Special Issue:

Veterinary Medicine between Sustainable Development and Public Health to Confront Global Changes

Ameliorative Effect of Dietary Silybum marianum on the Exposure of Oreochromis niloticus to Sublethal Doses of Lead Acetate

Abd El-Alim F. Abd El-Alim1, Abdelhakeem El-Murr2*, Tahsein Hasan1

1Pharmacology Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44511, Egypt; 2Department of Aquatic Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44511, Egypt.

Received | August 14, 2024; Accepted | September 25, 2024; Published | October 07, 2024

*Correspondence | Abdelhakeem El-Murr, Department of Aquatic Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44511, Egypt; Email: [email protected]

Citation | Abd El-Alim FA, El-Murr A, Hasan T (2024). Ameliorative effect of dietary Silybum marianum on the exposure of Oreochromis niloticus to sublethal doses of lead acetate. Adv. Anim. Vet. Sci. 12(s1): 161-172.

DOI | https://dx.doi.org/10.17582/journal.aavs/2024/12.s1.161.172

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Oreochromis niloticus is one of the most important commercial species that has become a global staple in aquaculture in Egypt (Moyo and Rapatsa, 2021). Roughly 80% of all tilapia farmed worldwide is produced by the Oreochromis niloticus (Naylor et al., 2021).

Nile tilapia fish are considered a good model for study due to their widespread breeding in most countries due to their ability to withstand the climatic conditions of most African countries, in addition to providing abundant production to fill the deficit in animal protein (Chan et al., 2014). Oreochromis niloticus able to withstand a wide variety of temperatures and adverse environmental conditions implies that it could be a valuable bio-indicator of water contamination and toxicity studies (Henson et al., 2018; Yan et al., 2020).

The large-scale emissions of heavy metal into aquatic life results in interfering with several physiological, metabolic, and cellular processes in fish and upset the ecological balance (Arisekar et al., 2020). The higher concentrations pose a threat to aquatic environments, as the biological system is frequently unable to eliminate such toxins on its own rapidly (Abbas et al., 2021; Eroglu et al.,2015).

Lead (Pb), considered as a serious risk to aquatic life and is also the cause of the decline in ecological health in these habitats (Paul et al., 2017). It has been observed that prolonged exposure to the Pb toxicant increases the generation of reactive oxygen species (ROS), which results in oxidative damage and aberrant free radical growth (Patra et al., 2011; Kiran et al., 2021).

Fish physiology is disturbed, blood cells are damaged, and major changes to bodily tissues result from lead poisoning. Increased lead buildup in water sources may result in increased lead permeability in fish flesh, which may have harmful consequences on the health of consumers (Abbas et al., 2021). Thus, it would be crucial to identify effective strategies for lessening the detrimental impacts of heavy metals on aquatic life. A small number of research suggests that natural remedies such as herbal extracts and oils might lessen the harmful physiological effects of pollutants in fish (Dawood et al., 2020; Khafaga et al., 2020; Ahmadifar et al., 2021).

The therapeutic benefits of Silybum marianum have long been acknowledged as a medicinal herb (Křen and Walterová, 2005). Strong antioxidant activity is exhibited by silymarin, a polyphenolic molecule that was isolated from S. marianum (Kvasnička et al., 2003).

The mechanism of action is a reduction of the free radicles formed by toxins that damage the cell membranes (lipoperoxidation) and competitive inhibition through hepatocyte external cell membrane modification. Silymarin forms a complex that impedes the entrance of toxins into the interior of liver cells. Additionally, silymarin metabolically stimulates hepatic cells and activates the RNA synthesis of ribosomes to stimulate protein formation (Abou Zid, 2012). In addition to dietary S. marianum, a medicinal herb, boosted certain non-specific immune responses in fish (Alishahi et al., 2011).

Fish that are exposed to heavy metals showed a broad range of physiological and histological abnormalities due to oxidative stress (Chowdhury and Saikia, 2020). Biochemical alterations in the liver and blood are typically employed in toxicological investigations to assess the mode of action of toxicants (Chupani et al., 2017; Chupani et al., 2018). Hepatic and plasmatic enzymes offer a thorough picture of the state of immunity and were a good indicator of environmental stresses (Yousafzai et al., 2011).

