J.Egypt.Ger.Zool. Vol.(63A): Comparative Physiology, 155-187.ISSN 1110-5321 The 19th International Conference 30 April-2May, Faculty of Science, Beni-Sueif University, July 2011.

Physiological studies on the antioxidant effect of lupine on oxidative stress in liver and muscle in the fish Oreochromis niloticus

exposed to neem oil

M Bassam Al-Salahy and Ashraf A El-BadawiZool. Dept., Fac. Sci., Assiut University1, Central Lab For Aquaculture Res

Abbassa Abo-Hammad, Sharkia2, Egypt.Abstract

This work aimed to study the toxicity of seed neem oil (NO) on oxidative stress in liver, white muscle and blood serum in the fish Oreochromis niloticus and the antioxidant effect of lupine supplementation (LS) against it. Healthy sixty six fish were selected. Two doses of neem oil; 1/20 LC50: 56.3 (NO1) and 1/10 LC50:112.5 PPM (NO2) and three periods of one, two and three weeks were used. The results showed that NO induced marked hyperglycemia and significant elevations in oxidative biomarker total peroxide as well as significant inhibition of antioxidants like catalase and superoxide dismutase activities in most experimental periods in the liver, white muscle and serum. In turn, administration of LS could abolish or at least, improve the adverse effect induced by NO exposure. This study suggests that sublethal doses of NO on fish led to generate oxidative damage in liver and white muscle. In addition, the antioxidant role of lupine seeds may be mainly due to its antihyperglycemic effect, as well as its contents of some natural antioxidants.

Key wards: Neem oil, fish, oxidative stress, Tilapia , lupine, muscle, liver.Introduction

Pesticides are directly used in agricultural field mainly to get red of pests and to improve growing of crops. These pesticides finally find their way into water channels and cause harm to aquatic flora and fauna. Due to the pollutant effect of chemical pesticides, organophosphates and organochlorides cause heavy environmental pollution , so there is trend worldwide to use herbal pesticides (biopesticides) like neem instead of the chemical ones. Neem, Azadirachta indica, provides many useful compounds that are used as pesticides and could be applied to protect stored seeds against insects. However, as the biopesticides neem started to widespread, some toxic effects were showed on animal and fish organs. Neem extract is less toxic to P. lineatus than other synthetic insecticides used in fish-farming (Winkaler and Santos 2007). The authors showed a decrease in liver catalase activity at all neem concentrations and the detoxifying enzyme glutathione-S-transferase was activated in fish exposed to the high dose of 5.0 g L− 1.accumpanied with tissue damage in gill and kidney. Extract of Azadirachta indica (neem) leaf induces apoptosis in rat oocytes cultured in vitro (Shail et al. 2006). In mice, also, crude ethanol extract of neem leaf induced mitosis disruptive changes in metaphase chromosomes of bone marrow cells on days 8, 15 and 35of treatment (Awasthy et al. 1999). Boeke et al. (2004) revealed that the non-aqueous extracts of neem appear to be the most toxic products.

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It has been showed that most sublethal dose of pesticides induced hyperglycemia in most different species of fish such as Labeo rohita (Cypermethrin: Das and Mukherjee 2003), Rhamdia quelen (Cypermethrin: Borges et al. 2007), Cyprinus carpio (2,4Diamin: Oruc and Uner 1999) and Clarias batrachus (malathion: Mukhopadhyay and Dehadrai 1980). Hyperglycemia induced by diabetic, pollutions or glucose infusion and glucose load are important factors to increase reactive oxygen species , oxidative stress and metabolic perturbation in fish and mammals (Al-Salahy 2005; Cuncio et al. 1995; Reddy et al. 2009 and Lai et al. 1995 ). In addition, hyperglycemia induced by glucose infusion causes hepatic oxidative stress and activates a low-grade systemic inflammation in rats in liver (Ling et al. 2003)

Van Heerden et al. (2004) found that the exposure of rainbow trout to copper developed hypoxia in gill tissues. Also, the dissolved oxygen content of water decreased with increasing doses of biopesticides (Radi et al. 1988; Debashri et al. 2007). Hypoxia enhanced the generation of TBARS in the carp liver and induced oxidative stress in the fish organs, generating peroxides (Lushchak et al. 2005; Al-Salahy 2006). However, tilapia species have a high tolerance to hypoxic and/or anoxic conditions (Ishibashi et al. 2005)

