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Kilka fish oil
a Vitamin A (as acetate) 3000 IU; vitamin D3, 1500 IU; menadione sodium bisulphite, 10; choline chloride, 2000; niacin, 50; riboflavin, 20; hpyridoxine, 10; thiamin mononitrate, 10; pantothenic acid, 40; folic acid, 5; vitamin B12, 0.02; biotin, 1; inositol, 400; vitamin C, 200. b Mineral mixture (g/100 g): NaCl, 1.0; MgSO4_7H2O, 15.0; NaH2PO4_2H2O, 25.0; KH2PO4, 32.0; Ca(H2PO4)2_H2O, 20; Fe-Citrate, 2.5; Ca-Lactate, 3.5; Trace element mixture*, 1.0
(*Trace element mixture (g/100 g): ZnSO4_7H2O, 35.3; MnSO4_4H2O,16.2; CuSO4_5H2O,
3.1; CoCl2_6H2O, 0.1; Cellulose, 45.0). Vitamin mixture: The mixture was diluted in cellulose and provides the following vitamin activities in mg or IU kg_1 of diet.
2. Fish and experimental condition
Juvenile (mean initial body weight 68.5+0.50 g) were obtained from Sturgeon Propagation and Rearing Complex of ShahidMarjani (Gorgan, Iran). Each dietary treatment was replicated three times, with 8 fish per replicate. Fish were randomly distributed into tanks of 350 L capacity. water quality parameters were monitored daily such as temperature, pH, Dissolved Oxygen and salinity. The Average daily water temperature was 27 + 2 °C. Fish were fed to satiation with an amount of 4% of biweekly body weight three times a day by hand (06:00, 12:00 and 18.00) over 7 weeks. fish were weighed at 1th and 49th days. Growth performance was calculated according to Mohseniet al. (2009).
3. Statistical analysis
All data were subjected to one-way analysis of variance (ANOVA) to test the effects of experimental diets.
Results and Discussion
The growth performance and survival rates of beluga juveniles fed the experimental diets are displayed in Table 2. Juveniles fed 0% soybean meal dietary displayed significantly higher final weight, weight gain,SGR and lower FCR compared to other group (P<0.05). However, survival rates did not significantly differ between the treatments (P>0.05). The results indicate that adding soybean levels supplemented with lysine and methionine in the diets decrease final weight, weight gain, SGR and increase FCR compared to the control treatment (P<0.05). Although no significant differences (P>0.05) were observed between Control, SBM1 and SBM2 treatment in weight gain(WG), specific growth rate (SGR) and feed conversion ratio (FCR). Results of the present study indicate that Beluga has a limited ability to utilize SBM as a protein source in practical diets. This study showed that the partial substitution of fish protein with SBM significantly decreased the growthperformance in agreement with findings by Mohseni et al. (2009) on the Beluga and other species as rainbow trout (Sugiura et al., 2001).
Beluga has a limited ability to utilize soybean meal as a protein source in practical diets (Khajepour andHosseini, 2012).
Table 2: Growth parameters of juvenile beluga fed 7 weeks with diets containing different levels of soybean meal.
Initial weight (g) 550+0.23
Final weight (g) 1380.75+8.24
WG (%) 150.7+1.6
The suppression in overallgrowth performance in fish fed diet SBM may be attributed to the presence of antinutrional factors in SBM. Although, the fish fed the diets supplemented with coated methionine and lysine trended to higher weight gain compared to fish fed the diet without supplementation of the amino acids (Alametal., 2005). This study showed apparent feed intake and survival were not influenced by the supplementation of methionine and lysine.
Alam, M.S.,Teshima, S., Ishikawa, M., Koshio, S., 2005. Supplemental effects of coated methionine and/or lysine to soy protein isolate diet for juvenile kuruma shrimp, Marsupenaeusjaponicus. Aquaculture.248, 13- 19.
Berge, G.E., Goodman, M., Espe, M., Lied, E., 2004. Intestinal absorption of amino acids in fish: kinetics and interaction of the in vitro uptake of L-methionine in Atlantic salmon (Salmosalar L.). Aquaculture. 229, 265-273.
Khajepour, F.,Hosseini, S.A., 2012. Citric acid improves growth performance and phosphorus digestibility in Beluga (Husohuso) fed diets where soybean meal partly replaced fish meal, Animal Feed Science and Technology. 171, 68-73.
