Introduction

Increases in tilapia production (Oreochromis Spp.), the most commonly consumed freshwater aquaculture species, are needed to cover the increase in the human population (El-Sayed 2020; Ayyat et al. 2022). It is the first species bred in Egypt and the third in the world due to its fast development, excellent food utilization, and favorable edible qualities (Almarri et al. 2023; Ayyat et al. 2024). As a tropical warm-water fish, Nile tilapia (O. niloticus) can withstand a wide variety of temperatures (8–42 °C; Leonard and Skov 2022). It cannot develop well at temperatures beneath 18 °C and cannot endure at temperatures beneath 10 °C for almost a few days (Wu et al. 2019).

The vulnerability of tilapia to cold temperatures is an urgent economic concern, as it restricts growing seasons and causes enormous over-winter death (Liu et al. 2022). Cold water stress (CWS) is a prevalent cause of fish sickness and death, which has reduced the production of tropical aquaculture systems (Almarri et al. 2023). Low temperatures could increase cortisol levels, decrease metabolism, alter blood enzymes, and impair fish immunological responses (Lima de Almeida et al. 2019). During CWS, fish swim deeper into ponds, gather in huge groups, inhibit feeding, become sluggish, and could easily exposed to predation (Crawshaw et al. 2022). Low water temperatures may also reduce the digestibility of nutrients through decreasing digestion, lengthening gut transit time, and decreasing gastrointestinal emptying speed (Zhang et al. 2023). To satisfy the growing energy needs and metabolic changes, CWS-exposed fish require a considerable oxygen amount, resulting in stored electrons at particular sites of the respiratory chain, which drives the reactive oxygen species (ROS) generation (Cheng et al. 2018) and lowers the antioxidant enzymes activity (Yilmaz et al. 2021). Moreover, hypothermia suppresses or delays the production of important innate immune components in adult fish in response to pathogen mimics or pathogens, blocking proinflammatory or antiviral responses (Abram et al. 2017). Studies have found histological abnormalities in the gills (short secondary lamellae, thin gill filament tips, absence of secondary lamellae, and increased cartilage tissue in the gill filament) and livers (severe hepatocytes degeneration and necrosis and severe hemolysis and infiltration) of fish reared under CWS (Refaey et al. 2022).

The significance of dietary approaches in mitigating the adverse effects of CWS on various fish species has recently been explored (Ibrahim et al. 2019a; Peng et al. 2022). Alpha-lipoic acid (ALA) is a lipid and water-soluble antioxidant that can easily cross biological membranes (Li et al. 2021). It exhibits antioxidant effects by directly absorbing metal ions and free radicals and boosting intracellular glutathione (Rezaei Zonooz et al. 2021). ALA showed antioxidant activities in aquatic organisms like grass carp (Shi et al. 2017), Pacific white shrimp (Silva et al. 2018), and Nile tilapia (El-Houseiny et al. 2023). Dietary ALA also boosted grass carp growth, fatty acid β-oxidation, and lipolysis, enhanced protein deposition, augmented immunity and antioxidant effectiveness, mitigated the inflammatory response, and lessened lipid oxidative damage (Ou et al. 2023). ALA can reduce inflammation by inhibiting the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling (Liu et al. 2018). Furthermore, ALA has many metabolic advantages, including anti-obesity, insulin sensitization, and lipid-lowering properties (Xiong et al. 2022). ALA supplementation activates the lipid and carbohydrate metabolisms, increasing non-protein energy availability and decreasing protein breakdown as an energy source (Rifai et al. 2022a).

Therefore, this study aimed to determine the effect of ALA addition to the Nile tilapia diet on their survival, growth performance, feed utilization, intestinal enzyme activities, hematological parameters, serum biochemical and immune indices, behavioral response, and expression of stress, antioxidant, apoptosis, and inflammation-related genes under chronic CWS.

Materials and methods

Diet preparation

ALA (C8H14O2S2Thiotacid) was supplied by the Egyptian International Pharmaceutical Industries Company (10th of Ramadan City, Egypt). Two isoenergetic and isonitrogenous experimental diets, a control diet consisting of a basal diet and an ALA diet in which the basal diet was supplemented with 600 mg ALA/kg diet, were formulated in the Fish Research Unit, Faculty of Veterinary Medicine, Zagazig University to cover the nutrient requirements of Nile tilapia (NRC 2011). The feed components were finely powdered and well mixed before adding water to form a homogenous paste. The meals were minced using an electric meat mincer into small pellets (2.0 mm in diameter), then air-dried at room temperature, and stored in airtight plastic bags at 4 °C. Table 1 shows the proximate chemical analysis of the basal and ALA diets.

Table 1 Ingredients and proximate chemical analysis of the experimental diets (g/kg)
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The fish were fed the experimental nutritional regimen at a rate of 3% of their total biomass twice daily (8:00 am and 3:00 pm) for 60 days. The introduced food was changed every 2 weeks based on the alteration in fish weight, and the wasted diet was dried and deducted from the supplied feed for an exact measurement of feed intake.

Fish rearing and experimental design

The study was performed at Aquatic Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, Egypt. The Animal Ethics Committee of Zagazig University (ZU-IACUC) approved the experimental methodology with an approval number ZU-IACUC/2/F/276–2023. Nile tilapia (Oreochromis niloticus) (N = 240 with average initial weight 31.03 ± 0.07) were bought from the Fish Research Unit at Zagazig University’s Veterinary Medicine Faculty. The fish were placed in 80 × 40 × 30 cm glass aquaria that contained 60 L of dechlorinated tap water. Fish were subjected to a control diet for 2 weeks just before the start of the study. The water criteria were investigated throughout the experiment and remained within the prescribed ranges (pH = 7.1 ± 0.4, dissolved oxygen = 6.25 ± 0.5 mg/L, ammonia = 0.02 ± 0.001 mg/L, nitrite = 0.016 ± 0.001 mg/L). This study was carried out in December and January during the cold seasons. Throughout the experiment, the water temperature was less than 18 °C and adjusted by the heater to 18 °C and 25 °C for all study groups. Water was exchanged twice weekly with pre-warmed water, and the aquaria were cleaned regularly. The behaviors of fish were observed during the exposure period.

Fish were arbitrarily distributed into four quadruplicate groups (15 fish/replicate, N = 60 fish/group). CNT was fed a basal diet without any supplementation and reared at 25 °C. The ALA group was fed a basal diet containing 600 mg ALA/kg and reared at 25 °C. The selected ALA dietary level was chosen based on earlier studies on different species of fresh water fish, including hybrid tilapia (Xiong et al. 2012), grass carp (Shi et al. 2018b), and Nile tilapia (Lu et al. 2019a). The CWS group was fed on a basal diet and reared at 18 °C (Ibrahim et al. 2019b). The ALA + CWS group was fed a basal diet encompassing ALA and reared at 18 °C.

