Aquaculture is one of the fastest growing food industries in the world(1) and continues to expand to accommodate the increase in seafood demand from a growing human population. However, this expansion has resulted in large-scale, intensive production facilities that often expose fish to stressful conditions and thus making the fish more susceptible to various diseases(Reference Balcazar, de Blas and Ruiz-Zarzuela2). Traditionally, antimicrobial agents have been used to treat disease outbreaks; however, there have been concerns over the misuse of antimicrobials leading to the evolution of antimicrobial resistance in numerous bacterial pathogens and thus prompting research for alternative methods(Reference Balcazar, de Blas and Ruiz-Zarzuela2, Reference Buentello, Neill and Gatlin3).
A relatively new alternative to using antibiotics in aquaculture has been to supplement prebiotics in the diet(Reference Buentello, Neill and Gatlin3–Reference Burr, Hume and Ricke8). Prebiotics are defined as ‘non-digestible food ingredients that beneficially affect the host by selectively stimulating growth and/or activity of one or a limited number of bacteria in the intestinal tract’(Reference Gibson and Roberfroid9). Although applications of prebiotics to human subjects and terrestrial livestock have been studied more extensively(Reference Cummings and Macfarlane4, Reference Kelly6, Reference Gibson and Roberfroid9–Reference Yang, Iji and Kocher12), information on the use of prebiotics in aquatic animals is rapidly accumulating(Reference Burr, Gatlin and Ricke5, Reference Li and Gatlin13–Reference Zhou, Buentello and Gatlin19).
Recent reviews(Reference Merrifield, Dimitroglou and Foey20–Reference Yousefian and Amiri22) in prebiotics have documented positive effects in various fish on growth and immune parameters from prebiotic supplementation. In particular, research in two prominent fish species, red drum (Sciaenops ocellatus) and hybrid striped bass (Morone chrysops× M. saxatilis), has demonstrated that dietary inclusion of prebiotics can increase feed efficiency, enhance nutrient and energy digestibility and reduce mortality of these two species(Reference Buentello, Neill and Gatlin3, Reference Burr, Hume and Ricke8, Reference Burr, Hume and Neill23). These benefits may be due to the ability of prebiotics to modify the gastrointestinal microbial community to promote fermentation and immune responses(Reference Burr, Gatlin and Ricke5, Reference Burr, Hume and Ricke8). Research also has reported changes in morphological characteristics in the intestine, such as villus height, and changes to digestive enzyme activities in terrestrial animals(Reference Xu, Zou and Hu10, Reference Xu, Hu and Xia11) and fish(Reference Xu, Wang and Li7) that seem to correspond with the positive effects seen in growth from prebiotic supplementation. At the present, there are several reports on the effects of prebiotics on the morphological characteristics of the intestine in several fish species(Reference Genc, Yilmaz and Genc15–Reference Dimitroglou, Merrifield and Spring18, Reference Pryor, Royes and Chapman24, Reference Yilmaz, Genc and Genc25) but only one study has presented changes in the intestine in red drum(Reference Zhou, Buentello and Gatlin19) and none for hybrid striped bass. Also, to date, there is only one study on prebiotics that reports effects on digestive enzymes in fish(Reference Xu, Wang and Li7), and none is available for these two species. As the changes in the morphology of the intestine and digestive enzymes may be complementary to the effects seen in growth, more research is needed to assess the morphological changes in the intestine and digestive enzymes in these two species for better understanding of the effects of prebiotics.
Therefore, the aim of the present study was to further investigate whether enhanced feed efficiency and digestibility associated with prebiotic supplementation may be due to changes in digestive enzymes and/or the morphology of the intestine. To this end, the present comparative study was conducted to evaluate the effects of commercially available fructo-oligosaccharide (FOS) in the form of inulin, Bio-MOS®, containing mannanoligosaccharide (MOS), transgalacto-oligosaccharide (TOS) and GroBiotic®-A (GBA) on the activities of pepsin, trypsin, chymotrypsin, aminopeptidase, α-amylase, lipase, and both acid and alkaline phosphatases in red drum and hybrid striped bass, as well as potential changes in fold length, and both enterocyte and microvillus height of different sections of the gastrointestinal tract (GIT) of these fish.