Mohseni, M.,Bahmani, M., Pourali, H., Poudeaghani, M., Bae, J.Y., Bai, S.C., 2009.Effect of soybean meal as a fish meal replacement without and with dietary lysine and methionine supplementation in great sturgeon Husohuso. In: 6th International Symposium on Sturgeon, Hubei Province, China, pp. 25-31.
Sugiura, S.H.,Gabaudan, J., Dong, F.M., Hardy, R.W., 2001. Dietary microbial phytase supplementation and the utilization of phosphorus, trace minerals and protein by rainbow trout (OncorhynchusmykissWalbaum) fed soybean-meal based diets.
Aquac. Res. 32, 583-592.
Comparative growth and survival of Gavkhuni Artemia (Artemia urmiana) in water treated with three fertilizers
Maryam Yaghubi1*, Samad Bahrami 1, Eisa Ebrahimi2
1 Department of Fisheries Sciences, Master student of Natural Resources, Isfahan University of Technology, Isfahan, Iran.
2 Assistant professor of Fisheries Sciences. Faculty of Natural Resources, Isfahan University of Technology, Isfahan, Iran.
Food supply and appropriate feeding in all of fish life stages play the basis role in increasing the production yield. Live food production with high quantity and quality for larval stage is so important when larvae is going to start external food, because the prosperity in larval stage, certified better growth, high survival rate and more health in the further stages. Artemia culture depends on biotic and abiotic parameters and biological responses. It means that Artemia abundance depends on the salinity, temperature and the accesable food (1). The most important factors in Artemia culture is temperature (18-25), PH (7/8-8/5), dissolved oxygen (2/5mg/L), salinity (100-150ppm) and the most recommended food is Dunaliella as a main food and Diatoms like Nitzschiaaz a secondary food (2). Artemia production is in low oxygen density (<2/5mg/L) (3). Nutritional like phosphor and nitrogen are the cramp factor in phytoplankton bloom. Accesable phosphor and nitrogen increase phytoplankton abundance and diversity and make the situation proper for Artemia culture. Among various live foods, Artemia is noteworthy because of it's high fecundity, high density, short maturation period and it's nutrtious like high protein source (52%) and fat (20%). On the other hand, Artemia can be used as a vitamine, drug and nutrients carrier as a boosting technique (4).
The study was conducted in aquaculture unit in Isfahan University of Technology in August, 2012. Artemia were collecting from Gavkhuni swamp. For disinfection, they transfer to the sodium hypochloride solution (20 ppt) for about 15 minute. Then they were placed in the three treatments and three replications. The qualifications was adjusted as a standard situation (salinity- 35ppt, temperature- 25, pH>8, photoperiod= 12D/12L). Neat dung, hen dung and beet ross were packed in the cloth and then put them in the water for one week before the investigate sterted. 500g of each fertilizer put in the cloth. The clothes were kept in the water until the water color become brown. If the brownish water color decrease because of food consumption, we add fertilizer again to make water color brown. We use Tertiolekna as a Artemia food. This algae can play a role in Nitrogen Absorbation result.
ANOVA data measured mean lengths repeated three treatments of Artemia fed with cow manure, chicken manure and beet pulp showed that the mean of the three treatments with 95% confidence level have significantly diffrence (05/0> p) but does not show significant difference in the mean survival with 95% confidence level.
Results show that using chicken manure to increase the size, have significantly difference. In this stady length changes in Artemia fed the chicken manure was7/7-8 mm ,cow manure was6/8-7/6 mm and samples with beet pulp was 5/8-6/5 mm. Comparison of the three groups shows that feeding chicken manure is better and cause faster grow(0364/0 - p). As is clear from the data, using chicken manure in Artemia meal relative beet pulp and cow manure to increase survival rates showed no significant difference. Survival rates range Artemia fed with cow manure was 85 -77% and in chicken manure was 97% -93% fed with broiler. Comparison of the three groups shows feed with chicken manure dosenot have significant difference compared to the groups fed with cow manure and beet pulp.
