Introduction

Aquaculture is a rapidly growing industry that satisfies the global demand for reliable, protein-rich food sources, particularly in developing countries (FAO et al. 2023). Oreochromis niloticus (L.), also known as the Nile tilapia, is one of the most popular fish species in the world. This fish species can utilize various protein sources, adapt to different environmental conditions, grow quickly, and have high consumer acceptability (El-Sayed 2019). In the last decade, the aquaculture industry in Egypt has greatly progressed, especially tilapia production, which represents more than 88% of the total aquaculture production (GAFRD 2019). Achieving the maximum economic return requires the following: keeping high water quality for as long as possible, stocking fish at a high rate, raising feed efficiency, and controlling pathogens (Kaleem and Bio Singou Sabi 2021; FAO et al. 2023). On the other hand, due to high stress levels brought on by high stocking density and the resulting poor water quality, tilapias maintained in intensive production systems may be more vulnerable to diseases which leads to a reduction in fish production (Mahmoud et al. 2021; Maulu et al. 2021). This occurred because of declining water quality and rising ammonia levels combined with inadequate fish pond management (Mehrim and Refaey 2023). Because stress can lead to oxidative responses, immune suppression, cell damage, and growth decline, it is critical to develop methods for reducing these detrimental effects on fish (Rajabiesterabadi et al. 2020; Lee et al. 2022).

Elevating the feed efficiency is one method for decreasing these hazards, thereby decreasing wastes in culture water and preventing the deterioration of water quality (Takase 2023). Many substances have been used as feed additives to improve the health status of fish, which achieved positive results (Bilen et al. 2020; Busti et al. 2020; Marimuthu et al. 2022; Vijayaram et al. 2023). One of these additives in aquafeeds is organic acids (acidifiers) that can be very significant in the future of aquaculture diets (Agouz et al. 2015). Organic acids (OAs) are classified into short-chain fatty acids, volatile fatty acids, and weak carboxylic acids. OAs are produced through the microbial fermentation of carbohydrates in the digestive system of animals (Kim et al. 2005). Two main pathways are involved in the potential modes of action of OAs in the digestive tract. Firstly, OAs lower the stomach and small intestine pH by delivering H+ ions. Secondly, OAs prevent the growth of Gram-negative bacteria by causing the acids to dissociate and generating anions inside bacterial cells (Lückstädt 2008; Asriqah et al. 2018). Many studies have confirmed that dietary supplementation of OAs plays a great role in controlling pathogenic bacteria, stimulating growth, fish gut health, and immune system function (Tran-Ngoc et al. 2019; das Neves et al. 2021; da Silva et al. 2023). Additionally, OAs are promising alternatives for growth-promoting antibiotics (Ng and Koh 2011; Lim et al. 2015).

Most studies have estimated the feed additives as health promoters under suitable conditions for tilapia rearing. For example, previous research recorded that feed or water additives presented the in pond water reduced levels of nitrogenous wastes and enhanced water quality (Abdel-Tawwab et al. 2022; El-Kady et al. 2022). Moreover, the stress condition (high stocking density or unchanged water) badly altered the water quality and fish health condition, and these impacts were improved by dietary herbal and watery probiotics supplements (Ayyat et al. 2022; Kord et al. 2022). However, the data on the palliative role of OAs against unchanged water-induced stress is still scarce in fish. Consequently, this study was designed to evaluate the potential functional interaction of feed additives [formic acid (FA), lactic acid (LA), and commercial organic acids mix (COM)] on growth performances, biochemistry, and pathology under a stressful condition (water exchange).

Materials and methods

Ethical statement

All experimental procedures, management conditions, handling, and sampling were approved by the National Institute of Oceanography and Fisheries (NIOF, Egypt) Committee for Ethical Care and Use of Animals/Aquatic Animals (Code: NIOF-AQ1-F-23-R-050).