Growth performance parameters and survivability

Individually weighed fish from each replicate at the onset of the trial for estimating the starting body weight (IBW) and at the termination of the feeding period (60 days) to investigate the final body weight (FBW). The following formulas were used to determine the feed utilization and growth metrics, as well as the survival rate:

$$\begin{array}{c}\mathrm{Total\ weight\ gain\ }({\text{TWG}}) ({\text{g}}/{\text{fish}}) = (\mathrm{WT }-\mathrm{ WI})\mathrm{\ where\ WT}=\mathrm{ final\ fish\ weight\ }({\text{g}})\mathrm{\ and\ WI }=\mathrm{ initial\ fish\ weight }({\text{g}}).\\ \mathrm{BWG \% }= (\mathrm{FBW }-\mathrm{ IBW})/\mathrm{IBW }\times 100.\\ \begin{array}{c}\mathrm{Specific\ growth\ rate\ }({\text{SGR}}) (\mathrm{\% per\ day}) = 100\ times\ (\mathrm{ln\ WT }-\mathrm{ ln WI})/{\text{duration}}/{\text{day}}.\\ \begin{array}{c}\mathrm{Feed\ conversion\ ratio\ }({\text{FCR}}) =\mathrm{\ total\ feed\ intake\ }({\text{g}})/\mathrm{total\ gain\ }({\text{g}}).\\ \mathrm{Survival \% }= (\mathrm{number\ of\ remaining\ fish\ at\ the\ end\ of\ the\ day\ feeding\ period\ per\ group}/\mathrm{initial\ fish\ number\ per\ group}) \times 100.\end{array}\end{array}\end{array}$$

Blood and tissue sampling

After a feeding session and before collecting blood, the fish were starving for a single-day at the finish of the experiment. The fish was sedated with 100 mg/L of benzocaine (Neiffer and Stamper 2009) to reduce handling stress. Blood was taken from the caudal blood vessels of the fish without using any anticoagulant and centrifuged at 1750 × g for 15 min to separate the serum (three samples per replicate; 12 samples per group). The collected sera were used to assess some immune and biochemical indices. Another blood sample was drained with a 1-mL heparinized syringe for erythrogram and leukogram measurement. Then, the fish were aseptically necropsied, and the liver, spleen, and intestine specimens were collected for histological analysis. Intestinal enzyme activity (amylase, lipase, and protease) was measured in intestinal samples containing pyloric caeca. The liver, spleen, and intestine parts were taken and stored in 10% neutral buffered formalin for histological investigation. Another fish’s spleen and liver tissues were taken and rinsed in ice-cold phosphate-buffered saline before being stored at − 80 °C for gene expression analysis.

The chemical composition of fish body

Fish (three samples per replication; 12 samples per group) were taken randomly and stored at − 20 °C for proximate examination of the entire body. The estimated moisture, lipids, protein, and ash contents were determined using AOAC (2005) protocol.

Hematological analyses

Red blood cells (RBCs), total and differential white blood cell counts (WBCs), hemoglobin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were measured using an automatic cell counter (XE-2100 Sysmex, Kobe, Japan) in line with the protocol of Fitzgerald and Haylock (1998).

Biochemical assays

For assessing serum cholesterol and triglycerides, enzymatic colorimetric diagnostic kits (Biodiagnostic Co., Giza, Egypt, Cat. No. CH 12 20 and TR 20 30, receptively) were used. Likewise, total serum proteins, albumin, and glucose concentrations have been established utilizing the commercial colorimetric Biodiagnostic kits (Giza, Egypt) with Cat. No. TP 20 20, AB 10 10, and GL 13 20, respectively. Total globulins were identified by deducting albumin from total protein concentration. Serum levels of cortisol hormone were estimated using the ELISA commercial kit of MyBioSource Inc., San Diego, CA, USA (Cat. No MBS704055).

Serum immune indices

Complement 3 (C3, Catalogue No.; MBS281020) was measured using kits received from MyBioSource Co. (CA, USA) according to the manufacturer’s directives. The serum nitric oxide was estimated in line with the protocol of Bryan and Grisham (2007). The method of Ghareghanipoora et al. (2014) was used to assess serum lysozyme activity through lysing Micrococcus lysodeikticus (Sigma Co., USA). The technique was modified with M. lysodeikticus suspension (pH = 6.2, 0.2 mg/mL in 0.05 M PBS) for 5 min at 25 °C. The optical density was measured at 540 nm every minute for the next 5 min using a 5010 Photometer (BM Co. Germany). To determine the serum’s lysozyme concentration, a calibration curve was developed by serially diluting lyophilized chicken egg-white lysozyme (Sigma Co., USA).

Serum oxidants/antioxidants biomarkers and acetylcholine esterase (AChE) activity

Serum antioxidant enzymes activities, including superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT), were detected using commercial kits of Biodiagnostic Co., Cairo, Egypt (SD 25 21, GP 2524, and CA 25 17, respectively). Besides, reduced glutathione (GSH) and malondialdehyde (MDA) levels were quantified by Bio-diagnostic Co. (Giza, Egypt) kits (Cat. No. GR 25 11 and MD 25 29, respectively). Furthermore, using a commercial kit of MyBioSource Inc. (San Diego, CA, USA), serum acetylcholine esterase (AChE) activity was spectrophotometrically (Cat. No MBS280290) estimated at a 450-nm detection wavelength.

Determination of intestinal enzyme activities

The intestinal tissues were collected, placed on ice-cold dishes, rinsed with sterile saline, dried on filter paper, and then frozen at − 20 °C. Following Teflon homogenization, 100 mg of tissue was centrifuged at 6000 × g for 5 min at 4 °C in a container containing 1 mL of a buffer (pH = 7.0 containing 10 mM phosphate/20 mM tris). After centrifugation, the supernatants were collected and stored at − 80 °C until analysis. Commercial MyBioSource Inc. (San Diego, CA, USA) colorimetric kits (Giza, Egypt) were employed concerning the manufacturer’s instructions to assess the activity of lipase (Cat. No MBS2540464) and α amylase (Cat. MBS9719214) enzymes in the intestinal homogenates from different groups. The intestinal protease activity was estimated as per the protocol of Bezerra et al. (2005).