Experimental methods
Diet formulation
The basal diet for both red drum and hybrid striped bass was formulated and analysed to contain, on a DM basis, 40 % crude protein from a menhaden fishmeal and cooked soyabean meal, 10 % total lipid primarily from menhaden fish oil, and dextrin as the soluble carbohydrate to provide approximately 14·2 kJ digestible energy/g diet. The basal diet was formulated without prebiotic supply (Table 1)(Reference Moon and Gatlin26). Experimental diets for red drum were prepared by supplementing the basal diet with FOS, MOS, TOS and GBA at 1 % (w/w) of diet in place of cellulose. For hybrid striped bass, GBA was provided at both 1 and 2 % (w/w), in place of cellulose.
Composition of the basal diet
* Omega Protein Corporation.
† Rangen, Inc.
‡ USB Corporation.
§ Same as Moon & Gatlin(Reference Moon and Gatlin26).
∥ Same as Moon & Gatlin(Reference Moon and Gatlin26) but prepared by MP Biomedicals LLC.
¶ Inulin; Encore Technologies.
** Bio-MOS®; Alltech, Inc.
†† Vivinal; Friesland Foods Domo.
‡‡ International Ingredient Corporation.
§§ Analysed values are means of two replicate samples.
Feeding trials
Procedures used in the present study were reviewed and approved by the Texas A&M University System Animal Care and Use Committee. We conducted two separate feeding trials, one for each species, at the Texas A&M University Aquacultural Research and Teaching Facility in 110-litre aquaria maintained indoors in a climate-controlled laboratory. Water flow in the recirculating system was maintained at approximately 1 litre/min and biological/mechanical filtration was used to maintain adequate water quality for red drum and hybrid striped bass culture. Salinity was maintained at 6–8 g/l by mixing synthetic sea salt mixture (Fritz Industries) with well water. Low-pressure electrical blowers provided aeration via air stones to maintain dissolved oxygen levels near air saturation. Water temperature was maintained at 25 ( ± 2)°C throughout each trial by controlling ambient temperature with dual air-conditioning units. A 12 h light–12 h dark photoperiod was maintained with fluorescent lights controlled by automatic timers.
For each trial, fish with no visual signs of disease were selected, graded by size (10·9 (sd 0·2) and 5·1 (sd 0·3) g for red drum and hybrid striped bass, respectively), and groups of fifteen fish were stocked into each aquarium. Red drum and hybrid striped bass were subjected to a 1-week conditioning period during which the basal diet was fed to satiation based on visual cues of the fish eating. After the conditioning period, triplicate aquaria were randomly assigned to each dietary treatment. The fish were fed close to satiation with pre-weighed rations based on growth and visual cues of the fish eating (initially 5 % of body weight and gradually reduced to 3 % of body weight), twice daily (morning and evening), 7 d/week. Fish in each aquarium were group-weighed every week and feed rations adjusted accordingly. For both fish species, the feeding trials continued for 8 weeks.
Histological sample collection and analysis
Processing for histological analysis was the same in both experiments. At each sample period, three randomly selected fish were taken from each aquarium in both trials. For red drum, samples were taken at 8 weeks and for hybrid striped bass, samples were taken at 4 and 8 weeks. To evaluate changes in histological structures of the intestinal mucosa, GIT were dissected from the gastro-pyloric region to the anal region, and tied at both ends with cotton twine. Davidson's solution was injected into the intestinal lumen for preventing autolytic changes of the mucosa. Intestinal samples were kept in Davidson's fixative for 24 h and then transferred to 70 % (v/v) ethanol for conservation until processing for slide preparation.
For histological analysis, two cross-sectional rings of approximately 0·5 cm were cut from each of the anterior, mid and posterior regions of the intestine, together with 0·2 cm rings from the most medial region of four pyloric caeca. The anterior region was identified as the region within 1 cm after the pyloric portion of the stomach and the posterior region was identified as the region within 1 cm before the anus. Intestinal regions were embedded in paraffin, and 5 μm sections were made for glass slide mounting and haematoxylin–eosin staining. All slides were evaluated in an Olympus BC-2 series light microscope linked to a digital camera. For each region, three fields at 4 × and five fields at 40 × objectives were captured. Images were then analysed under ImageJ (version 1.4g) Software (National Institutes of Health, freeware). Variables measured were fold length and both total enterocyte and microvillus height for the anterior and posterior intestine, for the red drum trial, whereas for the hybrid striped bass trial, variables measured were fold length for the anterior, mid and posterior intestine, and both enterocyte and microvillus height for the previous regions plus the pyloric caeca; in all cases, ninety measurements per variable in each treatment were made for each sample point. Only appropriately oriented folds were used for measurements.