White muscle mass occupies approximately 60% of the total fish body weight (Ozório et al. 2005). White muscle in the fish contains few mitochondria resulting in prevalent anaerobic oxidation during hypoxia (DeZwann et al. 1991; Virani and Rees 2000 and Lushchak et al. 2005]. In general fish liver possesses a powerful antioxidant potential (Lushchak et al. 2001). The liver is the main organ for detoxification (Dutta et al. 1993). El-Sokkary et al. 1999 suggested that liver normally contain higher amounts of melatonin as antioxidant than do most other organs. The control levels of white muscle oxidative damage products were the lowest of all tissues tested in fish carp ( Lushchak et al. 2005).

Superoxide dismutase catalyzes dismutation superoxide of anion radical to hydrogen peroxide which detoxified by the CAT activity into water (Dimitrova et al. 1994) Catalase activity was significantly decreased in tissues : liver, muscle, gills and brain after 1,2,3 and 4 days of lethal and sublethal sodium cyanide treatment in the fish carp (David et al. 2008). On the other hand, Total peroxide includes hydrogen peroxide and other derivatives of peroxides, produced physiologically in organisms and occur in higher concentrations in some pathological conditions (Harma et al. 2003). Total peroxide measured by xylenol orange, induced in early stage of long chain of reactions, which ultimately leads to form lipid peroxidation as terminal products (Lushchak et al. 2005).

It is suggested that lupine seeds may have higher antioxidant activity in lipid-soluble substances (Rubio and Seiquer 2002 and Oomah et al. 2006) The digestion of legume protein resulted in high amount of arginine, aspartate and glycine. El-Missiry et al. (2004) found that the amino acid arginine could ameliorate the oxidative stress in alloxan-treated rats. Also, Alpha-, gamma- and delta-tocopherols were found in the lupine oil antioxidant activity was found both in the flours and in the hulls (Lampart-Szczapa et al. 2003). It was found that aqueous suspension of lupine seeds have a prophylactic effect against prolonged hyperglycemia and its damaging effect on the kidney of the fish Clarias gariepinus Al-Salahy and Mahmoud (2004).

This study aimed to evaluate the toxicity of neem seed oil on powerful antioxidant organ, liver and other organ of less containing antioxidants, white muscle the main bulk of the body mass. In addition, to evaluate antioxidant power of lupine seed supplementation against oxidative stress generated by neem oil in these organs,

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through measurements of both antioxidant enzymes (CAT and SOD) and destructive oxidative stress parameter, (TP) in the fish, Oreochromis niloticus.

Materials and MethodsHealthy sixty six fish Tilapia (Oreochromis niloticus) of both sexes weighing 31-37 g /fish (13 – 16 cm length) were collected from the nursery ponds of Central Laboratory for Aquaculture Research at Abbassa, Abo-Hammad, Sharkia, Egypt in month July 2009. On arrival at the laboratory, fishes were immediately released into special five glass tanks (40 x 70 x 60) containing tap water and then maintained there for 7 days for acclimatization condition. The fish groups were three. The first one was subdivided into three subgroups exposed to neem oil (NO) for one week: control, low dose (NO1) and high dose (NO2). The second group was also subdivided into three subgroups exposed to NO for two weeks: control, NO1 and NO2. The third group was subdivided into five subgroups exposed to NO for two weeks: control, NO1, NO2, NO1+ lupine supplementation (LS) and NO2 + LS. The number of each fish subgroup was six.

The average body weight and total length were 35.1 ± 4.2 g and 14.2 ± 1.6 cm respectively. Air compressor was used for oxygenation of water. Dissolved oxygen concentrations ranged from 6.1 to 6.6 mg L−1. The ambient water temperature range was 20–23 °C, pH range was 7.1–7.4. Portable oxygen meter (DO-980) and Hanna Instrument HI 2210 Benchtop PH meter W/Temperature Compensation meter, were used.

