Effect of Probiotic Supplimentations on the Gut Histoarchitecture of Stinging Catfish, Heteropneustes fossilis

The study was conducted to evaluate the effect of probiotics on the gut histology of stinging catfish, Heteropneustes fossilis. The experiment was conducted in 15 ponds, each with 0.75 decimal and stocking density were 550 fingerlings/decimal (6.44 ± 0.054 gm) and cultured for 90 days (May to August). Commercially available gut probiotic ZYMETIN, water probiotic pH FIXER and soil probiotic Super PS were used in the experiment. T₁ was designed with the recommended dose of Super PS (soil probiotic). T₂ was supplemented with the recommended dose of gut probiotic (ZYMETIN). T₃ was designed with combined application of Super PS, ZYMETIN and pH FIXER at recommended doses. T₄ was designed with the recommended dose of water probiotic (pH FIXER). Only basal feed was applied for T₅ (control). Water quality parameters, morphometric measurements, body weight of the experimental fish were measured, and fish gut samples were collected for histological study at fortnightly intervals. The results showed that all water quality parameters in pH FIXER treated ponds were more favorable for fish culture compared with others. In combined probiotic treated groups (T₃) histoarchitecture of the gut were almost normal. Less pathological signs were observed in probiotic treated groups (T₁, T₂, and T₄) compared with T5 (control). From analysis of gut revealed that fold length, fold width and epithelial layer thickness were increased significantly (P < 0.05) in combined probiotics treated fish. Fish from T2 and T3 exhibited larger fold length, width and epithelial layer thickness than those of T₁, T₄, T₅ (control). Probiotic addition can improve intestinal structure of H. fossilis which may increase the nutrients absorption in fish. Hence, digestion capacity was increased that ultimately helps to improve the overall health condition of stinging catfish, H. fossilis.

Introduction

Aquaculture is one of the quickest food-yielding sector with the largest potential to achieve demand of aquatic food and nutrition (FAO, 2006). With the development and intensification of production in aquaculture, diseases and degradation of environmental conditions are major problems in fish culture. For prevention and control of diseases, antibiotics used as traditional strategy during the last decades for the fish growth (Uddinet al., 2019). However, the improvements of non-antibiotic agents are more suitable for health management in fish farming. Dietary supplements such as probiotics act as growth promoting factors for fish farming (FAO, 2010). Gradual declining of fish production is due to lack of proper and sustainable fish health management. In this case, probiotics is necessary to maintain sustainable balance of aquatic food production and keep the nature safe (Uddin & Nur-A-Sharmin Aktar, 2022).

Shing (Heteropneustes fossilis) is native stinging catfish of South-East-Asia. The species is not only known for its excellent taste, unique flesh quality and commercial value. This native stinging catfish is also highly considered from nutritional and medicinal properties of view and fairly high quantity of calcium compared to other freshwater fish species (Saha & Guha, 1939).

The word probiotic was first introduced by Lilly and Stillwell (1965) to describe “substances secreted by one microorganism that stimulate the growth of another.” The term probiotic comes from the Greek “pro bios,” which means “for life” (Soccolet al., 2010). Probiotics are microscopic organisms that are applied orally at optimum amount to alter the microbiota of the specific host and lead to benefits for the host’s health (Akhteret al., 2015). Probiotics was first applied in 1986, to trial ability to show great impact and increase growth rate of hydrobionts. Later, it is used to enhance quality of water, control of infectious bacteria and microorganisms. It shows a new measurement of disease counteraction and better water quality in aquaculture industry (Wanget al., 2005). The purpose of using probiotics is to sustain a friendly relationship with pathogenic microorganisms (Minghettiet al., 2017). The effect of probiotic organisms is acquired by improving the immune system of cultured species (Sayeset al., 2017) and restrain the pathogenic microorganisms from the formation of diseases in the host body (Ali, 2000).