Diets preparation

FA (molecular formula: CHOOH; molecular weight: 46.03) and LA (molecular formula: C3H6O3: molecular weight: 90.08) were obtained from Finar (ACETO), India. Meanwhile, COM (1,2-propanediol, E200 sorbic acid, E236 FA, E260 acetic acid, E270 LA, E280 propionic acid, and E284 ammonium propionate) was obtained from Selko®, Trouw Nutrition, a Nutrico Company (Fylax Forte-HC liquid). The FA, LA, and COM were added at 1% (10 mL/kg diet) and 2% (20 mL/kg diet) of the basal diet (FA1, FA2, LA1, LA2, COM1, and COM2), respectively. The doses of FA and LA were according to Reda et al. (2022) and El-Dakar et al. (2022) approaches, but no recorded trial on the dietary addition of COM in Nile tilapia.

The basal diet was formulated to cover the nutrient requirements of Nile tilapia, according to NRC (2011). The proximate composition of the basal diet used in the feeding trial was shown in Table 1. All ingredients were mixed well for 15 min before adding oil and water to make a moist, doughy mass for fish feeding. Then, the dough mass was pelleted without steam, yielding 2-mm-diameter sinking pellets. The pellets were then dried at room temperature and stored in clean, sterile plastic bags at −4 °C until use. During the acclimation period and 75 days of the trial, the fish were manually fed every other day (alternating-day feeding) three times daily at 8:00, 12:00, and 17:00 (four days/a week) with a feeding rate of 4% of the total fish biomass. The remains of uneaten feed and fish wastes were removed from the pond bottom using a small suction pump. Every 2 weeks, a sample of fish per replicate was weighed to adjust feed intake.

Table 1 Ingredients and proximate composition of the basal diet (% on dry matter basis)
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Fish rearing and experimental design

This study was conducted at a fish-feeding laboratory of the Fish Research Station, National Institute of Oceanography and Fisheries, Fayoum Governorate, Egypt. The fish were obtained from a commercial fish farm on the south coast of Qarun Lake (29°30°N, 30°40°E). They were transferred in aerated plastic bags and acclimatized to laboratory conditions for 15 days, where the fish were randomly distributed into indoor concrete ponds of 1 m3 water capacity (70 juveniles/pond). The average initial body weight (IBW) of the fish was 3.95 ± 0.05 g (with an age of 2 months).

For 75 days, the fish were randomly housed into eight groups (n = 210 mono-sex male tilapia juveniles; 70 fish/replicate; 3 replicates/group) and fed on the tested diets (control, FA1, FA2, LA1, LA2, COM1, and COM2). The first (negative control, NC) and second (positive control, PC) groups were fed on a basal diet without additives with water exchange at 20% of the water volume every 2 days for the first group and without water exchange for the second. The other six groups (FA1, FA1, LA1, LA2, COM1, and COM2) were fed on OAs-supplemented diets without water exchange throughout the trial. The clinical observation of the fish was performed to record any mortality and clinical signs during the 75 days. The loss of water volume due to evaporation or by taking fish samples every 15 days to adjust the feeding rate was compensated.

Water quality analysis

Water samples were collected from fish ponds, and the water quality criteria were measured as water temperature, pH, dissolved oxygen (DO) concentration, unionized ammonia (NH3), and nitrite (NO2) levels using chemical methods (APHA 2005).

Determination of growth indices and survival rate

After 30 and 75 days of the feeding trial, the final body weight (FBW) was estimated. The growth metrics in terms of weight gain (WG, g), specific growth rate (SGR, %/day), and feed conversion rate (FCR), as well as fish survival rate (SR, %), were determined using the following formulas:

$$\begin{array}{l}{\text{WG}}=\mathrm{FBW }\left({\text{g}}\right)-\mathrm{ IBW }\;({\text{g}})\\ {\text{SGR}}=(\mathrm{ln \;FBW}-\mathrm{ln \;IBW}\times 100)/\mathrm{time }\;({\text{days}})\\ {\text{FCR}}=\mathrm{Feed \;intake }/{\text{WG}}\\ {\text{SR}}=({\text{No}}.\mathrm{ of \;fish \;survived \;throughout \;the \;trial }\;(75\mathrm{ days}) /\mathrm{ Total \;number \;of \;fish \;in \;each \;group}) \times 100\end{array}$$

Proximate chemical composition

The chemical composition of the whole fish body (15 fish/group) was estimated according to standard methods (AOAC 2005). Moisture was estimated using a hot air oven, while crude protein (N × 6.25) was analyzed using the Kjeldahl method. The crude fat was measured using the Soxhlet method with ether extraction. Ash content was determined by adding ash at 550 °C for 16 h in a muffle Furance 6000 (Thermolyne, USA).

