Effect of trivalent chromium on haemato-biochemical and histopathological parameters at higher doses in broilers Raut S.S.1, Ganorkar A.G.1,1, Bhandarkar A.G.1,*, Jangir Babu Lal1, Mahaprabhu R.1, Kurkure N.V.1 1Department of Pathology, Nagpur Veterinary College, Maharashtra Animal and Fishery Sciences University, Seminary Hills, Nagpur-440006 *Corresponding author:
Abstract An experiment was conducted in equally distributed 7 groups of 140, day old broilers to study the effect of trivalent organic chromium picolinate (Cr-Pic) @ 40, 80, 120mg/kg and inorganic chromium chloride (CrCl3) @ 80, 120, 200mg/kg feed in group B,C,D,E,F and G, respectively. Group A was kept as control. The hematological and biochemical parameters were studied at weekly interval, whereas, patho- morphological studies was conducted on day 21 and 42. Experimental birds did not exhibit any clinical signs of toxicity during the period of observation. The groups B and D receiving Cr-Pic showed higher body weights while the lowest body weights was recorded in the groups F and G receiving CrCl3 @ 120mg and 200mg/kg respectively. Haematological observations revealed anaemia in all the treatment groups receiving CrCl3 on the other hand groups received Cr-Pic showed increased TEC, Hb and PCV values. Mild heterophillia with relative lymphopenia was observed. The serum glucose and cholesterol was decreased significantly in all the groups receiving organic and inorganic chromium with increased levels of serum creatinine and AST levels. Histo-pathologically, liver showed granular degenerative changes, periportal and perivascular necrosis with bile duct hyperplasia in birds receiving higher doses of chromium. Various degenerative changes in tubular epithelium and accumulation of hyaline cast in kidney tubules were noticed. Hyalinization of glomeruli and interstitial nephritis along with fibrous tissue proliferation were evident in group receiving higher dose of CrCl3. Present investigation suggest that organic and inorganic trivalent chromium salt produces histo-pathological alterations in liver and kidney. Top Keywords Chromium, Broiler, Haematobiochemical, Histopathological. Top |
INTRODUCTION Trivalent chromium (Cr3+) is a well known essential trace element for human and animals1. It involved in carbohydrate, lipid, protein and nucleic acid metabolic function2,3. Cr3+ has been shown to have antioxidative properties in vivo and it is integral in activating enzymes and maintaining the stability of proteins and nucleic acids. Its primarily metabolic role is to potentiate the action of insulin through its presence in an organometallic molecule called the glucose tolerance factor (GTF)1. It improves insulin effectiveness by enhancing its binding to receptors and the sensitiveness of the target cell4. Its role in stress condition in animals and birds is more appreciated in which it helps to reduce the negative influence of environmental and nutritional stress. It has been shown that organic form of chromium such as Cr-Pic, Cr nicotinate is absorbed more efficiently, about 25–30% more than inorganic compounds like CrCl3 which are poorly absorbed (1–3 %) as a dietary Cr5,6. Absorbed Cr circulates in blood, bound to the b-globulin plasma fraction and is transported to tissues. The Cr3+ ion becomes toxic only at extreme doses. Cr acts as gastric irritant rather than as toxic element that adversely affects the physiology and metabolism7. The major source of chromium for animals and humans is food, such as vegetables, meat, unrefined sugar, fish, vegetable oils and fruits. |
Cr deficiency is characterized by glucose intolerance, glycosuria, hypercholesterolemia, decreased longevity, decreased sperm counts, impaired fertility and signs resembling those of diabetes mellitus. Cr deficiency may retard the growth by affecting the activities of enzymes involved in protein synthesis8,9. Poultry diets may be deficient in chromium; because Cr concentration of plant products is low and it is lost as a result of grinding of grains largely used in poultry diets10. Clinical and experimental research data show that chromium supplementation within the recommended safe and adequate intake levels are relatively non-toxic. The most common systemic effects of Cr were parenchymatous changes in the liver and kidney11. Renal failure has been reported in a patient consuming high amount of Cr-Pic per day for weight loss, but renal function returned to normal upon discontinuation of the supplement12,13. The present study was formulated to study the effect of organic and inorganic Cr3+ in broilers at different higher doses. |