Behavior evaluation

The behavior measures were conducted throughout the study between 9:00 am and 3:00 pm hours using a stopwatch and a video camera. Throughout the experiment, all behavioral patterns were noted at (15-min intervals over 6 h each week. The following behavior patterns were identified: feeding behavior: the definite amount of food consumed during the feeding period (Khalil et al. 2022). Foraging behavior: looking for food (Ramadan et al. 2018). Eliminative behavior: Fish feces that have fallen or been hung from the anus (Ramadan et al. 2018). Surfacing behavior: measured by the incidence of fish air pipping adjacent to the water’s surface in aquariums with low dissolved oxygen (Noga 2010). Swimming behavior: refers to the observation of fish moving quickly or slowly without engaging in any movement at the aquarium surface, middle, or bottom, according to the protocol established by Chen et al. (2001). Aggressive behavior: According to Ramadan et al. (2018) protocol, several behaviors were observed to measure fish aggression, comprising the straight movement of one fish toward another (approach), the rate at which fish swim rapidly after one another (chasing), fishes escaping from the adversary by swimming (fleeing), the rate at which two fish stand with their mouths open against one another (mouth pushing), and all of a fish’s fins extend out or expand (spreading of fins). Resting behavior: Fish have wide eyes and are immobile (Aziz et al. 2022).

Histological evaluation of the liver, spleen, and intestine

For histological analysis, the intestines’ livers, spleens, and mid segments (three samples/replicate) were collected, trimmed, and fixed for 24 h in 10% neutral buffered formalin. Post-fixation, the tissue specimens were washed in distilled water, dehydrated by immersing in ascending grades of ethyl alcohol, cleared in two changes of dimethyl benzene, infiltrated, and embedded in paraffin wax, sectioned by Leica RM2125 RTS manual rotary microtome at 5-μm thick, and stained with Harris hematoxylin and eosin Y stains following the guides of Suvarna et al. (2018). The stained sections were examined microscopically, where any histological changes were recorded. The area fractions occupied by (1) melanomacrophage centers (MMCs) in the hepatic and splenic tissues, (2) cytoplasmic vacuolations in the hepatocytes, and (3) the areas not occupied by any cellular or extracellular elements in the splenic tissue were determined using the Java-based image processing software (Image J v1.53t) (Schneider et al. 2012). In addition, quantitative measurements of the intestinal villus width [(villus apical width + villus basal width)/2], villus height (from the tip to base), and villus surface area [(villus apical width + villus basal width)/2 × villus height] were determined in 10 complete, full-sized villi not showing any bending, mechanical damage, or fixation and/or processing artifacts following the guides of Wilson et al. (2018). All measurements on the intestinal villi were performed using the AmScope ToupView v4.8.15934 software (AmScope, Irvine, CA, USA). Finally, the results were presented as percentages (means ± SE).

Hepatic and splenic stress, apoptosis, antioxidant and inflammatory gene expression

The total RNA was extracted from 50 mg (liver and spleen) for the transcriptome study using 1 ml of Quiazol (Qiagen, Germany) (4 fish/replicate). The extracted RNA efficiency was assessed using the NanoDrop® ND-1000 UV–Vis Spectrophotometer (Thermo Scientific, Waltham, MA, USA), which analyzed the quantity of extracted total RNA at wavelengths 260 and 280. The extracted total RNA acceptable for gene expression was in the range (1.8–2). The cDNA280 was synthesized using a high-capacity reverse transcriptase kit from Applied Biosystem, Foster City, CA, USA (Khamis et al. 2021).

Caspase-3, heat shock protein 70 (hsp70), tumor protein p53 (p53), proliferating cell nuclear antigen (pcna), superoxide dismutase (sod), catalase (cat), interleukin-1 beta (il-1β), and tumor necrosis factor-α (tnf-α) RT-qPCR were assessed by the specified primers (Sangon Biotech, Beijing, China) as listed in Table 2. The sample was first heated to 95 °C for 10 min, then subjected to 40 cycles of melt curve analysis (denaturation for 10 s at 95 °C, annealing for 15 s at 60 °C, and extension for 15 s at 72 °C). Gene expression is often quantified by calculating the fold change in expression in relation to that of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (gapdh) (Schmittgen and Livak 2008).

Table 2 Oligonucleotide primer sequences and real-time PCR conditions
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Statistical analysis

Kolmogorov–Smirnov (Berger and Zhou 2014) and Levene’s tests (Nordstokke and Zumbo 2010) were used to check the data for normality and variance homogeneity, respectively. When normality assumptions were met, using IBM SPSS Statistics, version 21 (IBM; Armonk, NY, USA; Spss 2011), data were analyzed by one-way analysis of variance (ANOVA) to statistically define the variation between groups, followed by Tukey’s multiple range post hoc test for pairwise comparisons (Benjamini and Braun 2002). The data has been displayed as means ± SE for each group. At P < 0.05, mean differences were considered significant. Moreover, the GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA) was used for data presentation (Prism 2019).

Results

Clinical observation

Throughout the feeding trial, fish that reared at 25 °C and fed a basal diet or ALA-fortified diet displayed a normal appetite and normal swimming behavior during the experimental period. In contrast, fish groups that reared under 18 °C and fed on the control diet suffered from lethargy and anorexia, and reduced swimming behavior. In contrast, fish reared at 18 °C and fed a supplemented diet showed normal appetite and swimming behavior to a great extent.

Growth performance, feed utilization, and survival of fish

Table 3 shows that the CNT, ALA, and ALA + CWS groups had the best survival rates of any group, reaching 100%. The CWS group had the highest mortality rate (10%). In ALA-supplemented groups, there was a significant increase in final body weight (13.16%), weight gain (20.68%), weight gain % (17.46%), and SGR (11.19%) than the CNT group (P < 0.05) while feed intake value was comparable to the CNT. In contrast, FCR displayed a significant reduction (16.78%) in the ALA-supplemented group compared to CNT (P < 0.05).

Table 3 Effect of alpha-lipoic acid (ALA) supplementation on growth performance and whole body composition (% dry matter basis) of O. niloticus exposed to cold water stress (CWS) for 60 days
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A significant reduction in final body weight, weight gain, weight gain, feed intake, and SGR values was recorded in fish exposed to cold stress water at 18 °C for 60 days by 21.54%, 35.64%, 33.93%, 7.94%, and 25.17%, respectively, but a significant increase in FCR by 42.28% than the control fish. Remarkably, the ALA + CWS group elicited a significant (P < 0.05) and similar enhancement in final body weight, weight gain, weight gain %, feed intake, SGR, and FCR values than the CWS group and very relative to the CNT group (P < 0.05).

