https://doi.org/10.52973/rcfcv-e362916 Revista Cientíﬁca, FCV-LUZ / Vol. XXXVI Recibido: 27/02/2026 Aceptado: 28/04/2026 Publicado: 22/05/2026 1 of 9 Arzu Özlüer-Hunt¹ * , Ferbal Özkan-Yılmaz² Growth, body composition and digestive enzyme activities of rainbow trout fed with dietary Beta-glucan containing mannan oligosaccharide Crecimiento, composición corporal y actividades de enzimas digestivas en truchas alimentadas con una dieta de beta-glucano que contiene manano oligosacáridos ¹Fisheries Faculty, Aquaculture Dept., Mersin University, 33160, Mersin, Turkey. ²Fisheries Faculty, Basic Sciences Dept., Mersin University, 33160, Mersin. ⃰Corresponding author: huntarzu@mersin.edu.tr/arzuhunt51@gmail.com RESUMEN Palabras clave: Prebiótico; Oncorhynchus mykiss; tasa de crecimiento; conversión alimenticia; eﬁciencia proteica. This study investigated the impact of a yeast containing the prebiotic β-glucans, on the growth parameters and enzyme activities of rainbow trout (Oncorhynchus mykiss) at various dosage levels. The fish were randomly placed in plastic tanks (120 x 50 x 32 cm) in three trial groups and replicates, with an average weight of 73.37 ± 0.21 g (n = 90). Three different diets were prepared; 0 g.kg-¹ in group G-1 (control), 0.5 g.kg-¹ of the prebiotic (mannan oligosaccharides + β-glucan) in group G-2 and 1 g.kg-¹ ratio prebiotic (mannan oligosaccharides + β-glucan) were added to group G-3. The fish were fed these experimental feeds for 60 days. By the end of the experiment, the G-2 group had achieved the highest average live weight and weight gain, with values of 155.38 ± 0.19 g and 81.76 ± 0.16 g respectively. Feed conversion ratio, specific growth rate, protein efficacy ratio and survival rate showed the best values (1.13 ± 0.03, 1.24 ± 0.003, 1.91 ± 0.001, and 93.33 ± 3.33, respectively) were observed in the G-2 group (P < 0.05). Pepsin activity was also signiﬁcantly higher in the G-2 group (163.94 ± 2.23 U/mg protein; P < 0.05). Similarly, the highest trypsin, amylase, and lipase activities were recorded in the G-2 group, with values of 1.09 ± 0.05, 5.31 ± 0.22, and 4.38 ± 0.11 U/mg protein, respectively (P < 0.05). No signiﬁcant differences were detected among the groups with respect to muscle proximate composition (P > 0.05). Both β-glucan–supplemented yeast groups positively influenced the growth performance and digestive enzyme activities of trout. However, the group receiving 0.5 g.kg-¹ β-glucan supplementation exhibited signiﬁcantly superior results compared to the other groups (P < 0.05). En esta investigación se analizaron los efecto de una levadura que contiene el prebiótico β-glucano, sobre los parámetros de crecimiento y las actividades enzimáticas en la trucha arcoíris (Oncorhynchus mykiss) a diferentes proporciones. Los peces se colocaron aleatoriamente en tanques de plástico (120 x 50 x 32 cm) (73,37 ± 0,21 g; n = 90) en tres grupos experimentales con réplicas. Se prepararon tres dietas diferentes: 0 g/kg en el grupo G-1 (control), 0,5 g/kg manano oligosacaridos + β-glucano en el grupo G-2 y 1 g/kg de prebiótico manano oligosacaridos + β-glucano en el grupo G-3. Los peces fueron alimentados con estas dietas experimentales durante 60 días. Al ﬁnal del experimento, los mayores valores de peso y ganancia de peso fueron 155,38 ± 0,19 g y 81,76 ± 0,16 g, respectivamente, en el grupo G-2 al comparar el peso vivo promedio y la ganancia de peso. El índice de conversión alimenticia, la tasa de crecimiento especíﬁco, el índice de eﬁciencia proteica y la tasa de supervivencia mostraron los valores más altos en el grupo G-2 con 1,13 ± 0,03; 1,24 ± 0,03; 1,91 ± 0,001; y 93,33 ± 3,33, respectivamente (P < 0,05). La mayor actividad de pepsina se observó en el grupo G-2 (P < 0,05) con 163,94 ± 2,23 U/ mg proteína. Las actividades más altas de tripsina, amilasa y lipasa también se encontraron en el grupo G-2 con 1,09 ± 0,05; 5,31 ± 0,22; y 4,38 ± 0,11 U/mg proteína, respectivamente (P < 0,05). No hubo diferencias signiﬁcativas entre los grupos en cuanto a los componentes nutricionales del músculo (P > 0,05). Se ha determinado que la adición de levadura con el prebótico β-glucano afecta positivamente tanto los parámetros de crecimiento de la trucha como todas las actividades enzimáticas, pero el grupo suplementado con 0,5 g/kg de β-glucano mostró diferencias significativamente mayores (P < 0,05) que los demás. ABSTRACT Key words: Prebiotic; Oncorhynchus mykiss ; growth rate; feed conversion; protein efﬁciency.