Sampling

At the end of the trial period (75 days), five fish from each replicate were anesthetized using 100 mg/L benzocaine solution (Neiffer and Stamper 2009). Two blood samples (15 fish/group) were withdrawn using 3-mL syringes from the caudal blood vessels and emptied into two tubes containing anticoagulants for hematological assays. The other tubes did not contain anticoagulants for serum separation and measuring the biochemical, oxidant/antioxidant, and immunological assays. Moreover, fish were euthanized by decapitation, and representative tissue specimens were collected from the gills, liver, and intestine for histopathological analysis.

Hematological and biochemical assays

The hematological indices, such as white blood cell count (WBCs, 103/mm3), hemoglobin (Hb, g/dl), red blood cell count (RBCs, 106/mm3), and hematocrit (Hct, %), were determined according to the standard methods as described by Blaxhall and Daisley (1973). Serum levels of creatinine (catalog No.:7D64-20), urea (catalog No.:319-001), glucose (catalog No.:250-007), alanine aminotransferase (ALT, catalog No.:292-007), aspartate aminotransferase (AST, catalog No.:291-007), albumin (catalog No.:7D53-20), and total protein (catalog No.: 7D73-20) were measured spectrophotometrically using commercial kits of Egyptian CO. for Biotechnology with a semi-automated analyzer (3000 Evolution), Biochemical System International, Arezzo, Italy. The globulin level was calculated by subtraction of the albumin value from the total protein value.

Oxidant/antioxidant and immunological assays

The level of malondialdehyde (MDA; catalog No.:MBS268427) and the activity of superoxide dismutase (SOD; catalog No.: CSB-E08555r) and catalase (CAT; catalog No.:MB2600683), as well as the total immunoglobulin M level (IgM; catalog No.:CSB-E07978r), were measured using ELISA kits available from Biosource Inc. (San Diego, CA, USA) that were manufactured by CUSABIO BIOTECH CO., Ltd. In addition, lysozyme activity was assayed using the protocol of Ellis (1990).

Histopathological analysis

Specimens from the gills, liver, and intestine of tilapia fish per group (15 fish/group) were taken and instantly fixed in 10% neutral buffered formalin solution for 48 h and dehydrated in a series of graded ethyl alcohol (70%, 80%, 90%, and 100%; 1 h each). The dehydrated specimens were cleared in two changes of xylene (1 h each), impregnated and embedded in melted paraffin wax, microtomed at 4–5-μm sections, stained with hematoxylin and eosin (H&E) following standard procedures (Suvarna et al. 2018). Moreover, the intestinal morphometric indices (villus width, villus heights, and goblet cell hyperplasia) were scored according to Bernet et al. (1999) approach. Concisely, five non-overlapping randomly selected microscopic fields per intestine per fish were photographed, and the images were then analyzed.

Statistical analysis

To check the norm homogeneity of the data, Bartlett and Kolmogorov–Smirnov analyses were performed. The data were statistically examined by one-way analysis of variance (ANOVA) using Statistical Package for the Social Sciences (SPSS, version 20, IBM Corp.) following the approach of Dytham (1999). The differences among the treatment groups were determined using the Tukey test at the significance level of P < 0.05.

Results

Water quality indices

Table 2 shows the means of the water quality parameters assessed during 75 days of the trial. The water temperature showed no significant alteration among all groups. There was a marked decline (P < 0.05) in the DO level in the PC group compared to the control group (NC). No significant changes were observed in the DO level between the PC group and other groups that fed-OAs incorporated diets. The highest significant (P < 0.05) levels of pH, NH3, and NO2 were observable in the PC group compared to the NC group. In addition, these variables declined in the other experimental groups without significant variations.