Top MATERIALS AND METHODS Experimental Design One hundred forty day old broiler chicks were divided randomly in seven groups comprising 20 birds each. Birds were maintained in deep litter system with standard managemental conditions. Group A was fed with standard poultry ration, whereas group B, C, D, E, F and G were fed with feed containing Cr-Pic @ 40, 80, 120 mg/kg and CrCl3 @ 80, 120, 200mg/kg feed, respectively. The haematological and biochemical parameters were studied at weekly interval upto the day 42 of experiment. The pathomorphological examination was conducted in six experimental birds from each group on day 21 and 42. Clinico-pathological/Parameters The birds were monitor two times in day throughout the experiment to observe the clinical symptoms and mortality any. Weight of individual bird was recorded on weekly basis up to day 42. Blood was collected from six birds in each group in a clean dry vial containing with and without EDTA for haematology and serobiochemistry. Hb, PCV, TEC and TLC were studied14,15. Blood smears were also prepared at the same time and stained with Wright's stain for differential leucocyte count (DLC). Further, absolute heterophils and lymphocyte count was calculated. Individual serum sample was analyzed for serum glucose, cholesterol, creatinine and AST (Aspartate transaminase) by using commercial diagnostic kits (Ranbaxy Fine Chem. Ltd., Gurgaon). Six birds from each groups sacrificed on day 21 and 42 of the experiment. The lesions in different organs were carefully recorded and tissues from liver and kidney were collected in 10% formol saline solution. Preserved samples were processed in ascending grade of alcohol and cleared in xylene, embedded in paraffin, sectioned at 4–5 μm thickness and stained with haematoxylin and eosin method (H & E)16. Statistical analysis was done as per Snedecor and Cochran17 using Completely Randomized Design at particular dose (ANOVA). Top RESULTS Clinical parameters Experimental birds did not exhibit any untoward clinical symptoms during the period of experiment. At end of experiment, the groups receiving Cr-Pic showed numerically higher body weights while significantly lower body weights were observed in the groups receiving CrCl3 in dose dependent manner (Table1 Heamatobiochemical studies Haematological observations revealed reduction in TEC, Hb and PCV values in all CrCl3 treatment birds (P<0.05), on the other hand, Cr-Pic treatment birds showed increased TEC, Hb and PCV values on day 21 and 28 onwards. The TLC values were significantly (P<0.05) increased in birds of C, D, F and G groups at the end of experiment. Mild heterophilia with relative lymphopenia was observed in treated groups as compared to control (Table 2). The serum glucose and cholesterol values were decreased significantly (P<0.05) in all the treated groups in comparison to control group. Serum creatinine values were significantly increased while AST (P<0.01) values were also found elevated in treated birds on day 42 (Table 3). Pathomorphological studies On gross examinations visceral organs were found normal except enlarged kidneys in group G. Histo- pathologically, section of liver of group B did not show any changes on both period of observation. Liver showed pronounce focal necrotic changes along with degenerative changes in hepatocytes with increased intensity noticed in dose dependent manner on day 21. In group D and G, liver showed marked proliferation of kupffer cells and perivascular degenerative changes in liver parenchyma respectively Fig 1. On day 42, liver showed congestion, haemorrhages and accumulation of leucocytes in periportal and centrilobular area Fig 2. Group F showed congestion, vacuolar changes in hepatocytes and necrosis. Group G showed vacuolar degenerative changes in hepatocytes, bile duct hyperplasia and prominent periportal necrosis Fig 3. At both observation periods in group B, sections of kidneys revealed increased cellurarity in glomeruli and varied type of