2 of 9 Prebiotic eﬀects on growth and enzyme activity in rainbow trout/Özlüer-Hunt and Özkan-Yılmaz. INTRODUCTION Feed costs constitute the majority of production expenses in aquaculture, and fish meal has the highest share in feed input. In order to ensure the production of fishmeal obtained by hunting, as caught ﬁsh stocks began to decrease, the sector began to search for alternative protein sources and alternative feed additives to accelerate mixed feed studies and confirm sustainability [1]. As a result of the data obtained in the light of microbiological, biotechnological and engineering studies carried out with modern technology, it is aimed to ensure more production in less unit area and to produce this valuable protein source with the least mortality rates of enterprises through sustainable cultivation [2]. For this purpose, the use of antibiotics in the sector for the prevention and treatment of diseases and for increasing body resistance has begun, so that over time, antibiotic use has become routine practices due to its effects on the growth performance of individuals with high body resistance [3]. The damage caused by the antibiotics and other chemicals used to the natural ecological environment has been ignored for a long time. However, over time, these antibiotics used not only caused the development of bacterial resistance in animals, but also began to threaten human health with the residues they left in animal products. This is why the use of antibiotics in animal feed is prohibited in the European Union since 2006 [3]. Although aquaculture research on prebiotics is increasing day by day, it still cannot reach the desired intensity. Additionally, to studies on the growth and immune system performance of fish species that were fed prebiotic additives, many studies have attempted to observe that these oligosaccharides have a positive effect on the beneﬁcial bacteria in the intestinal flora and have digestive and immune system regulating effects [4].However, in the light of these studies, factors such as the type, age, and application dose of the fish to be applied prebiotics should be taken into consideration. Because prebiotics have different effects or do not cause any reactions depending on the species [4]. Prebiotics are mainly effective in regulating intestinal flora and in the activity of probiotics. In other words, it is deﬁned as a fermented and non-digestible component that allows speciﬁc changes in activity and / or composition in the gastrointestinal flora which beneﬁt the host's health and well-being [5]. Recently, there has been a great interest in the use of prebiotics as a functional food to regulate the composition of the gastrointestinal microbiology to benefit the host health [5, 6]. The host should have beneficial and systemic effects and must be hydrolyzed in the colon microflora and have a positive effect on the host. Prebiotics affect the composition and activity of microflora in organisms. They regulate bowel movements and increase the absorption and bioavailability of minerals such as calcium and magnesium. They also have a positive effect on blood cholesterol and triglyceride levels and reduce the risk of developing intestinal and extraintestinal infections by limiting the proliferation of pathogenic microorganisms [5]. There are positive aspects such as strengthening the immune system. According to previous studies, various types of prebiotics and their combinations have been shown to effectively enhance the growth performance of different aquatic species. Among them, mannan oligosaccharides (MOS), galactooligosaccharides, fructooligosaccharides, and other oligosaccharides are the most generally used prebiotics in aquaculture today [4, 7, 8]. One example of a prebiotic mixture is Immunogen (β-glucan + MOS), a commercially available product proven to boost the growth of common carp (Cyprinus carpio) [9]. MOS is obtained from the cell wall of the yeast. The cell wall of this yeast consists of 30 % mannan, 30 % glucan and 12.5 % protein. MOS is derived from the amino acids on the surface of the yeast cell wall and provides a binding site to the bacteria. Pathogen organisms follow to the MOS instead of following to the intestinal epithelial cells, thus moving in the intestinal tract without being colonized [6, 9, 10]. After the ban of the use of antibiotics as a feed additive, many researchers have intensified their research on natural additives which may be an alternative to antibiotics. One of these natural substances was β-glucan obtained from the cell wall of Saccharomyces cerevisiae [1]. The use of β-glucans as a feed additive to enhance the immune system and to reduce the risk of death against bacterial and viral diseases that are fatally infected and to support the immune and performance of animals, and draw attention as a good alternative in light of the studies [1]. In a study on Nile tilapia (Oreochromis niloticus), MOS was added to feed rations at 0, 2, 4, and 6 g.kg-1 and research was conducted on survival, growth and strengthen the immune system. Compared with the control group, fish fed the MOS- supplemented diet did not show signiﬁcant differences in weight gain, body length, survival rate, or feed conversion ratio (FCR). Nevertheless, these ﬁsh demonstrated a signiﬁcantly enhanced resistance to infection by Streptococcus agalactiae [11]. Another study conducted on