The effect of Pediococcus acidilactici probiotics supplementation in the feed of Nile Tilapia (Oreochromis niloticus) on water quality, growth performance, Hematological parameters, antioxidants, immunological responses, enzymatic Activity, and histology.

doi:10.21203/rs.3.rs-1739135/v1

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The effect of Pediococcus acidilactici probiotics supplementation in the feed of Nile Tilapia (Oreochromis niloticus) on water quality, growth performance, Hematological parameters, antioxidants, immunological responses, enzymatic Activity, and histology.

https://doi.org/10.21203/rs.3.rs-1739135/v1

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The effect of adding lactic acid bacteria (Pediococcus acidilactici) to the diet on water quality, feed utilization, growth, body composition, Hematological parameters, intestinal and liver histology of Nile tilapia, haemato-biochemical parameters, antioxidants, immunological responses, and Enzymatic Activity was investigated. Fish was distributed into Four groups; the control group (T0), T1 group (2.0 g), T2 group (2.5 g), and T3 group (3.0 g) of Pediococcus acidilactici per kg of diet. 25% of Water was changed daily and feed with 31% protein was added three times daily for 56 days. In probiotics-treated groups, growth performance, feed utilization, and fish biomass increased significantly compared to control. The biochemical and immunity parameters (Amylase, Lipase, and Protease activity) and villi height were significantly improved in probiotics groups. In conclusion, adding probiotics to fish diet improved all evaluated parameters

Pediococcus acidilactici

O.niloticus

Growth performance

body composition

Water quality

blood biochemical parameters

Intestinal and liver histology

During the last few decades, aquaculture has made significant contributions to the production of animal protein in all countries (Jahangiri and Esteban, 2018). However, the intensive system of aquaculture increased environmental pollution due to organic and inorganic discharges and increased incidence of disease outbreaks (Gatesoupe, 2004; Martínez Cruz et al., 2012; Hai, 2015). The emergence of diseases in aquaculture are further aided by several factors including water contamination, excessive stock densties, overfeeding, and damaging fishing technique (Abarike، Jian et al. 2018، Kuebutornye، Abarike et al. 2019، Hasan and Banerjee 2020). This could lead to a huge financial losses to the farmers and seek the use of antibiotics to combat diseases. Nevertheless, the use of antibiotics has many disadvantages including inassured longevity, development of antibiotic resistance, harming the living biota and the exposure of consumers to antibiotics (Cabello, Godfrey, et al. 2016, Watts, Schreier, et al. 2017, Elsabagh, Mohamed et al. 2018, Won, Hamidoghli, et al. 2020; Kavitha, Raja et al. 2018, Huerta-Rábago, Martínez-Porchas et al. 2019, Mamun, Nasren et al. 2019, Wang, Guo et al. 2020).

Several new production methods are being investigated to overcome the drawback of the intensive aquaculture (Badiola et al., 2012; Aly et al., 2016; Abdel-Rahim, 2017; Kibria and Haque, 2018; Abdel-Rahim et al., 2019; Fayed et al., 2019; Huerta-Rábago et al., 2019; Elhetawy et al., 2020;) which included the application of probiotics (Aly et al., 2016). The addition of single or combined type of beneficial bacteria in probiotics to aquaculture is considered a hopeful technique that is capable for substitution of traditional antibiotics (Daniel and Nageswari, 2017; Huerta-Rábago et al., 2019). Probiotics is also ecofriendly which have no harm to the natural beneficial bacteria (Ahmad, Rani, et al. 2017, Zibiene and Zibas 2019, Eissa et al., 2022).

Several definitions of probiotics were described which mainly emphasized on the cecept that they are nonharmful and beneficial microorganisms (FAO, 2001; Jahangiri and Esteban 2018). Probiotics can improve the microbial balance of the aquatic environment with a subsequent enhancement of the general health of the farmed species and aquaculture productivity (Dawood & Koshio, 2016; Eissa et al., 2021; Eissa et al., 2022).

Tilapia (Oreochromis niloticus) remains one of the commonly cultured species (Rahman, 2012). Nile tilapia is omnivorous fish, easily adapts to artificial feed and intensive production systems, and is well accepted by consumers with a high market value ( Brendan J et al.,2016).

Consequently, this study investigates the effect of adding probiotic on growth performance, feed utilization, whole-body proximate composition, water quality, blood biochemical, immunity, the histology of intestine and liver in Nile tilapia fish.

2.1. Experimental site

This study was carried out at a private fish farm, Kafr El-Sheikh Governorate, Egypt, under the supervision of the Faculty of Fish and Fisheries Technology, Aswan University, Egypt.

2.2. Experimental fish and cultural conditions

Apparent healthy 240 O. niloticus fingerlings weighing 3.9–4.2 g/fish were used in this experiment. Fish was raised in Twelve (1 × 1 × 1 m) open concrete ponds of 1000 liters capcity filled with untreated Nile agricultural irrigation water under natural environmental conditions. After acclimatization of Fingerlings for 14 days, 20 fingerlings were placed in each cubic meter. The study ended after 56 days. Tanks were equipped with an aerator. The water was changed daily at a rate of 25% per day.