Silymarin previously used in in aquaculture to mitigate the malathion toxicity in some fish species, Banaee et al. (2015), nickel oxide nanoparticles (Nazdar et al., 2018), Streptococcus agalactiae infection (Owatari et al., 2018). Lead is considered as water contaminant and up our knowledge there is no research article on the effect of SME on the O. niloticus exposed to lead toxicity. The current work conducted to study the effects of S. marianum on the growth, serum enzymes, cortisol, protein, stress markers, and hematological parameters of O. niloticus challenged with 1.45 mg of lead acetate L-1.

Materials And Methods

Detection Of Lead Acetate 96-H Lc50

From nursery ponds at the Aquaculture Research, Oreochromis niloticus fingerlings were obtained. In order to allow the fish to become used to the indoor wet laboratory, they were moved there and housed in fiberglass tanks for a period of 14 days. Fluorescent light were used to keep the photoperiod at 12–12 hours. As a source of lead acetate [Pb(C2H3O2)2], El-Gomhouria Company (Cairo, Egypt) was the supplier used. Dissolving lead acetate in deionized water produced newly generated Pb concentrations. Acclimatized fish (32.3–36.4 g) were divided into 10 fish per 100-L tank and subjected to 0.0, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 mg Pb/L for 96 hours in duplicate in order to calculate the half-lethal concentration (LC50) of Pb (Table 1 and 2). Fish mortality was tracked every day for ninety-six hours at each Pb concentration. Utilizing the techniques outlined by Behreus and Karber (1953), the 96-hour limit of detection (96-h-LC50) for lead was found to be 14.5 mg/L. In the current investigation, a sub-lethal Pb concentration (1/10 of the LC50) of 1.45 mg/L was employed. The methodology and management of the experiment followed the directive and national legislation pertaining to animal care, as fish were reduced to a minimum without compromising the aims of the research, and procedures eliminated any needless pain, suffering, or discomfort to the fish. In addition to, the protocol of work approved by the institutional animal cate and use committee, Zagazig university (ZU- IACUC/2/F/150/2024).

 

Table 1: Zero and hundred % mortalities in O. niloticus exposed to different concentration of lead acetate for 96 hours.

Conce (mg/L)	Time/day				Dead fish No.
	1	2	3	4
0	0	0	0	0	0
5	0	0	1	1	2
10	0	2	2	3	7
15	1	5	5	3	14
20	1	4	5	5	15
25	2	4	5	5	16
30	2	6	5	5	18
35	3	4	6	7	20
40	3	3	6	8	20
45	3	7	5	5	20
50	3	5	6	6	20

 

Table 2: Actual estimation of 96 hour LC50 of lead acetate in O. niloticus.

Conce (mg/L)	Dead fish Np. for 96h	a	b	axb	£ axb
0	0
5	2	5	1	5
10	7	5	4.5	22.5
15	14	5	10.5	52.5
20	15	5	14.5	72.5
25	16	5	15.5	77.5
30	18	5	17	85
35	20	5	19	95	410

 

96hrs LC50=highest dose -∑axb/n; 96 h LC50 of lead acetate =35-410/20=14.5 mg/L; a=constant factor between 2 successive doses; b=mean of dead fish between two groups; ∑axb=sum of axb

 

Feed Preparation

Fish meal (72% crude protein) (85), soybean meal (45% crude protein) (465), wheat bran (183), ground corn (100), corn oil (20), cod liver oil (20), mineral mixture (30), vitamin mixture (30), and starch (67) make up the basic diets in grammes per kilogram. To form a dough, the feed ingredients including S. marianum extract were combined with an appropriate quantity of water. The dough was ground into 1.5-mm pellets by first running it through a meat mincer. After that, the diets were produced and stored at 4 ◦C until they were needed.

