The effects of different levels of sodium diformate on growth performance, Immunological respond, digestive enzyme activity and intestinal histomorphology in juvenile Siberian Sturgeon Acipenser baerii

Material And Methods

2.1. Fish

All experiments were conducted in accordance with standard ethical guidelines and approved by the Animal Ethics Committee of Shahid Chamran University of Ahvaz (Approval number: EE/98.11.2.51020/scu.ac.ir). A total of 360 juvenile Siberian Sturgeon (mean weight: 34 ± 4.5 g) with no clinical signs of disease, were obtained from one of the Ahvaz fish farms and transferred to the aquatic animal health research laboratory of the faculty of veterinary medicine, Shahid Chamran University of Ahvaz. Fish were acclimatized to laboratory conditions (with a 12 h light/ 12 h dark photoperiod) for two weeks in 2000-L tanks and fed with a commercial food twice a day during the acclimation period. The formulation and components of a commercial extruded basal diet are shown in Table 1.

After the acclimation period, fish were randomly distributed in 12 tanks with similar conditions of water volume, quantitative, and qualitative factors in the form of 4 treatments (30 fish in each replication). During the experimental period, the physicochemical factors of water were as follows, temperature (°C): 22.5 ± 0.7, dissolved oxygen (mg/L): 7 ± 0.3, salinity (ppt): 1.2, pH: 7.8 ± 0.3, total hardness (ppm): 250 ± 27.

Table 1

Ingredient of the experimental diets (g kg^− 1 dry diet).
Ingredients^a	G1	G2	G3	Control
NaDF^h	0. 5	1	1.5	0
Crude protein	42.3	42.3	42.3	42.3
Crude lipid	14.2	14.2	14.2	14.2
Ash	8.80	8.80	8.80	8.80
Fiber	3	3	3	3

^hFormi® NaDF, Addcon Nordic AS, Porsgrunn, Norway.

2.2. Diet preparation and rearing period

For preparing the experimental diets, the basal diet (FFS2 and GFS1, Alltech Coppens Co, Germany) were formulated and supplemented with the Sodium diformate (Addcon Nordic AS, Porsgrunn, Norway) according to the methods reported in previous studies (Hoseinifar et al., 2017; Ng and Koh, 2017). Briefly, acidifiers were weighed with the desired concentrations (0.05, 0.1 and 0.15 g/Kg) and then dissolved in physiological serum and added to food with gelatin. The resultant doughy mixture was pelleted using a meat grinder. Then the pellets were air dried at room temperature for 1 h, packaged and stored in a refrigerator at 4°C until used. During the study, fish were fed 3% of body weight day^− 1, three times daily at 08:00, 14:00 and 20:00 for 60 days. Feed pH was measured according to the method proposed by Baruah et al. (2005). Briefly, five grams of feed was poured into a china plant and mixed in 50 ml of deionized water for 1 minute using a magnetic stirrer. After feed homogenization, the pH of the solution was measured. Experimental diets were include control (without supplementing NaDF), NaDF 0.05 (0. 5 g/kg sodium diformate), NaDF 0.1 (1 g/kg sodium diformate), NaDF 0.15 (1.5 g/kg sodium diformate).

2.3. Sampling

Three fish from each tank were randomly collected and anaesthetized (0.5 ml l^− 1 of 2-phenoxyethanol) and taken for blood samples and digestive enzyme analyses. Blood samples (3 fish per replicate, a total of 15 fish per treatment) were withdrawn from the caudal vein by a 2.5 ml sterile syringe and used to obtain serum. Then, the blood samples were kept at ambient temperature to clot and afterward centrifuged (3000 g for 10 min at a temperature of 4°C) for serum separation (Molayemraftar et al., 2022). Afterward, all the samples were stored at -80°C until analysis.

2.4. Growth measurement

At the beginning (0 days), middle (30 days), and end of the trial (60 days), total fish biomass in every tank was anaesthetized (0.5 ml/L of 2-phenoxyethanol) and their weight (g) and length (cm) were measured individually. The data obtained from each group were used to calculate the feed utilization and growth parameters. Daily weight gain (DWG), weight gain (WG), specific growth ratio (SGR), condition factor (CF), feed conversion ratio (FCR), and protein efficiency ratio (PER) was calculated for each group as follow (Chelemal Dezfoulnejad and Molayemraftar, 2021; Mohammadian et al., 2021):

WG (%) = [(W_F – W_I) / W_I] × 100

DWG = (WF – WI) / days

SGR (% body weight / days) = [(Ln W_F − Ln W_I) / t] × 100

CF = (FW × 100) / standard length3 (cm)

FCR = feed intake (g) / weight gain (g)

PER = protein intake (g)∕weight gain (g)

Where W_I is initial body weights; W_F is final body weights (g); and t is the trial duration in days.