Table 2 Changes in water quality indices of O. niloticus fed diets containing organic acids and with/without water exchange for 75 days
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Growth performance, SR (%), and clinical observations

Figs. 1 and 2 show the growth metrics regarding FBW, WG, SGR, and FCR of Nile tilapia after 30 and 75 days of the trial. After 30 days, there were significant (P < 0.05) declines in the FBW, WG, and SGR and an elevation in the FCR of the fish reared without water exchange (PC group) compared to the NC fish. Dietary OAs significantly enhanced (P < 0.05) the FBW, WG, and SGR and improved the FCR compared to the PC group, except for the FA1 and COM1. The highest values were recorded in the LA1 group.

Fig. 1
figure 1

Final body weight (FBW, g) and weight gain (WG, g) of O. niloticus fed diets containing organic acids and with/without water exchange for 30 and 75 days. A FBW (P = 0.025 and 0.002). B WG (P = 0.026 and <0.001). The bars with different superscripts significantly differ at P < 0.05

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Fig. 2
figure 2

Specific growth rate (SGR, %/day) and feed conversion ratio (FCR) of O. niloticus fed diets containing organic acids and with/without water exchange for 30 and 75 days. A SGR (P= 0.041 and 0.03). B FCR (P= 0.001 and 0.015). The bars with different superscripts significantly differ at P < 0.05

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After 75 days, the FBW, WG, and SGR recorded marked decreases with an increase in the FCR of the PC group compared to the NC group. Dietary LA addition significantly enhanced (P < 0.05) the FBW, WG, and SGR and improved the FCR compared to the PC group, and the dietary LA1 recorded the best results. In contrast, dietary FA1 and COM1 lowered the growth performance compared to the PC group.

Table 3 reveals that the PC group had a significantly (P < 0.05) lower SR (%) than the NC group. The SR (%) was decreased in the OAs groups (FA1, FA2, and COM1) compared to the PC group. Meanwhile, the other OAs groups (LA1, COM2, and LA2) revealed a significant (P < 0.05) increase. The LA1 group recorded the highest SR (%) among OAs groups. Moreover, the fish of PC and OAs (FA and COM) exhibited a decreased activity, skin darkness, and fin rot, and some fish showed moderate body hemorrhages. These observations were recorded from the second month of experiment till the end (75 days) and also were ameliorated in the fish of LA group.

Table 3 Survival rate (SR) % and proximate body composition indices (on dry matter basis) of O. niloticus fed diets containing organic acids and with/without water exchange for 75 days
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Proximate body composition indices

Table 3 exhibits the results of body composition indices, where no significant differences were observed between the PC and NC groups except for a decline in the moisture level. Marked elevations in the moisture, crude protein, and ash levels along with a lowering in the crude fat value were observed in the fish-fed diets containing OAs compared to the PC group, except for the ash content of the FA1 group. The highest level of crude protein was obvious in the LA1 group. Meanwhile, the COM2 group recorded the highest content of Ash. In contrast, the lowest value of crude fat was noted in the LA1 group.

Hematological and protein profile indices

As shown in Table 4, there were substantial decreases (P < 0.05) in the WBCs count, Hb value, RBCs count, and Hct (%) as well as protein profile indices (total protein, albumin, and globulin) of the PC group compared to the NC group. The highest count of WBCs was obvious in the LA groups. Meanwhile, the lowest count was in FA groups. No significant alterations were observed in the Hb value, RBCs count, Hct (%), and albumin level between the OAs and PC groups, and the LA1 group recorded the highest values.

Table 4 Hematological and protein profile indices of O. niloticus fed diets containing organic acids and with/without water exchange for 75 days
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The total protein and globulin values were substantially increased (P < 0.05) in the LA1 group, followed by the LA2, COM1, and COM2 groups relative to the PC group. The FA groups showed no marked alteration in the total protein and globulin levels with the PC group.

Biochemical indices

Table 5 reveals that serum biochemical indices (creatinine, urea, ALT, AST, and glucose) were significantly elevated (P < 0.05) in the PC group compared to the NC group. Dietary OAs did not significantly alter the creatinine and urea relative to the PC group. However, the lowest values were obvious in the LA1 group. The highest levels of ALT and AST were obvious in the FA2 group, while the LA1 group recorded the lowest value. Dietary OAs significantly reduced (P < 0.05) blood glucose compared to the PC group except for FA and COM2 groups, with the lowest level noted in the LA1 group.