Experimental Design

Fish weighing between 32.3 and 36.4g were placed into 100 L glass aquariums (10 fish/aquarium) and allowed to acclimate to the laboratory environment for 14 days Using an aquarium air pump and air-stones, compressed air was added to each tank. During the acclimatization period, fish were given the control meal three times a day until they were satisfied. For 20 days, acclimated fish were split into five groups of five duplicates. five experimental groups the negative control without any challenges in water and positive control water challenged with 1.45 mg lead acetate/liter for 20 days exposure both fed on the basal diet. The T1, T2, and T3: water challenged with 1.45 mg lead acetate/liter for 20 days exposure; T1: feed on basal diet supplemented with 50 mg/kg SME; T2: feed on basal diet supplied with 100 mg/kg SME; T3: feed on basal diet supplemented with 200 mg/kg SME, all of positive control. Every two days, the water in each tank was completely emptied out and refilled with fresh water that had the same Pb acetate. All water quality parameters are maintained according to Boyd (1984). The Lead concentrations were 0.17–0.19 and 8.01–8.07 μg/L in aquariums that were Pb-free and Pb-exposed, respectively. The following growth and feed consumption data were ascertained after the experimental trial by gathering, tallying, and group-weighing all the fish in each tank:

W2 − W1

Where;

W1 and W2 are the starting and ending weights, respectively, equals weight gain (g). Fish number in aquarium divided by total feed consumed equals feed consumption (g feed per fish).

Feed input / fish weight increase equals the feed conversion ratio (FCR). (Final fish number/Initial fish number) = 100 is the fish survival percentage.

Sampling Methods

Fish are fasted for twenty-four hours before their blood are sampled. To reduce handling stress on the fish tested, a 0.02 percent benzocaine solution was used to anesthetize the fish. Using 2-mL sterile hypodermic needles, blood samples were extracted from the caudal vein of three fish, per tank (15 fish/treatment). Two sections of the drawn blood samples were separated. For hematological analysis, a fraction was put into Eppendorf tubes filled with heparin. After being placed into Eppendorf tubes, the second part was centrifuged for 15 minutes at 1500 × g after clotting for 30 minutes at room temperature. Following collection, the serum was stored at 20 ◦C to facilitate further biochemical analyses. Fish were dissected after blood collection, liver tissues were collected, and the liver tissues were homogenised in cold physiological buffer saline (PBS) using a Potter-Elvehjem glass/Teflon homogenizer. After filtering and centrifuging for 10 minutes at 4 oC at 1600 × g, the resultant supernatant was stored at 20 oC for further analysis.

Haemato-Biochemical Assay

The counts of white blood cells (WBCs) and red blood cells (RBCs) were calculated using Brown’s (1980) techniques.

 

Table 3: Effect of Silybum marianum on growth performance, feed conversion ratio and feed intake of O. niloticus challenged with 1.45 mg lead acetate L-1.

Parameters	Time	-ve control	+ve control	T1 (50 mg)	T2 (100 mg)	T3 (200 mg)
Weight	Initial	35.21 ± 0.32	35.16 ± 0.30	35.33 ± 0.31	35.06 ± 0.31	35.40 ± 0.31
	20 days	56.44 ± 0.53a	52.40 ± 0.78b	56.73 ± 0.80a	56.20 ± 0.51a	56.65 ± 0.50a
	20 days	71.44 ± 1.32a	62.16 ± 1.04b	71.06 ± 0.90a	71.60 ± 1.24a	72.20 ± 0.51a
Weight gain	10 days	21.24 ± 0.78a	17.23 ± 1.06b	21.40 ± 1.11a	21.13 ± 0.80a	21.16 ± 0.90a
	20 days	15.54 ± 0.91a	9.76 ± 0.37b	14.33 ± 0.85a	15.40 ± 0.92a	15.63 ± 0.68a
FCR	10 days	1.22 ± 0.02b	1.34 ± 0.02a	1.23 ± 0.01b	1.23 ± 0.01b	1.22 ± 0.02b
	20 days	1.25 ± 0.06b	1.34 ± 0.02a	1.24 ± 0.02b	1.24 ± 0.03b	1.23 ± 0.01b
Feed intake	10 days	25.87 ± 1.11	23.11 ± 1.86	26.18 ± 1.53	26.06 ± 1.09	25.95 ± 0.96
	20 days	19.72 ± 1.16a	13.14 ± 0.27b	17.85 ± 0.78a	19.20 ± 1.12a	19.79 ± 0.66a

 

(a,b,c) different superscript letters in the same row indicate significant differences (p < 0.05).