At present, there are some commercial probiotic products prepared from numerous bacterial species such as Lactobacillus sp., Bacillus sp., Enterococcus sp., Carnobacterium sp., and the yeast species Saccharomyces cerevisiae among others, and their use is limited by careful administration and recommendations (Boyd & Massaut, 1999). As for probiotic strains Lactic Acid Bacteria have been largely used which are commonly present in the gut of fishes such as the Lactobacilli and Bifidobacteria. Bacillus, Enterococcus, Streptococcus are gram-positive bacteria act as a general probiotic strains which are the major gastrointestinal microorganisms (Pandiyanet al., 2013). Commercially available probiotics viz., ZYMETIN (Advance Pharma Co. Ltd., Bangkok, Thailand) functions as enzyme producer, organic decomposer and produce unfavorable condition for pathogenic bacteria. Super PS and pH Fixer maintains optimum water quality. Monitoring of water quality parameters is very important which directly regulate the production of H. fossilis. Thus, control of water quality parameters has become vital task for good health management and boost production.

Gut is one of the most important organ of fish which could definitely relates to changes in nutrient absorption of fish. Villi length is very important to determine the efficiency of the nutrient absorption of fishes (Purushothamanet al., 2016). Nutrient absorption in villi may impact on the overall fish health (Sweetmanet al., 2008). Toxic substances cause damage of normal gill tissue structure and histopathological degradations in fish body (Olojoet al., 2005). Histological method is one of the most important procedure for diagnosis of fish diseases to its tissue level. Practically this method has been used across the world. But in Bangladesh this process has used for diagnosis of fish in a limited extent (Ahmedet al., 1998).

Materials and Methods

Study Area and Duration

The experiment was carried out at the research ponds of the Faculty of Fisheries, Bangladesh Agricultural University (BAU), Mymensingh, Bangladesh for 90 days’ duration (May–August).

Pond Preparation

Fifteen (15) ponds were selected for the present experiment. Each pond size was 0.75 decimal. Depth of each pond were 1.0 m to 1.3 m. At first ponds were dried for two weeks, pond dykes were repaired and then filled with underground water. After that lime and salt were applied in each pond at a dose of 1 kg/decimal.

Experimental Design

The research was conducted with five treatments and each treatment was designed with three replications. In first treatment (T₁) commercial feed was used and soil probiotic mixed with sand was applied at a dose of 15 litre/hectare for 1–30 days, 30 litre/hectare for 31–60 days, 40 litre/hectare for 61–90 days. Second treatment (T₂) was prepared with gut probiotic ZYMETIN supplemented at a dose of 10 g/kg of basal feed. For third treatment (T₃) combination of all three probiotics (Super PS, ZYMETIN and pH FIXER) were applied at recommended doses. For fourth treatment (T₄) normal commercial feed was used and pH FIXER applied in water at a dose of 13 g/ponds/weeks. Fifth treatment (T₅) was designed to use basal feed (control) only.

Stocking of Fish

Healthy stinging catfish (H. fossilis) fingerlings (average weight of 6.44 gm ± 0.05 gm) were stocked at a density of 413/0.75 decimal. Fish were fed with the commercial feed (SMS Feeds Ltd.) supplemented with mentioned above probiotics. Control fish were provided same commercial feed without probiotics. Water exchange (30%) was done monthly.

Preparation of Probiotic Supplemented Feed

ZYMETIN (Advance Pharma Co. Ltd., Bangkok, Thailand) composed mainly with Streptococcus faecalis, Clostridium butyricum, Bacillus mesentericus, protease, lipase and beer yeast used in feed as gut probiotic and mixed with feed to increase the immunity and stop the growth of pathogenic organisms in gut; Water additives probiotic pH FIXER (CPF private limited, India) maintains optimum water quality parameters, which is composed of concentrated strain of beneficial Bacillus bacteria; Super PS (CPF private limited, India) is a soil probiotic which contains Rhodobacter spp. and Rhodococcus spp. is used to improve bottom condition of pond, diminish harmful bacteria and keep the suitable environmental condition for aquaculture. All the probiotic was selected based on the composition and purchased from registered local agent to use in the experiment. After probiotic supplementation, feeds were dried for overnight then stored in the laboratory at room temperature for daily feeding.