Whole body proximate composition

As shown in Table 3, dietary supplementation of ALA for fish reared at 25 °C significantly increases the crude protein content (6.60%) compared to the CNT group. The crude protein content decreased (9.72%) significantly in Nile tilapia-fed basal diet and reared at 18 °C compared to those of the CNT group (P < 0.05). Crud protein in the ALA + CWS group showed non-significant change (P > 0.05) compared to the CNT group. The groups had no statistically significant differences (P > 0.05) in the amounts of crude lipid, moisture, or ASH.

Hematological indices

Table 4 summarized the hematological changes in Nile tilapia when they were exposed to cold water stress and/or supplemented with ALA in their diet for 60 days. Compared with the control one, the counts of RBCs (12.99%), HB level (25.33%), PCV % (21.48%), MCV (9.70%), MCH (14.23%), and MCHC (4.97%) notably declined in the fish fed basal diet and reared at 18 °C (P < 0.05). These hematometric values characterize the anemia presented by the CWS-exposed fish as microcytic and hypochromic. On the contrary, the cold-stressed fish supplemented with ALA showed a substantial improvement in RBC count, HB level, PCV %, and MCV value compared with the CWS group and very similar to the CNT group. Moreover, MCH did not improve in the ALA + CWS group, continuing to be comparable to that detected in the CWS group.

Table 4 Effect of alpha-lipoic acid (ALA) supplementation on the hematophysiological parameters of O. niloticus exposed to cold water stress (CWS) for 60 days
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The WBCs, heterophils, lymphocytes, eosinophils, and monocytes counts showed a trend toward increase, but non-significant (P > 0.05), in the ALA group by 6.10%, 9.03%, 3.18%, 8.82%, and 10.14%, respectively, compared to the CNT group. Cold stress exposure at 18 °C for fish-fed basal diet in the CWS group elicited a significant decrease in all previous WBC counts by 12.15%, 12.74%, 8.27%, 27.27%, and 17.91%, respectively, compared to the CNT group (P < 0.05). In the ALA + CWS group, there were significant improvements in the total and differential WBC counts compared to the CWS group and very near the CNT group (P < 0.05).

Serum biochemical indicators

Blood protein profiles (total protein, albumin, and globulin); lipid profiles (cholesterol and triglycerides); and stress markers (cortisol and glucose) levels of Nile tilapia in response to cold water stress exposure and/or dietary supplementation with ALA for 60 days are presented in Table 5. ALA at dose concentrations of 600 mg/kg for fish reared at 25 °C significantly enhanced the total protein (18.49%), albumin (23.81%), and globulin (14.76%) in comparison with the CNT group (P < 0.05). On the other hand, a trend toward a decrease, but non-significant (P > 0.05), was recorded in total protein, albumin, and globulin in the CWS-exposed fish by 12.61%, 10.20%, and 14.76%, respectively, than the control group. On the other hand, a trend toward an increase of the protein profile but non-significant (P > 0.05) was recorded in the ALA + CWS compared to the CWS group.

Table 5 Effect of alpha-lipoic acid (ALA) supplementation on the serum biochemical parameters of O. niloticus exposed to cold water stress (CWS) for 60 days
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ALA supplementation (mg/kg) markedly decreased the levels of serum cholesterol (16.15%) and triglycerides (6.90%) of fish reared at 25 °C (P < 0.05). Relative to the control group, rearing at 18 °C with feeding a basal diet markedly increased serum cholesterol (22.69%) and triglycerides (22.03%) compared to the CNT group (P < 0.05). Compared with the CWS group, a significant decrease in cholesterol and triglyceride levels in serum was found in the ALA + CWS group (P < 0.05), similar to the CNT group.

As for serum glucose and cortisol, the ALA group revealed a marked decrease in serum glucose (23.35%) and cortisol (13.22%) concentrations compared with CNT, and all these values were significantly elevated (37.74%) and (52.65%), respectively, in the CWS group compared to CNT (P < 0.05). On the other hand, the ALA + CWS group displayed enhanced values of serum glucose and cortisol compared to the CWS group and near-to-control values (P < 0.05), as shown in Table 5.

Immunological parameters

Figure 1 demonstrates the changes in Nile tilapia’s immunological parameters in response to cold water stress exposure and/or dietary supplementation with ALA for 60 days. A marked elevation in the level of serum immune parameters (lysozymes (47.10%), complement 3 (44.58%), and nitric oxide (45.09%)) in the ALA group compared with the CNT one (P < 0.05). However, cold stress exposure in the CWS group revealed a significant decrease in these immune parameters (30.40%), (34.18%), and (32.68%), respectively, in comparison to the CNT group (P < 0.05). In contrast, the ALA + CWS group indicated better serum immune component values than the CWS group, nearly to control values (P < 0.05).

Fig. 1
figure 1

Immune parameters of Nile tilapia fed diets supplemented with 600 mg ALA/kg diet during 60 days and submitted to water temperature stress. CNT: Fish fed a basal diet without any supplementation at 25 °C. Alpha lipoic acid (ALA) group: Fish fed a basal diet supplemented with 600 mg ALA/kg diet at 25 °C. Cold water-stressed group (CWS): Fish fed on a basal diet and reared at 18 °C. ALA + CWS group: Fish fed a basal diet containing ALA and reared at 18 °C. Lysozyme (A), complement 3 (B), and nitric oxide (C). Values are expressed as means ± SE (N = 12/group). The bars with different superscripts differed significantly (P < 0.05, one-way ANOVA)

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Serum oxidative stress and AChE biomarkers

The alterations in serum oxidative stress markers of Nile tilapia after 60 days of exposure to cold water stress and/or dietary treatment with ALA are displayed in Fig. 2. Supplementing fish diets with ALA improved their antioxidant defenses. ALA group had the uppermost levels of antioxidants, including SOD (29.46%), CAT (48.58%), GPx (26.89%), and GSH (71.61%) but the lowest MDA levels (12.50%) relative to the CNT group (P < 0.05). Conversely, the only cold stress exposure at 18 °C elicited a significant reduction in antioxidants, including SOD (39.57%), CAT (40.37%), GPx (25.47%), and GSH (27.37%) (P < 0.05). In comparison, serum MDA levels were the highest (119.56%) among all fish groups. The ALA + CWS group significantly improved the changes in serum antioxidant variables and MDA levels compared to the CWS group and near the CNT group (P < 0.05). Compared to the non-cold stressed groups (CNT and ALA), the CWS group showed significantly lower AChE enzyme activity. The fish that consumed an ALA-rich diet during CWS exposure showed a significant (P < 0.05) increase in AChE activity compared to the CWS group, as presented in Table 5.