2.4. Experimental design

Three levels of Bactocell PA10 probiotics was used. The fish was divided into 4 experimental groups which are:

T0: with the addition of 0.0 gm Pediococcus acidilactici per kg diet.

T1: with the addition of 2.0 gm Pediococcus acidilactici per kg diet.

T2: with the addition of 2.5 gm Pediococcus acidilactici per kg diet.

T3: with the addition of 3.0 gm Pediococcus acidilactici per kg diet.

Commercial probiotics (Bactocell PA10) were used which contained encapsulated lactic acid bacteria (Pediococcus acidilactici)

2.5. Feeding protocol

The diet used for feeding fingerlings contained 31% protein. Feeding of fish was carried out three times daily and at a rate which was adjusted according to the fish biomass (6% in the first 4 weeks and 5% in the second 4 weeks of fish biomass). (Table 1).

Table 1

Composition and chemical analysis of experimental diets.
Components	Diets
Components	T0	T1	T2	T3
Fish meal (72% CP)	110	110	110	110
Soybean meal (45% CP)	360	360	360	360
Wheat bran	200	200	200	200
Yellow corn	60	60	60	60
Rice bran	200	198	197.5	197
Pediococcus acidilactici	0.0	2.0	2.5	3.0
Fish oil	15	15	15	15
Soybean oil	15	15	15	15
Dicalcium phosphate	10	10	10	10
Vitamins mixture¹	10	10	10	10
Minerals mixture²	10	10	10	10
Carboxymethyl cellulose	10	10	10	10
Total	1000	1000	1000	1000
Chemical analysis
Dry matter	91.5	91.3	91.8	91.2
Crude protein	31.3	31.3	31.3	31.3
Ether extract	8.1	8.1	8.1	8.1
Total ash	7.33	7.37	7.30	7.31
Crude fiber	6.60	6.68	6.65	6.62
Nitrogen-free extract	46.7	46.4	46.7	46.5
Gross energy (kcal/g)³	4.71	4.72	4.74	4.70
¹ Vitamin premix (per kg of premix): thiamine, 2.5 g; riboflavin, 2.5 g; pyridoxine, 2.0 g; inositol, 100.0 g; biotin, 0.3 g; pantothenic acid, 100.0 g; folic acid, 0.75 g; para-aminobenzoic acid, 2.5 g; choline, 200.0 g; nicotinic acid, 10.0 g; cyanocobalamine, 0.005 g; a-tocopherol acetate, 20.1 g; menadione, 2.0 g; retinol palmitate, 100,000 IU; cholecalciferol, 500,000 IU.
² Mineral premix (g/kg of premix): CaHPO₄.2H₂O, 727.2; MgCO₄.7H₂O, 127.5; KCl 50.0; NaCl, 60.0; FeC₆H₅O₇.3H₂O, 25.0; ZnCO₃, 5.5; MnCl₂.4H₂O, 2.5; Cu(OAc)₂.H₂O, 0.785; CoCl₃.6H₂O, 0.477; CaIO₃.6H₂O, 0.295; CrCl₃.6H₂O, 0.128; AlCl₃.6H₂O, 0.54; Na2SeO₃, 0.03.
³ Gross energy (GE) was calculated from NRC (2011) as 5.65, 9.45, and 4.11 kcal/g for protein, lipid, and carbohydrates, respectively.

2.6. Measured parameters

2.6.1. Water quality parameters

At 3 pm everday, water temperature, dissolved oxygen, pH, and salinity were measured by SensoDirect150 MultiMeter (Lovibond, Tintometer Limited, Amesbury, UK) whereas total ammonia nitrogen (TAN) was assesed by HANNA HI 96715-11 Ammonia Medium-Range photometer (HANNA, Nusfalau, Romania). The TAN, temperature, and pH values was used to calculate the unionized ammonia (NH3). Nitrate was assessed using HANNA HI 708 (HANNA, Nusfalau, Romania).

2.6.2. Growth performance parameters and survival percentage

The growth performance was evaluated every 15 days. Calculation of the Bodyweight gain (WG), average daily gain (ADG), specific growth rate (SGR), survival %, and condition factor was performed based on the following equations:

Bodyweight gain (WG) = Final weight (g)-Initial weight (g).

Body weight gain % (WG%) = [(Final weight - Initial weight) - Initial weight]×100.

Average daily gain (g/fish/day): ADG = Wt - W0/n

Where: W0: the initial mean weight of fish in grams.

Wt: the final mean weight of fish in grams.

Where: n: duration period.

Feed Intake (g/fish): The amount of feed given or supplied during the experimental period /fish (g).

Feed conversion ratio (FCR) = Feed intake (g) / Weight gain (g).

Specific growth rate (SGR) = 100 × (lnW2-lnW1) / T

Where ln is the natural logarithm, W1 is the initial weight, W2 is the final weight (g), and T is the number of days in the feeding period.