Feeding

Experimental diets were applied twice daily in the morning at 8:00 am and in afternoon at 5:00 pm at the rate of 10% of body weight. Gradually feeding rate was reduced with increasing growth of H. fossilis and given 5% of body weight after one month of culture and continued till termination of experiment.

Sampling of Fish

After fifteen days of experiment fish were sampled by net and body weight of the ten individual number fish was measured by using a weighing machine.

Monitoring of Water Quality Parameters

Water quality parameters are recorded and monitored during study period. Dissolved oxygen (DO) (mg/ L), free ammonia (NH₃), water temperature (°C), alkalinity and pH values were taken after fifteen days throughout the study period.

Histological Procedure

For histological observation gut segments were sampled from three fish from each treatment group and immediately fixed with 10% formalin (buffered). Automatic Tissue Processors were used to dehydration, clearing and infiltration of the sample. Samples were sectioned and stained with eosin and hematoxylin. Photomicrograph of the stained sections was done by a photo microscope (Primo Star ZEISS). At the end of experiment, assessment on fold width, fold length, and epithelial layer thickness of gut and villi structures were observed through ZEN 2.3 Lite software from the treatments as well as from control. Then analyzed data of fold length, width and epithelial layer thickness of gut villi were considered to evaluate effects of probiotics on those structures (Treatmentwise).

Results and Discssion

Water Quality Parameters

The water quality parameters of stinging catfish (Heteropneustes fossilis) in the rearing ponds were recorded.

Water Temperature (°C)

Water temperature were ranged from 27.86°C ± 0.07°C to 33.26°C ± 0.27°C during the study period. The maximum temperature was recorded as 33.26°C ± 0.27°C on 18 August in T₄, whereas, the minimum was 27.86°C ± 0. 70°C on 17 July, 2019 in T₃.

Dissolved Oxygen (mg/L)

The values of dissolved oxygen were varied from 3.33 ± 0.17 to 7.00 ± 0.29 mg/ L. The highest dissolved oxygen value was 7.00 ± 0.29 mg/ L on 26 May in T₁ and on 9 June in T₅, whereas, the lowest value was 3.33 ± 0.17 mg/ L on 21 July, 2019 in T_5.

pH

The recorded water pH were ranged from 7.00 ± 0.00 to 8.00 ± 0.15 during the study period. The highest pH value was 8.00 ± 0.15 on 26 May in T₁ while the lowest pH value was 7.00 ± 0.00 on 18 August, 2019 in T₅.

Free Ammonia (mg/L)

The values of free ammonia were varied from 0.00 ± 0.00 to 0.41 ± 0.08 mg/L. The highest free ammonia value was 0.41 ± 0.08 on 18 August in T₁ and the lowest value was 0.00 ± 0.00 mg/L on 21 July, 2019 in T_3.

Alkalinity

The values of alkalinity were ranged from 96.66 mg/L ± 6.67 mg/L to 173.33 mg/L ± 3.33 mg/L. The highest value was 173.33 ± 3.33 mg/L on 4 August in T₁ and the lowest value was 96.66 mg/L ± 6.67 mg/L on 4 August, 2019 in T_5.

Histopathological Observation of the Gut of H. fossilis

Histopathological changes occurred in the gut of H. fossilis at the start, middle, and end of the experiments are described below.

At the Start of the Experiment

At the start of the experiment cross section of gut of H. fossilis had pathological signs in almost all treatment. There were vacuum and disrupted layer of gastrointestinal (GI) tract in T₁ (Fig. 1a). Gut of T₂ had lost villi and clubbing (Fig. 1b). Almost normal structure of villi except vacuum was found in T₃ (Fig. 1c). Partly clubbed villi and necrosis were found in T₄ (Fig. 1d). But gut of T₅ villi were partly lost, clubbed and necrosis (Fig. 1e).