Because chicken manure is alkaline, increase the possibility of Artemia fed with chicken manure may be higher pH. In different sources, different concentrations of fertilizer in order to develop and increase Anemia have been reported. For example Coutteau in 1992, have been proposed the amount of 18 gr/L of water of cow manure is suitable for the cultivation of artemia (5). Accordingly Manaffar in 2001, the same concentration used for Artemia enrichinging (6). In another study on rotifer feeding, have been proposed the amount of 45 gr/L of water a cow manure for the survival and growth of rotifers. As indicated, different concentrations of the fertilizers used in the cultivation of aquatic animals including Artemia.Apart from the concentration of fertilizer, phosphate and nitrate nutritional value is important . The nutrient value of manure can be a very effective agent in reducing feeding, protect water quality and enhance growth. Nowadays most researches in this field is to enhance the nutritional value of Artemia. For this reason, using of fertilizers in different concentrations in the Artemia breeding is important.
Dhont, j., Levens, p., Sorgeloos, p., 1993, preparation and use of Artemia as food for shrimp and prawn larvae, In: CRC Handbook of mariculture. Crustacean Aquaculture Vol1. 2nd Edition,CRCPress.BocaRatan. USA.
Dhont, j. and Levens, P. , 1993. Tank Production and use of on grown artemi laboratory of aquaculture and relevance center university of Gent, Beligum. Pp. 16-194.
Dhont, J.; Levens, P. and Sorgeloos, P., 1993. Preparetion and use of Artemia as food for shrimp and prawn larvae, In: CRC Handbook of mariculture. Crustacean
Aquaculture Vol 1. 2nd Edition, CRC Edition, CRC Press. Bocaratan. USA.
Dhont, J. and Levens , P., 1993. Tank production and use of on grown Artemia laboratory of aquaculture and relevance center university of Gent. Beligum. PP.
Coutteau, P.; Brendonck, L.; Lavens, P. and Soregeloos, P., 1992. The use of manipulated baker's yeast as an algal substitute for the laboratory culture of Anostraca. Hydrobiologia. Vol. 234, PP.25-32.
Manaffar, R. Maleki, R. Atashbar, B and Agh, N., 2006. Use of Azadirachta indica against invasive ciliated in Dunaliella tertiolekna culture.
Study of effects of nano-silver particles on some vital tissues of Zebra fish (Danio rerio) fed via oral administration
Tahereh yazdanparast*1, Issa sharifpour*2, Mehdi soltani3
1Faculty of Fishery, Islamic Azad university of Science and research branch, Tehran, Iran. 2Department of aquatic animal health and diseases, Iranian fisheries research
organization, Tehran, Iran. 3Department of aquatic animal health, faculty of veterinary medicine, university of
Tehran, Tehran. Corresponding author email address: firstname.lastname@example.org
This study was initiated to enhance our insight on the health and environmental impact of silver nanoparticles (Ag-np). The increased use of silver nanoparticles in consumer and medical products has led to elevated human and environmental exposures. After characterizing the AgNPs using TEM, EDX, UV-Vis Spectroscopy, XRF and SEM methods, their effects on some vital tissues have been tested successfully in vitro.
In this study, 300 Zebra fish with mean weight of (2± 0.5) grams were used. Tests were performed statically based on instructions of OECD under fixed water quality conditions at the temperature 28 and pH 6.8-7 in a completely random trial with nine concentrations treatments of AgNps (0,10, 50, 100, 200, 400, 600, 800 and 1000 mg/gr of food) in three replications. According to the results of acute tests, the 96hr LC50 values were 195.208, 323.696, 486.637 and 804.601mg/L AgNPs for the Zebra fish. Accordingly, investigated colloidal AgNPs are classified as "toxic" to this fish.
In this test, clinical signs such as hunched spinal column, thrilling, clot in caudal fin and skin, and irregular swimming
were observed in the studied fish specimens. According to the results of chronic toxicity tests, fed via oral administration of AgNPs significantly made the histopathological effects. Also the most important histopathological effects of AgNPs were observed in the liver (vasculature and exposure, vacuolization and degeneration of some hepatocytes), intestine (Increase the submucosa layer, Narrowing of the intestinal lumen, fusion of parts of the mucosal layer and reduced intestinal absorption ), gills (clubbing of gill secondary lamaleas, hyperplasia gill primary and secondary lamaleas, Hyperemia primary and secondary lamaleas gills and shorten the primery lamaleas gills) and kidney (degeneration in urinary tubes, high increasing in interstitial cells and dilatation of Bowman's space of glomeruli), respectively.