Table 5 Biochemical indices of O. niloticus fed diets containing organic acids and with/without water exchange for 75 days
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Oxidant/antioxidant and immunological indices

Table 6 shows a substantial increase in the MDA level and a decrease in the activity of SOD, CAT, and lysozyme, as well as the total IgM level of the PC group relative to the NC group. Dietary FA1 and COM1 significantly increased the MDA (P < 0.05) value compared to the PC group, while the lowest value was noted in the LA1 group. Values of SOD, CAT, lysozyme, and total IgM were markedly enhanced (P < 0.05) in LA groups relative to the PC group. Meanwhile, the other groups (FA and COM) did not significantly differ from the PC group.

Table 6 Oxidant/antioxidant and immunological indices of O. niloticus fed diets containing organic acids and with/without water exchange for 75 days
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Histopathological investigation

Fig. 3 reveals the histopathological alterations in the gills of O. niloticus fed diets containing different levels of OAs and with/without water exchange for 75 days. The control fish (NC) showed normal histological architectures with no morphological alterations. The gills of the fish kept without water exchange (PC group) were severely affected and displayed numerous lesions, including epithelial hyperplasia with a fusion of secondary lamellar and capillary congestion. Dietary OAs addition did not protect the gills from the impact of stress (non-water exchange) except for moderate changes attained by dietary LA1. The gills of the FA groups revealed an interlamellar hemorrhage, lamellar necrosis, and epithelial desquamation (FA1 group), as well as capillary telangiectasia, lamellar sloughing, and epithelial hyperplasia (FA2 group). The gills of the LA groups displayed inflammatory cell infiltrate, and capillary telangiectasia (LA1 group), as well as epithelial hyperplasia with lamellar fusion, capillary dilatation, and hemorrhage with hemosiderosis (LA2 group). The gills of the COM groups exhibited necrosis of lamellar epithelial, epithelial uplifting as well as congested capillaries (COM1 group), capillary telangiectasia, and epithelial uplifting (COM2 group).

Fig. 3
figure 3

Photomicrograph (H&E) of gills sections of O. niloticus. Fish of the NC group show a normal histological picture. Fish of the PC group show epithelial hyperplasia with lamellar fusion (arrowheads) and capillary congestion (arrows). Fish of the FA1 group show interlamellar hemorrhage (arrows), lamellar necrosis (red arrowhead), and epithelial desquamation (black arrowhead). Fish of the FA2 group show capillary telangiectasia (arrow), lamellar sloughing (red arrowhead), and epithelial hyperplasia (black arrowhead). Fish of the LA1 group inflammatory cell infiltrate (red arrow), capillary telangiectasia (black arrow), lamellar necrosis (red arrowhead), and epithelial uplifting (black arrowhead). Fish of the LA2 group show epithelial hyperplasia with lamellar fusion (arrowhead), capillary dilatation (black arrow), and hemorrhage with hemosiderosis (red arrow). Fish of the COM1 group show congested capillaries (black arrow), necrosis of lamellar epithelial (black arrowheads), and epithelial uplifting (red arrowhead). Fish of the COM2 group showing capillary telangiectasia (arrow) and epithelial uplifting (red arrowhead). Scale bar: 30 and 100 μm

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The histopathological alterations in the hepatic tissues were presented in Fig. 4, where the liver of the NC group displayed normal histological architectures with no histopathological changes. The liver of the PC group exhibited a wide array of hepato-pathic histological alterations, including focal coagulative necrosis. Dietary addition of OAs worsens the hepato-pathic changes as more severe lesions were noted. However, moderate alterations were obvious by LA1. The livers of the FA groups showed individualization of hepatic cells, vascular congestion, sinusoidal dilatation, hyperplastic melanomacrophages (FA1 group), and thrombosis in a central vein (FA2 group). The livers of the LA groups showed vascular congestion and focal mononuclear-cell aggregation (LA1 group), and extensive vacuolation with single-cell necrosis (LA2 group). The livers of COM groups exhibited vascular congestion, focal mononuclear-cell aggregation (COM1 group), and focal coagulative necrosis (COM2 group).