 

Van Kampen and Zijlstra’s (1961) cyanmethemoglobin technique was used to measure hemoglobin (Hb). Fresh blood was placed in capillary glass tubes, spun for 10 minutes in a microhematocrit centrifuge, and the packed cell volume was measured to calculate the hematocrit (Ht) values right away after the sample (Brown, 1980). In accordance with Dacie and Lewis (1984), The mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC) of the generated blood indices were calculated.

As directed by the manufacturer, the biochemical, oxidative stress, and immunological biomarkers were identified using the diagnostic reagent kits (Biodiagnostic Co., Cairo, Egypt). Cortisol and blood glucose levels were measured using the procedures outlined in Foster and Dunn (1974) and Trindern (1969), respectively. The colorimetric techniques outlined by Wotton and Freeman (1982) and Henry (1964) were used to assess serum total protein (TP) and albumin (ALB), respectively. By deducting albumin from the total protein in the sample, serum globulin (GLO) was computed. Reitman and Frankel (1957) reported that the activity of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured colorimetrically.

Statistical Analysis

The normality and homogeneity of variances were assessed using Barlett’s test and significant differences between the various experimental groups, one-way ANOVA was employed. At P < 0.05, significant differences across groups were evaluated using Duncan’s test. The statistical software version 20 (SPSS, Richmond, VA, USA) was used for all statistical analyses (Dytham, 2011).

Results and Discussion

One-way ANOVA analysis demonstrated that exposure to lead acetate significantly decrease the weight (62.16 ± 1.04 g) and weight gain (9.76 ± 0.37 g) in positive control group on comparing with negative control and S. marianum treated groups (p< 0.05) after 20 days exposure. In addition, supplementing diets with S. marianum induced significant (P < 0.05) decreases in feed conversion ratio on treated groups T1 (1.24 ± 0.02), T2 (1.24 ± 0.03) and T3 (1.23 ± 0.01). The addition of S. marianum to the diet, induce a noticeable increase in the amount of feed that the fish consumed; however, after 20 days, exposure to lead acetate significantly (P < 0.05) decreased the amount of feed that the fish consumed (Table 3). When lead acetate exposure (T1, T2, and T3) was combined with feeding S. marianum to O. niloticus, the feed consumption was considerably higher than in the positive control. On the tenth day of exposure to lead acetate, there were not statistically significant (P > 0.05) changes in feed consumption among the various treatments (Table 3). One-way ANOVA analysis revealed that AST and ALT were notably (P < 0.05) higher due to Pb toxicity in control positive group. Meanwhile, the inclusion of 50 mg (T1), 100 mg (T2) and 200 mg (T3) of S. marianum in O. niloticus diets for Pb-exposed fish significantly restored their values in comparison to control (Table 4). The creatinine level in positive control group was 1.42 ± 0.14 mg/dL that significantly higher than (P< 0.05) negative control 0.72 ± 0.06 mg/dL and S. marianum treated groups T1 (0.92 ± 0.08 mg/dL), T2 (0.81 ± 0.07 mg/dL) and T3 (0.75 ± 0.06 mg/dL). The urea level in O. niloticus serum was significantly lower in all S. marianum treated groups T1 (31.65 ± 1.06 mg/dL), T2 (28.12 ± 0.89 mg/dL) and T3 (23.69 ± 0.98 mg/dL) in comparing to positive control group (38.62 ± 1.12 mg/dL). The finding in Table 4 demonstrated that the positive control group’s cortisol and glucose levels were 9.16 ± 0.89 ng/mL and 84.67 ± 2.97 mg/dL considerably (P < 0.05) higher than those of the negative control 4.49 ± 0.62 ng/mL and 76.16 ± 3.36 mg/dL, respectively. Giving O. niloticus a meal containing 100 mg or 200 mg S. marianum increases protein synthesis, whereas giving it a negative control group resulted in a substantial decrease in total protein, albumin, globulin,

 

Table 4: Effect of Silybum marianum on aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, urea, glucose and cortisol of O. niloticus challenged with 1.45 mg lead acetate L-1.