After biological measurement, heavy metals were measured by spectrum photometry reveal, The greatest bioaccumulation of silver occurred in the liver, gills and muscle of fish respectively.
In conclusion, with respect to observed damages by fed via oral administration of AgNPs, its direct application as antimicrobial agent in aquaculture and also its release into the environment should no longer be allowed.
Keywords: Silver Nanoparticles (AgNPs), Oral administration, LC50, Bioaccumulation, Histopathology, Zebra fish (Danio rerio)
Nano-toxicology is relatively a new and expanding science. With the increasing use of nanotechnology in science (Chen et al, 2008), there are concerns about the potential risks of nanoparticles issues in human health
and environmental. There is currently great interest in the safety issues of production processes like consumption and release of nanoparticles in aquatic ecosystem (Rushton et al, 2010). There are few reports of silver nanoparticles toxicity in the aquaculture industry (Griffitt et al, 2008).
There are many consumer products and applications utilising Ag NPs. The Woodrow Wilson Database (http://www.nanotechproject.org), although not exhaustive, has listed 1015 consumer products presently on the market incorporating NPs, with 259 containing Ag NPs (May 2010), exploiting their strongly bactericidal action and making Ag NPs the largest and fastest growing class of NMs in product applications (Yoon &
Commercial applicable programs of nanoparticles have been associated with the lack of rules and regulations regarding safety and toxicological data particles (Asharani and colleagues, 2008), also researchers have found that nano size particles can cause more harmful than larger particles at the same concentration (Morones et al, 2005).
The results of this research can be a starting for the direct use of nanosilver in fish fed in aquaculture centers; and also these Results can be important for standardized and acceptable AgNPs concentration in fresh water fishes. Unfortunately in few studies have been done about using AgNPs in aquaculture systems, have been less attention about toxicity and mortality effects on aquatic and human.(Vaseeharan et al, 2010; Soltani et al, 2011)
Most of the nanotoxicology studies were focused on in vitro models. Only a few research groups have dealt with aquatic in vivo systems (Obserdorster, 2004). Toxicology studies in in vivo systems carry greater significance pertaining to their diversity in physiology and anatomy. Experiments on medaka fish using fluorescent solid latex nanoparticles confirmed a homogeneous distribution of the particles (Kashiwada et al,
2006).Earlier reports (Braydich-Stolle et al, 2005; Skebo et al, 2007)
proved that silver nanoparticles are more lethal to cell-based in vitro systems than other metal nanoparticles screened. In the present study, zebrafish were chosen as model systems for testing ecotoxicity of the silver nanoparticles.
We have used different concentrations of silver nanoparticles colloid in their food with spray method. Moreover, use of organic solvents and other toxic chemicals may yield highly toxic products that hinder bio-applications(Kashivada et al, 2006). The nanoparticles employed in this study were highly stable and water soluble. Transmission electron microscopy (TEM) of the Ag-BSA treated fish showed a significant concentration of nanoparticles inside the nucleus in liver.
Because AgNPs can have accumulation and cause damage vital tissues and change in various fish tissues, the goal of this research is about determination of LC50 AgNPS, Histopathological effects and accumulation nano silver in Zebar fiish tisses.
Materials and methods
1. Characterization of nanoparticles
1.1. TEM analysis of the nanoparticles.
Stock solutions of the nanoparticles were used for TEM analysis. TEM images showed that Ag-colloid (figure 1(A)) nanoparticles have an average size of 4-164 nm. These images have been take at Azad university, Sciences and research branch material department, and also TEM device worked with 125kV.
1.2. UV-vis spectrum of silver nanoparticles.
Silver nanoparticles exhibit an intense brown color due to the surface plasmon resonance (SPR), which results from collective oscillations of their conduction band electrons in response to electromagnetic waves. This is well studied and reported earlier by other groups (Thomas et al, 2008). Under the UV region, Ag-nps give a characteristic absorbance band due to the excitation mode of their surface plasmons which is dependent on the nanoparticle size. These SPR bands undergo red shift or blue shift depending on the quantum size effects (Thomas et al, 2008).
Hence, absorbance peaks can be used as tools to predict particle size and stability. Smaller silver nanoparticles will have an absorbance maximum around 400 nm which increases with size and disappears when
particle size falls outside nanodimensions. To study the optical properties of our silver nanoparticles, the absorbance maximum of silver nanoparticles was measured. The device UV-Vis Spectra-MAX-PLUS 384 (Molecular Devices) present at Sharif University has been read nano particles.