degenerative changes along with detachment of tubular epithelium from basement membrane. On day 21, Group C showed interstitial haemorrhages, proliferation of round cells and detachment of tubular epithelium from basement membrane Fig 4. Group D showed severe interstitial nephritis. Group E showed haemorrhages and varied type of degenerative changes. Group F and G showed haemorrhages, tubular degenerative changes and hyaline cast in tubular lumen Fig 5. On day 42, Group C showed interstitial haemorrhages and congestion and swelling of the glomerular tuft with adhesion ot Bowman's capsule. Group D showed interstitial haemorrhages and interstitial nephritis, necrosis and adhesion of glomerular tuft to Bowman's capsule. Glomeruli showed hyaline degenerative changes with increased Bowman's space. Group E showed interstitial nephritis with vacuolar degeneration. Group F showed severe interstitial nephritis, degenerative changes and occlusion of tubular lumen and proliferation of round cell in interstitial space. Group G showed severe interstitial nephritis and adhesion of glomerular tuft to Bowman's capsule. Glomeruli showed hyaline degenerative changes. The tubular epithelium showed granular degenerative changes which led to occlusion of tubular lumen. The fibrous connective tissue proliferation was also evident in this group indicating severe destructive changes in kidney at higher dose level (Fig.6). Top DISCUSSION Increased in body weight and improve haematological parameters in present study might be due to beneficial effects Cr-Pic and were in accordance with the observations of earlier workers2,18, while decreased in body weights (Gr. G) might be due to the toxic effects of CrCl3 reported by previous workers19,20. At high levels, chromium may compete with iron for transferrin binding and deplete iron levels12,13. Therefore, the anaemia in present study was due to toxic effect of inorganic CrCl3 as reported in previous studies19,21. Cerulli et al.22 found decreased in Hb and TEC values in a woman who ingested 1200–1400 μg/day. Increased in TLC values and slight lymphocytopenia were reported by earlier workers20,23. Cr°+ in present experiment showed hypoglycaemic and hypocholesteremic effects at higher doses as reported by previous workers9,24. A plausibly explanation of cellular alterations would be that the Cr induces significant increase in membrane cholesterol level as well as significant decrease in membrane phospholipids level in chromium exposed rat which led to structural alterations of both liver and kidney plasma membrane25. The membrane becomes more permeable with water and glucose entrance in hepatocyte cytoplasm. Mammals need Cr3+ to maintain balanced glucose metabolism, and thus chromium may facilitate insulin action26,27. The findings of bile ducts proliferation was similar to that observed in rabbits by Tandon et al.28 and similar histopathological findings also reported by Silva et al.23 Pechova and Palvata29 reviewed that hepatic parenchyma develops necrosis only at very high Cr3+ doses. Hepato-toxicity of the Cr3+ in present study was well supported by increased in AST values12. On other hand Anderson et al.30 did not observe changes in rat liver when fed 100 mg/kg of Cr for period of 20 wks. The kidneys changes like cellular degeneration and hyaline casts in the lumen of the renal tubules, desquamation of tubular epithelium and interstitial nephritis were similar to the findings of previous workers31,32. Gabardi et al.33 reported three cases of renal dysfunction secondary to the excess ingestion of Cr-Pic. A renal biopsy revealed necrotic tubular epithelium with intra-luminal debris and severe chronic active interstitial nephritis. Increased in creatinine values and extensive histopathological alterations in kidneys indicate that Cr at high doses caused nephro-toxicity22. |
Top ACKNOWLEDGEMENTS Authors are thankful to the Associate Dean, Nagpur Veterinary College, MAFSU, Nagpur for providing necessary facilities to carried out research work. |
Top Figures | Fig. 1.: Liver showing Kupffer cell proliferation and degenerative in group D on 21st day. H&E ×400.
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| | Fig. 2.: Liver showing congestion and leucocyte infiltration in perivascular area in group E on 42nd day. H & E ×400.