Enzyme sample collection and analysis
For the red drum and hybrid striped bass trials, two fish were randomly selected from each aquarium and euthanised at weeks 4 and 8. All fish were sampled 4 h after being fed to ensure that the enzymes would be active from digestion. The stomach and the intestine were aseptically dissected and then separated from each other. The entire intestine was then flash-frozen in liquid N2 and stored at − 80°C until analyses.
Frozen intestinal samples were homogenised in cold 50 mm-2-amino-2-hydroxymethyl-propane-1,3-diol (Tris)–HCl, 20 mm-CaCl2 buffer, and supernatants were stored at − 20°C before enzyme analyses. The concentration of the soluble protein in the samples was determined by the Bradford method (BioRad Protein Assay), using bovine serum albumin as a standard. The activities of pepsin, trypsin, chymotrypsin, aminopeptidase, α-amylase, lipase, and both acid and alkaline phosphatases were assayed spectrophotometrically in triplicate for each of the two intestinal samples per aquarium per time period.
Pepsin activity was assayed based on a method described by Anson(Reference Anson27) using 0·5 % (w/v) Hb in 0·1 m-glycine–HCl buffer (pH 2). The samples were incubated at 37°C for 30 min and then the reaction was stopped with 20 % (w/v) TCA. The samples remained at 4°C for 15–30 min and were then centrifuged. The optical density of the supernatants was measured at 260 nm using deionised (DI) water as a blank.
Trypsin activity was assayed based on the method described by Erlanger et al. (Reference Erlanger, Kokowsky and Cohen28), using N-α-benzoyl-dl-arginine 4-nitroanilide hydrochloride in dimethyl sulfoxide and 50 mm-Tris–HCl, 10 mm-CaCl2 buffer (pH 8·2) as the substrate. The samples and the substrate were incubated at 37°C for 30 min, and then the reaction was stopped with 30 % (v/v) acetic acid. Trypsin was measured at 410 nm against a blank containing the substrate, acetic acid and DI water in place of the sample.
Chymotrypsin was assayed based on the method described by Asgeirsson & Bjarnason(Reference Asgeirsson and Bjarnason29), using 5 mm-N-benzoyl-l-tyrosine ethyl ester in dimethyl sulfoxide and 44·4 mm-Tris–HCl, 55·5 mm-CaCl2 buffer (pH 7·8). The samples remained at room temperature for 10 min and then incubated at approximately 100°C for 15 min. The samples were measured at 256 nm against a blank containing N-benzoyl-l-tyrosine ethyl ester and buffer.
Aminopeptidase was assayed using l-leucine p-nitroanilide in dimethyl sulfoxide and 50 mm-Na3PO4 (pH 7·2) as the substrate. The samples and substrate were incubated at 37°C for 10 min, and then the reaction was stopped with 30 % (v/v) acetic acid. Aminopeptidase activity was measured at 410 nm against DI water as the blank.
α-Amylase was assayed based on Vega-Villasante et al. (Reference Vega-Villasante, Nolasco and Civera30), using 50 mm-Tris–HCl buffer (pH 7·5) and 1 % soluble starch as the substrate. The samples were incubated for 10 min at room temperature. To initiate the reaction, 2 m-sodium carbonate and reactive dinitrosalicylic acid were added and then the samples were incubated for 15 min at approximately 100°C. DI water was added and then the samples were read at 550 nm against DI water as the blank.
Lipase was measured using the method described by Versaw et al. (Reference Versaw, Cuppett and Winters31). Sodium cholate hydrate, 50 mm-Tris–HCl buffer (pH 7·2) and β-naphthyl-caprylate were used as the substrate. The samples were incubated for 30 min at room temperature and then 100 mm-Fast Blue BB Salt was added. After 5 min of incubation at room temperature, the reaction was stopped using 0·72 m-TCA and then ethanol–ethyl acetate (1:1, v/v) was added to clarify the solution. The samples were read at 540 nm against DI water as a blank.