Fig. 2
figure 2

Serum oxidative/anti-oxidative status of Nile tilapia, fed diets supplemented with 600 mg ALA/kg diet during 60 days and submitted to water temperature stress. CNT: Fish fed a basal diet without any supplementation at 25 °C. Alpha lipoic acid (ALA) group: Fish fed a basal diet supplemented with 600 mg ALA /kg diet at 25 °C. Cold water-stressed group (CWS): Fish fed on a basal diet and reared at 18 °C. ALA + CWS group: Fish fed a basal diet containing ALA and reared at 18 °C. Superoxide dismutase (SOD) (A), catalase (CAT) (B), glutathione peroxidation (GPx) (C), reduced glutathione (GSH) (D), and malondialdehyde (MDA) (E). Values are expressed as means ± SE (N = 12/group). The bars with different superscripts differed significantly (P < 0.05, one-way ANOVA)

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Intestinal digestive enzymes’ activities

Changes in intestinal digestive enzyme activity of Nile tilapia exposed to cold water stress and/or supplemented with ALA for 60 days are depicted in Fig. 3. The α amylase (153.04%), lipase (74.83%), and protease (158.83%) activities increased significantly in Nile tilapia fed on basal diet supplemented with ALA. It was reared at 25 °C compared to those fed on the control diet (CNT group) (P < 0.05). Compared with the control one, these intestinal enzymes notably declined by 27.83%, 13.72%, and 26.87% in the fish-fed basal diet and reared at 18 °C (P < 0.05). Exposure to cold stress at 18 °C concurrently with ALA supplementation ameliorated the intestinal enzyme activities compared to the CWS group nearly to CNT values (P < 0.05).

Fig. 3
figure 3

Intestinal digestive enzymes of Nile tilapia fed diets supplemented with 600 mg ALA/kg diet during 60 days and submitted to water temperature stress. CNT: Fish fed a basal diet without any supplementation at 25 °C. Alpha lipoic acid (ALA) group: Fish fed a basal diet supplemented with 600 mg ALA/kg diet at 25 °C. Cold water-stressed group (CWS): Fish fed on a basal diet and reared at 18 °C. ALA + CWS group: Fish fed a basal diet containing ALA and reared at 18 °C. α-amylase (A), lipase (B), and protease (C). Values are expressed as means ± SE (N = 12/group). The bars with different superscripts differed significantly (P < 0.05, one-way ANOVA)

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Behavioral observations

The impacts of ALA food supplementation on O. niloticus behavioral performance parameters after 60 days of CWS exposure are shown in Table 6. Compared to the CNT group, there were significant (P < 0.05) decreases in feeding behavior, surfacing, and middle swimming behavior in the CWS groups. Moreover, CWS groups showed considerably (P < 0.05) lower rates of aggression indications (approach, chasing, mouth pushing, fleeing, and spreading of fins). But in contrast, to control fish, CWS groups revealed considerably (P < 0.05) higher rates of resting and bottom swimming behavior. On the other hand, compared to fish-fed non-fortified diets, adding ALA to O. niloticus diets significantly (P < 0.05) enhanced all behavioral indicators except for resting and bottom swimming behavior. Furthermore, all behavioral indicators are significantly affected (P < 0.05) by combining CWS and ALA feed supplementation.

Table 6 Effect of alpha-lipoic acid (ALA) supplementation on the behavioral responses of O. niloticus exposed to cold water stress (CWS) for 60 days
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Liver, spleen, and intestinal histopathology

The postmortem examination of the fish of different experimental groups did not reveal obvious PM changes except for the liver enlargement of CWS-exposed fish only. Normal histological pictures were seen in the examined hepatic tissue sections of all tilapias of the CNT group as displayed in Fig. 4A, with a non-significant increase in the area fractions occupied by both hepatocytes’ cytoplasmic vacuolations and melanomacrophage centers (MMCs) of all tilapias of the ALA group as shown in Fig. 4B. The basic effects of the cold stress on the hepatic tissue of the CWS group included a sharp reduction in the hepatocytes’ lipoidal cytoplasmic vacuolations with significant reductions in the sizes of the MMCs (Fig. 4C). Alpha lipoic acid supplementation moderately increased the lipoidal cytoplasmic vacuolations in the hepatocytes of the ALA + CWS group yet did not regain the normal values. Still, it significantly increased the areas occupied by MMCs compared to the CWS group (Fig. 4D).

Fig. 4
figure 4

A–D Representative light micrographs of the Hand E-stained hepatic tissue sections show normal histological pictures in the CNT (A) and ALA tilapias (B), with notable and moderate reductions in both hepatocytes’ cytoplasmic vacuolations and MMCs in the CWS (C), and ALA + CWS (D) tilapias, respectively. The scale bar equals 25 µm. E–H Representative light micrographs of the Hand E-stained splenic tissue sections show normal histological pictures in the CNT (E) and ALA tilapias (F), with notable and moderate reductions in both the cellular and MMCs in the CWS (G) and ALA + CWS (H) tilapias, respectively. The scale bar equals 25 µm. I–L Representative light micrographs of the Hand E-stained intestinal tissue sections show normal histological pictures in the CNT (I), and ALA tilapias (J), with significant reductions in the villus width, height, and surface areas, and the numbers of the mucosal-associated lymphocytes in both the CWS (K), and ALA + CWS (L) tilapias. The scale bar equals 100 µm. Symbols|black arrowhead; hepatocytes cytoplasmic vacuolation, black arrow; MMCs, blue arrowhead; mucosal-associated lymphocytes, green arrowhead; goblet cells. CNT: Fish fed a basal diet without any supplementation at 25 °C. Alpha lipoic acid (ALA) group: Fish fed a basal diet supplemented with 600 mg ALA/kg diet at 25 °C. Cold water-stressed group (CWS): Fish fed on a basal diet and reared at 18 °C. ALA + CWS group: Fish fed a basal diet containing ALA and reared at 18 °C. (N = 12/group)

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The spleens of the CNT (Fig. 4E) and the ALA (Fig. 4F) groups showed normal histological pictures without any histological differences. On the contrary, all splenic tissue sections of the CWS tilapias exhibited a substantial rise in the area fractions not occupied by cellular or extracellular elements due to notable depletion in the cellular elements, including the lymphoid, erythroid, and MMCs (Fig. 4G). The spleens of the ALA + CWS tilapias appeared much more cellular, with a hyperactivation to the MMCS compared to the CWS group (Fig. 4H).