2.5. Digestive enzyme activity

The activity of chymotrypsin, trypsin, α-amylase, lipase, protease, and ALP was assayed in triplicates for each tank (using pooled samples from each tank) after 60 days feeding with different levels of Sodium diformate. Intestinal samples were completely homogenized with PBS buffer (1:5 w/v) on ice water bath. Then, the mixture was centrifuged at 12000 rpm for 20 min at 4°C and the supernatant was separated and used for the analysis of digestive enzyme activity (Regoli et al. 2012). Bovine serum albumin was used as a standard for the Bradford method to measure the total proteins in the crude enzyme extracts (Bradford, 1976). The levels of chymotrypsin and trypsin activity were kinetically measured using N-Benzoyl-L-tyrosine ethyl ester (BTEE) and Nα-Benzoyl-L-arginine ethyl ester hydrochloride (BAEE) as substrates, respectively (Hummel, 1959; Bergmeyer, 1974). The level of alpha-amylase activity was measured using soluble starch as the substrate hydrolyzable to maltose when it was reacted with 3,5-Dinitrosalicylic acid solution (Bernfeld, 1951). The activity of lipase enzyme in crude enzyme extract was determined by hydrolysis of p-nitrophenolemyristate using spectrophotometer. The release of fatty acids as a result of enzymatic hydrolysis of triglycerides to glycerol in the stabilized emulsion of olive oil, Fluka TM (Borlongan, 1990), was used to assess lipase activity. Protease enzyme activity was measured using 2% azocasein substrate solution in 50 mM Tris / Hcl buffer at pH = 7.5 and using a spectrophotometer (Garcia-carreno et al., 1993). The activity of intestinal ALP was quantified using p-nitrophenyl phosphate (PNPP) as substrate by a commercially colorimetric kit (Pars Azmoon Co., Tehran, Iran).

2.6. Serum Immunity parameters

Total protein, albumin and globulin levels as well as lysozyme activity and alternative complement (ACH₅₀) and bactericidal activities were detected in the sera samples.

Micrococcus lysodeikticus was used to determine serum lysozyme activity according to the turbidometric assay (Sharifuzzaman & Austin 2009). Briefly, sodium phosphate buffer (0.02 M, pH = 5.8, Sigma–Aldrich) was used. The phosphate buffer-free serum sample was applied as a negative control. The absorbance was recorded at 450 nm and expressed in the unit of lysozyme per ml serum when causing a reduction of 0.001 per min at 22°C.

Total protein (TP) content was determined according to Biuret method. The basis of this method is a formation of a Cu²⁺-protein complex in alkaline reagent and then measuring optical density at 540 nm by a spectrophotometer. Serum albumin (Alb) was also measured at 540 nm using bromcresol green complex (Pars Azmun, Iran). Finally, total globulin was calculated by subtracting of Alb from TP.

Agarose plates containing rabbit red blood cells were applied for detecting the activity of ACH₅₀ (give refenece here). Several holes (diameter = 3 mm) were punched on a plate and then filled with 15 µl of serum. After 24 h of incubation at room temperature, the zone of lysis was measured and expressed as an arbitrary unit per ml of serum (Barta 1993; mohammadian et al 2021).

Serum bactericidal activity was determined by incubating (90 min at 25°c) the mixture of the diluted sera and L. garvieae as previously described by Gisbert et al. (2015). The bactericidal activity of serum was expressed as a percentage of the ratio of CFU in the experimental group to those in the control group.

2.7. Intestinal histomorphology

Intestinal histomorphology

At the days 30 and 60 from the start of the experiment, the intestine of fish (n = 3) were dissected immediately out following euthanizing. The samples were then divided into three different sections, including proximal, middle and posterior parts and separately fixed in 10% neutral phosphate buffered formalin (pH = 7.2) and processed using the standard protocol for histopathological examination. After embedding the sample with paraffin wax, three separate cross sections with the thickness of ~ 5 µm were prepared using a microtome (Microtec CUT4050) and then have been stained with hematoxylin and eosin (H&E) for further histopathological investigations. The villi height, villi width and the thickness of epithelium, lamina propria and muscularis layers were determined under Nikon light microscope (Eclipse E600) by using of AxioVision 8.4 microscope software from Carl Zeiss (Oberkochen, Germany).