Fig. 4
figure 4

Photomicrograph (H&E) of hepatic tissue sections of O. niloticus. Fish of the NC group show a normal histological picture. Fish of the PC group show focal coagulative necrosis (ellipse). Fish of the FA1 group show individualization of hepatic cells, vascular congestion (black arrows), sinusoidal dilatation (black arrowhead), and hyperplastic melanomacrophages (red arrow). Fish of the FA2 group showing thrombosis in a central vein (arrowhead). Fish of the LA1 group showing vascular congestion (arrow) and focal mononuclear-cell aggregation (arrowhead). Fish of the LA2 group show extensive vacuolation with single-cell necrosis (arrowheads). Fish of the COM1 group showing vascular congestion (arrow) and focal mononuclear-cell aggregation (arrowhead). Fish of the COM2 group showing focal coagulative necrosis (ellipse). Scale bar: 30 μm

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Fig. 5 displays the histopathological alterations in the intestinal tissues, where the intestine of the NC group displayed normal histological architectures with no histopathological alterations. The intestine of the fish kept without water exchange (PC group) was severely affected and displayed numerous lesions, including necrotic enterocytes and epithelial desquamation. Dietary OAs additions exhibited no significant protective effects on intestinal histology except in the LA1 group, which showed mild lesions. The intestine of the FA groups revealed hyperplastic goblet cells (FA1 group) and coagulative necrosis of the villous tips (FA2 group). The intestine of the LA groups displayed leucocytic infiltration at the villous tip (LA1 group) and enterocytes necrosis and desquamation at the villous tips (LA2 group). The intestine of the COM groups exhibited villous curling (COM1 group) and villous necrosis with epithelial desquamation (COM2 group).

Fig. 5
figure 5

Photomicrograph (H&E) of intestinal tissue sections of O. niloticus. Fish of the NC group show a normal histological picture. Fish of the PC group show necrotic enterocytes (arrow) and epithelial desquamation (arrowhead). Fish of the FA1 group showing hyperplastic goblet cells (arrowheads). Fish of the FA2 group showing coagulative necrosis of the villous tips (arrowheads). Fish of the LA1 group show leucocytic infiltration at the villous tip (arrowhead). Fish of the LA2 group show enterocytes necrosis and desquamation at the villous tips (arrowheads). Fish of the COM1 group showing villous curling (arrowhead). Fish of the COM2 group showing villous necrosis (arrowhead) with epithelial desquamation (arrow). Scale bar: 100 μm

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Moreover, there were marked reductions in the villous width and length with marked increases in goblet cell hyperplasia frequency of the PC compared with the NC group as shown in Table 7. Dietary LA significantly elevated (P < 0.05) villous width and length and decreased the goblet cell hyperplasia frequency relative to the PC group. However, the other groups (FA and COM) did not significantly differ from the PC group (Table 7).

Table 7 Intestinal morphometric indices of O. niloticus fed diets containing organic acids and with/without water exchange for 75 days
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Discussion

The effectiveness of employing OAs on fish performance is variable and based on various variables, including fish species, the types, concentrations of OAs employed, water quality, and culture management (Paul et al. 2024). This study intended to evaluate how Nile tilapia reared without water renewal responded to dietary OAs in terms of growth rate and physiological functions.

Water quality variables have acceptable limits in which aquatic organisms function optimally (Verma et al. 2022). In our study, the level of DO declined and NH3 and NO2 increased in the PC group. Reduced DO levels could have been caused by the microbial decomposition of accumulated organic wastes in unchanged water (Obirikorang et al. 2019). The low DO levels resulted in ion loss in freshwater fish, negatively affected osmoregulation, and can lead to fish mortalities (Samaras et al. 2023). In the OAs-fed groups, NH3 and NO2 declined, but their values were not acceptable for the optimum Nile tilapia growth (Makori et al. 2017). The considerable decreases in the release of excreted nitrogen compounds caused by the increased feed digestion and nutrient absorption may cause these outcomes (Fabay et al. 2022). OAs markedly improved water quality by reducing nitrogen compounds excretion (NH3 and NO2). According to earlier research, adding OAs to the diets of gilthead seabream (Sparus aurata) and Nile tilapia decreased nitrogen compound excretion and improved water quality in pond water (Abdel-Tawwab et al. 2022; El-Dakar et al. 2022).