seabass (Dicentrarchus labrax) fish with MOS-supplemented feeds, MOS addition significantly reduced the lipogenic enzyme activity in the liver and more regular morphological structure of the hepatocytes. This could be linked to improved energy utilization and feed efﬁciency, as well as reduced feed intake [12]. The cell walls of yeasts contain high levels of two substances: β-glucan and MOS. Their immunostimulant properties and ability to reduce harmful organisms by strengthening the intestinal barrier are well known [13]. As a result of feeding, the findings showed that there was a significant improvement in feed conversion and the activities of amylase, lipase, and protease were higher in ﬁsh fed beta glucan and MOS mixture than in the control group in carp [13]. A study was conducted on flounder (Platichthys stellatus) to observe their feeding behavior, growth rate, and physiological parameters when fed a diet supplemented with β-glucan/MOS for 56 days. Individuals fed with β-glucan/MOS supplemented feed showed significant (P < 0.01) growth and development compared to the control groups [14]. For many years, the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have been widely utilized in biotechnological applications, particularly in the production of food and beverages. In recent years, however, non-conventional

3 of 9 Revista Cientíﬁca, FCV-LUZ / Vol. XXXVI Fish experiment design and growth performance Fish fed ad libitum two times for morning (09:00-10:00 h) and afternoon feeding (17:00-18:00 h) during the 60 day grow out period. The experiment was conducted in a climate-controlled laboratory at the Fish Culture Unit. The water temperature was kept between 13 ± 0.3 °C, and a 12-h light : 12-h dark photoperiod was maintained throughout the study. Dissolved oxygen concentrations were measured every other day using a YSI Model 58 oxygen meter (Yellow Springs Instruments, Yellow Springs, Ohio, USA) and maintained at ≥ 6 mg.L-¹ throughout the experiment. The pH was monitored twice weekly using an electronic pH meter and recorded 7.89 ± 0.65 (Fisher Scientiﬁc, Cincinnati, Ohio, USA). Total nitrate (NO₃), levels were determined weekly and recorded 0.70 ± 0.21 mg.L-¹ using a Merck Spectroquant® Nova 60A (Germany) system. In order to analyze the growth indices of the rainbow trout, all fishes from each tank was determined once every 15 d during the 60-d experiment, at least 12 h after the last feed. The ﬁsh were weighed individually by a digital scale (ME503 T/ A00, Switzerland) after they had been anesthetized. At the end of the experimental period, body weight gain (BWG), percent body weight gain (PBWG), specific growth rate (SGR), FCR, protein efﬁciency ratio (PER), and survival rate were determined according to the equations provided below. Growth performance was evaluated using the formulas cited in the literature and listed below [18, 19]. BWG = Wt - W0 PBWG = (Wt-W0)/W0×100 SGR = (lnWt-lnW0) / t ×100 FCR = Feed given (g) / wet weight gain (g) PER = Wet WG (g) - protein intake (g) Survival rate = (Nt / N0) / 100 In the above formulas, Wt denotes to the mean final body weight (g), whereas W0 indicates the mean initial body weight Biometric parameters yeast species have gained increasing attention, with Pichia guilliermondii emerging as an important alternative [15]. This material was used for the ﬁrst time in this study for trout. Functional foods such as β-glucan and MOS, a prebiotic additive, can rise efficiency in the feed industry and meet the needs of the fish farming sector by falling antibiotic use, developing digestive activity and improving feed efﬁciency. The main objective of in this research was to value the effects of addition a mixture of β-glucan and MOS to extruded trout feed on growth performance, fish body chemical composition, and digestive enzymes. The demonstration of the economic beneﬁts of this approach was also observed. In this study, nine semi-recirculated rearing tanks (120cm x 50cm x 32cm) were used. Each tank contained 10 ﬁsh (10/0.19 m³). The 90 trout used in the study were randomly selected from a stock of 400 fish and placed in the. A total of 90 fish were weighed (73.37 ± 0.21 g) individually before being placed in experimental tanks. The experiments were conducted with triplicate. Three different diets were prepared considering the energy requirements of the trout during the study period. Diets used for this experiment: Group G-1 (control): 0 g.kg-¹, Group G-2: 0.5 g.kg-¹ prebiotic (MOS + β-glucan) Group G-3: 1 g.kg-¹ prebiotic (MOS + β-glucan). A crude protein content of 46–47 % was achieved in test feeds formulated using fish meal, corn gluten, fish oil, trace minerals, vitamin supplements, antioxidants, binding agents, and beta-glucan. Prepared feeds of β-glucan + MOS mixed (ADM Alliance Nutrition, USA) contribution were used. prebiotic contained 38 % protein, 1.5 % crude oil, 10 % cellulose and 8 % moisture. A yeast mannan, is a proprietary from yeast (Pichia guilliermondii) supplement for all life stages and classes of livestock, companion animals, and poultry. It is a coproduct of citric acid manufacturing. Due to the use of high-quality raw materials and the single production site has a consistent composition and is cost effective [16], this product does not contain viable microbial cells. The yeast used contained 16 % MOS and 16 % β-glucan. The design and chemical composition of the research diets are shown in TABLE I. Nitrogen-Free Extract was calculated as the remainder of crude protein + crude lipid + ash. Calculated based on standard physiological energy values: 19 kJ.g-¹ for protein, 36 kJ.g-¹ for lipid and 15 kJ.g-¹ for carbohydrate [17]. MATERIALS AND METHODS