Fish were fed on artificial feed twice daily a total of 2% of mean body weight of dry pellets of 2.5 mm. Food ratio of dry weight composed of 60% protein, 18% fat, 10 % corn starch, 11% ashes.Plant materials

Lupine seeds (dose was 500 mg/kg wt) could improve the metabolic disturbances in Clarias lazera (C. gariepinus) treated with glucose and alloxan (Mahmoud and Al-Salahy 2005). Seeds of lupine (Lupinus termis) were washed, kept in the incubator at 37°C to dryness for 24 h and ground well , then lupine powder was added by 5 % to the paste of normal ratio of fish diet before dryness. This food containing lupine was prepared to be tested as antioxidant effect as well as against oxidative stress in fish treated with bioinsecticide; neem oil of different doses and durations.

Seed neem oil: We purchased seed neem oil from Trifolio-m company. The recorded LC50 of neem oil for the tilapia was 1124.6 PPM (Jacobson 1995). Therefore, the used neem oil doses used in this experiment were 1/10 LC50 (112.5 PPM: high dose) and 1/20 LC50 (56.3 PPM: low dose). Before use, the neem oil was emulsified by using emulsifier agent (SiSi-6) and the emulsifiable concentrate was 50%.

Design of the experiment

Design was conducted as followed table: Table 1: Design of fish groups with different treatments

For one week For two weeks For three weeksGroup1 Group2 Group3

Each SG was 8

SG1 SG2 SG3 SG1 SG2 SG3 SG1 SG2 SG3 SG4 SG5

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fishCont + + +NO1 + + +NO2 + + +NO1+LS +NO1+LS +

NO1: Neem oil of 56.3 PPM, NO2: Neem oil of 112.5 PPM, SG: SubgroupHealthy six fish were chosen from each SG. n = 6.

At the end of the experiment, blood sample was taken from the caudal peduncle by suction, then fish were sacrificed and were dissected. Samples of liver and white muscle (under the dorsal fin) were excised. Ten percentage homogenates (w/v) were made in phosphate buffer (pH 7.4) using homogenizer (model IKA- WERKE, D118 BASIC, Germany). Then, homogenates were centrifuged at 5,000 rpm for 15 min to separate the homogenate. Blood samples were left to coagulate and centrifuged at 3000 rpm. Separated serum was kept at -40 °C till used. All samples were 250 L-aliquotted into Eppindorff’s tubes and stored at -40 °C till used to avoid repeated freeze-thaw cycles in different assays, except aliquots of total peroxide were assayed quickly after homogenization of tissues for more accurate estimation.

Chemicals: Absolute ethanol, Ferrous sulphate, Butylated hydroxytoluene, NADPH, EDTA, catalase, hydrogen peroxide, HCL, H2SO4, xylenol orange, epinephrine folin-ciocateau phenol, and hydrogen peroxide (Fluka , Merk and Sigma-Aldrich Companies). All other chemicals were of the highest quality available.Biochemical assays:

Catalase activity was measured by method of Beers and Sizer (1952). The reaction mixture contained 50 mM phosphate buffer (pH 7.0) and 50 mM H2O2. The reaction rate was measured at 240 nm. Sigma catalase enzyme was used as standard. Superoxide dismutase (SOD) activity was determined based on its ability to inhibit the autoxidation of epinephrine in alkaline conditions as described by Misra and Fridovich (1972). Sigma SOD enzyme was used as standard. OD was measured at 480 nm. Total peroxide (TP) was assayed according to the method of Harma et al. (2005). The reagent was prepared as follows: 9.8 mg ammonium ferrous sulphate was added to 10 ml of 250 mM H2SO4. This solution was added to 90 ml absolute methanol containing 79.2 mg butylated hydroxytoluene, then 7.6 mg xylenol orange was dissolved. The absorbance was measured after incubation of homogenate sample with the reagent for 30 min at 560 nm. H2O2 was used as standard. Haemoglobin (Hb) content was also, assayed by cyanomethaemoglobin method (Jain 1986).Total protein in the tissue homogenates was measured using the folin reagent Lowry et al.(1951). All assays were performed in triplicate. Homogenizer (Yellow line Di18basic Germany) and cooling centrifuge (Mikro 200R Hettich Zentrifugen Germany) were used. Spectrophotometer, UNICAM, Helios Gamma, No. UVG 060609, England was used for all biochemical assays.

Statistical analysisThe data were expressed as mean + SEM. The results were analyzed

statistically using column statistics and one way ANOVA with Newman-Keuls Multiple comparison test as a post-test. These analyses were carried out using computer statistics prism 3.0 package (Graph pad software, Inc, San Diego. (A. USA). The minimum level of statistic significance was set at P < 0.05.