The intestine of the CNT (Fig. 4I) and the ALA (Fig. 4J) groups showed normal histological pictures without significant histological differences between them. Still, the ALA group’s intestines showed more mucosal-associated lymphocytes and surface epithelial intermingled goblet cells. The intestines of the CWS tilapias (Fig. 4K) showed significant reductions in the villus width, villus height, and villus surface areas compared to the CNT or ALA groups. In addition, marked reductions in the numbers of mucosal-associated lymphocytes were evident. The effects of ALA supplementation on the intestinal histology were very weak as a non-significant increase in the villus width, villus height, and villus surface areas were observed in the intestines of the ALA + CWS tilapias (Fig. 4L) compared to the CWS group. All the statistical data of the histological parameters in all groups’ hepatic, splenic, and intestinal tissues were summarized in Table 7.

Table 7 Effect of alpha-lipoic acid (ALA) supplementation on the Histological criteria of liver, spleen, and intestine of O. niloticus exposed to cold water stress (CWS) for 60 days
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Genes expression levels

Hepatic stress, apoptosis, antioxidant, and inflammatory gene expression

Stress- and apoptosis-related genes such as hsp70, caspase-3, p53, and pcna were significantly elevated in the hepatic tissues of CWS group by 2.51-, 2.82-, 1.84-, and 1.81-fold, respectively, compared to the hepatic tissues of the control ones as shown in Fig. 5. Dietary ALA supplementation significantly reduced this change, with expression levels of the hsp70, caspase-3, and pcna genes significantly lower in the ALA + CWS group compared to the CWS group, though not reaching the levels measured in the CNT, with fold changes of 1.41-, 1.49-, and 0.76-, respectively. Moreover, P53 gene expression did not improve in the ALA + CWS group (1.47-fold), comparable to that detected in the CWS group. In contrast, the cat gene was significantly downregulated (0.50-fold) in the CWS group compared to the CNT group. Also, the CAT mRNA expression levels were significantly increased (1.86-fold) in healthy fishes reared at 25 °C (P < 0.05) when fed an ALA diet. Although the CAT gene expression in the ALA + CWS group did not reach the levels seen in the CNT group (P < 0.05), it did increase by 1.21-fold when compared to the CWS group. SOD mRNA gene expression revealed non-significant change between all groups (P > 0.05).

Fig. 5
figure 5

Quantitative real-time PCR expression of hepatic stress, apoptosis, antioxidant and inflammation-related genes (heat shock protein 70 (HSP70), caspase 3, tumor protein p53 (P53), proliferating cell nuclear antigen (PCNA), superoxide dismutase (SOD), catalase (CAT), interleukin 1 beta (IL 1β) and tumor necrosis factor α (TNF α)) of Nile tilapia fed diets supplemented with 600 mg ALA/kg diet during 60 days and submitted to water temperature stress. CNT: Fish fed a basal diet without any supplementation at 25 °C. Alpha lipoic acid (ALA) group: Fish fed a basal diet supplemented with 600 mg ALA/kg diet at 25 °C. Cold water-stressed group (CWS): Fish fed on a basal diet and reared at 18 °C. ALA + CWS group: Fish fed a basal diet containing ALA and reared at 18 °C. Values are expressed as means ± SE (N = 12/group). The bars with different superscripts differed significantly (P < 0.05, one-way ANOVA)

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For inflammation-related genes, a significantly higher expression (P < 0.05) of il-1β (1.78- fold) and tnf-α (1.72- fold) were observed in the CWS group than in the CNT one. However, there was a substantial decrease in the expression of the genes mentioned above in the ALA + CWS group compared to the CWS group, with fold changes (P < 0.05) of 0.81- and 0.55- for il-1β and tnf-α, respectively, as presented in Fig. 5.

Splenic stress, apoptosis, antioxidant, and inflammatory gene expression

Genes involved with stress and apoptosis, including Hsp70, caspase-3, p53, and pcna, were significantly upregulated in the splenic tissues of Nile tilapia in the CWS group by 5.90-, 2.89-, 1.83- and 1.54- fold, respectively, than the control fish as shown in Fig. 6. Such change was significantly mitigated by dietary ALA supplementation, with expression levels of the caspase-3, hsp70, and p53 genes being significantly decreased in ALA + CWS group than the CWS group, though not reaching the levels dignified in CNT with fold changes 2.61-, 1.56- and 0.98-, respectively (P < 0.05). Interestingly, dietary ALA significantly decreased PCNA mRNA gene expression either in control fish reared at 25 °C (0.38-fold) or in cold-stressed fish reared at 18 °C (0.40-fold) compared to the CNT group (P < 0.05).

Fig. 6
figure 6

Quantitative real-time PCR expression of splenic stress, apoptosis, antioxidant and inflammation-related genes (heat shock protein 70 (HSP70), caspase 3, tumor protein p53 (P53), proliferating cell nuclear antigen (PCNA), superoxide dismutase (SOD), catalase (CAT), interleukin 1 beta (IL 1β) and tumor necrosis factor α (TNF α)) of Nile tilapia fed diets supplemented with 600 mg ALA/kg diet during 60 days and submitted to water temperature stress. CNT: Fish fed a basal diet without any supplementation at 25 °C. Alpha lipoic acid (ALA) group: Fish fed a basal diet supplemented with 600 mg ALA/kg diet at 25 °C. Cold water-stressed group (CWS): Fish fed on a basal diet and reared at 18 °C. ALA + CWS group: Fish fed a basal diet containing ALA and reared at 18 °C. Values are expressed as means ± SE (N = 12/group). The bars with different superscripts differed significantly (P < 0.05, one-way ANOVA)

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Antioxidant enzyme (sod and cat) mRNA expression values were significantly reduced in the CWS group (0.59-fold and 0.66-fold, respectively) compared to the CNT group (P < 0.05). Whereas, in the fish-fed ALA-containing diet group, these values were significantly elevated in healthy fishes reared at 25 °C (3.41- and 2.91-fold, respectively) (Fig. 6). Yet, the expression of the cat and sod genes significantly increased in ALA + CWS group (1.62- and 1.92-fold, respectively) than the CWS group, though not reaching the values of CNT group (P < 0.05) (Fig. 6).

For inflammation-related genes, a significantly higher expression (P < 0.05) of il-1β (1.71-fold) and tnf-α (1.52-fold) were observed in the CWS group than in the CNT one. Yet, the expression of the genes mentioned above significantly reduced in the ALA + CWS group compared to the CWS group, which were near to the values of the CNT group where (0.89-) and (0.59-) for il 1β and tnf-α, respectively, were fold changes (P < 0.05). Interestingly, dietary ALA significantly decreased TNF α mRNA gene expression in control fish reared at 25 °C (0.37-fold) compared to the CNT group (P < 0.05) (Fig. 6).