Fig. 1. Photomicrograph of gut of H. fossilis during first sampling in (T₁) in May with disrupted layer (DL) of gastrointestinal tract and vacuum (V). H & E × 125 (a), Section of gut of H. fossilis during first sampling in (T₂) in May having partly lost villi (VL) and clubbing (CB). H & E × 125 (b), Cross-section of gut of H. fossilis during first sampling in (T₃) in May with almost normal structure except vacuum (V). H & E × 125 (c), Photomicrograph of gut of H. fossilis during first sampling in (T₄) in May having clubbed villi (CB) and necrosis (N). H & E × 125 (d), Cross-section of gut of H. fossilis at first sampling in (T₅) in May with clubbed villi (CB), partly lost villi (VL) and necrosis (N). H & E× 125 (e).

At the Middle of the Experiment

Treatments containing probiotic (T₁, T₂, T₃ and T₄) have a better result than the control one (T₅). Gut of T₁ had clubbed villi (CB), hemorrhages and partly lost villi (Fig. 2a) which treated with gut probiotic. In T₂ clubbed and partly lost villi were present (Fig. 2b). In T₃ gut section had almost normal structure (Fig. 2c). In T₄ had partly lost villi, clubbed villi and necrosis (Fig. 2d). And gut section of T₅ (control) contained vacuum, necrosis and disrupted layer (Fig. 2e).

Fig. 2. Photomicrograph of gut of H. fossilis at the middle of experiment (T₁) in July with clubbed villi (CB), hemorrhage (H) and partly lost villi (VL). H & E × 125 (a), Cross-section of almost normal gut of H. fossilis at the middle of experiment (T₂) in July with clubbed villi (CB) and partly lost villi (VL). H & E × 125 (b), Section of almost normal structure of gut of H. fossilis at the middle of the experiment (T₃) in July with necrosis (N) in GI. H & E × 125 (c), Photomicrograph of gut of H. fossilis at the middle of experiment (T₄) in July with clubbed villi (CB), necrosis (N) and partly lost villi (VL). H & E × 125 (d), Cross-section of gut of H. fossilis at in control group (T₅) in July had disrupted layer (DL) of GI, necrosis (N), vacuum (V) and partly lost villi (VL). H & E × 125 (e).

At the End of the Experiment

At the end of the experiment, gut of H. fossilis in T₁ and T₂, T₃ had comparatively improved structure of villi except necrosis and clubbing (Figs. 3a and 3b). Among five treatments T₃ had more or less normal structure (Fig. 3c). Gut section of T₄ had necrosis, clubbed villi and lost villi (Fig. 3d), whereas, T₅ (control) had necrosis (N), clubbed villi (CB) vacuum (V) and lost villi (Fig. 3e).

Fig. 3. Photomicrograph of normal gut of H. fossilis at the end of experiment (T₁) with necrosis (N) and clubbed villi (CB) in August. H & E × 125 (a), Cross-section of almost normal gut of H. fossilis at the end of experiment (T₂) with clubbed villi (CB) in August. H & E × 125 (b), Section of almost normal gut of H. fossilis at the end of the experiment (T₃) except necrosis (N) in August. H & E × 125 (c). Photomicrograph of gut of H. fossilis at the end of experiment (T₄) in August with necrosis (N), clubbed (CB) and lost villi (VL). H & E × 125 (d). Cross-section of gut of H. fossilis at the end of experiment (T₅) in August with necrosis (N), clubbed villi (CB) vacuum (V) and lost villi (VL). H & E × 125 (e).

Effect on Gut of H. fossilis

All histological measurements including fold length (villus height), fold width (villus width), and epithelial layer thickness (mucosa width) of the midgut of H. fossilis samples in response to the dietary administration of different host-associated probiotics are showed in Tables I–III.

