gave a similar ranking profle for the Verrucomicrobia and Spirochaetes as seen for the FM diet. While the ranking of Synergistetes dropped, the ranking of Bacteriodetes, Fusobacteria and Actinobacteria was higher Fig. 5 Relative abundance (%) of the main bacterial phyla present in in fsh fed SBM-based diets compared to the FM diet. Die- the gut of striped catfsh fed experimental diets for 12 weeks tary GOS and YβG lowered the position of Bacteriodetes to almost the same as that for the FM diet. SBM diet (7.40 and 1.3%, respectively) compared to the other diets. The highest percentage of Synergistetes was in fsh fed the FM diet (15.30%), whereas the percentage of Discussion Bacteriodetes was highest in fsh fed the unsupplemented SBM diet (24.70%). Another four phyla were found at rela- The limitations of soybean as a dominant protein source in tively low percentages and varied in abundance, including fsh feeds is well documented, and in some fsh species this Spirochaetes (0.30–1.20%), Actinobacteria (0.10–0.40%), disadvantage can be overcome by the inclusion of probiotics Lentisphaerae (0.10–0.60%) and Planctomycetes and prebiotics, e.g., in red drum (Burr et al. 2008), Atlantic

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530 Fisheries Science (2018) 84:523–533 salmon (Grisdale-Helland et al. 2008), sharpsnout seabream of the fsh (Dimitroglou et al. 2009; Daniels et al. 2010; (Piccolo et al. 2013) and rainbow trout (Sealey et al. 2010). Zhou et al. 2010). The analysis of intestinal morphology in These studies also indicate that the infuence of these sup- this study showed that GOS or YβG supplementation of the plements depends on the species, as well as the probiotics SBM diet signifcantly increased villi length in both the ante- and prebiotics used. rior and posterior gut regions, and a signifcant improvement In striped catfsh, preliminary studies to investigate the in microvilli length was also observed in the posterior gut. infuence of probiotic and prebiotics in diets containing FM Since nutrient uptake is related to microvilli length, which and casein as the main protein source showed that GOS and determines absorptive surface area, an increase of microvilli YβG signifcantly enhanced both growth performance and length would have resulted in better nutrient absorption, uti- feed utilization to levels better than the unsupplemented FM lization, and retention (Daniels et al. 2010; Sang and Fotedar diet (Sutriana 2017). In this study, feeding striped catfsh 2010) and consequently induced the improvement of nutrient with SBM-based diets containing GOS and YβG, respec- utilization and growth performance of striped catfsh in this tively, resulted in signifcantly better growth and feed utili- study. Anguiano et al. (2013) observed similar increases in zation compared to the FM-SBM diet, but were similar to villi and microvilli length in red drum (Sciaenops ocella- those of the FM diet. tus) and hybrid striped bass (Morone chrysops × M. saxa- These positive efects of GOS and YβG supplementa- tilis) fed dietary GOS. To date, the efect of dietary YβG tion on growth and feed efciency may be associated with on fsh microvilli length is not known; however, fsh fed a improved nutrient digestibility. In this study, signifcant diet supplemented with either β-glucan or MOS (prebiot- decreases in ADM and APD were found in fsh fed the ics derived from yeast cell walls) show signifcant increases unsupplemented SBM diet. These observations are consist- in villus and microvillus lengths compared to those fed an ent with Phumee et al. (2011), who reported that APD in unsupplemented diet (Yilmaz et al. 2007; Dimitroglou et al. striped catfsh was highest in a diet containing FM as the 2009; Daniels et al. 2010; Kühlwein et al. 2014). Moreover, only protein source compared to a diet containing SBM. Abu-Elala et al. (2013) observed that tilapia treated with This was attributed to the diferent protein structures found Saccharomyces cerevisiae showed an increase in the length in SBM, in that the SBM diet contains mainly compact and and density of the intestinal villi. globular proteins that are generally hydrolyzed and absorbed The gut microbiota form a complex community of micro- more slowly than FM proteins (Yamamoto et al. 1998). In organisms within the gastrointestinal tract of animals and this study, supplementation with either GOS or YβG showed are important for host metabolism (Velagapudi et al. 2010; signifcant improvement in APD and ADM, which is in line Backhed 2011) as they provide energy through various with previous studies (Burr et al. 2008; Grisdale-Helland metabolic pathways such as fermentation and absorption of et al. 2008), indicating that these supplements are able undigested carbohydrates (Gill et al. 2006). There is increas- to make nutrients more accessible by increasing nutrient ing evidence that the microbial community of the intestinal digestibility