The growth performance decreased in the PC group after 30 and 75 days of the trial compared to the control group with a lower SR (%). The higher deposition of fish wastes and metabolites and the reduced oxygen level from unchanged water tanks are the causes of the slower growth (Katsika et al. 2021). Since oxygen is essential for the production of energy in aerobic metabolic activities, a decrease in DO availability may, over time, limit the use of dietary energy for growth and metabolic processes. This finding follows a prior study (Obirikorang et al. 2019), which found that fish in culture can experience persistent hypoxia, impairing their ability to consume food and grow.

Dietary OAs (LA) improved these indices and augmented SR except for the FA and COM1. The highest values were obvious in the LA1 group. The positive effect of dietary LA can be attributed to their ability to reduce pH in the fish gastrointestinal tract, stimulating pepsin activity, thus increasing protein metabolism and mineral intake (Asriqah et al. 2018). The lowered pH inhibits the pathogenic bacteria and reduces the immune system’s involvement in consuming dietary proteins to form antibodies to combat the pathogenic bacteria, allowing this protein to contribute to muscle development (Fabay et al. 2022). Furthermore, OAs can promote the colonization of beneficial microflora in the gastrointestinal tract and nutrient utilization and absorption (Busti et al. 2020). Moreover, the improved intestinal histomorphometric indices as observed in our findings by dietary LA which indicated the improved absorption capacity of nutrients can be another rationale. Therefore, employing OAs especially LA in this circumstance will undoubtedly increase muscle mass and growth in the fish (Sobhy et al. 2018). Similar outcomes reported the growth and survival-enhancing effects of the dietary OAs, including LA in fishes (Pelusio et al. 2020; da Silva et al. 2023).

Concerning body composition, the crude protein and ash were positively affected by adding dietary OAs (LA and COM). This may be attributed to that OAs improve the fish digestive system to utilize dietary protein and mineral absorption (Chen et al. 2024). In the same trend, Soltan et al. (2017) and Omosowone et al. (2018) observed an enhancement in the crude protein value of Nile tilapia by dietary OAs.

Hemato-biochemical measurements are significant biomarkers for improper environmental circumstances (Hastuti and Subandiyono 2018). The findings revealed that hematological variables of the PC group and OAs-fed groups exhibited a decline. These variables are vital indicators of fish’s ability to breathe adequately and reflect the presence of anemia (Seibel et al. 2021). According to Hastuti and Subandiyono (2018), the hematological indices decrease when fish are exposed to stress. In contrast, the LA1 group recorded the highest values among these groups, demonstrating a significant improvement in general health. The enhancement could be related to LA supplementation that maximizes the liberation of vital minerals from the diet ingredients (Shafique et al. 2018). In addition, because of the entrance of LA into the gastrointestinal tract, LA may undergo dissociation, releasing lactate, an important substance for supplying energy to all body cells, including blood cells. This will elevate the number of blood cells and the Hb content (Lall 2022). Similar outcomes were retrieved by Hoseini et al. (2022) and Mirghaed et al. (2023).

Liver and renal function activities are considered crucial diagnostic indicators as they reflect the overall nutritional state and exposure to water pollutants in aquaculture (Shahid et al. 2022). It is observed that the fish kept without water exchange had lower levels of protein fractions and higher renal-hepatic biomarkers (creatinine, urea, ALT, and AST) levels. This outcome reflected a protein deficiency, altered hepato-renal function, and lowered immunity (Ghelichpour et al. 2017). The histopathological alterations observed in the liver in our study induced by the unchanged water confirmed the hepatic dysfunction. In addition, the increase in gluconeogenesis in response to the increased demand for energy under the stress situation may cause elevated hepatic enzymes (Rasal et al. 2020). Concurrently, Abd Elnabi et al. (2018) and Elshopakey et al. (2023) reported elevating in the hepato-renal indices of red tilapia (Oreochromis sp) and Nile tilapia exposed to stress (ammonia toxicity). Moreover, the OAs-fed fish exhibited decreases in these variables except for the FA group. The decrease in liver and kidney function indices implies improved health status, confirming the protective function of LA. Similarly, Sobhy et al. (2018) and Hussein et al. (2023) observed a reduction in the ALT and AST values of Nile tilapia and gilthead sea bream-fed OAs-supplemented diets.