2.9. Statistical analysis

The statistical analysis of this study was conducted using SPSS-20 software (SPSS Inc, USA). The normality and variance homogeneity of the data was checked using the Kolmogorov–Smirnov and Levine test. The data were analyzed using the one-way analysis of variance (ANOVA), followed by Tukey’s test. The significance level was accepted at P < 0.05. All data are expressed as mean ± standard error (SE) for each experimental group.

Results And Discussion

Currently, there is a great interest in using organic acids and their salts as natural feed additives. Recently, many studies have been carried out to determine the effects of organic acids and their salts on the growth performance, use of nutrients and resistance to diseases in different populations and important farms for breeding various fish species, including gilthead seabream. Rainbow trout, salmon, tilapia, Asian sea bass and carp have been done (Mohammadian et al 2020; Reyshahri et al. 2019; kalantarian et al. 2019).

4.1. Growth performance

To our knowledge, no study had previously investigated the impacts NaDF on growth parameters in this species. The results of growth parameters in different groups are presented in Table 2. Most of the growth parameters in this research were affected by NaDF, so that the specific growth rate, weight gain percentage, protein efficiency ratio in the group 0.1 g/Kg NaDF increased significantly compared to other groups and the control at 30 days (P < 0.05). In our study, fish fed the 0.1 g/Kg NaDF showed the favorable FCR, SGR, PER value, While the worst value of (P < 0.05) FCR were recorded in the NaDF 0.15 group. At the end of the feeding experiment, there was no significant difference in the WI, SGR, FER, and PER of A. baerii (P > 0.05). In our study, feeding A. baerii with diets containing 0.05 g/kg NaDF for 60 days had the worst value compared to other groups. Our results are in accordance with those of Nermeen et al. (2015) who observed the highest growth performance in Nile tilapia (Oreochromis niloticus) fed with 0.2 and 0.3% KDF. Sudagar et al. (2010) also announced in a study that the use of citric acid (organic acid) as an absorbent in the diet of juvenile Huso huso at the rate of 5, 10 and 15 gr/kg of diet increases the final weight and improves Daily growth rate, specific growth rate, condition index and reduction of food conversion factor are significant. Various studies show that the use of organic acids due to improving the metabolism and digestibility of proteins and minerals in the intestine, improves growth and nutrition beside enhanced appetite and changed the composition, diversity, and/or activity of the population of beneficial bacteria in the gut microbiota while inhibiting pathogenic bacteria in aquatic species (Sarker, 2005; Sarker et al., 2012; Hoseinifar et al., 2016; Ng and Koh, 2017; Wassef et al., 2019; Pelusio et al., 2020).

It is worth mentioning that this increasing trend of growth in the present study did not continue until the end of the experimental period and showed a decreasing trend. In the meantime, the noteworthy point is that in the second 30 days of the experiment, the growth indices measured compared to the first 30 days of the experiment had a relatively significant decrease and followed a slower trend. The improvement of growth performance in the first 30 days can be due to reasons such as the role of organic acids in reducing the microbial load in the diet (Owen et al., 2006), the use of organic acids in the diet and the start of the digestion process in the diet (Zhou et al. ., 2009), decreased stomach pH and better performance of pepsin (Hossain et al., 2007), decreased intestinal pH and decreased harmful bacteria and intestinal microbial flora balance (Pandey and Satoh, 2008). With increasing age in all fishes, the growth rate decreases and the Siberian fish is no exception. It is possible that long-term feeding with the amount of 0.1% NaDF in the diet will reduce the improving role of acidifiers and reduce the growth rate of fish due to internal interactions with the normal physiological functions of the fish's digestive tract microbiota.