Table I. Mean Thickness and Standard Deviation of the Fold Length (μm) of the Middle Portion of the Intestine of H. fossilis at the Start, Middle and End of the Experiment

Treatments	Start of the experiment	Middle of the experiment	End of the experiment
T1	36.36 ± 1.26	40.97 ± 1.53	47.40 ± 2.33
T2	38.33 ± 1.40	42.34 ± 2.10	53.11 ± 3.60
T3	38.79 ± 1.91	44.31 ± 1.55	59.20 ± 1.84
T4	37.70 ± 0.78	41.31 ± 2.39	48.99 ± 0.98
T5	36.18 ± 1.22	39.49 ± 1.98	44.87 ± 0.90

Table II. Mean Thickness and Standard Deviation of the Fold Width (μm) of the Middle Portion of the Intestine of H. fossilis at the Start, Middle and End of the Experiment

Treatments	Start of the experiment	Middle of the experiment	End of the experiment
T1	7.93 ± 0.32	9.85 ± 0.58	12.35 ± 0.92
T2	8.09 ± 1.07	10.30 ± 1.39	13.71 ± 1.26
T3	8.40 ± 0.54	11.09 ± 1.04	14.68 ± 0.60
T4	8.04 ± 0.54	10.08 ± 0.42	12.81 ± 0.27
T5	7.85 ± 0.40	8.39 ± 0.54	10.84 ± 0.53

Table III. Mean Thickness and Standard Deviation of Epithelial Layer Thickness (μm) of the Middle Portion of the Intestine of H. fossilis at the Start, Middle and End of the Experiment

Treatments	Start of the experiment	Middle of the experiment	End of the experiment
T1	3.97 ± 0.79	5.37 ± 0.61	6.59 ± 0.52
T2	4.12 ± 0.07	5.72 ± 0.73	7.76 ± 0.70
T3	4.41 ± 0.53	6.24 ± 0.24	8.41 ± 0.67
T4	3.98 ± 0.67	5.64 ± 0.58	6.91 ± 0.33
T5	3.96 ± 0.16	4.37 ± 0.43	5.30 ± 0.53

Effect on Fold Length (Villus Height)

At the start of the experiment, there were no significance differences of fold length (FL) among the treatments. However, in the middle and end of the experiment FW of probiotic treated groups T₁ (Fig. 4a), T₂ (Fig. 4b), T₃ (Fig. 4c) and T₄ (Fig. 4d) had increased when compared with control treatment T₅ (Fig. 4e). At the end of the experiment, the highest FL (59.20 μm) was observed in T₃ (Fig. 4c) in comparison with other treatments and the lowest FL (44.87 μm) was observed on T₅ (control) (Fig. 4e). Structure of gut in T₂ (Fig. 4b) had increased FL (53.11 μm) compared with T₁ (Fig. 4a), T₄ (Fig. 4d), and T₅ (Fig. 4e); T₄ (Fig. 4d) had increased FL (48.99 μm) compared with T₁ (Fig. 4a) and T₅ (control) (Fig. 4e) (Table I).

Fig. 4. Cross-section of gut of H. fossilis at the end of experiment (T₁) in August having 48.25 μm FL. H & E × 125 (a), Cross-section of gut of H. fossilis at the end of experiment (T₂) in August having 52.62 μm FL. H & E × 125 (b), Cross-section of gut of H. fossilis at the end of experiment (T₃) in August having 55.75 μm FL. H & E × 125 (c), Cross-section of gut of H. fossilis at the end of experiment (T₄) in August having 49.56 μm FL. H & E × 125 (d), Cross-section of gut of H. fossilis at the end of experiment (T₅) in August having 44.02 μm FL. H & E × 125 (e).

Effect on Fold Width (Villus Width)

Fold width showed 7.85 μm to 8.40 μm at the start and 8.39 μm to 11.09 μm at the middle of the experiment. At the end of the experiment fold width exhibited 10.84 μm to 14.68 μm where highest (14.68 μm) was in combined treated groups T₃ (Fig. 5c) and the lowest (10.84 μm) was in control treatment T₅ (Fig. 5e). At the end of the experiment gut of T₂ (Fig. 5b) exhibited increased FW (13.71 μm) compared with T₁ (Fig. 5a) (12.35 μm), T₄ (Fig. 5d) (12.81 μm), and T₅ (Fig. 5e) (10.84 μm); T₄ (Fig. 5d) (12.81 μm) had increased FW compared with T₁ (Fig. 5a) (12.35 μm) and T₅ (Fig. 5e) (10.84 μm) (Table II).