Survival rate (SR) = (Z/X) × 100, Where, Z is the surviving fish number and X is the initial fish number.

2.6.3. Fish and feed analytical methods

fish and feed samples were collected at the start and the finale of the experiment to determine the protein, lipid, moisture, and ash contents of diets and fish bodies. On the day of stocking, a single fish sample was used for body chemical analysis. Crude protein, and fat contents in addition to whole fish body moisture were evaluated according to AOAC ( 2000).

2.6.4. Blood sampling

Blood samples were collected from the caudal vertebral vein (Feldman et al. 2000). The erythrocytes and leukocytes counts assessed by hemocytometer and Natt- Herrik solution and Hemoglobin concentration using the cyanomet hemoglobin method Drabkin's solution were determined (Stoskopf, 1993). PCV% and differential leukocyte count was performed according to Dacie and Lewis (1991) and Stoskopf (1993). The absolute differential leukocytic count (DLC) was calculated according to Thrall (2004) with the following formula:

Absolute DLC = No. of each white cell x no. of total leukocytic count /100

2.6.5. Blood serum biochemical analysis

Total proteins and albumin in serum were measured at 540 nm and 550 nm wave length respectively (Doymas et al., 1981; Dumas and Biggs, 1972) and were used to calculate globulins content. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured colorimetrically at the wavelength 540 nm (Reitman and Frankel, 1957). Cholesterol (CHL) was determined with CHOD-PAP (commercial clinical kit) methods (Fynn-Aikins et al., 1992). Glucose level (mg/100 ml) was determined using glucose enzymatic PAP (Trinder, 1969) kits (Bio-Merieux, France). The amylase activity was measured in triplicates (Robyt and Whelan, 1968). Phagocytosis assay was carried out using fluorescent latex beads and flow cytometry-based on the slightly modified technique in Haugland et al (2012).

2.6.6. Histopathological examination

Liver and intestine tissue samples (3 fish/group) were collected in which 40% ethyl alcohol was used for anesthesia (Dawood et al., 2020). 10% neutral buffer formalin was used for fixation of tissue samples. Fixed samples were processed by ascending concentration of alcohol and xylene. Sectioning of tissue at a thickness of 5 µm was performed using a rotary microtome (Leica 2025, Germany), and stained with Hematoxylin and Eosin (H&E) stain. A light microscope equipped with digital camera was used for examiniation (Bancroft and Gamble, 2007).

2.7. Statistical analysis

One-way analysis of variance (ANOVA), followed by pot hoc test (Duncan test) (SPSS software program, Version 26) were used to detect the significance between different groups. Data were presented as a mean value ± standard error (Steel and Torrie 1980).

3.1. Water quality

No variation in temperatures were detected between different goups (Table 2). (26.59 ± 0.01, 26.53 ± 0.01,26.58 ± 0.01, and 26.63 ± 0.01°C, respectively). In probiotic treatments except T3, the pH value of water was significantly decreased (p < 0.01) (7.96 ± 0.00, 7.91 ± 0.00, respectively) less than in control (8.01 ± 0.00). Salinity also varied in which it increased in T1, and T2 (0.51 ± 0.00, 0.51 ± 0.00, respectively) compared with T0 (0.50 ± 0.01 ppt) and a decrease in T3 (0.49 ± 0.00 ppt). The dissolved oxygen in water was similar in control and probiotics groups. The ammonia of both control and probiotics diets was similar or more. (Table 2)

Table 2

Effect of probiotic use on some water parameters of rearing water of Nile Tilapia (*Oreochromis niloticus*).
Parameters	Treatment
Parameters	T0	T1	T2	T3
T (C^o)	26.59 ± 0.01^b	26.53 ± 0.01^c	26.58 ± 0.01^b	26.63 ± 0.01^a
Salinity(ppt)	0.50 ± 0.01^a	0.51 ± 0.00^a	0.51 ± 0.00^a	0.49 ± 0.00^a
pH	8.01 ± 0.00^a	7.96 ± 0.00^b	7.91 ± 0.00^c	8.01 ± 0.00^a
DO (mg/l)	6.01 ± 0.01^a	6.01 ± 0.01^a	5.98 ± 0.00^b	6.01 ± 0.00^a
NH4	0.32 ± 0.00^b	0.33 ± 0.00^a	0.33 ± 0.00^a	0.32 ± 0.00^c
NO2	0.02 ± 0.00	0.02 ± 0.00	0.02 ± 0.00	0.02 ± 0.00
NO3	0.22 ± 0.00^b	0.21 ± 0.00^c	0.22 ± 0.00^a	0.22 ± 0.00^b
The values with a different superscript in the same row are significantly different (p ≤ 0.05).

3.2. Growth performance and feed utilization

In probiotic groups, the growth performance and fish biomass were significantly (p ≤ 0.05) improved compared with the control group (Table 3).