4 of 9 Prebiotic eﬀects on growth and enzyme activity in rainbow trout/Özlüer-Hunt and Özkan-Yılmaz. Feed cost was incorporated into the economic evaluation of the experimental groups. The cost of feed required to produce 1 kg of biomass was calculated based on the feed price and FCR. The Economic Conversion Ratio (ECR) and Economic Proﬁt Index (EPI) were calculated according to the following formulas: ECR (US.$/kg) = Feed cost (US.$/kg) x Feed conversion ratio (kg diet/kg ﬁsh), The Economic Proﬁt Index [EPI (US$ /ﬁsh) = ﬁnal weight (kg/ ﬁsh) x ﬁsh sale price (US$ /kg)-ECR (US$/kg) x weight increase (kg)] [20]. At the end of the trial, 27 ﬁsh in total (3 ﬁsh from each tank, corresponding to 9 ﬁsh per treatment) were randomly selected from every treatment group. The fish were then dissected, and tissue samples were obtained for biochemical analysis. Fish ﬁllets were subjected to proximate composition analysis as follows: moisture content was determined by drying the samples at 60 °C until a constant weight was achieved; crude protein was measured by determining Kjeldahl nitrogen (×6.25) using an automated distillation unit (Velp DK20, DK6, UDK126; Italy) crude lipid was extracted with a chloroform/methanol solvent system; and ash content was quantiﬁed by incineration in a muffle furnace (Protherm Furnaces 110, Germany) at 550 °C for 18 h [21]. To evaluate the effect of yeast-based β-glucan on digestive enzyme activity, six fish were sampled from each tank at the end of the experiment, and crude enzymatic extracts were subsequently prepared according to experiment protocol [22]. Pepsin activity (E.C.3.4.23.1; Enzyme Commission, Pepsin A) was assayed using 2 % hemoglobin from bovine blood in HCl as a substrate. Trypsin activity (E.C.3.4.21.4; Enzyme Commission, Trypsin) was measured [23] and the measurements modified using benzoyl-DL-arginin-p-nitroanilide (BAPNA, Sigma) as a substrate [24] . Amylase activity (E.C. 3.2.1.1; Enzyme Commission, Alpha-amylase) was assayed by the dinitrosalicylic acid (DNS, Sigma®) procedure [25] using 1 % soluble starch (Sigma) as a substrate. Lipase activity (E.C.3.1.1.3; Enzyme Commission, Lipase) was measured spectrophotometrically (Analytikjena-SPECORD 50, Germany) [26] . Tissue protein content was determined following the method described by Lowry et al. [27]. Economic proﬁt analysis Sampling biochemical analyses and digestion enzyme assay All data were subjected to one-way ANOVA (SPSS 22.0). Duncan test was also used to compare means and significance was accepted at probabilities of 0.05 or less. Statistical analysis (g). The sign t represents the length of the experimental period expressed in days. N0 and Nt denote the number of fish at the beginning and at the end of the trial, respectively. After a 60 d study, the effects of adding different rates of yeast-derived beta-glucan to the feed on final weight, weight gain (WG), live weight gain (LWG), daily live weight gain (DLWG), percentage live weight gains (LWG %), SGR, protein efficiency ratio (PER) and survival rate were investigated, and the data are presented in TABLE II. Optimum growth was observed on G-2 diet, whereas the control diet (G-1) produced the lowest growth rate. Fish fed with yeast glucan diets final weight, WG, LWG (%) and SGR increased significantly (P < 0.05) when compared control diet no glucan added G-1 diet. There were significant changes in survival between control and yeast based glucan added diets (P < 0.05). The lowest FCR was obtained at 0.5 g.kg-¹ glucan (G- 2) supplementation. Fish fed the control diet showed a higher FCR. On the other hand, yeast based glucan supplementation improved nutrient utilization; moreover, ﬁsh fed G-2 diet showed highest PER. Yeast based glucan supplementation did not signiﬁcantly affected muscle ﬁsh composition (TABLE II). It is well established that when prebiotics are added to ﬁsh and shellfish diets, they grow better, use food more efficiently, are stronger against disease and have higher digestive enzyme activity [28]. In the large intestine, an increased production of short-chain fatty acids and lactic acid is stimulated by the fermentation of dietary prebiotics, which in turn stimulates the growth of beneﬁcial bacteria. This improves growth performance and the health of the host. The current investigation revealed that the growth rates of ﬁsh were enhanced when they were fed diets comprising yeast- derived glucan. Signiﬁcantly higher growth rates (P < 0.05) and higher weight gain were shown by ﬁsh fed the yeast-based glucan 0.5 g.kg-¹ diet than with other treatments. The yeast glucan supplementation groups gained more weight than the control group. Live weight gain percentage ranged from 82 % to 111 %, and was significantly affected by the yeast-based glucan. This study showed that adding 0.5 g.kg- ¹ beta-glucan yeast to trout diets could provide an effective RESULTS AND DISCUSSION