Blood glucose levels are crucial physiological markers for evaluating fish health (Jeong et al. 2021). Significant elevation in the level of blood glucose was noted in the PC group and OAs-fed groups except in the LA and COM1 groups. This observation may be related to the rise in glucose oxidation during chronic stress to fulfill greater energy needs, as reported by Raposo de Magalhães et al. (2020). Furthermore, fish must spend more energy on homeostatic processes, which decreases growth performance (Goodrich and Clark 2023).

Nonspecific immunity in fish is a crucial defense mechanism (Mokhtar et al. 2023). In this study, the fish kept under unchanged water had lower immune response manifested by a decline in WBCs count, globulin, lysozyme, and total IgM. The minimal drops in these variables are commonly associated with stress (Elshopakey et al. 2023). These results were harmonious with the findings of Obirikorang et al. (2019) and Rajabiesterabadi et al. (2020). In contrast, dietary LA improved these variables, indicating the augmented immune, which could enhance a better SR (%). OAs can affect indigenous intestinal flora, which is required to develop the gut immune system (Busti et al. 2020). Similarly, Torrecillas et al. (2021) reported a modulated immune response of European sea bass (Dicentrarchus labrax) by dietary OAs.

Antioxidant enzymes are crucial in reducing oxidative stress in fish by preserving redox equilibrium and regulating imbalances in the antioxidant system (Ighodaro and Akinloye 2018). Our finding cleared a substantial increase in the MDA level and a decrease in the SOD and CAT activity of the PC group. This result may be due to the elevated NH3 from the unchanged water that could accumulate in aquatic animals’ tissues, causing the release of reactive oxygen species (ROS), which in turn could disrupt cell membranes and produce lipid peroxides (Zhao et al. 2021). Dietary LA improved these variables, indicating a reduction of oxidative stress and augmenting the antioxidant capacity that enhances fish health and welfare. Previous reports confirmed these results (Hoseini et al. 2022; Hoseini et al. 2023).

Investigating the health status and stress exposure requires a thorough evaluation of the histopathological changes in fish tissues (Georgieva et al. 2021). Here, we reported numerous histopathological lesions in the gills, liver, and intestine of the fish kept without water exchange. These lesions may be induced by the high NH3 level resulting from unchanged water, which causes intracellular ROS, disrupts intracellular balance, and ultimately results in DNA damage and cell death (Liu et al. 2021). Furthermore, dietary OAs additions did not protect the tissues from the impact of stress (non-water exchange) except for LA1. Romano et al. (2016) and Ebrahimi et al. (2017) observed a hepatic injury of Nile tilapia and red hybrid tilapia fed on a high dietary level of OAs. Similarly, moderate histopathological alterations in the liver, intestine, and kidney of Nile tilapia-fed OAs diets were reported (Rabea et al. 2023).

However, the dietary LA1 revealed moderate lesions that resulted from the enhanced immune-antioxidant parameters and reduction in the release of ROS which shielded the tissues from the impacts of stress. Similar histopathological impacts were exerted by dietary OAs as reported by da Silva et al. (2023) and Nimalan et al. (2023).

Conclusion

Based on our findings, the unchanged water has a number of major negative consequences on the health status of Nile tilapia. Among these include a decline in growth performance, body composition, antioxidant-immune response as well as alterations in the physiological and histopathological pictures. Dietary FA and COM did not protect fish from these hazards and gave worse results. In contrast, dietary additions of LA at a dose of 1% could alleviate these alterations and promote the health status of fish. As long as the water quality is within allowable bounds, OAs generally work well; however, when the quality of the water drops, they constitute another source of stress. In low-water situations, LA is thought to be less damaging than OAs. Future research is necessary to examine the impact on a molecular level and in different fish species.