Parameters	-ve control	+ve control	T1 (50 mg)	T2 (100 mg)	T3 (200 mg)
AST(IU/ L)	49.66 ± 1.78c	72.45 ± 1.86a	52.17 ± 1.46b	48.45 ± 1.35bc	44.35 ± 1.54c
ALT(IU/ L)	35.21 ± 0.92c	57.36 ± 1.02a	42.61 ± 1.22b	38.14 ± 1.35b	36.27 ± 1.08c
Creatinine (mg/dL)	0.72 ± 0.06c	1.42 ± 0.14a	0.92 ± 0.08b	0.81 ± 0.07bc	0.75 ± 0.06c
Urea (mg/dL)	22.36 ± .82b	38.62 ± 1.12a	31.65 ± 1.06ab	28.12 ± 0.89ab	23.69 ± 0.98b
Glucose (mg/dL)	76.16 ± 3.36b	84.67 ± 2.97a	79.36 ± 2.65a	78.89 ± 2.16ab	77.28 ± 3.11b
Cortisol (ng/mL)	4.49 ± 0.62c	9.16 ± 0.89a	7.56 ± 0.85ab	5.79 ± 0.93b	4.87 ± 0.81c

 

(a,b,c) different superscript letters in the same row indicate significant differences (p < 0.05).

 

 

and albumin globulin ratio (Figure 1). When the positive group was exposed to lead acetate in water for 20 days, their levels of catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) significantly increased (P < 0.05) in comparison to the negative control group (Figure 2). Conversely, following exposure to 1.45 mg lead acetate/L, the activity of total antioxidant capacity (TAC) showed a significant reduction (P < 0.05). Feeding on diet containing 50, 100 and 200 mg S. marianum tolerates the effect of lead acetate decreasing oxidative stress and enhanced total antioxidant capacity (Figure 2). After 20 days of trial, the positive control showed substantially lower levels of RBCs, Hb, PCV, platelets, and MCV compared to the negative control (p<0.05). Meanwhile, there were no significant changes in MCH or MCHC across all groups. The S. marianum-treated groups are comparable to the negative control groups, particularly the group treated with 200 mg of S. marianum (Table 5). The count of WBCs significantly reduced (P < 0.05) in positive control group in comparison to negative control and all S. marianum treated groups as well as the differential leukocyte count (Table 6).

 

Table 5: Effect of Silybum marianum on heamatological parameters of O. niloticus challenged with 1.45mg lead acetate L-1.

Parameters	-ve control	+ve control	T1(50 mg)	T2(100 mg)	T3(200 mg)
RBCs count (×106/cm)	2.84 ± 0.26a	1.82 ± 0.19c	2.14 ± 0.21b	2.56 ± 0.23ab	2.75 ± 0.28a
Hb g/dl	8.52 ± 0.67a	5.89 ± 0.74c	6.75 ± 0.65b	7.36 ± 0.85ab	8.24 ± 0.59a
PCV%	25.65 ± 2.13a	17.23 ± 1.52c	20.21 ± 2.33c	22.78 ± 2.49c	24.28 ± 1.98c
platelets count (×103/cm)	71.45 ± 4.24a	37.36 ± 3.16c	42.38 ± 1.45bc	55.78 ± 2.93b	68.27 ± 2.75a
MCV (fl)	101.23 ±6.35a	87.29 ± 5.24b	88.49 ± 6.27ab	96.24 ± 5.22a	98.87 ± 5.47a
MCH (pg)	29.96 ± 3.14a	30.26 ± 2.34a	30.41 ± 3.24a	30.45 ± 2.39a	30.12 ± 2.94a
MCHC (g/dl)	32.24 ± 3.75a	32.25 ± 3.45a	31.42 ± 2.65a	30.58 ± 3.21a	31.42 ± 3.47a

 

(a,b,c) different superscript letters in the same row indicate significant differences (p < 0.05).