5 of 9 Revista Cientíﬁca, FCV-LUZ / Vol. XXXVI When the ECR values were examined at the end of the research, when the values of yeast-derived beta glucan feeds were compared, it was seen that the best values were 1.47±0.004 in the G-2 group and 1.49 ± 0.004 in the G-3 group. A statistical difference was determined between these two groups and the G-1 group (P < 0.05). When examined in terms of EPI, the lowest was found to be 0.40±0.002 in the G-1 group, and the highest was 0.49 ± 0.001 in the G-2 group. Statistically, it was determined between the groups fed with β-glucan-derived yeast and the control (P < 0.05) and the most proﬁtable feed group was determined to be the G-2 group. At the end of the research, a proﬁtability of 0.45 $/kg was determined in terms of feed cost and accordingly, a feed cost proﬁt of 23.43 % was calculated for 1 kg of feed. Feed prebiotic supplement without negatively affecting fish growth performance. In former research, the effects of MOS on the growth performance of aquatic species have been investigated, showing varying results. For example, MOS supplementation had no encouraging effect on growth parameters in Atlantic salmon (Salmo salar) [29] and cobia larvae (Rachycentron canadum) [30] thus, the inclusion of prebiotics in feed rations was not recommended. However, in another study on Oreochromis niloticus, ﬁsh were fed diets containing MOS at levels of 0, 2.5, 3.5, and 4.5 g.kg-1 for sixty d. The results showed that ﬁsh fed the diet supplemented with 3.5 g.kg-¹ MOS exhibited improved growth, feed conversion ratio, protein efficiency ratio, and nutrient composition [8] . Similarly, in D. labrax fed diets containing 2 and 4 g.kg-¹ MOS, a better growth rate was obtained, with approximately 10% higher growth compared to the control group [31]. In rainbow trout, fish fed with 2 g.kg-¹ MOS also demonstrated better growth performance than the control group [32]. In agreement with these results, Persian sturgeon, Acipenser persicus . [33] , large yellow croaker Pseudosciaena crocea [34], Lobeo rohita fingerlings [35], white shrimp Litopenaeus vannamei [36], juvenile rainbow trout (Oncorhynchus mykiss) [37], Nile tilapia, O. niloticus [38], Showed differences in the effects of MOS, which may be attributed to factors such as yeast strain, ﬁsh species, or feeding duration [39]. In our study, MOS supplementation had positive effects on growth parameters at both doses, with the 0.5 g/kg-¹ MOS (G-2) level showing the most pronounced effect. However, the effective dose of MOS in the diet may vary depending on the feeding duration, fish species, age, nutritional status, and physiological condition. The bio-economic values of trout fed with feed containing beta-glucan-enriched yeast are presented in TABLE III. input constitutes approximately 40-50 % of the trout production cost. When examined from this perspective, the annual feed cost brings a proﬁtability of 9.2-11.5 %. At the end of the 60-day study, the effects of feeding trout with different amounts of yeast-derived beta-glucan on stomach pepsin activity (A), intestinal trypsin (B), amylase (C) and lipase (D) activities are presented in FIG. 1. FIGURE 1. Pepsin activity in stomach tissues (A), Trypsin (B), Amylase (C) and Lipase (D) activities (U.mg-1 protein) in intestine tissues of rainbow trout fed different levels of β-glucan supplementation. Each value represents the mean ± SEM. (n = 6). The different letters indicate signiﬁcant (P < 0.05) difference between diet groups Subsequent to being administered varying quantities of yeast supplementation over a 60-day period, the mean pepsin and trypsin digestive enzyme activities of the G-2 treatment group exhibited a signiﬁcant discrepancy from those of the control and G-3 groups (FIGS. 1A and 1B). However, ﬁsh fed with yeast based glucan added groups were significantly different than control group for amylase and lipase activity (P < 0.05) (FIGS. 1C and 1D). The results of the analysis of digestive tract enzyme activity showed that diets supplemented with beta-glucan yeast significantly increased the activity of gastric pepsin, intestinal trypsin, lipase and amylase enzymes. The highest digestive enzyme activities were generally observed in the G-2 group. Pepsin, trypsin, amylase, and lipase activities in this group were signiﬁcantly higher compared to the other groups (P < 0.05). No direct studies have been found on the effect of yeast- based prebiotics used as feed additives on enzyme activity. However, other research has shown that different prebiotic

6 of 9 Prebiotic eﬀects on growth and enzyme activity in rainbow trout/Özlüer-Hunt and Özkan-Yılmaz. CONCLUSION supplements and feeding regimes can influence digestive enzymes. For example, feeding white seabream (Diplodus sargus) and meagre (Argyrosomus regius) with diets containing 1–2% brewer’s spent yeast (BSY) (Saccharomyces pastorianus) affected amylase, lipase, and protease activities differently between species, White seabream showed lower proteolytic but higher amylase and lipase activities than meagre. In white seabream, enhanced amylase and protease activities were observed in the pyloric caeca and intestine, and lipase activity was increased in the pyloric caeca, following BSY administration. In meagre, an improvement in amylase activity in the pyloric caeca was seen with the 2 % BSY diet [40]. Similarly, common carp fed diets with 0.5–1.5 g.kg-¹ Biomin® IMBO synbiotic showed increased trypsin and chymotrypsin activities, while α-amylase, lipase, and alkaline phosphatase were not signiﬁcantly affected [41]. Another