Acid and alkaline phosphatases were measured using 2 % (w/v) 4-nitrophenylphosphate as the substrate in either sodium citrate dihydrate (pH 4·8) for acid phosphatase or 30 mm-sodium carbonate (pH 9·8) for alkaline phosphatase. The samples were incubated for 30 min at 37°C and then the reaction was stopped using 0·05 m-NaOH. The samples were read at 405 nm using a blank containing the substrate, NaOH and DI water in place of the samples.
Statistical analysis
Data from the histological and enzyme analyses were subjected to one-way ANOVA, and mean separation was assessed by Duncan's multiple range test using SAS (version 9.2, SAS Institute). Differences in treatment means were considered to be significant at P≤ 0·05.
Results
After feeding red drum the experimental diets for 8 weeks, intestinal histological structures were affected by prebiotics (Table 2). All structures in the anterior intestine were consistently affected by TOS, significantly (P< 0·05) increasing the length of intestinal folds and the height of both enterocyte and microvilli. Similarly, MOS had a significant effect on intestinal fold length and microvillus height, whereas GBA increased only the microvillus height. In contrast, FOS had a significant detrimental effect on anterior intestinal structures, decreasing the fold length and enterocyte height. Conversely, in the posterior intestine, GBA had a significantly greater effect on histological structures, augmenting the length of folds and the height of the microvilli, whereas TOS significantly affected only the microvillus height.
Histological parameters (μm) of the gastrointestinal tract of red drum fed different prebiotics for 8 weeks(Mean values with their pooled standard errors)
GBA, GroBiotic®-A; FOS, fructo-oligosaccharides; TOS, transgalacto-oligosaccharides; MOS, mannan-oligosaccharides; PSE, pooled standard error.
a,b,c Mean values within a row with unlike superscript letters were significantly different (P< 0·05; Duncan's multiple range test).
For hybrid striped bass, intestinal histological structures were measured at 4 and 8 weeks after being fed graded levels of GBA. These structures were positively affected by the addition of this prebiotic to the diet (Table 3). At 4 weeks, all structures of all intestinal sections were affected by 2 % GBA inclusion, significantly increasing the length of folds and the height of both enterocyte and microvilli. Dietary inclusion of 1 % GBA only significantly affected the enterocyte and microvillus height in the anterior intestine. Conversely, after 8 weeks of the feeding trial, the anterior intestine was not affected by the treatments, whereas the inclusion of 1 % GBA significantly increased the fold length of both mid and posterior intestine and the enterocyte height of the mid intestine. In addition, 2 % GBA significantly increased the fold length of the mid intestine and the microvillus height of pyloric caeca.
Histological parameters (μm) of the gastrointestinal tract of hybrid striped bass fed graded levels of GroBiotic®-A (GBA) for 4 and 8 weeks(Mean values with their pooled standard errors)
PSE, pooled standard error.
a,b,c Mean values within a row with unlike superscript letters were significantly different at week 4 or 8 (P< 0·05; Duncan's multiple range test).
Intestinal samples obtained from red drum and hybrid striped bass at weeks 4 and 8 were analysed to measure the specific enzyme activities. For red drum, MOS, in general, had higher activities of the analysed enzymes at week 4. Aminopeptidase activity in fish fed the MOS diet was significantly increased at week 4 compared with the values obtained from fish fed the basal and the GBA-supplemented diets (Table 4). The inclusion of MOS in the diet also increased the activities of alkaline phosphatase and α-amylase in a significant manner when compared with those of fish fed the basal, GBA and FOS diets. Similarly, the TOS diet tended to produce numerically higher values for aminopeptidase, acid and alkaline phosphatases, and α-amylase compared with fish fed the FOS, GBA or basal diet, although these changes were not significantly different. Conversely, GBA, in general, had lower activities of all analysed enzymes except for pepsin and lipase.