2. Prepare nano particle silver solution with different concentration
Nanosilver colloid, with a concentration of 4000 mg/L was bought from the NanoNasb Pars Company (Tehran - Iran). To provide concentrations of 100, 400, 600 and 800 mg/L has been used 25, 100, 150 and 200 cc nano particle colloid, respectively with dionized water. Then after dilution and production these solution put them in bath sonication for 30 min to became suspension.
3-Preparing food coated with silver nanoparticles
In this experiments, the consume food were prepared by Anousheh Arab Iran with brand Byvmar with size 0.8. Before addition of silver nanoparticles in food, the food was divided into four equal 5 grams parts. then placed into glass plates and were sprayed nanoparticles prepared with 100, 400, 600 and 800 mg /kg(food) concentrations on it, and finally were dry with oven on 45 ° C for 24 hr.
The food without nanosilver particles were studied as controls sample. Using scanning electron microscope (SEM) equipped with energy diffuse X-ray analysis (EDX) to determination how coverage food with nanoparticles and AgNps charactrictics.
4 - Preparation of Location experiments and investigation of acute and chronic toxicity of silver nanoparticles colloid in Zebra fish
The study was done at the Laboratory of Fisheries Science and Research Branch of Azad university. In this study to determine LC50 Silver Nanoparticles in Zebra fish (2 ± 0.5 g) using 15 tanks. Their capacity wrer 20 liters and put 20 Zebra Fish for each one that the first aquarium was used as control sample (Cnps=0) and others has been fed by 10, 50, 100, 200, 400, 600, 800 and 1000 mg NPs/kg food (OECD, 2000). Aquariums water temperature was around 28 ° C and pH was
almost neutral about 6.8, water hardness was 120 mg/L of oxygen intake was carefully controlled by air pump (8 mg per liter).
During the experiment, fish were fed with food containing silver nanoparticles. Mortality was not observed during adaptation. Before starting, aquariums were completely washed by concentrated salt water 70% and potassium permanganate due to became any dirt and disease-free environment for the fishes.
Then tanks were filled with dionized water. So that the fish affected by low concentrations of silver nanoparticles (10) to high (1000) and then the loss of 100 percent of fish in 1000 mg/ kg food while There were no mortality in 10 to 100 mg/ kg food concentration. Bases of this experiment concentrations were chosen for main studies. Every 24 hours, Aquarium were investigated and the mortality rate was recorded. These works were done for 96 hours (4 days) and water exchange were done every morning at 10 am.
Also to invesigation histopathological effects of silver nanoparticles, 15 aquariums were provided and 20 fishes have been put in each aquarium supply which fed with lethal concentrations (100, 400, 600 and 800 mg) that all chemical and physical conditions were equally for all aquariums.
All fishes which were fed with food contain of silver nanoparticles and control samples were taken for pathological tests. Each sample of tissues was placed in buffered formalin 10%. Then section tissues were cut by 5-micron-thickness, before hematoxylin and eosin staining were done normaly (Kumar et al, 1992).
TEM analysis method for localization of nanoparticles in the liver was used. Liver sections 0.5 mm were prepared. Sample livers were fixed for 30 min in 2.5% gluteraldehyde and dehydrated. The samples were embedded in resin (Spurr's low viscosity resin) and sectioning was done using a Reichert Jung Ultracut instrument. Ultrathin sections of tissues of interest were selected using microscopy. TEM analyses were done using a JEOL JSM 3010F and JEOL JSM 2010F .
Finally, to assess the bio-accumulation of silver nanoparticles from 4 sub-lethal concentrations of 100, 400, 600, 800 mg/kg food in 30 and 56
days, 45 fishes were chosen (three samples from each treatment and 3 samples in each aquarium) randomly.
Then liver, muscle and gill tissues were digested by nitric acid and hydrogen peroxide in the automatic microwave digestion system. For determination of silver metal, the spectrum photometry reveal (Elmer Aanalyst GFAAS AA800 Perkin) was used (Moopam.et al, 1993).
TEM microscope images (Fig. 1(A)) revealed that silver nanoparticles used in this study have had spherical shape. The images illustrated that largest particles size had 129 nm diameter. According to the results of optical spectroscopy in the range of visible and ultraviolet wavelength, a typical resonance surface Plasmon were centered at about 410 nm (Fig. 1(E)).