study, conducted over 60 day in rainbow trout, investigate the effects of replacing ﬁsh meal with a nucleotide- supplemented prebiotic on growth and digestive enzymes. The results indicated that the prebiotic supplementation positively influenced both growth and digestive enzyme activities. At the end of the study, pepsin, trypsin, and amylase activities were found to be signiﬁcantly different in all prebiotic-supplemented groups compared to the control group [42]. Studies examining how prebiotics affect enzyme activities in fish are still limited. Fish fed with 100 mg.kg-¹ of prebiotic xylooligosaccharides (XOS) showed better growth performance than those fed with 50 or 200 mg.kg-¹ XOS. Similar to this research, no positive correlation was observed between the XOS level and fish growth. Moreover, there was no significant difference in final weight gain between the 200 mg.kg-¹ XOS group and the control group, suggesting that higher XOS levels might have negatively affected nutrient digestibility, leading to reduced growth performance [43]. The microbial communities in the ﬁsh digestive system play a crucial role in maintaining host health and improving the balance of the normal gut flora [44]. In the studies, it was determined that prebiotic additives increased the fermentation of short- chain fatty acids in the intestine. Digestive enzymes increase the efﬁciency of feed use in ﬁsh [45]. The characterization of these enzymes provides information on the digestive capacity of ﬁsh. It also provides information on the enzymes' ability to hydrolyse carbohydrates, proteins and lipids. [46]. Prebiotics increase intestinal viscosity and increase the efficiency of the digestive system in many organisms [47] and enhance the solubility and absorption of minerals such as calcium, magnesium, and iron ions [48], and the bioavailability of trace elements [48], by lowering the intestinal pH. As is common knowledge, prebiotics have very valuable effects on the digestive systems, most notably with the morphological and microbiological systems of organisms [47]. Therefore, fish fed with prebiotics tend to develop better enzyme activity due to the modulation of the beneﬁcial microbial population, which enhances trypsin, amylase, and lipase activities. Consequently, it is thought that growth efﬁciency also improves in relation to increased enzyme activity [49]. In this study, the growth performance, enzyme activities, and feed cost of rainbow trout were investigated by adding a prebiotic substance consisting of a mixture of MOS and glucan, used as a yeast source, to the feed at different levels (0, 0.5 and 1 g.kg-1). When the results were analyzed, the best growth parameters and digestive enzyme activities were obtained in the group (G-2; 0.5 g.kg-1) fed with feed prebiotic. Considering that enzymes are the most important substances for the digestive function to occur, the results obtained suggest that the appropriate amount of prebiotic supplementation stimulates enzyme activity showed better digestion, thereby enabling to achieve better growth values. However, further studies are needed to determine whether prebiotics can be used in different ﬁsh species or in different growth stages of the same species, and if so, in which stages and in what quantities. This study was supported by Mersin University, Department of Scientific Research Projects (BAP-2017-2-TP2-2081). The authors would like to thank ADM Alliance Nutrition for supplying yeast supplement free of charge. ACKNOWLEDGMENTS The authors declare no conflicts of interest regarding this article’s research, authorship, and/or publication. This research was conducted following the approval of the animal experiment by the Mersin University Local Ethics Committee (Decision number: 14/46/2016). Conﬂict of interest Ethical approval Boyd CE, McNevin AA. Aquaculture, resource use, and the environment. Oxford: Wiley-Blackwell. [Internet]. 2015; doi: https://doi.org/q62q [2] Serra V, Pastorelli G, Tedesco DEA, Turin L, Guerrini A. Alternative protein sources in aquafeed: Current scenario and future perspectives. [Internet]. Vet. Anim. Sci. 2024; 25:100381. doi: https://doi.org/hbgbxh BIBLIOGRAFIC REFERENCES [1] Ringø E, Van Doan H, Lee SH, Soltani M, Hoseinifar SH, Harikrishnan R, Song SK. Probiotics, lactic acid bacteria and bacilli: interesting supplementation for aquaculture. J. Appl. Microbiol. [Internet]. 2020; 129:(1):116-136. doi: https://doi.org/ggpwtr [3]

Revista Cientíﬁca, FCV-LUZ / Vol. XXXVI 7 of 9 Wee W, Abdul-Hamid NK, Mat K, Raja-Khalif RIA, Rusli ND, Rahman MM, Kabir MA. Wei LS. The effects of mixed prebiotics in aquaculture: A review. Aquac. Fish. [Internet]. 2024; 9(1):28-34. doi: https://doi.org/q62x [4] Saad N, Delattre C, Urdaci M, Schmitter JM, Bressollier P. An overview of the last advances in probiotic and prebiotic ﬁeld. LWT. Food Sci. Technol. [Internet]. 2013; 50(1):1-16. doi: https://doi.org/gfwkkr [5] Dawood MAO, Koshio S. Recent advances in the role of probiotics and prebiotics in carp aquaculture: A review. Aquaculture. [Internet]. 2016; 454:243-251. doi: https:// doi.org/q623 [6] Özkan-Yılmaz F, Özlüer-Hunt A, Berköz M. Investigation on paraoxonase enzyme activity and malondialdehyde level in liver of Oreochromis niloticus fed with MOS supplemented diet. [Internet]. Turk. J. Agric. Food Sci. Technol. 