Table 3

Growth performance and feed utilization of Nile tilapia (*O. niloticus*) fed diets supplemented with *Pediococcus acidilactici* for 56 days.
Parameters	Treatment
Parameters	T0	T1	T2	T3
Initial weight (g)	4.00 ± 0.06^a	4.07 ± 0.03^a	4.03 ± 0.09^a	4.00 ± 0.06^a
Final weight (g)	18.80 ± 0.78^d	24.50 ± 0.38^c	26.87 ± 0.29^b	28.63 ± 0.46^a
Weight gain	14.80 ± 0.72^d	20.43 ± 0.38^c	22.83 ± 0.20^b	24.63 ± 0.49^a
Weight gain %	369.66 ± 12.85^d	502.52 ± 9.89^c	566.43 ± 7.31^b	616.28 ± 18.84^a
Feed intake (g feed/fish)	23.61 ± 0.65^c	27.03 ± 0.35^b	28.66 ± 0.37^a	29.20 ± 0.22^a
Feed conversion ratio	1.51 ± 0.03^a	1.32 ± 0.01^b	1.26 ± 0.01^c	1.19 ± 0.01^d
SGR (%/day)	2.76 ± 0.05^d	3.21 ± 0.03^c	3.39 ± 0.02^b	3.51 ± 0.05^a
ADG (g/fish/day)	0.27 ± 0.01^d	0.36 ± 0.01^c	0.41 ± 0.00^b	0.44 ± 0.01^a
Fish biomass (g/m³)	376.00 ± 15.53^d	490.00 ± 7.57^c	537.33 ± 5.81^b	572.67 ± 9.26^a
Fish survival (%)	100.00 ± 0.00	100.00 ± 0.00	100.00 ± 0.00	100.00 ± 0.00
Means having different letters in the same row are significantly different at P < 0.05 (Duncan test).

T3 achieved the best significant (p ≤ 0.05) results, T2, T1, respectively in comparison to the control group. No mortalities were recorded in the experiment. The data indicated that Nile Tilapia in T1, T2, and T3 groups suplemented with probiotics had an improved significant final body weight, weight gain, weight gain rate, and specific growth rate (SGR) (p < 0.05, 0.01) compared to fish in the control group (18.80 g, 14.80 g, 369.66 g and 2.76%/day, respectively). The best FCR was found in T3 (1.19). The better fish biomass was in T3, T2, T1 (572.67 g, 537.33 g, 490.00 g per m³, respectively) than in the control group (376.00 g per m³).

3.3. Whole-body chemical composition

The whole-body composition like dry matter, protein, and ash content of juvenile tilapia was improved significantly in all probiotic groups (p ≤ 0.05) compared with the control. While the content of lipids was decreased in probiotic group compared with the control group, indicating a significant increase in the carcass energy content (Table 4).

Table 4

Effects of some probiotics on the body composition of Nile tilapia *(O. niloticus)* fed diets supplemented with *Pediococcus acidilactici* for 56 days.
parameters	initial	Final
parameters	initial	T0	T1	T2	T3
Dry matter	23.99 ± 0.06^d	25.35 ± 0.05^c	27.02 ± 0.06^b	27.20 ± 0.08^ab	27.33 ± 0.10^a
Crude protein	53.11 ± 0.06^d	57.11 ± 0.58^c	59.63 ± 0.07^b	60.77 ± 0.47^a	60.96 ± 0.06^a
Ether Extract (%)	20.65 ± 0.04^c	25.52 ± 0.57^a	24.38 ± 0.07^b	24.62 ± 0.05^b	24.60 ± 0.05^b
Ash (%)	18.87 ± 0.05^a	14.47 ± 0.04^d	15.18 ± 0.04^c	15.50 ± 0.05^b	15.58 ± 0.05^b
Carcass Gross Energy (Kcal/100g)	493.73 ± 4.68^b	562.46 ± 1.02^a	565.33 ± 0.54^a	568.54 ± 0.66^a	568.76 ± 0.61^a
The values with a different superscript in the same row are significantly different (p ≤ 0.05).

3.4. Blood and serum biochemical analyses

3.4.1. Activities of digestive enzymes

Diets fortified with probiotics significantly (P < 0.05) increased the activities of digestive enzymes compared to control diet (Table 5). The best concentration of dietary Pediococcus acidilactici probiotics that enhanced the secretion of digestive enzymes was a 3 g/kg diet.

Table 5

Activities of serum amylase, lipase, and protease of Nile tilapia (*O. niloticus*) fed diets supplemented with *Pediococcus acidilactici* for 56 days.
Parameters	Treatment
Parameters	T0	T1	T2	T3
Amylase (IU/L)	13.53 ± 0.05^c	15.83 ± 0.26^b	16.31 ± 0.18^b	17.03 ± 0.12^a
Lipase (IU/L)	21.65 ± 0.11^c	23.40 ± 0.15^b	25.83 ± 0.18^a	26.17 ± 0.12^a
Protease (IU/L)	19.67 ± 0.09^d	23.23 ± 0.15^c	24.57 ± 0.09^b	25.83 ± 0.22^a
Means having different letters in the same row are significantly different at P < 0.05

3.4.2. Haemato-biochemical parameters

The results of hematological parameters in different groups are shown in Table 6. The RBCs and WBCs count, as well as Hb concentration and Ht level of fish, fed a 3 g probiotic/kg diet were statistically (P < 0.05) increased than the control group. It is noticed that lymphocytes, neutrophils, and monocytes increased significantly in fish-fed probiotic supplemented diets with Pediococcus acidilactici for 56 days (P > 0.05; Table 6).