 

Bioaccumulation is a characteristic aspect of toxicity caused by Pb exposure. Toxic effects are induced in fish due to Pb exposure affecting its biochemical and physiological functions. Exposure pathway (dietary and waterborne), environmental factors (salt-water or fresh water) and Pb binding capacity with protein, SH and sulfur group decide accumulation pattern of Pb exposure. The accumulated lead in fish flesh is considered a serious issue to human health (Hussein et al., 2023). The 96 h-LC50 of lead acetate in the present study was 1.45 mg/L. In previous studies LC50 value of lead acetate for O. mossambicus is 20.03 mg/l (Meena et al., 2017), 18.7mg/l (Latif et al., 2013). For the same species of fish, different concentrations of the same heavy metal were obtained. This might be because to variations in sex, age, and eating habits, in addition to the experimental setup (Witeska et al., 1993; Elora et al., 2011). The exposure of O. niloticus to sub lethal dose of lead acetate for 20 days retarded its growth performance, as shown

 

Table 6: Effect of Silybum marianum on total and differential leukocyte count of O. niloticus challenged with 1.45 mg lead acetate L-1.

Parameters	-ve control	+ve control	T1(50 mg)	T2(100 mg)	T3(200 mg)
WBCs (×103/cm)	15.8 ± 0.41a	5.78 ± 0.57c	8.81 ± 0.62b	13.52 ± 0.67ab	14.26 ± 0.48a
Lymphocytes	10.80 ± 0.13a	3.09 ± 0.32b	4.84 ± 0.37b	9.02 ± 0.42ab	10.06 ± 0.41a
Heterophils	3.35 ± 0.11a	2.28 ± 0.21b	2.74 ± 0.25b	3.41 ± 0.26ab	2.96 ± 0.31a
Monocytes	0.86 ± 0.09a	0.22 ± 0.11b	0.33 ± 0.14b	0.67 ± 0.14b	0.80 ± 0.13a
Eosinophils	0.33 ± 0.03a	0.13 ± 0.03b	0.18 ± 0.11b	0.29 ± 0.09a	0.30 ± 0.07a
Basophils	0.16 ± 0.02a	0.06 ± 0.01b	0.09 ± 0.01b	0.13 ± 0.03a	0.14 ± 0.06a

 

(a,b,c) different superscript letters in the same row indicate significant differences (p < 0.05).

 

by decreased weight and weight gain and increased feed conversion ratio. These declines in fish performance might be linked to the harmful effects of lead poisoning on many metabolic processes in fish exposed to lead. According to Bengani et al. (2015), lead (Pb) is a very hazardous element that negatively impacts the biological functions of inebriated organisms. Regarding this, Ikeogu et al. (2016) recorded that fish exposed to lead poisoning had significant mortality rates and reduce development in some fish species. In addition, Kumar et al. (2021) recorded that Pb-exposed O. niloticus showed a significant reduction in growth when compared to the control group. Contrariwise, dietary S. marianum increased the growth rate as recorded in pervious works (Hassaan et al., 2019; Abdel-Latif et al., 2023). The digestion-related enzyme activities may be enhanced by the increased growth and feed intake following dietary supplementation with S. marianum levels (Abdel-Latif et al., 2023). When S. marianum is fed to fish together with Pb exposure, the harmful effects of Pb acetate on O. niloticus growth are retrieved. Dietary S. marianum may have a mitigating effect on lead acetate toxicity because of its high antioxidant content 92.25 and 123 mg/ kg dry weight, of flavonolignans, which activated the enzymatic system to accelerate protein synthesis (Banaee et al., 2011). Hassan et al. (2019) reported comparable results when S. marianum was used to treat Pb-induced toxicity in O. niloticus. In addition, fish fed a diet enriched with S. marianum seeds showed the lowest FCR in comparison to positive control. The FCR decreased in poultry and quail feed silymarin for 4 weeks (Khaleghipour et al., 2019). The obtained results might be explained by the presence of bioactive components in S. marianum, which affect protein retention and increase feed efficiency. The active components of plant extract might enhance the availability and digestibility of nutrients, leading to increased levels of protein synthesis and feed utilization (Citarasu, 2010). In line with this, silymarin extract was reported to have a positive impact on the growth performance and feed consumption efficiency of fish species (Jia et al., 2013; Xiao et al., 2017). Meanwhile, Carassius auratus growth performance and feed consumption were unaffected by the addition of silymarin extract (Yi et al., 2012). These disparities in the outcomes might be caused by various fish species, culture circumstances, or dietary components. ALT is dispersed throughout the hepatic cells and bile duct, whereas AST is often found in the mitochondria of hepatocytes. Fish with elevated blood AST and ALT levels may be exhibiting increased hepatic tissue production of enzymes or enzyme leakage through damaged plasma membranes (Yang and Chen, 2003). Accordingly, serum AST and ALT activity are employed as critical markers to represent the liver’s health and the fish’s activities (Zhai et al., 2014). They may also be used to evaluate the fish’s health and serve as stress markers (Satheeshkumar et al., 2010). In the present investigation, a better trend was seen in the liver cells of O. niloticus as serum ALT and AST activity reduced with increasing dietary level of S. marianum extract. Additionally, as previously stated by Kvasnicka et al. (2003) and Akrami et al. (2015), the lowest level of AST and ALT may be related to the strong antioxidant activity of S. marianum and their hepatoprotective properties, which led to prevent lipid peroxidation of cell membranes and ultimately suppress the release of liver damage enzymes like AST and ALT into plasma. According to Davila et al. (1989), naturally occurring antioxidant flavonoids such silybin and catechin increased liver function and strengthened its resistance to oxidative stress and tissue damage. Moreover, Banaee et al. (2011) found that include S. marianum extract in meals controlled the activities of ALT and AST in rainbow trout plasma. The action of SME attributed to increasing the regenerative ability of the liver cells by enhancing the synthesis of DNA and RNA, as silymarin has a steroid structure; Altering the structure of the hepatocyte external membrane, that prevents entrance of the xenobiotics into the cell (Karimi et al., 2011).