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ResultsLiver: Regarding to liver antioxidants, table 1 shows that biopesticide, neem

oil (NO) significantly decreased catalase (CAT) activity after exposure to low dose 56 ppm for one week (W)by 24% and to high dose (112 ppm) for two and three weeks by 34 and 50 % respectively. NO of the low dose significantly reduced activity of superoxide dismutase (SOD) at periods of one and three W , and high of NO significantly diminished SOD activity in the liver after two and three W by 32, 31, 49 and 36% (Table 1 and Fig 1: a, b). On the other hand, the destructive oxidative stress indicator, total peroxide showed marked rise by percentages ranged from 138 to 300% at all periods of the experiment with both doses except of the low dose at the first W which did not show any significant change (Table 2 and Fig 1:a).

At the third week, lupine supplementation (LS) significantly enhanced CAT activity in liver of the fish exposed to both doses low and high by 24 and 29 % vs control group, respectively in fish-exposed to NO (Table 1 and Fig 1: a, b). LS normalized liver SOD activity in the fish exposed to both doses of NO. Oxidative stress indicator TP was significantly reduced after treatment with LS in the fish exposed to both doses of NO in comparison with NO-treated fish showing improvement but TP level in fish treated with NO plus LS was not be normal. It is shown that percentage of rises vs control in liver of fish-exposed to NO decreased from 300 and 190 % to 51.5 and 116% after LS in NO-treated fish (Table 2 and Fig 1:a).

Muscle: The current data show that the low dose of the first W induced marked rise by 83% , while the high dose of the second and third W significantly decreased CAT activity by58 and 38 % respectively in white muscle (Table 1 and Fig 1: c, d). The muscle SOD activity shows marked rise in all periods and both doses by percentages ranged from 90 to 160 % (Table 1 and Fig 1: c, d). On the other hand, pronounced rise in TP level in muscle was showed in response to all periods and both doses by percentages ranged from 86.5 to 195 % (Table 2 and Fig 1:b).

At the third week, LS significantly counteracts the increase rate (in comparison to control) of TP level in muscle of fish treated with low and high dose of NO from 189 and 182 to 85.5 and 80.1% , respectively (Table 2 and Fig 1:b). There are significant differences between value of TP of LS plus NO group and that of both control and NO groups. In addition, LS recovered activities of both CAT and SOD in muscle of the fish –exposed to NO ( Table 1 and Fig 1: c, d).

Serum: CAT activity shows a significant rise in the first W by 79% followed by pronounced decrease in both second and third W by 24 and 37% , respectively, in response to the high dose, while low dose only decreased it in the third W by 32% in response to NO ( Table 1 and Fig 1: e). In turn, oxidative stress parameter, TP level shows significant rise in all period of high dose by 50.1, 149 and 130%, respectively (Table 2 and Fig 1:c).

Also, at the third week, LS significantly curtails the rise rate (in comparison to control) of TP level in serum of fish treated with NO of high dose from 130 to 64.8 %. There are significant differences between value of TP of LS plus NO group and that of both control and NO group of high dose . In addition, LS normalized the activities of both CAT and SOD in serum of the fish –exposed to NO ( Table 1 and Fig 1: f ).Glycemia: Table 3 and Fig 3 pointed out that both doses of NO significantly induced hyperglycemia, while treatment with LS in the fish –exposed to NO of both doses induced normal glycemia.

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Linear regression ( r2) analysis showed that most activities of antioxidant CAT and SOD were negatively correlated with the destructive indicator, TP in the liver and muscle. However, this correlation was weak between antioxidant enzymes and TP in blood serum (Table 4 and Figs 4-9). Also, Linear regression ( r2) analysis mostly revealed positive correlation between hyperglycemia and the oxidative stress biomarker, TP in the liver and muscle (Table 4 and Figs 10-12).