Table 2

Growth performance of *Acipenser baerii* fed feed supplemented with different levels of Sodium diformate for 60 days. IW: initial weight, FW: final weight, SGR: specific growth rate, FCR: feed conservation ratio, DWG: daily weight gain, FER: feed efficiency ratio, PER: protein efficiency ratio
Parameter	Treatment	Day30	Day60
IW	0/5% sodium diformate	34/3 ± 0/1^A,b	149/86 ± 0/51^B,a
	1%sodium diformate	36/6 ± 0/1 ^A,b	162/95 ± 0/18 ^A,a
	1/5%sodium diformate	34/7 ± 0/1 ^A,b	139/24 ± 1/8 ^B,a
	Control	33/1 ± 0/13 ^A,b	137/17 ± 0/5 ^B,a
FW	0/5% sodium diformate	149/86 ± 0/51 ^B,b	314/07 ± 0/2 ^B,a
	1%sodium diformate	162/95 ± 018 ^A,b	352/5 ± 0/58 ^A,a
	1/5%sodium diformate	139/24 ± 1/8 ^B,b	321,41 ± 0/83 ^B,a
	Control	137/17 ± 0/5 ^B,b	311/58 ± 0/63 ^B,a
SGR	0/5% sodium diformate	4/92 ± 0/01 ^A,a	2/47 ± 0/15 ^A,b
	1%sodium diformate	4/98 ± 0/003 ^A,a	2/57 ± 0/017 ^A,b
	1/5%sodium diformate	4/63 ± 0/03 ^A,a	2/79 ± 0/012 ^A,b
	Control	4/75 ± 0/013 ^A,a	2/73 ± 0/042 ^A,b
FCR	0/5% sodium diformate	1/45 ± 0/01 ^A,b	1/9 ± 0/02 ^B,a
	1%sodium diformate	1/3 ± 0/015 ^B,b	1/64 ± 0/02 ^A,a
	1/5%sodium diformate	1/57 ± 0/02 ^A,a	1/7 ± 0/02 ^A,a
	Control	1/56 ± 0/02 ^A,a	1/78 ± 0/02 ^A,a
DWG	0/5% sodium diformate	3/85 ± 0/01 ^B,b	5/47 ± 0/001 ^B,a
	1%sodium diformate	4/21 ± 0/09 ^A,b	6/32 ± 0/004 ^A,a
	1/5%sodium diformate	3/48 ± 0/002 ^B,a	6/07 ± 0/002 ^A,b
	Control	3/47 ± 0/07 ^B,a	5/81 ± 0/007 ^{AB, b}
FER	0/5% sodium diformate	69/07 ± 0/15 ^A,a	52/51 ± 0/12 ^A,b
	1%sodium diformate	77/14 ± 0/04 ^A,a	60/93 ± 1/46 ^A,b
	1/5%sodium diformate	63/64 ± 0/12 ^A,a	58/71 ± 0/9 ^A,a
	Control	63/96 ± 0/22 ^A,a	56/10 ± 1/12 ^A,a
PER	0/5% sodium diformate	1/72 ± 0/05 ^A,a	1/4 ± 0/12 ^A,b
	1%sodium diformate	1/81 ± 0/04 ^A,a	1/59 ± 0/46 ^A,b
	1/5%sodium diformate	1/58 ± 0/02 ^B,a	1/57 ± 0/359 ^A,a
	Control	1/57 ± 0/12 ^B,a	1/5 ± 0/23 ^A,a

*Significant differences between values (means ± S.D n = 15), when compared with control groups, were characterized by alphabet symbol (p < 0.05). Different capital letters indicate significant differences (p < 0.05) between the experimental groups at each sampling time. Different lower case letters show significant differences (p < 0.05) in each of experimental groups among various intervals.

4.2. Digestive enzymes

Table 2 represents the effects of supplemented NaDF foods on the activity of the digestive enzyme of A. baerii. On the 30th day of the experiment, there was a significant increase in the trypsin, protease, Chymotrypsin, and ALP levels of A. baerii in all supplemented NaDF groups compare to the control group (P < 0.05). There was a significant increase (p < .001) in the Lipase level of the NaDF 0.15 groups compared to the control group (P < 0.05). On the 60th day of the experiment, the activity of all digestive enzymes showed a significant decrease in all treatments compared to the 30th day (P < 0.05). At the end of the feeding experiment, there was a significant increase in the trypsin, protease, Chymotrypsin, and ALP levels of A. baerii in NaDF groups compared to the control group (P < 0.05). Moreover, a remarkable increase of α-amylase and Lipase level were observed in the NaDF 0.05, and 0.15 groups compared to the control group (P < .05).