Discussion

The study findings demonstrated an elevated stress response after fish exposure to CWS (18 °C) for 60 days on the hemato-biochemical, immunological, behavioral, and molecular markers. The current study discovered a lower survival rate in Nile tilapia rearing at 18 °C for 60 days. The increased mortality rate caused by CWS could be due to fish immune system depression (Hassaan et al. 2019). Cold shock and stress can be fatal if fish cannot acclimatize or survive with temperature shifts, and temperature drops may result in lower survival rates (Reid et al. 2022). ALA dietary supplementation reduced CWS in fish maintained at 18 °C and boosted survival rate, possibly due to its increased antioxidant ability (Xiong et al. 2022). Additionally, Mahmoudi-Nezhad et al. (2021) found that including ALA in a prawn, diet can increase glycolysis, support aerobic oxidation, and result in the aerobic oxidation of carbohydrates, which can lessen damage caused by oxidative stress. This finding may also explain why ALA consumption is associated with a higher survival rate.

In the present study, Nile tilapia subjected to low temperatures (18 °C) demonstrated a substantial decrease in final body weight, weight gain %, feed intake, and SGR with increased FCR. This current finding is congruent with the of Refaey et al. (2023) findings, who attributed the drop to fish fasting during CWS and using stored energy to resist challenging conditions. This pushes the body to consume its conserved energy, which manifests as a loss of body weight (Zhu et al. 2020). Herein, the reduced SGR, the link between feeding rate and feeding efficiency, indicates that low temperatures may diminish the feeding axis (Pang et al. 2016). When ALA (600 mg/kg diet) was added, the fish had enhanced growth performance indices than the control. The enhanced growth rate could be attributable to ALA-induced lipid utilization, which allows for more energy provision for growth (Xu et al. 2019). Increased appetite with feed intake could be generated by ALA’s participation in adenosine monophosphate-activated protein kinase enzyme activation, which provided an alarm to the cells for energy uptake and hunger-full control (Rifai et al. 2022b).

Compared to control fish, fish-reared under CWS had significantly lower intestinal digestive enzymes, indicating the impact of CWS on the entire metabolic processes, resulting in suppression of enzyme activity (Li et al. 2023a). Instead, adding ALA to the diet minimized the reduction of digestive enzymes, showing that ALA played essential roles in metabolism adjustment and energy transformation (Serhiyenko et al. 2018). Besides, the intestinal histological examination exhibited notable decreases in villus width, height, and surface areas compared to the CNT or ALA groups. Similarly, Xing et al. (2019) showed that CWS might alter the integrity of the intestinal mucus layer and disrupt the mucosal mechanical barrier of juvenile Trachinotus ovatus intestinal tissue, indicating CWS-induced deficits in body metabolism (Liu et al. 2022).

The current findings demonstrated a considerable drop in the crude protein of Nile tilapia reared under CWS, which explained alterations in conserved energy mobilization and reduced growth rate (Hossain et al. 2018). Oppositely, the consumption of ALA counteracted the decrease in crude protein induced by CWS in the current study. Similarly, Xu et al. (2019) demonstrated that genetically enhanced farmed tilapia fed various levels of ALA (600, 900, 1200, and 2400 mg/kg diet) significantly increased crude protein contents. In this regard, Shi et al. (2018a) discovered that ALA regulates the phosphorylated activated protein kinase and carnitine palmitoyl transferase 1α pathway to augment protein deposition.

In the present study, CWS induced apparent microcytic and hypochromic anemia. The alterations in hematological parameters were confirmed by splenic histopathological findings, which revealed a significant increase in the area fractions that were not occupied by any cellular or extracellular elements and notable depletion in the cellular elements, including the lymphoid, erythroid, and MMCs. In accordance with existing findings, CWS diminished splenic red blood cells in the spleen in the study of Liu et al. (2022). The same findings were previously reported by Jakiul Islam et al. (2021) in the European seabass, Dicentrarchus labrax. In this regard, CWS impairs erythropoiesis, resulting in decreased oxygen transport capability for organism tissue, and juvenile tilapia cannot keep the minimal number of RBCs required to ensure their health (Barros et al. 2009). Besides, CWS induces metabolism to reset to homeostasis rather than conservation, preventing nutrients from being acquired adequately for optimal HB production (Witeska 2015). Moreover, regarding the detected leukopenia, a possible explanation could be that during CWS, circulation was retarded, and consequently, leukocyte release from the myeloid sinuses into the bloodstream was hampered (Teległów et al. 2022). On the contrary, ALA dietary addition ameliorated CWS-induced hematological alterations in the present study, which is compatible with former studies that demonstrated that ALA significantly elevated Hb concentration and serum iron level (Elewa et al. 2020; Abdel Hamid et al. 2022).

Herein, CWS promoted immunosuppression in Nile tilapia, as evidenced by decreased WBC count, lysozyme, complement 3, and nitric oxide levels. Chronic stress and its key signaling mediator, cortisol, have been thought to decrease immunity (Shepherd et al. 2018; Ayyat et al. 2020). Similarly, decreased lysozyme activities were recorded in O. mossambicus (Ndong et al. 2007) and European seabass (Jakiul Islam et al. 2021) during CWS. The immunostimulant action of ALA may have improved those immunological measures in Nile tilapia grown at 18 °C and fed ALA-supplemented diets. ALA influences the immune system directly and indirectly (Liu et al. 2019). In Lu et al. (2019b) research on Nile tilapia dietary, ALA significantly increased lysozyme activity. Likewise, the ALA group’s total protein, globulin, and albumin contents were significantly higher, which could be attributable to ALA supplementation improving immune function (Ou et al. 2023). Similarly, ALA dietary supplementation in commercial broilers favorably increased total protein, albumin, and globulin (Sakr et al. 2020). The authors of the earlier study correlated the increased serum protein profile with increased body weight gain and enhanced liver function as the organ that synthesizes proteins. Moreover, ALA has been recorded to decrease urinary protein excretion (Kang et al. 2008).

The current study found a considerable rise in total cholesterol and triglycerides in CWS fish, indicating increased energy demands to cope with repair processes (Hu et al. 2022). Under CWS, a rise in total cholesterol has been documented in Lateolabrax maculatus to act as a critical energy source (Wang et al. 2022). Similarly to these findings, cold stress raised triglyceride content in a genetically improved farmed tilapia strain of Nile tilapia (Liu et al. 2011). On the contrary, fish fed an ALA-supplemented diet showed accelerated lipid metabolism and reduced lipid production. According to Xu et al. (2019), dietary ALA enhanced lipolysis dose-dependently by increasing mRNA expression of adipose triglyceride lipase, a rate-limiting enzyme in lipolysis (Li et al. 2018).