DiscussionIn the last decades, many studies confirmed the severe toxicity of chemical and

synthetic pesticide (organophosphates and organochlorides) on the environmental Safety on the animals particularly fish (Pena-Llopis et al 2003; Sweilum 2006 and El-Sayed et al 2007). These effects let to change the trends of researchers to look for a biopesticide of high safety instead of these chemicals [Boeke et al. 2004 ; Subapriya and Nagini 2005). Neem plant (Azadirachta indica) have been used, particularly as an antifeedant, antiattractant, or repellent (Sharma and Dhiman 1993). Azadirachta indica Syn, Melia Azadirachta, margosa of the family Meliaceae, are widely distributed in Asia, Africa and other tropical parts of the world. However, Boeke et al. (2004) revealed that the non-aqueous extracts of neem appear to be the most toxic products. The authors concluded that neem derived pesticides as an insecticide should not be discouraged. Moreover, the long exposure to low concentrations of the crude extract of neem (A. indica) delayed the growth of fish (Omoregie and Okpanachi 1992). Little literature was available concerning the effect of neem oil on oxidative stress in animals particularly fish.

Fish liver possesses a powerful antioxidant potential (Lushchak et al. 2001) and consider as the main organ for detoxification (Dutta et al. 1993). The alterations in liver due to toxicity impact are often associated with a degenerative necrotic condition (Olojo et al. 2005; Figueiredo-Fernandes et al. 2007). In the current study showed that neem oil (NO) induced significant decrease in both catalase (CAT) and superoxide dismutase (SOD) activities in liver by 24 to 50% and 31 to 49 % , respectively. The result showed that reducing effect of NO on CAT and SOD in liver in response to high dose was more than that of low one with high percentage recorded after three weeks.

The present study showed that NO caused significant increase in total peroxide (TP) by 138 to 300% with high percentage after three weeks of NO contamination. The reducing effect of NO on the antioxidant enzymes and the rise in the destructive oxidative stress parameter TP may be due to the toxicity of the NO resulted from the overproduction of reactive oxygen species (ROS) in liver tissue. Oxidative stress arises when there is an imbalance between radical-generating and radical-scavenging activity; it may therefore cause an increase in the formation of oxidation products (Gutteridge 1995).

The present study showed that NO exposure to the fish in general induced marked decrease in CAT activity by 38 to 58% and increase in SOD activity by 90 to 160% in white muscle. These effects were more potent in the first and second W and the reducing effect of high dose of NO on CAT activity in muscle was more obvious. In turn, NO induced significant increase in TP level in w muscle by percentages ranged from 85.5 to 195% with higher percentages in the first and third week of the experiment.

Current data showed that NO exposure to the fish in general induced pronounced decrease in CAT activity by 24 to 79% and increase in SOD activity by

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20 to 59% in serum. Similarly, SOD activity in control fish was highest in liver and progressively lower in muscle, the levels of lipid peroxides (LOOH) in control fish were highest in liver and lowest in muscle (Lushchak et al. 2005).

Serum TP level showed significant rise in response to both doses of NO by 50.1 to 149%. In spite of control values of antioxidant enzyme activities (CAT and SOD) and oxidative stress indicator (TP) in Oreochromis niloticus were higher in liver more than that of muscle and that of the later were higher than that of serum, the differences between the percentages of changes in response to NO were low. In general, the toxicity of the high dose may be more toxic due to its high effect in reducing activities of CAT and SOD and increasing TP in liver, muscle and serum. The depletion of SOD activity may be due the overproduction of superoxide anion while, reduced CAT may be due to overproduction of H2O2 and its consumption during detoxifying H2O2 into water.