Therefore, the enhancement of digestive enzymes activity was apparently one of the main reasons for growth-stimulatory effects of the NaDF used. According to some reports, elevated digestive enzyme activity facilitates macromolecule digestion and, therefore, nutrient absorption in the gut lumen (Jang et al., 2019). Similar results related to digestive enzymes were observed in hybrid tilapia fed with potassium diformate (KDF) (Zhou et al., 2009), in Oncorhynchus mykiss fed with Dietary PrimaLac® and potassium diformate (KDF) (Naderi Farsani et al., 2019), in Lateolabrax japonicus fed with citric, lactic, and phosphoric acids (Huang et al., 2021) and in Litopenaeus vannamei fed with microencapsulated organic acids blend (Romano et al., 2015). As a proteolysis enzyme, trypsin plays a role in feed consumption and fish growth, while chymotrypsin comes into play when food is limited or unavailable (Rungruangsak-Torrissen et al., 2006). Also, a higher intestinal ALP activity indicates a greater absorption of nutrients in the enterocytes of fish, which is important for carbohydrate as well as lipid absorption (Calhau et a., 2000; Gawlicka et al., 2000).

Table 3

The activities of digestive enzymes in *Acipenser baerii* fed feed supplemented with different levels of Sodium diformate for 60 days.
parameter	Treatment	Day 0	Day 30	Day 60
Chymotrypsin (U/mg)	0/5% sodium diformate	10/12 ± 1/19^A,b	21/3 ± 0/003^B,a	13/2 ± 1/01^B,b
	1%sodium diformate	11/3 ± 1/5^A,c	57/26 ± 4/082^A,a	24/21 ± 2/54^A,b
	1/5%sodium diformate	13/4 ± 2/2^A,b	25/41 ± 1/41^B,a	15/71 ± 1/5^B,b
	Control	11/42 ± 1/11^A,a	13/2 ± 1/6^B,a	13/8 ± 1/28^B,a
Trypsin (U/mg)	0/5% sodium diformate	0/55 ± 0/19^A,b	1/24 ± 0/13^A,a	1/53 ± 0/031^A,a
	1%sodium diformate	0/57 ± 0/51^A,b	1/85 ± 0/12^A,a	1/41 ± 0/44^{A, a}
	1/5%sodium diformate	0/5 ± 0/81^A,b	1/16 ± 0/41^A,a	1/42 ± 0/21^A,a
	Control	0/58 ± 0/11^{A, a}	00/66 ± 0/17 ^B,a	0/69 ± 0/18 ^B,a
Alpha amylase (U/mg)	0/5% sodium diformate	16/67 ± 2/6 ^A,a	17/36 ± 2/13 ^AB,a	17/53 ± 2/31 ^B,a
	1%sodium diformate	16/67 ± 3/05 ^A,b	25/14 ± 2/2 ^A,a	17/73 ± 1/14 ^B,b
	1/5%sodium diformate	14/1 ± 1/9 ^A,b	22/2 ± 1/11 ^{A, ab}	33/21 ± 3/15 ^A,a
	Control	14/66 ± 2/97 ^{A, a}	15/14 ± 1/23 ^B,a	14/24 ± 2/18 ^B,a
Lipase (U/mg)	0/5% sodium diformate	112/77 ± 18/19 ^A,b	166/6 ± 26/3 ^A,ab	182/38 ± 28/31 ^{A, a}
	1%sodium diformate	110/6 ± 19/05 ^A,b	168/07 ± 46/2 ^A,a	158/7 ± 14/14 ^AB,a
	1/5%sodium diformate	111/56 ± 19/13 ^A,b	180/08 ± 48/1 ^A,a	172/8 ± 29/15 ^A,a
	Control	114/6 ± 12/1 ^A,a	119/54 ± 19/3 ^B,a	112/84 ± 14/18 ^B,a
Protease (U/mg)	0/5% sodium diformate	48/93 ± 8/19 ^A,b	125/6 ± 27/3 ^B,a	59/53 ± 6/31 ^AB,b
	1%sodium diformate	45/08 ± 7/05 ^A,b	327/4 ± 34/32 ^A,a	79/7 ± 9/4 ^A,b
	1/5%sodium diformate	49/14 ± 10/13 ^A,b	293/7 ± 31/41 ^A,a	41/2 ± 6/15 ^B,b
	Control	50/75 ± 6/1 A,a	53/24 ± 7/03 ^C,a	48/84 ± 9/18 ^B,a
Alkaline phosphatase (U/mg)	0/5% sodium diformate	12/6 ± 1/31^A,b	36/71 ± 6/3^B,a	22/53 ± 1/31^AB,b
	1%sodium diformate	17/21 ± 1/05^A,C	52/4 ± 4/32^A,a	28/7 ± 1/14^A,b
	1/5%sodium diformate	16/31 ± 1/13^A,b	29/21 ± 9/41^B,a	27/2 ± 4/15^A,a
	Control	17/47 ± 1/1^A,a	18/24 ± 2.23^C,a	16/84 ± 1/38^B,a