Table 6

Hematological parameters of Nile tilapia (*O. niloticus*) fed diets supplemented with *Pediococcus acidilactici* for 56 days
Parameters	Treatment
Parameters	T0	T1	T2	T3
MCV	100.07 ± 2.19^a	92.06 ± 1.57^b	95.93 ± 1.13^ab	101.21 ± 1.81^a
PCV (%)	36.33 ± 0.33^d	37.33 ± 0.33^c	39.67 ± 0.33^b	42.00 ± 0.00^a
RBCs (1x10⁶ uL)	3.63 ± 0.07^b	4.06 ± 0.05^a	4.14 ± 0.08^a	4.15 ± 0.08^a
Hb (mg/dL)	11.50 ± 0.13^c	11.94 ± 0.06^b	12.84 ± 0.09^a	13.12 ± 0.05^a
MCH	31.68 ± 0.93^a	29.45 ± 0.41^a	31.05 ± 0.63^a	31.60 ± 0.49^a
MCHC	31.65 ± 0.41^a	31.91 ± 0.42^a	32.37 ± 0.45^a	31.24 ± 0.13^a
Ht (%)	38.00 ± 0.00^d	40.33 ± 0.88^c	43.67 ± 0.88^b	47.33 ± 0.67^a
WBCs (x103 uL)	29.35 ± 0.35b	30.43 ± 0.40ab	31.13 ± 0.62a	31.23 ± 0.23a
Lymphocytes (%)	22.67 ± 0.33^c	23.67 ± 0.33^b	24.00 ± 0.00^ab	24.67 ± 0.33^a
Neutrophils (%)	21.67 ± 0.33^c	26.67 ± 0.33^b	28.33 ± 0.33^a	28.67 ± 0.33^a
Monocytes (%)	11.33 ± 0.33^b	15.33 ± 0.33^a	15.67 ± 0.33_a	16.00 ± 0.00^a
Means having different letters in the same row are significantly different at P < 0.05.

The blood glucose levels, as well as total cholesterol and triglycerides levels, were significantly (P < 0.05) increased due to dietary (Pediococcus acidilactici) when compared to the control diet (Table 7). TP, GLO, and ALB were significantly (P < 0.05) higher in the serum of probiotic-fed fish than in the control group, especially in fish fed with a 3g Pediococcus acidilactici/kg diet (Table 7). Serum AST, ALT, and ALP activities as well as creatinine and urea levels were significantly affected by the dietary Pediococcus acidilactici (Table 7).

Table 7

Changes in haemato-biochemical parameters of Nile tilapia (*O. niloticus*) fed diets supplemented with *Pediococcus acidilactici* for 56 days
Parameters	Treatment
Parameters	T0	T1	T2	T3
Glucose (mg/dL)	13.70 ± 0.25^b	14.25 ± 0.21^ab	14.76 ± 0.11^a	15.16 ± 0.45^a
Total cholesterol (mg/dL)	77.16 ± 1.92^c	79.45 ± 0.28^bc	82.59 ± 1.03^ab	84.31 ± 0.90^a
Triglycerides (mg/dL)	154.22 ± 0.09^d	155.12 ± 0.05^c	155.65 ± 0.13^b	155.98 ± 0.01^a
Total protein (g/dL)	3.28 ± 0.04^d	3.88 ± 0.03^c	4.05 ± 0.04^b	4.48 ± 0.02^a
Albumin (g/dL)	1.49 ± 0.02^d	1.82 ± 0.02^c	1.93 ± 0.02^b	2.21 ± 0.02^a
Globulin (g/dL)	1.79 ± 0.02^c	2.06 ± 0.03^b	2.12 ± 0.02^b	2.27 ± 0.03^a
AST (IU/L)	80.21 ± 0.25^a	78.40 ± 0.31^b	76.61 ± 0.46^c	75.23 ± 0.16^d
ALT (IU/L)	8.44 ± 0.08^a	8.16 ± 0.06^ab	7.98 ± 0.12^b	7.63 ± 0.09^c
ALP (IU/L)	31.63 ± 0.66^a	31.22 ± 0.55^a	30.75 ± 0.05^a	30.21 ± 0.11^a
Creatinine (mg/dL)	0.37 ± 0.01^b	0.37 ± 0.00^b	0.39 ± 0.00^a	0.41 ± 0.01^a
Urea (mg/dL)	4.56 ± 0.04^a	4.62 ± 0.05^a	4.55 ± 0.07^a	4.47 ± 0.04^a
AST = aspartate aminotransferase; ALT = alanine aminotransferase; ALP = alkaline phosphatase
Means having different letters in the same row are significantly different at P < 0.05