Pesticide can influence body glucose homeostasis by several mechanisms including such as nitrosative stress, pancreatitis, inhibition of cholinesterase, stimulation of adrenal gland, and disturbance in metabolism of liver tryptophan (Rahimi and Abdollahi 2007). Most sublethal concentrations of pesticide induced hyperglycemia in different species of fish, such as carbaryl in Clarias batrachus (Jyothi and Narayan 1999), lindane in Anguilla anguilla (Ferrando and Andreu-Moliner 1991), Mystus vittatus Dalela et al. (1981) and Diamin induced hyperglycemia in Cyprinus carpio after exposure to concentrations 50 and 80 PPM for 1,2,3,4,15 and 30 days (Oruc and Uner 1999). Also, hyperglycemia was induced in fish Prochilodus lineatus exposed to sublethal dose of 2.5 g L− 1 and 5.0 g L− 1 neem leaf extract (Winkaler et al. 2007). On the other hand, hyperglycemia induced by glucose infusion causes hepatic oxidative stress and activates a low-grade systemic inflammation in rats in liver (Ling et al. 2003). Also, hyperglycemia is one of the important factors that causing overproduction of ROS inducing oxidative stress and lower antioxidants in tissue organs (Lai et al. 1995; Brownlee 2003). Also, NO reduces the oxygen uptake of fish fingerlings and causes mortality at a faster rate (Mondal et al. 2007). Hypoxia induced oxidative stress in fish (Lushchak et al. 2005). Based on these findings, the recoded hyperglycemia in the third week and probably hypoxia may be the main factors to induce overproduction of ROS causing oxidative stress and reduced antioxidants in the liver and muscle of Oreochromis niloticus exposed to NO especially resulted from the highest dose. In the current study, the negative correlations between antioxidant enzymes and TP as well as the positive correlation between hyperglycemia and the rise in TP in both liver and muscle in the fish exposed to NO, may confirm the role of this factor in the ROS generation and oxidative stresss. Seeds of lupine ( Lupinus termis) have a hypoglycemic action in diabetic animals (Abdel-Aal et al. 1993; Eskander and Won Jon 1995 ; Newairy et al. 2002) and in normal rats (Helmi et al. 1969; Abdel -Aal et al. 1993). Also, hypoglycemic effect of lupine was recorded in fish either treated by alloxan or loaded glucose Mahmoud and Al-Salahy (2005). In addition, lupine seeds have an ameliorating effect on glucose tolerance test in the fish Clarias gariepinus (Al-Salahy and Mahmoud 2004). Oomah et al. (2006) had suggested that lupine seeds might have higher antioxidant activity. Lupine seeds contain high amount of arginine, aspartate and glycine Oomah et al. (2006), Alpha-, gamma- and delta-tocopherols (Lampart-Szczapa et al. 2003), phenol and phospholipids (Tsaliki et al. 1999). Both arginine and tocopherol are considered as antioxidants (Kausalya and Nath 1998; Gupta et al. 2005) and may enhance lupine seeds to be free radical scavenger and more efficient as an antioxidant. The current

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study revealed that lupine supplementation (LS) normalized glycemic level. Based on these findings, the present work suggests that the ameliorating or normalizing effects of LS on the levels of destructive oxidative stress biomarker TP and antioxidant enzyme activities of CAT and SOD, may be associated mainly with its hypoglycemic effect in Oreochromis niloticus exposed to NO. Also, the hypoglycemic effect of lupine may improve glucose metabolism and probably increasing NADPH necessary for CAT activity which is consider as a powerful antioxidant in tissues. It has been demonstrated that NADPH protects catalase from inactivation (Gaetani et al. 1994), because each of the four monomers of catalase contains an NADPH-binding site necessary for enzymatic activity (Kirkman and Gaetani 1984; Kirkman et al. 1999). Hypoxia induced severe alterations in the liver and that antioxidant power of melatonin could exert beneficial role in restoring tissue alterations after subjection to hypoxia (El-Sokkary et al. 2006). Likewise, the antioxidant effect of lupine supplementation may increase the potency to resist the possible hypoxia resulted in response to NO exposure. In conclusion, this study suggest that sublethal doses of NO on fish generate oxidative damage in organ-containing powerful antioxidants like liver or organ-containing less antioxidants like white muscle. In turn, the antioxidant role of lupine seeds against NO induced oxidative stress in fish, Oreochromis niloticus, may be mainly due to its antihyperglycemic effect rather than to its contents of some antioxidants.References Abdel-Aal W.E., Abdel-Nabi I.M., Hanna R.A., Mahdy K.A., (1993) Biochemical

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العربى الملخص

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Table 1 Effect of lupine (L) on catalase (CAT) and superoxide dismutase (SOD) activities in liver, muscle and serum in the fish Oreochromis niloticus contaminated with neem oil (doses:NO1= 56 and NO2 =112 ppm ). بس جيد بشكل مراجع

Data are presented as means SEM. Columns in each period with different letters differ significantly (P < 0.05), while those with the same letters do not differ significantly. NO: neem oil, L: Lupine, n= 6