and contributing to the improvement in growth tract provides both nutritional benefts and protection against and feed utilization. pathogens in fsh (Ringø et al. 2010), therefore manipula- Nutrient digestibility of the supplemented SBM diet tion of the gut fora is an important mechanism to achieve was further enhanced by the increased digestive enzyme an increased feed efciency, growth, and health of fsh in activities possibly following the modulation of the bacte- aquaculture (Geraylou et al. 2013). rial populations in striped fsh intestines. There is evidence Firmicutes, the dominant phylum in the fsh gut together that prebiotics and probiotics can increase the benefcial with Verrucomicrobia, Bacteriodetes and Proteobacteria, intestinal microbiota (such as lactic acid bacteria) in vari- are associated with metabolic functions (Clavel et al. 2014) ous fsh species (Ringø and Gatesoupe 1998; Irianto and as they ferment non-digestible carbohydrates in the intes- Austin 2002; Ringø et al. 2010), and particular improvement tine (Gill et al. 2006; Turnbaugh et al. 2006; van Kessel in the number of lactic acid bacteria following feeding of a et al. 2011) and interact with the immune system (Kau et al. prebiotic has also been observed in striped catfsh (Akter 2011). Furthermore, Smriga et al. (2010) suggested that et al. 2015). These lactic acid bacteria may improve gut members of the Proteobacteria, Bacteroidetes, Firmicutes maturation and secrete protease and amylase enzymes in the and Fusobacteria may contribute to the digestive process digestive tract (Irianto and Austin 2002; Gullian et al. 2004; by providing fsh with a variety of enzymes, as observed in Wache et al. 2006; Askarian et al. 2011; Hoseinifar et al. parrotfsh, snapper or sturgeon. 2013); thus, contribute to the signifcantly higher enzyme The response of the gut microbiota profle to the difer- activities observed in this study (Fig. 3). ent diets was mapped by ranking the bacteria according to Further, increasing the number of lactic acid bacteria in their percentage abundance within each treatment. Replac- the intestine resulted in a higher concentration of the volatile ing the FM diet with 45% protein from SBM altered the fatty acids that benefcially afect the intestinal morphology relative abundance of six phyla of gut bacteria, while the

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Fisheries Science (2018) 84:523–533 531 pattern only difered for two and three of the gut micro- Acknowledgments Thanks are due to technical staf at the School of biota when the SBM diet was supplemented with GOS Biological Sciences, Universiti Sains Malaysia. We are grateful to Frie- sland Foods for supplying Vivinal GOS and to Biorigin, Macrogard for and YβG, respectively, compared to the control FM diet. the β-glucan. This study was supported by the Malaysian Ministry of The dominant bacteria afected were Verrucomicrobia, Higher Education ERGS grant no. 203/PBIOLOGY/6730134. Bacteriodetes and Proteobacteria. Verrucomicrobia is a mucolytic bacterium (Png et al. 2010) responsible for the degradation of mucin in the mucus layer (Kim and Ho References 2010; Ganesh et al. 2013). The drop in its position from 2nd to 3rd position in the SBM diet suggests gut infam- Abu-Elala N, Marzouk M, Moustafa M (2013) Use of diferent Saccha- mation due to a disruption of the mucus layer in fsh fed romyces cerevisiae biotic forms as immune-modulator and growth Oreochromis niloticus c Ver- promoter for hallenged with some fsh the SBM diet making it unfavorable for the growth of pathogens. Int J Vet Sci Med 1:21–29 rucomicrobia. Researchers have observed that inclusion of Akter MN, Sutriana A, Talpur AD, Hashim R (2015) Dietary sup- soybean in fsh feed is associated with distal gut infamma- plementation with mannan oligosaccharide infuences growth, tion characterized by changes in absorptive cells, shorten- digestive enzymes, gut morphology, and microbiota in juvenile striped catfsh, Pangasianodon hypophthalmus. Aquac Int. https​ ing of intestinal villi and microvilli, increase in the number ://doi.org/10.1007/s1049​9-015-9913-8 of infammatory cells in the lamina propria, and increase Anguiano M, Pholenz C, Buentello A, Gatlin DM III (2013) The efects or decrease in numbers of mucus-producing goblet cells of prebiotics on the digestive enzymes and gut histomorphology of (Baeverford and Krogdahl 1996; van den Ingh et al. 1996; red drum (Sciaenops ocellatus) and hybrid striped bass (Morone chrysops × M. saxatilis). Br J Nutr 109:625–629 Merrifeld et al. 2011). As a decrease of villi length and Association of Ofcial Analytical Chemists (AOAC) (1997) Associa- micro-villi length following SBM feeding was evident tion of Ofcial Analytical Chemists. Ofcial Methods of Analysis in this study (Table 3), it is assumed that SBM-induced of AOAC International, 16th edn, vol 1. 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