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| | Fig. 3.: Liver showing focal necrotic changes and bile duct hyperplasia in group C on 42nd day. H&E ×200.
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| | Fig. 4.: Kidney showing degenerative changes and leucocytic infiltration in group C on 21st day H&E ×400.
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| | Fig.5.: Kidney showing degenerative changes and accumulation of hyaline cast in tubule lumen on 21st day in group G. H&E ×400.
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| | Fig. 6.: Kidney showing hyalinization of glomeruli and interstitial fibrous connective tissue proliferation on 42nd day in group G. H&E ×400
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Tables | Table 1.: Comparison of weekly body weight (g) in broiler chicks of various experimental groups (n=6, values are Mean±SE).
| | Days | A | B | C | D | E | F | G | | 0 | 50.75±1.16 | 51.00±1.12 | 49.75±0.84 | 49.50±1.01 | 51.50±1.15 | 50.25±0.99 | 51.00±1.12 | | 7 | 126.00±2.65 | 128.50±2.32 | 127.50±3.31 | 132.00±3.04 | 133.00±2.62 | 128.00±3.52 | 126.00±3.50 | | 14 | 288.75±4.81b | 315.50±5.92ab | 320.00±6.32a | 324.75 ±6.68a | 310.00±7.73ab | 320.00 ±7.40a | 299.50 ±7.62ab | | 21* | 529.85±5.13abcd | 545.00±7.41abc | 525.00±4.27bcd | 505.00 ±10.50d | 535.00 ±4.96abc | 560.00 ±5.58a | 520.00±10.39cd | | 28* | 835.00 ±7.48abc | 865.00±6.71a | 835.00±7.48abc | 810.71 ±.47.00c | 835.00±7.48abc | 835.00 ±7.48abc | 815.00 ±8.45bc | | 35** | 1240.71 ±4.25a | 1265.71 ±7.68a | 1255.35±5.63a | 1225.00 ±3.99a | 1245.71±4.41a | 1230.00 ±4.09a | 1186.78 ±18.97b | | 42* | 1624.64 ±4.24abc | 1635.00±3.77a | 1625.00 ±4.16ab | 1635.00 ±3.77a | 1625.00 ±4.16ab | 1610.71±6.06bc | 1605.71 ±6.85c |
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| Figures with different superscript with in a row differ significantly. (* P < 0.05; **P<0.01) | | | Table 2.: Mean Values of Hb, PCV, TEC, TLC, Absolute heterophil and lymphocyte count in different experimental groups at various intervals (n=6, values are Mean±SE).