After one week After two weeks After three weeks

Cont NO1 NO2 Cont NO1 NO2 Cont. NO1 NO2 NO1+L NO2+L

Liver Cat activityU/min/mg protein

2.53a±0.11

1.91b ±0.13

2.14ab ±0.29

2.73a ±0.15

2.44ab±0.31

1.78bc ±0.25

2.51a ±0.16

2.39a ±0.27

1.25b ±0.10

3.10c ±0.12

3.25c ±0.23

liver SOD activityU/min/mg protein

4.31a±0.23

2.92b±0.20

4.94a±0.45 5.14a±

0.34

4.49a±0.43

2.64b±0.17 4.26a±

0.24

2.90b ±0.25

2.74b±0.27

3.94a±0.32

4.06a ±0.46

Muscle CAT activityU/min/mg protein

1.31a±0.06

2.41b±0.15

1.17a±0.02

1.59a±0.13

1.90ab±0.31

0.66b±0.09

1.49a±0.09

1.23a±0.11

0.92b±0.10

1.16a±0.08

1.34a±0.14

Muscle SOD activityU/min/mg protein

3.03a±0.56

6.85b±0.40

6.91b±0.90 3.53a±

0.65

8.66b±0.42

7.12b±0.89 2.49a ±

0.20

4.89b ±0.60

4.55b ±0.56

2.77a ±0.21

3.05a±0.44

Serum CAT activityU/min/mg protein

0.90a±0.15

0.96a±0.09

1.61b±0.07

1.26ab ±0.15

1.52a±0.17

0.95b±0.08 0.96a ±

0.12

0.65b ±0.07

0.60b±0.05

1.12a±0.11

0.87ab±0.05

Serum SOD activityU/min/mg protein

0.87 a±0.06

0.55b ±0.07

0.77a ±0.04

0.87ab ±0.07

0.75a ±0.03

1.04b±0.11 0.88a±

0.06

0.53bc±0.08

0.36c±0.03

0.73ab±0.08

0.79ab±0.09

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Table 2 Effect of lupine (L) on on total peroxide in liver ,muscle and serum in the fish Oreochromis niloticus contaminated with neem oil (doses:NO1= 56 and NO2 =112 ppm ).

After one week After two weeks After three weeks

Cont NO1 NO2 Cont NO1 NO2 Cont. NO1 NO2 NO1+L NO2+L Liver T Peroxide nMol/mg protein

16.58a ±0.92

20.11a±1.09

40.59b±7.97 15.80a±

0.93

46.25b±3.36

37.69b±8.70

17.48a±0.75

69.93b±5.74

50.86b±3.39

26.00c±2.60

37.79d±4.46

Muscle T peroxide nMol/mg protein

9.44a±1.34

28,88b±2,95

23,88b±1,90

14,04a±1,048

26,19b±2,643

27.19 b±1,806 11.44a±

1.38

33.16b±1.34

32,30b±3.46

19.74c±1.65

20.61c±1.31

Serum T peroxide nMol/mg protein

2.05a ±0.12

1.66a ±0.26

3.09b ±0.14

1.56a ±0.20

1.23a ±0.13

3.05b ±0.33

1.93a ±0.21

1.49a ±0.23

4.45b ±0.43

2.17a ±0.29

3.18c ±0.37

Data are presented as means SEM. Columns in each period with different letters differ significantly (P < 0.05), while those with the same letters do not differ significantly. NO: neem oil, L: Lupine, n= 6

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Table 3 Effect of lupine supplementation (LS) on blood sugar (glycemia) in the fish Oreochromis niloticus contaminated with neem oil (doses:NO1= 56 and NO2

=112 ppm ).

Control NO1 NO2 NO1+ LS NO2+ LS

Blood sugar mg/dL

16.00 ±1.378 38.00*± 2.550

28.60*± 1.030

23.00± 1.673

20.60± 1.435

Data are presented as means SEM. *: Significant at P<0.5.

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636637638639640641642643644645646647648649650651652653654655656657658659660661662663664665666667668669670

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Table4. Linear regression ( r2) analysis of paired data of individual in the fish Oreochromis niloticus exposed to NO and NO plus LS of TP in liver, muscle and

blood serum with either CAT and SOD activities in these organs or glycemic level.