Fig. 5. Cross-section of gut of H. fossilis at the end of experiment (T₁) in August having 11.29 μm FW. H & E × 425 (a), Cross-section of gut of H. fossilis at the end of experiment (T₂) in August having 13.90 μm FW. H & E × 430 (b), Cross-section of gut of H. fossilis at the end of experiment (T₃) in August having 15.38 μm FW. H & E × 430 (c), Cross-section of gut of H. fossilis at the end of experiment (T₄) in August having 12.50 μm FW. H & E × 430 (d), Cross-section of gut of H. fossilis at the end of experiment (T₅) in August having 10.30 μm FW. H & E × 420 (e).

Effect on Epithelial Layer Thickness (Mucosa Width)

Epithelial layer thickness showed 3.96 to 4.41 μm at the start and 4.37 to 6.24 μm at the middle of the experiment. At the end of the experiment fold width exhibited 5.30 to 8.41 μm where highest ELT was observed in T₃ (Fig. 6c) (8.41 μm) and the lowest ELT was observed on T₅ (Fig. 6e) (5.30 μm); gut of T₂ (Fig. 6b) (7.76 μm) showed increased ELT compared with T₁ (Fig. 6a) (6.59 μm), T₄ (Fig. 6d) (6.91μm), and T₅ (Fig. 6e) (10.84 μm); T₄ (Fig. 6d) provided larger ELT (6.91 μm) compared to T₁ (Fig. 6a) (6.59 μm) and T₅ (Fig. 6e) (10.84 μm) (Table III).

Fig. 6. Cross-section of gut of H. fossilis at the end of experiment (T₁) in August having 6.23 μm ELT. H & E × 420 (a), Cross-section of gut of H. fossilis at the end of experiment (T₂) in August having 8.52 μm ELT. H & E × 430 (b), Cross-section of gut of H. fossilis at the end of experiment (T₃) in August having 9.37 μm ELT. H & E × 430 (c), Cross-section of gut of H. fossilis at the end of experiment (T₄) in August having 7.05 μm ELT. H & E × 430 (d), Cross-section of gut of H. fossilis at the end of experiment (T₅) in August having 4.73 μm ELT. H & E × 425 (e).

Probiotic considered as a non-harmful microorganism with beneficial effects to the host body and culture environment, it ensures the betterment of host’s response to diseases resistance and the water quality parameters (Verschuereet al., 2000). Aquatic environment contains a huge of microorganisms in direct contact with the aquatic animals, with the gills and with the food supplied and having free access to the digestive tract of the aquatic animal. Gut is a valuable part in fish which could clearly relates to alter the absorption of nutrition in fish body (Purushothamanet al., 2016). Gut villi length, width, enterocyte height are very important part which could determine the efficiency of absorption of nutrition. The present study was performed to understand the effect of commercial probiotics on the gut histological structure of H. fossilis. In this investigation the commercial probiotics Super PS (soil probiotic), ZYMETIN (gut probiotic) and pH FIXER (water probiotic) were analyzed for their potential effects on gut histoarchitecture of shing, Heteropneustes fossilis.

For sustainable and successful aquaculture optimum values of water quality is very important. Improvement of water quality has proven in aquaculture by using water probiotics. During experimental period temperature of water were varied from 27.86°C to 33.26°C. Kohinooret al. (2012) monitored temperature in ponds water varied from 27.90°C to 27.49°C which were more or less similar to the recent study. Dissolved oxygen (DO) concentration in water varied from 3.33 mg/L. to 7.00 mg/L observed from the present study. According to Rahman (1992) dissolved oxygen value of an aquaculture pond should be 5.0 ppm or more. DoF (1996) reported that the value of suitable dissolved oxygen (DO) for fish farming would be 5.50 to 6.50 mg/L. So, it could be mentioned that the values of DO concentration monitored in the present study were suitable for aquafarming.

pH refers the condition of acidity-alkalinity in the water body. It is called the productivity index of aquatic environment. Optimum range of pH for fish farming is 6.50 to 8.50, whereas slightly alkaline pH is most suitable for fish culture. By contrast, acidic pH of water reduces the growth and metabolic rate as well as other physiological activities of fishes (Swingle, 1967). In the present research, the range of pH were varied from 7.00 to 8.00. Ahmedet al. (1996) observed that pH ranged from 6.50 to 8.50 and Kohinooret al. (2012) recorded pH range from 7.08 to 7.15 which is almost similar with the finding of the present experiment.