2015; 3(8):639-643. doi: https://doi.org/q624 [7] Özlüer-Hunt A, Berköz M, Özkan F, Yalin S, Erçen Z, Erdoğan E, Gündüz SG. Effect of mannan oligosaccharide on growth, body composition, and antioxidant enzyme activity of tilapia (Oreochromis niloticus). Isr. J. Aquac. Bamidgeh. [Internet]. 2011 [cited 20 Jan 2026]; 63(2):619-627. Available in: https://goo.su/GHxYUvU [8] Ebrahimi G, Ouraji H, Khalesi MK, Sudagar M, Barari A, Zarei-Dangesaraki M, Jani-Khalili KH. Effects of a prebiotic, Immunogen®, on feed utilization, body composition, immunity and resistance to Aeromonas hydrophila infection in the common carp Cyprinus carpio (Linnaeus) fingerlings. J. Anim. Physiol. Anim. Nutr. [Internet]. 2012; 96(4):591-599. doi: https://doi.org/ cnr69p [9] Martins de Brito J, Urbich AV, Pereira da Cruz T, Panczevicz PAP, Galioto-Miranda JA, Wernick B, Furuya VRB, Furuya WM. Xylanase and β-glucanase improve growth performance, gut barrier, and microbiota of pre-growout Nile tilapia, Oreochromis niloticus fed a vegetable-based diet. Aquaculture. [Internet]. 2022; 561:738653. doi: https://doi.org/q625 [10] Samrongpan C, Areechon N, Yoonpundh R, Srisapoome P. Effects of mannan- oligosaccaharide on growth, survival and disease resistance of Nile Tilapia ( Oreochromis niloticus, Linneaeus) Fry. 8 th Interntional Symposium on Tilapia in Aquaculture. [Internet]. 2008; 345-353 Available in: https://goo.su/jztuZC [11] Torrecillas S, Makol A, Cabellero D, Montero R, Gines J, Sweetmen M, Izquierdo M. Improved feed utilization, intestinal mucus production and immune parameters in sea bass ( Dicentrarchus labrax ) fed mannan oligosaccharides (MOS). Aquac. Nutr. [Internet]. 2011; 17:223–233. doi: https://doi.org/c3gv3h [12] Magouz FI, Salem MFI, Emara AEI, Hassan MM, Dawood MAO. A mixture of β-glucan and mannanoligosaccharide ameliorated the growth rate, digestive enzyme activity, intestinal morphometry, and immunity of common carp ( Cyprinus carpio). Annals Anim. Sci. [Internet]. 2021; 21(3):1027-1041. doi: https://doi.org/q626 [13] Schmidt J, Bischoff AA, Weiß M, Kim SK, Frickenhaus S, Slater MJ, Buck BH. Effect of beta-1-3-glucan and mannans on growth and fitness of starry flounder ( Platichthys stellatus): A potential new candidate for aquaculture in temperate regions. J. Fish. Sci. [Internet]. 2017; 11(3):17-25. doi: https://doi.org/q628 [14] Spencer JFT, de Spencer AR, Laluce C. Non-conventional yeasts. Appl. Microbiol. Biotechnol. [Internet]. 2002; 58:147-156. doi: https://doi.org/fvp9kc [15] ADM Animal Nutrition. CitriStim®. [Internet]. Quincy, Ilinois, USA: ADM Animal Nutrition. 2026 [cited 20 Jan 2026]; 0917. Avalabile from: https://goo.su/3tGUUF [16] Smith RR. Nutritional energetics. In: Halver JE, Editor. Fish Nutrition. (2nd ed.) New York, USA: Academic Press.. 1989; p. 2-31. [17] Ricker WE. Growth rates and models. In: Hoar WS, Randall DJ, Brett JR, Editors. Fish Physiology, VIII, Bioenergetics and Growth. New York, USA: Academic Press,. 1979; 677–743 pp. [18] Tarak RR, Yadav MK, Saxena S, Kher D. Effect of leaf powder of giloy ( Tinospora cordifolia) in fish feed on survival and growth of post larvae of Cyprinus carpio fingerlings. Int. J. Adv. Biochem. Res. [Internet]. 2025; 9(8):532-536. doi: https://doi.org/q63f [19] Martínez-Llorens S, Moñino AV, Tomás A, Pla M, Jover M. Soybean meal as a protein source in gilthead sea bream (Sparus aurata L.) diets: effects on growth and nutrient utilization. Aquac. Res. [Internet]. 2007; 38:82-90. doi: https://doi.org/c86w7r Official Methods of Analysis of Assocıatıon of Official Analytical Chemists (AOAC). Arlington, Virginia, USA: AOAC. [Internet]. 15th edition. 1990. [cited 20 Jan 2026]. 771 p. Available in: https://goo.su/aTWze [20] [21] Worthington CC, Worthington V, Worthington K, Decher L, Hackler D, Worthinton A. Worthington Enzyme Manual. Enzymes and Related Biochemicals Worthington Chemical. New Jersey, USA: Worthington Biochemical Corporation. [Internet]. 2016. [cited 20 Jan 2026]. 782 p. Available in: https://goo.su/ICc9U [22]

8 of 9 Prebiotic eﬀects on growth and enzyme activity in rainbow trout/Özlüer-Hunt and Özkan-Yılmaz. Erlanger B, Kokowsky N, Cohen W. The preparation and properties of two new cromogenic substrates of trypsin. Arch. Biochem. Biophys. [Internet]. 1961; 95(2):271–278. doi: https://doi.org/d8jjvr [23] Benjakul S, Visessanguan W, Thummaratwasik P. Isolation and characterization of trypsin inhibitors from some Thai legume seeds. J. Food Biochem. [Internet]. 2007; 24(2):107-127. doi: https://doi.org/d8qf4f Ai Q, Mai K, Zhang L, Tan B, Zhang W, Xu W, Li H. Effects of dietary β-1,3 glucan on innate immune response of large yellow croaker, Pseudosciaena crocea. Fish Shellﬁsh Immunol. [Internet]. 2007; 22(4):394-402. doi: https:// doi.org/d47mt2 [24] [34] Bernfeld P. Amylases, α and β. Methods in enzymol. [Internet]. 1995; 1:149-158. doi: https://doi.org/dc9pbx Misra CK, Das BK, Mukherjee SC, Pattnaik P. Effect of long term administration of dietary β-glucan on immunity, growth and survival of Labeo rohita ﬁngerlings. Aquaculture. [Internet]. 2006; 255(1-4):82-94. doi: https://doi.org/bwv2j8 [25] [35] Winkler UK, Stuckman M. Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exo-lipase by Serratia marescens. J. Bacteriol. [Internet]. 1979; 138(3):663-670. doi: https://doi.org/q63k Bai N, Zhang WB, Mai KS, Wang XJ, Xu W, Ma, HM. Effects of discontinuous administration of β-glucan and glycyrhizin on the growth and immunity of white shrimp Litopenaeus vannamei. Aquaculture. [Internet]. 