Digestive enzyme activities of red drum at 4 and 8 weeks(Mean values with their pooled standard errors)
GBA, GroBiotic®-A; FOS, fructo-oligosaccharides; TOS, transgalacto-oligosaccharides; MOS, mannanoligosaccharides; PSE, pooled standard error.
a,b,c Mean values within a column with unlike superscript letters were significantly different at week 4 or 8 (P< 0·05; Duncan's multiple range test).
* Specific activity/mg soluble protein.
The observed changes in enzymatic activities at week 4 for red drum were transient in nature because at the end of week 8, none of the selected enzymes showed any significant difference compared with fish fed the basal diet (Table 4). Interestingly, at week 8, the activities of pepsin, aminopeptidase, trypsin, α-amylase, and both acid and alkaline phosphatases were of higher magnitude in fish fed TOS than in those fed MOS or all other diets, although these values were not significantly different for any dietary treatment.
GBA supplementation at 1 % in the diets of hybrid striped bass for 4 weeks tended to increase the activities of chymotrypsin and alkaline phosphatase, while these activities tended to decrease with a supplementation level of 2 %. Conversely, GBA supplementation at 1 and 2 % elicited a significant decrease in the activity of α-amylase at 8 weeks (Table 5).
Digestive enzyme activities of hybrid striped bass at 4 and 8 weeks(Mean values with their pooled standard errors)
GBA, GroBiotic®-A; PSE, pooled standard error.
a,b Mean values within a column with unlike superscript letters were significantly different at week 4 or 8 (P< 0·05; Duncan's multiple range test).
* Specific activity/mg soluble protein.
Discussion
Prebiotic supplementation in the diets of hybrid striped bass and red drum elicited structural changes in the GIT of the fish. Conversely, prebiotic supplementation did not elicit many significant changes in the activities of digestive enzymes in both fish species. The present study yielded similar results to those reported by a previous study(Reference Refstie, Bakke-McKellep and Penn32), which demonstrated that Atlantic salmon (Salmo salar) fed inulin did not have any significant changes in trypsin, amylase, alkaline phosphatase and leucine aminopeptidase. However, these results are different from those seen in allogynogenetic crucian carp (Carassius auratus gibelio)(Reference Xu, Wang and Li7) which were fed xylo-oligosaccharide. In those studies, treated fish exhibited an increase in several enzyme activities and all also coincided with improvements in condition indices such as weight gain. Observed differences between these studies may be explained in part by the sharp contrast between carnivores (e.g. red drum, hybrid striped bass and Atlantic salmon) and omnivores/herbivores (e.g. crucian carp) in terms of the physiology and the architecture of their GIT. It is possible that the observed prebiotic effects on digestive enzyme activities in the latter correlate with the extended length and passage rate through the GIT of cyprinids, conceivably explaining the improved growth observed in the carp investigations.
Previous studies with prebiotics in red drum and hybrid striped bass have also shown enhancements in several performance parameters including weight gain, feed efficiency and survival(Reference Buentello, Neill and Gatlin3, Reference Li and Gatlin13, Reference Li and Gatlin14, Reference Zhou, Buentello and Gatlin19). Because the present data indicate that the prebiotic treatments had no persistent effect on the activities of various digestive enzymes, we cannot conclude that the increased weight gain and feed efficiency of red drum and hybrid striped bass observed in the previously mentioned studies are related to changes in endogenous digestive enzymes.