3.4.3. Antioxidant and immunity biomarkers

The antioxidant and immunity responses of Nile tilapia in different groups are shown in Table 8. At the end of the experiment, fish fed with Pediococcus acidilactici-enriched diets had significantly (P < 0.05) lower MDA levels compared to control group, whereas the least MDA levels were recorded in fish fed a 3 g/kg diet. On the reverse, the antioxidant enzymes activities (SOD, CAT, and GPx) were significantly elevated in fish fed Pediococcus acidilactici diets for 56 days (P < 0.05) than those of the control diet, and their highest (P < 0.05) activities were recorded at 3 g Pediococcus acidilactici/kg diet. It is also noticed that feeding fish on Pediococcus acidilactici-containing diets significantly (P < 0.05) triggered the serum LYZ activity and increased IgM levels than those of the control diet (Table 8). Additionally, the immunological values of fish fed 2, 2.5, and 3 g Pediococcus acidilactici/kg diet was significantly the highest compared with the control group.

Table 8

Serum antioxidants and immunological responses of Nile tilapia (*O. niloticus*) fed diets supplemented with *Pediococcus acidilactici* for 56 days.
Parameters	Treatment
Parameters	T0	T1	T2	T3
MDA (nmol/dL)	2.16 ± 0.04^a	2.02 ± 0.03^b	1.98 ± 0.01^b	1.95 ± 0.01^b
SOD (IU/L)	13.53 ± 0.318^c	14.77 ± 0.186^b	15.67 ± 0.27^a	16.23 ± 0.12^a
CAT (IU/L)	10.27 ± 0.15^c	11.40 ± 0.26^b	11.90 ± 0.15^ab	12.37 ± 0.24^a
GPx (IU/L)	19.53 ± 0.26^c	21.40 ± 0.25^b	22.77 ± 0.23^a	23.23 ± 0.12^a
LYZ (µg/mL)	1.39 ± 0.03^c	1.69 ± 0.05^b	1.77 ± 0.02^b	1.98 ± 0.01^a
IgM (mg/mL)	19.13 ± 0.12^d	20.23 ± 0.15^c	21.23 ± 0.15^b	21.73 ± 0.09^a
Means having different letters in the same row are significantly different at P < 0.05.

3.5. Histology of intestine and liver

Microscopy of the intestine revealed branched and markedly increased length of intestinal villi in T3 and T2 groups with mild desquamation of the mucosal epithelium at the apical part in the T3 group. The desquamation of the intestinal epithelial lining was prominent in T0 and T1 groups (Fig. 1: T0, T1, T2, and T3).

Microscopy of the liver of the four groups revealed a normal histological structure in which polyhedral vacuolated hepatocytes were arranged around the central vein (Fig. 2: T0, T1, T2, and T3).

Probiotics were reported to enhance survival, growth, and improve feed digestion, and enhance the immune system (Huerta-Rábago et al., 2019). As yet, most researchers investigating the effects of probiotics in aquaculture have used dietary additives (Jahangiri and Esteban, 2018). Aside from the laboratory groundwork of probiotics, commercial products are available. One of the essential bacteria species found in most commercial products is Pediococcus acidilactici isolates (Martínez et al., 2012). Many factors may affect the efficiency of probiotics, such as water temperature, salinity, source of probiotics, dose, and age of fish (Jahangiri and Esteban, 2018).

Good water quality is critical in aquaculture production. For optimal growth, survival, and production, a thorough understanding of the link between water quality and aquatic productivity is required. (Soundarapandian and Babu 2010, Hossain, Kamal et al. 2013).

Results of the current study indicate clear improvement in water quality, represented by a significant decrease in total and toxic ammonia levels in probiotics-treated groups. These results agree with many previous studies (Balcazar et al., 2006; Jó´zwiakowski et al., 2009; Zheng et al., 2011; Zhang et al., 2011; Banerjee and Ray, 2017; Jahangiri and Esteban, 2018).

Growth performance parameters are important elements in aquaculture because they reflect production yield and are controlled by environmental conditions, genetic factors, and feeding quantity and quality (Oduleye 1982). As a result, they are often employed to assess the efficacy of various diets and supplements on fish (Naiel, Ismael, et al. 2020). The research showed that as probiotic levels increased and feed conversion decreased, growth parameters were dramatically optimized. These findings corroborate the findings of Sharibi, Pour et al. (2015) and Adorian, Jamali et al. (2019). The best growth performance can be attributed to improving nutrient digestibility and availability to fish (Opiyo, Jumbe et al., 2019) by producing digestive enzymes (e.g., amylase, protease, and lipase), providing necessary growth factors (e.g., vitamins and amino acids), improving intestinal microbial flora, detoxifying potentially harmful compounds in feed, and stimulating the immune system (Li, Ringø et al. 2019, Opiyo, Jumbe et al. 2019). The increased lifespan of fish fed probiotics-supplemented diets could indicate improved health conditions, which is consistent with the findings of Welker and Lim (2011) and Abdel-Aziz, Bessat et al. (2020).