Unionized form of ammonia (NH₃) is fatal to fish, while the ammonium ion (NH₄⁺) is not toxic. In the present study free ammonia (NH₃) concentrations were varied from 0.01 mg/L to 0.41 mg/L. Santhosh and Singh (2007), stated that maximum limit of ammonia concentration for aquatic organisms is 0.10 mg/L while Bhatnagar and Singh (2010) recommended that ammonia levels of less than 0.20 mg/L are suitable for aquaculture. The concentration of unionized ammonia should not exceed more than 0.025 ppm (Jhingran, 1988). So, it could be stated that the values of ammonia concentration observed in the present research were suitable for fish culture.

From the present study alkalinity varies between 96.66 mg/L to 173.33 mg/L which is suitable for H. fossilis culture. Chakraborty and Nur (2012) indicated that better H. fossilis growth within the alkalinity range between 140.50 mg/L to 188.40 mg/L. Boyd (1982) mentioned that the total alkalinity value should be more than 20 mg/L in cultured ponds to increase the production. The variation of total alkalinity in all the treatments were within the productive range for aquaculture ponds which also coincided with the result of Wahabet al. (1995).

Based on observations made on histological section of gut, pathological signs like necrosis (N), vacuum (V), clubbed villi (CB) and partly lost villi (VL) were present in T₅ (control), however, gut structures of T₃ were almost normal except few vaccums. From the research findings of Akter (2019) gut structure of Thai pangas (P. hypophthalmus) in combined application of gut and water probiotics (T₃) was comparatively improved and normal than the start of the experiment; fish of T₄ (gut probiotic) had normal structure of intestine but fish of T₅ (control) showed hemorrhage, necrosis, clubbing, partly missing villi, and almost lost gastrointestinal tract. On the other hand, T₁, T₂ and T₄ had necrosis, partly lost and clubbed villi at the start of the experiment. But towards termination of the experiment very few pathologies in gut were observed in T₁, T₂, T₃ and T₄ compared the start of the experiment. According to research findings of Vishwakarmaet al. (2021) highest damage was noticed in the gastrointestinal tract layer with ruptured and fused microvilli, hyperplasia of villi and necrotic enterocytes. Other degenerative modifications might be the cause of haemorrhage inside the layers. According to Inmaculadaet al. (2021) significant damage was noticed in the gut tissue, mainly due to chemical effect and to adherent bacterial populations of the gut.