On the other hand, the present set of experiments indicates that intestinal histological structures were affected by prebiotics, which is in line with recent reports for other fish species(Reference Genc, Yilmaz and Genc15–Reference Dimitroglou, Merrifield and Spring18, Reference Pryor, Royes and Chapman24, Reference Yilmaz, Genc and Genc25). From a histological perspective, the most thoroughly evaluated prebiotic is MOS, in fish and other vertebrates. In the present study, MOS (1 %) was evaluated only in red drum, where it had a positive effect on the fold length and microvillus height of the anterior intestine. Consistent with the results reported in the present paper, a previous study observed that 1·5 or 3 % MOS in the diet improved fold length in juvenile rainbow trout (Oncorhynchus mykiss)(Reference Yilmaz, Genc and Genc25). However, lower inclusion levels (0·2 %) failed to elicit the same response in fish of similar age, but not in subadult trout(Reference Dimitroglou, Merrifield and Moate16). Correspondingly, in larval white sea bream (Diplodus sargus), MOS supplementation improved intestinal morphology by increasing the villous surface area and the microvillus height(Reference Dimitroglou, Davies and Sweetman17). In contrast, histological evaluation of the anterior intestine of gilthead sea bream (Sparus aurata) revealed that dietary MOS had no effect on villous morphology. However, morphological examination of MOS-treated fish indicated improvements in the absorptive area of the posterior intestine(Reference Dimitroglou, Merrifield and Spring18). Also, no effects on intestinal structure were observed in either hybrid tilapia (Oreochromis niloticus× O. aureus)(Reference Genc, Yilmaz and Genc15) or Gulf of Mexico sturgeon (Acipenser oxyrinchus)(Reference Pryor, Royes and Chapman24). The previously listed evidence points to obvious differences in the effects that dietary prebiotic supplementation has on GIT morphological features and these differences appear to be species-specific.
There are very few scientific reports on the intestinal effects available for the additional prebiotic products evaluated in the present experiment(Reference RingØ, Olsen and Gifstad21). Only one study has evaluated FOS and TOS in addition to MOS and reported increased microvillus height in juvenile red drum(Reference Zhou, Buentello and Gatlin19). In the present study, similar effects, in addition to longer mucosal folds, were observed for TOS and MOS. However, contrasting results were observed with FOS, where dietary inclusion had a detrimental effect on the red drum intestine in the present experiment.
GBA, a prebiotic proven to increase nutrient digestibility of red drum(Reference Burr, Hume and Neill23), seemed to have a tendency to affect more the posterior intestine of red rum and hybrid striped bass after 8 weeks of feeding rather than the anterior section. Similar results with MOS have been reported for gilthead sea bream(Reference Dimitroglou, Merrifield and Spring18) where supplementation for 9 weeks tended to have an effect on the posterior intestinal folds and microvillus height in both anterior and posterior intestine. Interestingly, increasing the inclusion level of GBA to 2 % elicited more rapid effects and of greater magnitude on intestinal structures in hybrid striped bass. However, these effects were transient, more accentuated and only significant at week 4 but not at week 8.
Further research is needed to elucidate whether the normal development of the GI tract in fish may have overshadowed possible prebiotic effects at week 8. This could help explain, in part, the observed transitory nature of these effects. It is also possible that prebiotic supplementation may have accelerated gut development, as indicated by the histological assessment at week 4. This finding may coincide with a previous study with MOS in larval cobia that suggests that MOS may drive the gut to develop more rapidly as evidenced by longer microvilli in treated fish(Reference Salze, McLean and Schwarz33).
The results from the present experiment, together with the enhanced growth observed in previous studies(Reference Buentello, Neill and Gatlin3, Reference Li and Gatlin13, Reference Li and Gatlin14, Reference Zhou, Buentello and Gatlin19), appear to correlate better with an improved nutrient absorption due to enhanced intestinal features than with possible increases in the activities of the evaluated digestive enzymes.
Acknowledgements
The authors would like to thank the International Ingredient Corporation for providing GBA and research funding. We would also like to thank the National Center for Foreign Animal and Zoonotic Disease Defense, a Department of Homeland Security Science and Technology Center of Excellence and Hispanic Leaders in Agriculture and Education (HLAE) for providing fellowship support for M. A. Funding for C. P. was provided in part by Consejo Nacional de Ciencia y Tecnología (CONACyT-México), Secretaría de Educación Pública (SEP, Mexican Government) and the Aquaculture Protein Centre (Norway). We would further like to thank the Histology Laboratory of the Department of Veterinary Integrative Biosciences at Texas A&M University for the histological processing and to the Bone Biology Laboratory in the Department of Health and Kinesiology at Texas A&M University for providing the image analysis equipment. Finally, the authors extend their gratitude to the staff at the Texas A&M Aquacultural Research and Teaching Facility. The authors have no conflicts of interest. D. M. G. and A. B. designed and coordinated the study. M. A. and C. P. were responsible for the data collection, analysis and statistical analysis. All authors contributed to the interpretation of the data. M. A. and C. P. wrote the first draft of the manuscript and all authors critically reviewed and revised the manuscript.