Lead exposure in current study increased the blood cortisol and blood glucose levels in O. niloticus, suggesting that the stress response to Pb poisoning is intensifying. Sayed et al. (2017) and Kumar et al. (2021) who reported that Pb exposure significantly raised the levels of cortisol and hyperglycemia in African catfish and O. niloticus, respectively made similar findings. Furthermore, S. marianum has hepatoprotective effects, maintaining hepatocytes from toxin-induced damage. Considering the significance of the liver in glucose metabolism, maintaining liver function in fish exposed to lead acetate by supplementing the diet with S. marianum may help maintain glucose homeostasis.

The waste product creatinine is created during muscle metabolism and is removed from the blood by the kidneys. As a result, increased plasma creatinine levels may be a sign of compromised renal function. In current study, dietary supplementation with S. marianum reduced serum urea and creatinine levels in serum. Fish exposed to lead acetate may experience oxidative stress, inflammation, and damage to their kidney tissues. These side effects may disrupt regular kidney function, which might have an impact on urea and uric acid metabolism and excretion. Purine metabolism produces uric acid, whereas urea is a consequence of protein metabolism (Banaee et al., 2011; Abdelkhalek et al., 2017).

Elevations in fish serum total protein, albumin, and globulin levels are often associated with a more robust innate immune response (Bernet et al., 2001). According to the data presented here, fish given all diets including S. marianum extract showed significant (P<0.05) enhancement of “total protein, albumin, and globulin.” This suggests that the rise in serum total protein level is likely closely related to the increase in protein synthesis in liver tissue. Fish have a robust immune system, as evidenced by the increased serum albumin content, which may be linked to the enhancing effect of silymarin flavonolignans on ribosome formation and stimulation of DNA and protein synthesis in liver tissues (El-Kamary et al., 2009; Akrami et al., 2015). According to Ahmadi et al. (2012), young Oncorhynchus mykiss with serum albumin levels greater than those of the control group were fed a diet supplemented with 0.1 g/ kg of S. marianum extract. The presence of antioxidant and radical-scavenging properties in silymarin promote its cytoprotective properties (Karimi et al. 2011). The inhibition and modification of cell transporters, pglycoprotein, estrogenic and nuclear receptors are among the putatively understood mechanisms of action of silymarin protection (Ardo et al., 2008).