In the current investigation, the higher serum glucose and cortisol levels of fish cultivated under CWS and fed a basal diet may be related to thermal adaptation to decreased enzymatic response rate (Dellagostin et al. 2022). It may also reveal a greater demand for energy to combat the effects of stress (Sun et al. 2019). In line with Rotllant et al. (2001), a drop in water temperature alters the pituitary-interrenal axis in gilthead seabream, causing cortisol release. In contrast, dietary ALA also considerably reduced the harmful effects of CWS on Nile tilapia blood glucose and cortisol. The lowering of serum glucose by ALA suggests that ALA increases glycolysis and the conversion of pyruvate to acetyl-CoA while not affecting gluconeogenesis. Besides, dietary ALA has been shown to lower serum cortisol levels (Kim et al. 2015).

According to the present findings, CWS decreased SOD, CAT, GPx, and GSH activity but increased MDA concentration in the serum of CWS-exposed fish. Several recent reports verified that fish’s antioxidant defense system was severely disrupted by CWS, which may have altered SOD, CAT, GSH-Px, and MDA-related enzyme activities and content (Li et al. 2023b; Refaey et al. 2023). In contrast, ALA supplementation at 600 mg/kg improved antioxidant parameters in fish maintained at 25 °C and reversed changes in antioxidant markers caused by cold stress at 18 °C. This suggests that ALA can protect against CWS-induced oxidative stress by increasing antioxidant enzyme activities, replenishing endogenous antioxidants, and scavenging excess ROS (Li et al. 2021). Similarly, the higher antioxidant capacity in genetically modified farmed tilapia-fed ALA diets can be attributed to its ability to suppress ROS production (Xu et al. 2019).

Fish exposed to CWS in the current experiment showed a marked fall in AChE activity, feeding, middle swimming, and aggression, indicating decreased food consumption and activity. CWS-induced decrease in fish activity in the ecosystem may make it easier for predators to catch fish larvae (Crawshaw et al. 2022). Additionally, the decreased activity and altered food-seeking behavior may harm their fitness and growth (Said et al. 2020). A shift in water temperature may influence the aggressive behaviors of fish species, as a decrease in water temperature is followed by a decrease in all kinds of aggressive behavior (Brandão et al. 2018). Moreover, it was noticed that the brain’s AChE-specific activity significantly decreased when tilapia fish were exposed to CWS (Hassan et al. 2013). Due to dietary ALA’s potential to lessen the oxidative stress caused by cold, AChE levels and behavioral responses in fish exposed to CWS were improved. By activating choline acetyltransferase and increasing glucose absorption, ALA promotes the production of acetylcholine (ACh) and provides more acetyl-CoA for ACh synthesis (Maczurek et al. 2008).

The current results revealed that cold stress exposure at 18 °C promoted upregulation of hepatic and splenic HSP70, caspase 3, P53, PCNA, IL 1β, and TNF-α with downregulation of hepatic and splenic SOD and CAT gene expression. Comparably, Mladineo and Block (2009) demonstrated the HSP70 upregulation in Bluefin tuna splenic tissue reared at 14 °C (Iwata et al. 2007). CWS-induced mitochondrial failure significantly induces mitochondria-mediated apoptosis and necrosis (Chapa-Dubocq et al. 2018). CWS-induced apoptotic gene patterns resulted in activating the initiator Caspase 9, followed by the proteolytic maturation of the executioner Caspase 3 (Han et al. 2022). Low temperature promoted apoptosis in puffer fish and grouper livers by upregulating caspase 3, 9, and p53 (Sun et al. 2019). P53 is easily triggered by lots of stress signals, particularly temperature fluctuations in aquatic animals (Qian et al. 2020). Furthermore, p53 can enhance apoptosis by upregulating pro-apoptosis genes (Perfettini et al. 2004). PCNA is a well-known protein that interacts with various enzymes and regulatory proteins to repair DNA in various pathological circumstances (Mailand et al. 2013). The augmented PCNA mRNA in cold-exposed fish muscle tissue could indicate a mechanism to offset muscle catabolism caused by skeletal muscle restructuring (Weber and Bosworth 2005). Similarly, cold shock altered the activity and mRNA expression of antioxidant genes, causing oxidative damage in zebrafish’s liver, gills, and brain (Wu et al. 2015). The present study has shown an increase in tnf-α and il-1β gene expression in the liver and spleen of fish exposed to cold conditions, consistent with Dellagostin et al. (2022). IL-1β has multiple functions within the inflammatory process (Dinarello 2009), while TNF-α is responsible for inducing critical cellular responses ranging from cellular proliferation via the NF-κB pathway to apoptosis induction (Cook et al. 2018).

Stress, apoptosis, and oxidative alterations in Nile tilapia were alleviated by ALA dietary supplementation, as seen by down-regulation of hepatic and splenic HSP70, caspase 3, P53, PCNA, IL-1β, and TNF-α expression, as well as up-regulation of SOD and CAT expression. ALA’s antioxidant, anti-apoptotic, and anti-inflammatory capabilities could explain these benefits. The ability of ALA to alleviate oxidative stress in fish caused by cold stress exposure may be linked to the activation of nuclear factor-erythroid factor 2 (Nrf2) signaling (Giuliani and Regoli 2014). The capacity of ALA to inhibit NF-κB signaling could explain its anti-inflammatory action (Li et al. 2022). ALA inhibited NF-κB signaling and reduced inflammatory responses in immature grass carp’s skin, head, kidney, and spleen against Aeromonas hydrophila (Liu et al. 2018). ALA’s ability to inhibit NF-κB is most likely connected to its ability to reduce inhibitor of nuclear factor kappa B degradation (Gomes and Negrato 2014).

Conclusion

This study concluded that cold exposure at 18 °C causes oxidative stress, cell apoptosis, and inflammation in Nile tilapia. However, dietary supplementation of ALA at 600 mg/kg meal can increase the fish’s development, digestion, and blood parameters, revealing its hemostatic efficacy on RBCs, HB, PCV, and total and differential WBCs. Furthermore, nutritional supplementation with ALA could boost fish innate immunity, antioxidant capability, and welfare, reducing cell apoptosis after cold stress. Moreover, dietary ALA may alleviate cold stress-induced oxidative damage, apoptosis, and inflammation in fish by modulating NF-κB signaling. As a result of ALA’s great antioxidant, anti-inflammatory, and anti-apoptotic activities, it could be recommended as a safe and ecologically acceptable aqua feed supplement (600 mg/kg diet) to alleviate the CSW-induced harmful effects in fish. Future research is required to assess the efficacy of ALA on the health and performance of different fish species, as well as its effect on various water stressors.