The beneficial role of Pediococcus acidilactici in removing organic matter from aquatic rearing ponds was documented (Luis-Villaseñor et al., 2011). Kuebutornye et al. (2019) emphasized that adding probiotics, e.g., Pediococcus acidilactici, to feed would increase beneficial bacteria and multiply them in the fish intestine. Another significant benefit of adding probiotics to feed, Xu et al. (2013) stated that Pediococcus acidilactici can produce extracellular enzymes and antimicrobial peptides that control pathogenic bacteria and improve water quality.

The quality of the aquatic environment directly affects the performance and welfare of farmed organisms (Hura et al., 2018). In the current study, probiotics significantly improved tilapia growth performance and feed utilization compared with the control group. The best results were in favor of T3 treatment. The positive effects of probiotics treatments are consistent with many studies in Tilapia, O. niloticus (Wang et al., 2008b; Zhou et al., 2010; Wang et al., 2017; Madani et al., 2018; Sutthi et al., 2018;), Gilthead sea bream, Sparus aurata (Lotfy et al., 2015), European sea bass Dicentrachus labrax (Aly et al., 2016), yellow perch, Perca flavescens (Shaheen et al., 2014), Asian catfish Pangasius hypophthalmus (Gobi et al., 2016), Abalone Haliotis discus hannai (Guo et al., 2017), and Pacific white shrimp Penaeus vannamei (Xia et al., 2014). Pediococcus acidilactici, exhibited a significant improvement in the feed conversion ratio and growth performance of O. niloticus. The ability of Probiotics to enhance growth performance is dose-dependent (Elsabagh et al., 2018; Madani et al., 2018).

Fingerlings showed better growth performance, feed utilization, condition factors, and survival percent compared with control. The positive effect of probiotics in improving the nutritional status of fish might be due to the positive role of beneficial microorganisms in improving the digestive system (Thiam et al., 2015; Lotfy, 2015), through the improvement of the intestinal microbial balance, stimulate appetite, production of vitamins, breakdown of indigestible nutrients (Irianto and Austin, 2002), reducing pathogenic microbes in the gastrointestinal tract (Lotfy, 2015), and consequently leading to improve feed absorption (Tovar et al., 2002).

The results of the current study are consistent with the results of previous studies regarding the positive effect of probiotics, whether water additive, feed additive, or even enrichment to live foods (rotifer, artemia, etc.) during larval stages and beyond (Kuebutornye et al., 2019; Lotfy, 2015, Eissa et al., 2022).

Probiotics used as feed additives were documented to positively affect the microbial groups of aquatic organisms and the environment. Therefore, this improves growth performance and enhances the immune system (Wang et al., 2017). The application of probiotics in this study significantly improved blood and serum analytical parameters of Nile tilapia. This result is consistent with many previous studies, such as in O. niloticus (Sutthi et al., 2018, Wang et al., 2008b), Cyprinus carpio (Gupta et al., 2016), Pangasius hypophthalmus (Gobi et al., 2016), and Dicentrarchus labrax (Schaeck et al., 2017). Hemoglobin levels and numbers of red blood cells (RBC) increased in Southern catfish, Silurus meriaionalis treated with probiotic (Wu et al., 2004). Sutthi et al. (2018) investigated the effects of probiotics on the performance and biochemical parameters of Nile tilapia. They found that fish reared using probiotics showed a synergistic effect that significantly decreased AST and ALT levels. Also, in Cyprinus carpio lysozyme activity, catalase, and superoxidase dismutase (SOD) activity improved in probiotics treated fish (Gupta et al., 2016).

Fish administered high dosages of probiotics had a significantly higher body lipid composition, Hassaan, Soltan, et al. (2014) and Yones, S Hussein et al. (2019) found similar results. Other body compositions, on the other hand, showed no significant variations but were improved by probiotic therapy. These findings confirm the findings of Morshedi, Nafisi Bahabadi, et al. (2015). The increased nutritional deposition could have caused the rise in protein content. As a result, the higher body protein content seen in this study could be linked to more proteins released by the probiotics and efficient conversion of consumed food into structural protein, resulting in the development of more muscle (Mehrabi, Firouzbakhsh et al. 2012, Lara-Flores and Olvera-Novoa 2013).

The health status of fish can be determined using plasma biochemistry measures (Vani, Saharan et al. 2011, Falahatkar 2015). They are also used as markers for assessing the health of fish after they have been fed a diet supplemented with probiotics and have been exposed to stresses in the fish farming process (Ahmadifar, Moghadam et al. 2019).