It is well known that heavy metals cause oxidative stress in aquatic organisms by inhibiting the activity of antioxidant enzymes. This increased cellular generation of reactive oxygen species (ROS) causes the oxidation of biomolecules. Lead is a redox inactive heavy metal that causes oxidative damage by changing the functions of enzymes that contain thiols (Pinto et al., 2003). All aquatic creatures have antioxidant defense mechanisms to counteract the harmful effects of ROS (Lushchak, 2011). Antioxidant defense systems, such as antioxidant enzymes, avert tissue oxidative damage (Hoseinifar et al., 2020). Fish are commonly utilized as non-specific immunological indices due to the synthesis of superoxide dismutase (SOD) and catalase (CAT), both of which are important markers of a cell’s capacity to withstand oxidative damage (Shiau et al., 2015). In current study CAT, SOD and MDA significantly increased (P< 0.05) in control positive group on contrary TAC decreased in the same group responding to the oxidative stress induced by the lead The S. marianum treated groups T1, T2 and T3 were tolerated the oxidative stress due to lead exposure, which may be attributed to the active silymarin molecule in S. marianum seeds contains flavonoids and vitamin E, which have a highly effective scavenging effect on free radicals inside tissues (Doehmer et al., 2008). Banaee et al. (2011) state that silymarin has healing capabilities that include “antioxidant, anti-inflammatory, anti-carcinogenic, and anti-fibrotic properties.” It also affects the efficacy of cellular protein manufacturing, the liver’s decreased glutathione (GSH) concentration, and the cell membrane’s enhanced stability. Moreover, Chand et al. (2011) found that the active silymarin in S. marianum seeds significantly raises the oxidative status of the liver and directly impacts immune cells. Furthermore, Astuya et al. (2017) observed that the pro-inflammatory enzymes’ relative expression in salmonid cell lines was dramatically decreased by the phenolic compounds that were isolated from Pinus radiata bark. Other phenolic substances have been shown in comparable experiments to have favorable effects on SOD activity under situations of oxidative stress (Subash and Jayanthi, 2010). According to the current study, O. niloticus fed diets supplemented with S. marianum showed improvements in its erythrocyte and leucocyte counts, especially in the T2 and T3 groups, suggesting that S. marianum has immunostimulant qualities. Nearly similar results obtained in fish fed dietary silymarin showed improvements in blood parameters erythrocyte count, white blood cell count, hemoglobin, and packed cell volume (Sherif et al., 2023). Deficit in the antioxidant system is directly linked to low erythrocyte and haemoglobin levels. The alterations in the erythrocyte profile point to the body compensating for an oxygen shortage brought on by lead exposure-related gill damage. Haematological traits are commonly used to identify fish contaminated with metals. (Shah and associates, 1995; Drastichova and associates, 2004). Metals typically have an impact on fish hematological parameters through osmotic changes that cause hemoconcentrations, or an increase in blood cell concentration brought on by the loss of plasma or water from the bloodstream, or hem dilution, which is an increase in plasma volume and a decrease in red blood cell concentration in blood (Tort and Torres, 1988). The current investigation found that lead exposure lowered hematological parameters. A prior study found that exposure to lead acetate significantly reduced hemoglobin, packed cell volume, RBC count, WBC count, and lymphocytes, especially after the tenth week (Ilesanmi et al., 2022). The spleen is typically to blame for this alteration. Because the spleen functions as a powerful blood storage organ, storing blood cells during rest and releasing them into the bloodstream in response to different forms of stress (Yamamoto, 1988). According to Bano and Hasan (1990), exposure to lead causes spleen damage, which lowers fish hematological and immunological indices.

Conclusions and Recommendations

Lead acetate decreases the growth performance parameters, antioxidants capacity and immunological indicators. Meanwhile, increase liver enzymes (AST and ALT), cortisol, glucose, CAT, SOD, and MDA. The addition of dietary S. marianum extract plays in the protection of O. niloticus from the harmful effects of Pb poisoning in water and enhance immunological biomarkers, antioxidant capacity, and performance of fish. Future studies are needed for evaluation of of other potential physiological parameters and lead residue in fish flesh.

Acknowledgments

The authors thank faculty of veterinary medicine, Zagazig University for their support of this research. This work has not been published or submitted to other journals previously is included.

Author’s Contribution

AFA designed the experiment, AE, TH carry out experiment, wrote the manuscript, analyzed the data and reviewed the manuscript.

Conflict Of Interest

The authors have declared no conflict of interest.

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