4.3. Immunological parameters

Our results showed that NaDF is able to change the immunity response of this species even following 30 days of experiments. 0.1 and 0.15% supplemented NaDF used in the present study, were provoked serum lysozyme activity compared to fish with other treatments. In previous studies application NaDF. for 60 days could boost lysozyme activity in L. calcarifer and Salmo trutta caspius (Reyshahri et al., 2019; Mohammadain et al. 2020). However, in parallel to our findings, also demonstrated increasing serum/plasma lysozyme in various fish species fed diets supplemented with acidifiers such as butyrate (0.54%, Alamifar et al., 2019) in L. calcarifer, butyric acid glycerides (1.0% Zarei et al., 2021), sodium propionate or acetate (0.5-1.0%, Sotoudeh et al., 2021) in yellowfin seabream, and sodium diformate (0.05%, Wassef et al., 2017) and butyrate (0.02%, Abdel-Mohsen et al., 2018) in European seabass.

On the 30th and 60th day of the experiment, there was NBT and bactericidal activity significant difference in the blood levels of A. baeri.in all supplemented NaDF groups compared to the control group (P > 0.05). Also, there was a significant increase in the serum Glb and ACH₅₀ levels of A. baeri.in all supplemented NaDF groups compared to the control group at the 30th (P < 0.05). On the 60th day, the amount of complement activity in NaDF treatments 0.1 and 0.15% was more than other experimental treatments. Moreover, there was a significant increase in the Serum lysozyme activities levels of A. baeri in 0.1 and 0.15% supplemented NaDF groups except NaDF 0.05% group compared to the control group (P < 0.05). Furthermore, the statistical analysis of results revealed that 0.1 and 0.15 NaDF significantly increased the Serum lysozyme activities compared to the control group at 60th day (P < .05). It has been suggested that NaDF can modulate the host’s immunocompetence by affecting gut microbiome and immune cells related to the gut-associated lymphoid tissue that have key roles in the host’s health condition (Zhou et al., 2009). Recent findings also showed that SCFA can trigger the immune responses by binding to G protein‐coupled receptor, GPR43, that profoundly affect innate immunity and inflammatory cells (Maslowski and Mackay, 2011).

4.6. Intestinal histomorphology

The effects of NaDF on the Intestinal histomorphology of A. baeri were presented in Table 6. Higher villi height in proximal area has been observed in T3 as compared with control at day 30 while all acidifier treatments led to significant increase in this parameter at day 60. The villi height was elevated in the distal parts of intestine following acidifier treatment (T1,T2 and T3) at day 30 while in other areas the similar pattern has not been observed (T2 and T3).

The T2 and T3 led to significant changes in the villi width in different parts of intestines when compared with the control. While all acidifier treatments led to significant increase in this parameter at day 60. The T1 and T3 treatment did not show any significant changes in this parameter in middle part of intestine. The most changes in the case of muscle thickness in acidifier treatment have been observed in the proximal and middle parts of intestine following 30 and 60 days treatment with T1 and T2. The number of goblet cells in proximal area has been observed in T2 and T3 as compared with control at day 30 while all acidifier treatments led to significant increase in this parameter at day 60. The numbers of goblet cells were elevated in the distal parts of intestine following acidifier treatment (T1 and T3) at day 30 and 60.

Acidifiers could improve the intestinal wall thickness, villus height and villus density in animals. In our study, dietary administration of NaDF histologically influenced the intestinal tract including villus height, villus mid-width and villus area (Hossain et al., 2007, Ng et al., 2009, Klalantarian et al, 2020; Mohamadian et al 2020; Reyshahri et al 2020). Probably, it can be concluded that NaDF can show better performance to improve health status of fish. Increasing the diameter and height of the villi, indicates an increase in intestinal absorption (which depends on the villi size and number) and indirectly an increase in the efficiency of dietary intake. Probably the reason for the increase in villus height in treatments T2 and T3 caused by the production of organic acids such as butyric acid is to increase the villus height. In parallel with our results, in silver catfish (Rhamdia quelen) fed an organic acid salts diet showed a significant increase in width, height, number of villi, and numbers of goblet cells compared to fish fed the control diet (Pereira et al. 2019).