Scanning electron microscope (SEM) images of covered food with silver nanoparticles have been showed that silver nanoparticles had spherical particles with an average diameter of 45.51 ± 90.26 nm (Fig. 1(B)). In the X-ray diffraction spectra patterns, a clear difference between ordinary food and samples coated with silver nanoparticles was observed (Figure 1(D)),that were showed the two peak in silver diffraction with 81 and 51.8 that there were not in ordinary food sample.
According to the results of acute testes ,the 96hr LC50 values were 804.601, 486.637, 323.696 and 195.208 mg/ kg of food values, for 24, 48, 72 and 96 hours, respectively. According to the results of chronic toxicity tests, the most important histopathological effects of AgNps were observed in the liver, intestine, gill and kidney. Results of microscopic studies were as follows:
Also the most important histopathological effects of AgNPs were observed in the liver, intestine, respectively.
Histologic effects were not observed of silver nanoparticles in the control samples, but silver nanoparticles effects on gill tissues including clubbing of gills secondary lamaleas, hyperplasia primary and secondary gill lamaleas, Hyperemia primary and secondary gills lamaleas and shorten the secondary lamaleas gills (Figure 4).
According to the results were observed of the liver, hyperemia and sinusoidal congestion and vacuolization and degeneration of some liver hepatocytes were existed, especially in the higher concentrations (Figure 5).
The intestinal tissue's damages in Zebra fishes which were fed with different concentrations of silver nanoparticles was shown that tissue damages including high fusion parts of the mucosal layers and intestinal tract, the intensification, narrowing of the intestinal lumen and submucosa layer vacuolization.(Figure 7).
About kidney tissue, degeneration, high necrosis in urinary tubes, high increasing in interstitial cells and dilatation of Bowman's space of glomeruli was seen in the high concentrations (Figure 8).
The bio-distribution of nanoparticles using TEM analysis showed that the AgNps has an affinity for the nucleus. Most of the nanoparticles were deposited inside the nucleus of the cells (figure 6), whereas the cytoplasm had only a few nanoparticles (figure 6). The magnified view of the nucleus showed small clumps of nanoparticles. However, clumping of Ag-nps was observed inside the liver.
According to the results of bioaccumulation tests, the greatest bioaccumulation of silver was occurred in the liver, gills and muscle of fish respectively
In the present study, although fishes were fed (100 mg /gr) with lowest concentration of silver nanoparticles, the silver concentration was observed in tissues, liver, muscle and gill. Treatments were exposed with silver nanoparticles, there were silver concentrations in liver tissue as twice or more as control ones.
Figure 1. Characterization of nanoparticles: TEM image of AgNps clloid (A) and food coated by nanoparticle silver (B). EDS of AgNps showing the presence of silver (C). EDS of food showing the presence of silver in food (D). UV-vis spectrum (E) of AgNps showed maximum absorbance at 400 nm.
Table 1- lethal concentration values (about 95%) in Zebra fish within 96 hours fed via oral administration with nanosilver colloid.
Table 2- the silver concentration in the liver, gills and muscle Zebra fish during 56 days feeding with a food containing different concentrations of AgNps colloid.
Silver Silver concentrations in tissue ug/gr
in food (mg/gr)
First international Larviculture Conference in iran
Figure 2-. Compare accumulation of silver nanoparticles in tissues, liver, gills and muscle in 30th day.
Figure 3-. Compare accumulation of silver nanoparticles in tissues, liver, gills and muscle in 56 day.
Figure 4- clubbing of secondary lamaleas gills (arrows), hyperplasia primary and secondary lamaleas (H), Hyperemia primary(Star) and secondary lamaleas gills (C) and shorten the primery lamaleas gills (S) were observed.
Figure 6- TEM image of ultrathin section of the liver tissue. Deposition of the AgNps in the cytoplasm and nucleus of the cells.
Figure 7- Increase the submucosa layer (I), Narrowing of the intestinal lumen (L), fusion of parts of the mucosal layer (F) and reduced intestinal absorption (L) were observed.
Figure 8- degeneration and necrosis of kidney tubular (D), increasing in interstitial tissue (I) and edema of Bowman capsules of kidney tissues (E), were seen.