Linear regression (r2) of: NO1 NO2 NO1+LS NO2+LS

Liver TP with CAT activity

r2=0.7456*P=0.0267NC

r2= 0.6856*P=0.0418NC

r2=0.9518**P=0.009NC

r2=0.7583*P=0.0240NC

Liver TP with SOD activity

r2=0.7105*P=0.0351NC

r2=0.7030*P=0.0370NC

r2=0.8412*P=0.0110NC

r2=0.7376*P=0.0285NC

Muscle TP with CAT activity

r2=0.7903*P=0.0178NC

r2=0.8797**P=0.0057NC

r2=0.8402*P=0.0111NC

r2=0.1198P=0.5016Non Sign

Muscle TP with SOD activity

r2=0.7123*P=0.0346NC

r2=0.7159*P=0.0337NC

r2=0.7385*P=0.0283NC

r2=0.3941P=0.1821Non Sign

Serum TP with CAT activity

r2=0.06667P=0.6200Non Sign

r2=0.3304P=0.2327Non Sign

r2=0.6615*P=0.0490NC

r2=0.4808P=0.1266

Serum TP with SOD activity

r2=0.2322P=0.1710Non Sign

r2=0.7585*P=0.0239NC

r2=0.9647***P=0.0005NC

r2=0.5006P=0.1158Non Sign

Liver TP with glycemia r2=0,8373*P=0,0105PC

r2=0,8784**P=0,0058PC

r2=0,7044*P=0,0367PC

r2=0,8134*P=0,0140PC

Muscle TP with glycemia

r2=0,4939P=0,1194Non Sign

r2=0,7395*P=0,0280PC

r2=0,7849*P=0,0188PC

r2=0,7614*P=0,0143PC

Serum TP with glycemia r2=0,0005P=0,9653Non Sign

r2=0,8757**P=0,0053PC

r2=0,02565P=0,7618Non Sign

r2=0,8189P=0,131Non Sign

Data are presented as means SEM. *: Significant at P<0.5.*: Significant at P<0.5, **: Significant at P< 0.01 and ***: Significant at P< 0.001LS: Lupine supplementation, NO: neem oil, NC: Negative correlation, PC: Positive correlation. , n=6.

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676677678679680681682683684685686687688689690691692693694695

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لالكسدة الترمسكمضاد تأثير على دراساتفسيولوجيةالبلطى اسماك وعضالت كبد فى االكسدة علىجهد

النيم بذور لزيت المعرضة النيلىالصلاحى. ¹ بسام محمد محمد . ـــ¹ د البدوى احمد اشرف ²د

جامعة- كلية – الحيوان علم قسم-1 اسيوط العلوم

بالعباسة -2 السمكية الثروة لبحوث المركزى الشرقية –المعملالملخص

جهد على النيم نبات بذور زيت سمية دراسة الى هدف العمل هذاسمكة فى الدم ومصل البيضاء والعضالت الكبد فى االكسدةالضافة لالكسدة المضاد التأثير دراسة وكذلك النيلى البلطى

المبيد لهذا المعرضة االسماك لعليقة بذورالترمس .مسحوقوهما بتركيزين االسماك معاملة من 1/20تم LC₅₀ (56 ppm) و

من 1/10 LC₅₀ ( 112 ppm). استخدمت مقسمة 66وقد سمكة . 11على احدث قد النيم بذور زيت ان النتائج اظهرت وقد مجموعة

جهد دليل مستوى فى وارتفاع الدم جلوكوز فى معنوية زيادةمستوى فى تثبيط حدث وكذلك الكلية البيروكسيدات وهو االكسدةاكسيد السوبر وانزيم الكتاليز انزيم نشاط مثل االكسدة مضادات

والعضالت الكبد من لكل التجربة فترات معظم فى ديسميوتيز . لعليقة الترمس مسحوق اضافة عند المقابل فى الدم ومصل

االقل على تحسن او اختفاء حدث النيم لزيت المعرضة االسماكاالكسدة جهد دالئل من كل على المبيد لهذا السلبى للتأثير

. ومن التجربة هذة فى المستخدمة لالعضاء االكسدة ومضاداتلمبيد مميتة التحت الجرعة ان نقترح ان يمكن الدراسه تلك خالل

الكبد من كل فى تأكسدى تلف توليد الى تؤدى النيم بذور زيت , ان الدراسة تقترح لذلك اضافة البلطى السماك البيضاء والعضالت

لعليقة المضافة الترمس بذور لمسحوق لالكسدة المضاد الدورالتأثير الى اساسا يرجع ان يمكن المبيد لهذا المعرضة االسماك

لما بالترمسوكذلك المعاملة عند الدم جلوكوز الرتفاع المضادلالكسدة المضادة الطبيعة ذات المواد بعض من .يحويه

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706707708709710711712713714715716717718719720721722723724725726727728729730731732733734735

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