Table 4

Histomorphometric comparison of the anterior intestine between the investigated treatments in different stages of sampling (results are reported based on Means ± SD).
Parameter	Treatment	Day 0	Day 30	Day 60
pile height (micrometer)	0/5%sodium diformate	5/12 ± 322/7	2^* /12 ± 329/3	^&*#4/12 ± 337/2
	1%sodium diformate		8^* /13 ± 332/4	^&8^*#/13 ± 339/4
	1/5%sodium diformate		^*&2/14 ± 337/2	^&2^*#/11 ± 342/6
	Control		2 ^*/12 ± 328/3	5 ^*#/13 ± 330/5
Bristle width (micrometer)	0/5%sodium diformate	2/11 ± 9/141	5 /11 ± 142/1	5 ^*#&/11 ± 145/1
	1%sodium diformate		8 ^*&/10 ± 144/3	8 ^*/12 ± 145/8
	1/5%sodium diformate		8^*#&/12 ± 146/2	8 ^*#&/12 ± 148/2
	Control		5 /12 ± 142/6	5 ^*/12 ± 143/6
The thickness of the muscle layer (micron)	0/5%sodium diformate	9/3 ± 105/6	8^* /9 ± 108/7	8 ^*#/10 ± 110/7
	1%sodium diformate		6 ^*&/11 ± 111/3	5 ^*&/12 ± 112/2
	1/5%sodium diformate		8 ^*&/11 ± 113/1	3 ^*&/12 ± 115/5
	Control		2 ^*/10 ± 107/4	2^*#/9 ± 109/4
Goblet cell density (square micrometer)	0/5%sodium diformate	1/2 ± 46/4	6/2 ± 48/2	5^*#&/3 ± 53/2
	1%sodium diformate		3^*&/2 ± 50/3	4^*#&/3 ± 55/3
	1/5%sodium diformate		8^*&/3 ± 53/6	2^*#&/4 ± 56/6
	Control		3^*/1 ± 48/6	3^*#/2 ± 51/6

*Significant differences between values (means ± S.D n = 15), when compared with control groups, were characterized by alphabet symbol (p < 0.05).

Table 5

Comparison of mid gut histomorphometry between the investigated treatments in different stages of sampling (results are reported based on Means ± SD).
Parameter	Treatment	Day 0	Day 30	Day 60
pile height (micrometer)	0/5%sodium diformate	271/1 ± 13/8	5^*& /10 ± 278/6	2^*# /11 ± 285/3
	1%sodium diformate		5^*&/12 ± 280/8	5 ^*#&/13 ± 287/8
	1/5%sodium diformate		5 ^*&/11 ± 285/2	4 ^*#&/12 ± 292/2
	Control		2 ^*/11 ± 276/4	2^*# /12 ± 284/4
Bristle width (micrometer)	0/5%sodium diformate	139/4 ± 12/6	7 /11 ± 140/4	5^*/12 ± 142/6
	1%sodium diformate		8^*& /12 ± 143/6	8^*#& /12 ± 145/6
	1/5%sodium diformate		2^*&/13 ± 145/3	2^*#&/13 ± 148/3
	Control		5 /12 ± 140/2	5^*/10 ± 142/3
The thickness of the muscle layer (micron)	0/5%sodium diformate	2/3 ± 98/1	2^*&/10 ± 105/4	2 ^*/10 ± 106/2
	1%sodium diformate		7 ^*&/12 ± 105/6	7 ^*#&/12 ± 108/6
	1/5%sodium diformate		5 ^*&/9 ± 108/2	5 ^*#&/10 ± 111/3
	Control		8 ^*/9 ± 102/6	8^*# /9 ± 105/6
Goblet cell density (square micrometer)	0/5%sodium diformate	1/8 ± 35/3	8^*/1 ± 42/6	2^*#&/3 ± 46/5
	1%sodium diformate		3^*&/2 ± 45/8	5 ^*#&/2 ± 47/8
	1/5%sodium diformate		5^*&/3 ± 48/2	5 ^*#&/3 ± 52/2
	Control		6^*/1 ± 40/2	6 ^*/2 ± 42/5