In the present experiment, histological measurements of gut sections of H. fossilis showed that fish in treatments applied with probiotics had significantly increased fold length, fold width and epithelial layer thickness compared to the treatments with no probiotic. Based on measurement made, combined probiotics treated fish (T₃) showed the highest fold length (59.20 μm), Fold width (14.68 μm) and epithelial layer thickness (8.41 μm) followed by fish in T₁, T₂, T₄ and T₅ at the end of the experiment. Akter (2019) found that fish from T₃ and T₄ showed larger fold length, width and enterocyte height than that of T₁, T₂, T₅ (control). Thus, probiotic supplementations can improve the intestinal morphology of Thai pangas (P. hypophthalmus) which may increase absorption of nutrition in fish and thereby enance the digestion capacity that ultimately helps to improve the health status. From the present study, T₂ (FL 53.11 μm, FW 13.71 μm, ELT 7.76 μm) and T₄ (FL 48.99 μm, FW 12.81 μm, ELT 6.91 μm) had larger fold length, fold width and epithelial layer thickness than that of T₁ (FL 47.40 μm, FW 12.35 μm, ELT 6.59 μm) at the end of the experiment. The lowest value was found in T₅ (FL 36.18 μm, FW 7.85 μm, ELT 3.96 μm) at the start of the experiment. It could be due to the fact that more efficient nutrient absorption had occurred when fish treated with combind probiotic supplementation resulted to a physically healthy fish. Mayraet al. (2018) mentioned that after 109 days of feeding the probiotic supplimented feed to totoaba, histological measurements showed significantly larger FL (435.6 μm) in the proximal segment of the gastrointestinal tract compared to the treatments with no probiotic (382.3 μm) and fish fed the diet containing both probiotic and prebiotic, showed the largest FL (448.7 μm). Merrifieldet al. (2010) observed that dietary applications of P. acidilactici could significantly improve length of microvilli in O. mykiss compared to the control group. According to Akter (2019) noticed highest fold length (FL, 75.540 µm ± 0.35 µm), width (FW, 51.657 µm ± 0.12 µm), enterocyte height (EH, 23.584 µm ± 0.07 µm) in T₃ in comparison with other treatments and the lowest fold length (FL), fold width (FW), enterocyte height (EH) was observed in T₅ (control). Fish of T₄ also showed larger fold length (FL), fold width (FW), enterocyte height (EH) compare to T_1, T₂ and T₅. Fish of T₂ provided larger fold length (FL), enterocyte height (EH) and fold width (FW) compared to T₁ and T₅.

So, it could be stated that combind application of gut, soil and water probiotics provided the increased fold length, width and epithelial layer thickness than those of only supply of gut, soil or water probiotic treated fish. This showed the highest nutrient absorption occurred in the fish gut which can be reflected by better growth performance when compared to control. The fish from T₁, T₂, T₃, T₄ showed increased fold length, width and epithelial layer thickness than those of T₅ (control) at the end of the experiments which indicates that probiotic supplementation can improve the gut structure of H. fossilis which increase nutrient absorption capacity, thereby improve the digestion ability of fish. For this reason health status of H. fossilis improved in the probiotics applyed treatments.

From this investigation, it can be mentioned that probiotic supplementation can improve gut structure. Probiotic bacteria inhibit colonization and invation of pathogenic bacteria through hepatic portal system by improving epithelial layer thikness. Probiotic improves the intestinal morphology which are related to nutrients absorption of fish. The digestibility of fish can be increased through probiotic supplementation which results in improve health condition of cultured fish. Thus probiotics could be applied with fish feeds, soil and water to attain healthy fish as well as to a boost production in farming systems through improve immune and defence mechanism of fish.

Conclusion

Probiotic is one of the key tools to prevention of fish diseases and development of efficiency of nutrient absorption in aquafarming. Due to increasing demand for eco-friendly aquaculture system use of probiotics is becoming popular day by day. Based on observations made on histological section of gut less pathological sign was present in combined treated fish (T₃₎ at the end of the experiment. T_1, T₂ and T₄ had improved structure with less pathological sign than that of T₅ (control). T₂ (gut probiotic) had improved structure with less pathological sign compared to T₁, T₄ and T₅ (control). Most pathological sign was found in control group of fish (T₅₎. Under the experimental conditions, some differential responses were observed in the fold length, fold width and epithelial layer thickness of the gut morphology in the fish treated with probiotics. Highest fold length (FL), fold witdh (FW) and epithelial layer thickness (ELT) was observed in T₃ (combined probiotic supplementation of Super PS, ZYMETIN, and pH FIXER) followed by T₁ (Soil probiotic), T₂ (Gut probiotic)_, T₄ (Water probiotic)_, and the lowest was observed in T₅ (control). T₂ had larger FL, FW and ELT compared to T₁, T₄ and T₅ (control). Improved intestinal morphology results in more efficient nutrient absorption. It could be concluded that probiotic supplementation of single or combined way (ZYMETIN, pH FIXER, and Super PS) can improve the gut condition of fish which helps in regular digestion, more nutrient absorption and growth of fish.