The results showed that probiotic treatments had the highest levels of total protein, albumin, globulin, and glucose, indicating that probiotics-fed fish were healthier than the control group. These indicators also rose as the concentration of probiotics increased. The findings are similar to those of Kamgar and Ghane (2014), and Nargesi, Falahatkar et al. (2020). The improved protein profile could be attributable to the use of probiotics, which improve the intestinal environment, resulting in improved digestion and nutritional absorption (Eshak, Khalil et al. 2010). A significant immunological response in fish is indicated by the increase in total protein, albumin, and globulin (Al-Dohail, Hashim et al. 2009). Where a decrease in total protein might be an indication of a variety of disorders caused by liver disease, impaired protein absorption, or protein loss (Bernet, Schmidt et al. 2001).

Furthermore, the data revealed that T1, T2, and T3 had lower ALT and AST levels than the control. Furthermore, the inclusion rate of probiotics in their diets affected their activity. The liver enzymes ALT and AST are essential indicators of liver health and function. The key biochemical indicator that may be used to assess the influence of fish food supplements on metabolic activity and fish health is enzyme activity assessment (Fadl, El-Gammal et al. 2020, Xu, Zheng et al. 2020), As their levels increase in animal blood when liver cells are damaged (Kumar, Roy et al. 2016). The results of the present study are following the study of Hassanien, El-Moghazy et al. (2017) and Kurdomanov, Sirakov et al. (2019), who found that probiotics-treated diets reduced ALT and AST levels when compared to control diets, but disagree with those of Al-Hisnawi and Beiwi (2021) who found that probiotics-treated diets increased ALT and AST levels. The discrepancies across the studies could be due to the type of probiotics used, the concentration of probiotics utilized, the fish species studied, the time of administration, and/or environmental conditions.

The development of the internal organs, especially the intestine and liver, significantly influences the growth of fish and their resistance to diseases and stress factors and improves feed utilization. In the current study, histology of commercial probiotics treated tilapia showed remarked positive developments in the intestine and liver than the control group with the best results in favor of T3 followed by T2, and T1. The results of the current study are consistent with the results of previous studies regarding the positive effect of probiotics, whether water additive, feed additive, or even enrichment to live foods (rotifer, artemia, etc.) during larval stages and beyond (Kuebutornye et al., 2019; Lotfy, 2015). The addition of commercial probiotics was confirmed to improve the intestinal development of gilthead sea bream (Lotfy, 2015; El-Okaby, 2015).

According to the findings, Pediococcus acidilactici levels vary widely in efficacy when used as a feed additive to improve water, performance, fish health, and increased fish biomass/production per cubic meter. Pediococcus acidilactici improved blood biochemical and immunity parameters, such as WBCs, lysozyme activity, and phagocytic activity. Pediococcus acidilactici positively improved the development of internal fish organs, whether intestine and liver. Generally, the study confirms the importance of probiotics as a feed additive to improve water quality, fish performance, blood biochemical and immunity of Nile tilapia. As a result, the probiotics are necessary to be included in the aquaculture process. It's worthy to mention that addition of higher doses is better.

Acknowledgment: The authors would like to thank the Department of Aquaculture , Faculty of fish and fisheries Technology, Aswan University, Egypt and Dr.Ahmed A. El-Kady Kafrelsheikh governorate for providing facilities to carry out this experiment.

Code availability: Not applicable.

Author contribution:

EL- Sayed Hemdan Eissa: conceptualization,formal analysis, writing original draft, and follow up publication.

Fatima S. Alaryani : conceptualization, methodology,and nvestigation.

Marwa S. Khattab: histopathology,samples and slides.

Amal Elfeky: conceptualization, methodology,and investigation.

Moaheda E.H.Eissa: conceptualization,follow up and statistical analysis

Khaled Youssef AbouelFadl: conceptualization,methodology, and investigation

Funding : Open access funding provided by The Science,Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Data availability: Data of the present article are available under request.

Ethical approval: All applicable international, national, and/or institutional guidelines for the care and use of fish were followed by the authors.

Consent for publication: All authors review and approve the manuscript for publication.

Conflict of interest The authors declare no competing interests.

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The effect of Pediococcus acidilactici probiotics supplementation in the feed of Nile Tilapia (Oreochromis niloticus) on water quality, growth performance, Hematological parameters, antioxidants, immunological responses, enzymatic Activity, and histology.

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Experimental site

2.2. Experimental fish and cultural conditions

2.4. Experimental design

2.5. Feeding protocol

2.6. Measured parameters

2.6.1. Water quality parameters

2.6.2. Growth performance parameters and survival percentage

2.6.3. Fish and feed analytical methods

2.6.4. Blood sampling

2.6.5. Blood serum biochemical analysis

2.6.6. Histopathological examination

2.7. Statistical analysis

3. Results

3.1. Water quality

3.2. Growth performance and feed utilization

3.3. Whole-body chemical composition

3.4. Blood and serum biochemical analyses

3.4.1. Activities of digestive enzymes

3.4.2. Haemato-biochemical parameters

3.4.3. Antioxidant and immunity biomarkers

3.5. Histology of intestine and liver

4. Discussion

5. Conclusion

Declarations

References

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