2010; 306(1-4):218- 224.doi: https://doi.org/fsc3wb [26] [36] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J. Biol. Chem. [Internet]. 1951; 193(1):265-275. doi: https://doi.org/ ghv6nr Khodadadi M, Abbasi N, Adorian TJ, Farsani HG, Hedayati A, Hoseini SM. Growth performance, survival, body composition, hematological parameters, intestinal histomorphology, and digestive enzymes’ activity in juvenile rainbow trout (Oncorhynchus mykiss) fed dietary Immunogen®. J. Appl. Aquac. [Internet]. 2018; 30(2):174- 186. doi: https://doi.org/q64s [27] [37] Ringoe E, Olsen RE. Gifstad TØ, Dalmo RA, Amlund H, Hemre GI, Bakke AM. Prebiotic in aquaculture, review. Aquac. Nutr. [Internet]. 2010; 16:117-136. doi: https:// doi.org/dhb32j [28] Salze G, McLean E, Schwarz MH, Craig SR. Dietary mannan oligosaccharide enhances salinity tolerance and gut development of larval cobia. Aquaculture. [Internet]. 2008; 274(1):148-152. doi: https://doi.org/d6k3x7 [30] Torrecillas S, Makol A, Caballero MJ, Montero D, Robaina L, Real F, Sweetman J, Tort L, Izquierdo MS. Immune stimulation and improved infection resistance in European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides. Fish Shellfish Immunol. [Internet]. 2007; 23(5):969-981. doi: https://doi.org/fw379x [31] Grisdale-Helland B, Helland SJ, Gatlin-III DM. The effects of dietary supplementation with mannanoligosaccharide, fructooligosaccharide or galactooligosaccharide on the growth and feed utilization of Atlantic salmon (Salmo salar). Aquaculture. [Internet]. 2008; 283(1-4):163-167. doi: https://doi.org/b3dmz8 [29] Staykov Y, Spring P, Denev S, Sweetman J. Effect of a mannan oligosaccharide on the growth performance and immune status of rainbow trout (Oncorhynchus mykiss). Aquac. Int. [Internet]. 2007; 15:153-161. doi: https://doi. org/bm5vh5 [32] Aramli MS, Kamangar B, Nazari RM. Effects of dietary β-glucan on the growth and innate immune response of juvenile Persian sturgeon, Acipenser persicus. Fish Shellﬁsh Immunol. [Internet]. 2015; 47(1):606-610. doi: https://doi.org/f7zwbm [33] Brito JM, Urbich AV, Cruz TP, Panczevicz PAP, Miranda JAG, Wernick B, Furuya VRB, Furuya WM. Xylanase and β-glucanase improve growth performance, gut barrier, and microbiota of pre-growout Nile tilapia, Oreochromis niloticus fed a vegetable-based diet. Aquaculture. [Internet]. 2022; 561:738653. doi: https://doi.org/q625 [38] Welker TL, Lim C, Yıldırım-Aksoy M, Shelby R, Klesius PH. Immune response and resistance to stress and Edwardsiella ictaluri challenge in channel catﬁsh, Ictalurus punctatus, fed diets containing commercial whole-cell yeast or yeast subcomponents. J. World Aquac. Soc. [Internet] 2007; 38(1):24-35. doi: https://doi.org/d62qqr [39] Castro C, Pérez-Jiménez A, Coutinho F, Pousão-Ferreira P, Brandão T.M, Oliva-Teles A, Peres H. Digestive enzymes of meagre ( Argyrosomus regius) and white seabream (Diplodus sargus). Effects of dietary brewer’s spent yeast supplementation. Aquaculture. [Internet]. 2013; 416- 417:322-327. doi: https://doi.org/q64t [40] Dehaghani PG, Baboli MJ, Moghadam AT, Ziaei-Nejad S,. Pourfarhadi M. Effect of synbiotic dietary supplementation on survival, growth performance, and digestive enzyme activities of common carp (Cyprinus carpio) fingerlings. Czech J. Anim. Sci. [Internet]. 2015; 60(5):224–232. doi: https://doi.org/q64v [41]

Revista Cientíﬁca, FCV-LUZ / Vol. XXXVI 9 of 9 Özlüer-Hunt A, Özkan-Yılmaz F, Engin K, Berköz M, Gündüz SG, Yalın S, Şahin NÖ. The effects of fish meal replacement by yeast based nucleotides on growth, body composition and digestive enzyme activity in rainbow trout juveniles (Onchorchyncus mykiss). Isr. J. Aquac – Bamidgeh. [Internet]. 2014 [cited 20 Jan 2026]; 66:964- 973. Available in: https://goo.su/If9B [42] Xu B, Wang Y, Li J, Lin Q. Effect of prebiotic xylooligosaccharides on growth performances and digestive enzyme activities of allogynogenetic crucian carp (Carassius auratus gibelio). Fish Physiol. Biochem. [Internet]. 2009; 35(3):351-357. doi: https://doi.org/ bw9kzp [43] Ringø E, Strøm E, Tabachek JA. Intestinal microflora of salmonids: a review. Aquac. Res. [Internet]. 1995; 26:773–789. doi: https://doi.org/fd83m6 [44] Özlüer-Hunt A, Çetinkaya M, Özkan-Yılmaz F, Yıldırım M, Berköz M, Yalın S. Effect of dietary supplementation of Inulin on growth performance, digestion enzyme activities and antioxidant status of rainbow trout (Oncorhynchus mykiss). Turk J. Agric. Food Sci. Technol. [Internet]. 2019; 7(9):1344-1353. doi: https://doi.org/m4cq [45] Ringø E, Gatesoupe FJ. Lactic acid bacteria in fish: a review. Aquaculture. [Internet]. 1998; 160(3-4):177–203. doi: https://doi.org/fqcgjm [46] Lemieux H, Blier P, Dutil JD. Do digestive enzymes set a physiological limit on growth rate and food conversion efficiency in the Atlantic cod ( Gadus morhua)? Fish Physiol. Biochem. [Internet]. 1999; 20:293-303. doi: https://doi.org/bbvf64 [47] Bongers A, Van den Huevel EGHM. Prebiotics and the bioavailability of mineral and trace elements. Food Rev. Int. [Internet]. 2003; 19(4):397-422. doi: https://doi.org/ djrnpr Ganguly S, Paul I, Mukhopadhayay SK. Application and effectiveness of immunostimulants, probiotics, and prebiotics in aquaculture: A review. Isr. J. Aquacult. Bamidgeh. [Internet]. 2010 [cited 20 Jan 2026]; 62(3):130-138. Available in: https://goo.su/n6KDB [48] [49]