| | Days | A | B | C | D | E | F | G | | Haemoglobin (Hb) (g/dL) | | 7 | 8.45±0.60 | 8.95±0.96 | 8.65±0.56 | 8.60±0.72 | 8.85±0.64 | 8.80±0.52 | 8.30±0.37 | | 14 | 8.77±0.15 | 8.87±0.21 | 8.63±0.12 | 8.93±0.25 | 8.67±0.45 | 8.43±0.27 | 8.07±0.16 | | 21 | 8.17 ±0.19 | 8.13±0.12 | 8.07±0.19 | 8.03 ±0.15 | 8.10 ±0.15 | 8.00±0.21 | 7.90 ±0.15 | | 28* | 8.61±0.39ab | 8.91±0.23a | 8.64±0.20ab | 8.77±0.18ab | 8.76±0.26ab | 8.62±0.13ab | 8.18±0.12b | | 35* | 8.53±0.17a | 8.57±0.21a | 8.47±0.19abc | 8.00 ±0.19bcd | 8.43±0.20abc | 7.97±0.26cd | 7.80±0.19d | | 42* | 8.67 ±0.20a | 8.60 ±0.24a | 8.53±0.23a | 7.90±0.32bc | 8.63±0.24 a | 8.50±0.18abc | 7.70 ±0.24 c | | Packed cell volume (PCV) (%) | | 7 | 28.75±1.65 | 29.00±0.91 | 28.75±1.31 | 29.00±0.91 | 28.50±1.94 | 28.50±1.32 | 27.75±0.63 | | 14 | 27.00±0.82 | 27.33±0.67 | 27.50±2.40 | 26.83±0.95 | 26.33±0.42 | 26.17±0.70 | 25.67±0.67 | | 21 | 27.50±0.56 | 27.17±0.95 | 27.33±0.67 | 27.50±0.99 | 27.67±0.76 | 26.83±0.87 | 26.17±0.54 | | 28* | 26.67±1.43 | 26.33±0.49 | 26.17±0.95 | 26.67±0.61 | 26.50±0.99 | 25.17±0.87 | 24.67±0.80 | | 35* | 28.67±1.15a | 28.67±0.56a | 27.17±0.48abc | 27.17±0.48 abc | 27.33±0.71abc | 26.17±0.54bc | 25.67±0.84 c | | 42* | 27.33±0.76 a | 26.67±0.67ab | 26.17±0.48 ab | 25.83±0.60 abc | 26.50±0.99ab | 25.17±0.31bc | 24.17±0.60c | | Total erythrocyte count (TEC) (106/cumm) | | 7 | 2.17±0.05 | 2.14±0.05 | 2.11±0.13 | 2.11±0.07 | 2.13±0.04 | 2.06±0.10 | 1.90±0.03 | | 14* | 2.14±0.04a | 2.11±0.04ab | 2.11±0.03ab | 2.06±0.02abcd | 2.00±0.06cd | 2.01±0.06bcd | 1.99±0.03d | | 21* | 2.10±0.04abc | 2.14 ±0.02abc | 2.20±0.06a | 2.12±0.04abc | 2.07±0.03bcd | 2.03±0.06cd | 1.97±0.01d | | 28** | 2.13±0.05abcd | 2.28±0.07a | 2.11±0.04bcd | 2.09±0.03cd | 2.23±0.04abc | 2.17±0.06abc | 1.98±0.02d | | 35* | 2.07±0.05bcde | 2.25±0.07a | 2.13±0.07abcd | 2.07±0.05cde | 2.16±0.03abcd | 2.04±0.04de | 1.97±0.03e | | 42* | 2.10±0.03bcd | 2.24±0.05a | 2.15±0.07abc | 2.12±0.05abc | 2.16±0.03abc | 2.04±0.03cd | 1.98±0.05 e | | | | Total Leucocyte count (TLC) (103/cumm) | | 7 | 24.75±1.25 | 25.25±1.51 | 24.25±1.75 | 25.00±0.98 | 26.00±2.20 | 24.25±2.05 | 25.88±1.52 | | 14 | 24.83±0.71 | 24.58±0.45 | 25.58±0.90 | 26.92±1.56 | 26.75±1.39 | 28.75±1.53 | 30.02±1.87 | | 21* | 24.42±0.62e | 24.67±0.64de | 25.92±1.15abcde | 27.17±0.73 abc | 25.83±1.03bcde | 25.50±0.62cde | 27.92±0.55a | | 28* | 24.75±0.79f | 24.83±0.87ef | 26.00±0.53bcdef | 26.42±0.57abcdef | 24.83±0.75def | 25.57±0.43cdef | 27.83±0.77a | | 35* | 25.08±0.76d | 25.83±0.93bcd | 27.17±0.81abc | 27.50±0.62ab | 25.25±0.74cd | 26.75±0.50abcd | 28.25±0.94a | | 42* | 25.92±0.62cde | 24.92±0.80e | 26.08±1.36abcde | 27.83±0.81abc | 24.92±0.74de | 26.54±0.57abcde | 28.29±0.51a | | Absolute heterophil counts (103/cumm) | | 7 | 6.83±0.24 | 6.51±0.36 | 6.16±0.63 | 6.44±0.62 | 7.24±0.36 | 6.13±1.44 | 7.25±0.83 | | 14 | 7.10±0.40 | 7.21±0.55 | 7.22±0.40 | 7.89±0.98 | 8.06±0.96 | 9.68±1.42 | 10.32±1.07 | | 21 | 6.72±0.64 | 6.93 ±0.62 | 7.39 ±0.60 | 7.86 ±0.39 | 7.42 ±0.78 | 7.76 ±0.87 | 9.50 ±0.28 | | 28* | 5.94±0.53g | 6.91±0.37fg | 7.46±0.61cdefg | 7.57±0.38bcdefg | 6.98±0.58efg | 7.29±0.58defg | 9.64±1.13a | | 35* | 6.88±0.59d | 7.51±1.15 cd | 8.98±0.85abc | 9.02±0.51abc | 8.30±0.65bcd | 9.64±0.25ab | 10.09±0.54a | | 42** | 8.13±0.85g | 8.60±1.11ef | 9.87±1.08d | 10.73±0.52b | 8.29±0.83fg | 10.64±0.98c | 12.77±0.76a | | Absolute lymphocyte count (103/cumm) | | 7 | 17.17±0.38 | 17.74±1.41 | 17.09±1.43 | 17.55±1.10 | 17.75±0.84 | 17.12±1.64 | 17.62±1.15 | | 14 | 17.39±0.76 | 17.04±0.40 | 17.36±0.96 | 18.02±0.76 | 17.69±0.86 | 18.06±0.66 | 17.86±1.04 | | 21 | 18.36±0.34 | 16.73±0.43 | 17.53±0.88 | 18.30±0.87 | 17.40±0.54 | 16. 73±0.43 | 16.57±0.36 | | 28 | 18.14±0.42 | 16.91±0.95 | 17.54±0.45 | 17.85±0.79 | 16.87±0.63 | 17.27±0.38 | 16.86±0.88 | | 35* | 17.71±0.43a | 17.32±0.34abcd | 17.18±0.36abcd | 17.47±0.71abcd | 15.95±0.68bcd | 16.10±0.50cd | 15.82±0.46d | | 42* | 17.78±0.19a | 16.31±0.34bcdef | 16.21±0.50cdef | 17.10±0.71abcdef | 16.62±0.65def | 15.93±0.42ef | 15.51±0.50f |
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| Figures with different superscript with in a row differ significantly. (* P < 0.05; **P<0.01) | | | Table 3.: Comparison of serum glucose, cholesterol, creatinine and AST values in different experimental groups at various intervals (n=6, values are Mean±SE).
| | Days | A | B | C | D | E | F | G | | Serum glucose (mg/dl) | | 7 | 163.83±8.21 | 155.14±2.16 | 156.49±5.31 | 158.33±10.53 | 154.03±11.84 | 154.33±3.73 | 158.97±5.36 | | 14 | 162.28±7.45 | 152.66±5.07 | 151.26±5.91 | 147.76±6.73 | 152.00±6.20 | 149.67±8.24 | 145.00±3.50 | | 21 | 161.04±11.74 | 153.01±13.26 | 151.54±16.35 | 145.00±16.67 | 148.61±18.94 | 144.93±11.76 | 143.77±10.19 | | 28* | 149.78±8.35a | 122.71±6.39acdefg | 123.18±8.36bcdefg | 116.88±4.24efg | 118.32±8.41defg | 116.66±3.20fg | 113.16 ±4.09g | | 35* | 141.88±8.98a | 121.74±9.22bcdefg | 117.94±5.70cdefg | 113.85±3.80fg | 116.99±5.74defg | 114.31±6.57efg | 112.89±3.00g | | 42* | 143.84±8.02a | 115.72±6.41efg | 120.31±10.68bcdefg | 119.59±8.87cdefg | 117.41±2.00defg | 114.70±1.00fg | 113.13±2.76g | | Cholesterol (mg/dl) | | 7 | 131.35±10.58 | 124.22±4.45 | 120.67±10.95 | 126.16±9.17 | 125.09±19.64 | 132.38±21.86 | 126.81±5.44 | | 14 | 139.59±17.92 | 119.05±7.44 | 136.24±13.11 | 130.56±9.16 | 121.23±20.13 | 134.85±6.91 | 132.16±2.95 | | 21* | 144.67±4.80a | 127.38±5.99cdef | 129.00±5.52bcdef | 119.26±1.94f | 126.96±8.42def | 135.93±5.55abcd | 120.84±1.04ef | | 28* | 142.87±5.76a | 139.93±6.01a | 138.67±12.39a | 114.44±7.72de | 116.61±9.25cde | 119.18±1.58bc | 112.76±7.17e | | 35* | 143.06±10.72a | 131.06±7.66abcde | 116.53±3.86bcdef | 111.21±8.31ef | 114.50±4.67def | 114.64±12.17cdef | 109.95±2.36f | | 42* | 142.54 ±5.16a | 108.65±17.63efg | 112.67±3.12cdefg | 115.07±9.29bcdefg | 102.76±3.18g | 106.75±2.46fg | 109.80±7.12efg | | Creatinine (mg/dl) | | 7 | 0.75±0.04 | 0.76±0.05 | 0.75±0.05 | 0.76±0.03 | 0.74±0.03 | 0.75±0.03 | 0.78±0.09 | | 14 | 0.75±0.06 | 0.74±0.03 | 0.75±0.03 | 0.72±0.03 | 0.76±0.05 | 0.81±0.03 | 0.78±0.07 | | 21* | 0.77±0.03bcde | 0.73±0.02e | 0.74 ±0.05cde | 0.86±0.05a | 0.74±0.03de | 0.84 ±0.04ab | 0.85±0.02ab | | 28* | 0.74 ±0.02d | 0.74±0.03cd | 0.74 ±0.04bcd | 0.83±0.06 abcd | 0.79 ±0.03abcd | 0.85±0.04ab | 0.87 ±0.03a | | 35* | 0.74±0.04d | 0.75±0.02bcd | 0.74±0.04cd | 0.85±0.03ab | 0.80±0.03abcd | 0.82±0.04abcd | 0.87 ±0.02a | | 42* | 0.77 ±0.02de | 0.75±0.02e | 0.79±0.03bcde | 0.86±0.04ab | 0.78±0.03cde | 0.82±0.02abcde | 0.88±0.03 a | | Aspartate transaminase (AST) (IU/L) | | 7 | 207.14±7.38 | 208.65±20.26 | 242.54±15.99 | 214.08±5.95 | 231.22±28.60 | 242.54±15.99 | 256.45±7.71 | | 14 | 225.94±18.91 | 234.14±20.03 | 231.91±14.27 | 224.01±10.89 | 232.04±13.83 | 237.75±24.38 | 253.82±32.86 | | 21* | 246.24±7.58bcdef 241.14±10.21ef | 245.76±12.29cdef | 289.28±16.05abc | 233.87±29.58f | 243.91±9.46def | 298.36±19.53a | | 28* | 243.98±8.02 c | 244.70±14.08bc | 263.13±8.01abc | 271.42±8.06abc | 267.97±5.76abc | 289.36±14.79a | 283.95±14.11a | | 35* | 241.07±8.41d | 258.06±8.23abcd | 248.33±4.35 bcd | 286.67±12.08 a | 247.57±18.97cd | 271.90±10.62a | 281.76±12.61a | | 42** | 245.18±22.89b | 303.63±28.25a | 333.09±21.05a | 298.60±13.42a | 332.38±14.91a | 291.94±12.26a | 297.69±10.99a |
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| Figures with different superscript with in a row differ significantly. (* P < 0.05; **P<0.01) | |
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