Protective Effect of Sea buckthorn (Hippophae rhamnoides L.) Leaves on Ochratoxin-A Induced Hepatic Injury in Japanese Quail

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1 VETERINARY RESEARCH INTERNATIONAL Journal homepage: ORIGINAL ARTICLE Protective Effect of Sea buckthorn (Hippophae rhamnoides L.) Leaves on Ochratoxin-A Induced Vikram Patial 1,4, R.K. Asrani* 1, R.D. Patil 1, Naresh Kumar 2 and Rinku Sharma 3 1 Department of Veterinary Pathology, 2 Department of veterinary Physiology and Biochemistry, Dr G C Negi College of Veterinary and Animal Sciences, CSK Himachal Pradesh Agricultural University, Palampur , Himachal Pradesh, India 2 Indian Veterinary Research Institute, Regional Station, Palampur , Himachal Pradesh, India 4 Regulatory Research Center, CSIR - Institute of Himalayan Bio-resource Technology (CSIR-IHBT), Palampur , Himachal Pradesh, India *Corresponding Author: Dr. R K Asrani asranirk@gmail.com Received: 09/10/2015 Revised: 17/11/2015 Accepted: 21/11/2015 Abstract The present study was designed to study the efficacy of sea buckthorn (SBT) against ochratoxin-a (OTA) induced hepatic damage in Japanese quail. Day old quail chicks were divided into 6 groups and fed a basal quail chick mash containing 2% SBT-leaf powder (Group SX), OTA at a dietary level of 3 ppm (Group OX), 25 ppm L-β-phenylalanine with OTA (Group OP), 2% dietary level of SBT leaf powder plus with OTA (Group OS), SBT-leaf extract in drinking water with OTA (Group OSS) and a standard toxin-free feed (Group CX) for 21 days. OTA led to anorexia, diarrhea, heavy mortality and marked depression in body weight. Addition of SBT was found to lower the clinical signs mortality in birds treated with OTA. Alanine aminotransferase (ALT), uric acid, cholesterol, total protein and albumin values were improved by SBT in OTA treated birds. Grossly, there was significantly (P 0.05) enlarged and pale liver in OX birds on 21 day, whereas the severity of gross lesions found lower in OS, OSS and OP birds at this interval. Similarly, the severity of microscopic lesions such as degenerative, necrotic and inflammatory changes were less pronounced in OS, OSS and OP birds than in OX on 21 day. The addition of SBT in the water or diet of quail enhanced regenerative changes in the liver on 7 and 14 days. In conclusion, the inclusion of SBT-leaf powder and extract in water was found to reduce the intensity of OTA-induced hepatic injury in Japanese quail. Keywords: Ochratoxin A, Liver, Protection, Pathology, Japanese quail. 1. Introduction Ochratoxin-A (OTA) is a known nephrotoxic, hepatotoxic, immunotoxic and carcinogenic mycotoxin (Varga et al. 1996; Clark and Snedeker, 2006). Molds associated with the production of OTA include several species of genera Aspergillus and Penicillium. It is a wide spread natural contaminant and co-contaminant of various food and feed commodities, resulting in toxicosis in human and animal populations. It is more toxic than other ochratoxins (OTB and OTC) and has been reported to be more toxic than Aflatoxin B 1 (AFB 1 ) in poultry (Marquardt and Frohlich, 1992; Samson, 1992). Oxidative damage is one of the mechanisms of cellular damage in OTA toxicity. Reactive oxygen species (ROS) have a major role in the mediation of cell damage. OTA inhibits succinate dependent electron transfer in the electron transport chain, but at higher concentration it also inhibit electron transport at complex 1 (Wei et al., 1985; Aleo et al., 1991). OTA causes significant losses to the poultry industry due to its effects on performance and health. It causes a reduction in growth rate and feed consumption, poorer feed conversion and increased mortality (Peckham et al., 1971). OTA induces degenerative changes and an increase in weight of the liver and the kidneys, as well as a decrease in weight of lymphoid organs (Stoev et al., 2000; 2002a). Inhibition of protein synthesis by OTA is due to its structural homology to L-β-phenyalanine resulting in competition for the specific t-rna synthetase (Bunge et al., 1978; Creppy et al., 1979). This fact is supported by the protective effect of phenylalanine and

2 its structural analogue aspartame administration in earlier studies (Belmadani et al., 1998). In the present study, phenylalanine was thus used as a reference compound. Hippophae rhamnoides L., commonly known as Sea buckthorn (SBT) growing in North-West Himalayas at high altitude ( ,000 feet) is a dwarf to tall (3-15 feet), branched, and thorny nitrogen fixing deciduous shrub, native to Europe and Asia (Rousi et al., 1971). This plant, which belongs to the family Elaeagnaceae, is renowned for its versatile pharmacological applications due to the presence of many known and unknown bioactive substances mainly in its fruits and leaves. Fruit and leaves of SBT are quite rich in B vitamins, vitamins C, E, K and in other biologically active substances like organic acids, amino acids, carotenoids, tocopherols, flavonoids, sterols etc. (Upendra et al., 2008). The fruit and leaf extract of H. rhamnoides improves the antioxidant defense system of cells by increasing the intracellular GSH levels and inhibiting reactive oxygen species (ROS) production (Hsu et al., 2009). The oil extracted from the SBT berries has been shown to have hepatoprotective effect against AFB 1 in broiler chickens (Solcan et al., 2013). The leaf extracts of SBT, containing flavonoids, have been reported to have significant antioxidant, antibacterial, anti-viral, anti-inflammatory and anti-tumor activities (Tsybikova et al., 1983; Ganju et al., 2005). The phytoantioxidant property of SBT has been found to reduce the liver damage (Yang et al., 2011) thereby making it hepatoprotective (Geetha et al., 2008). In addition, SBT leaves extract stimulate lymphocyte proliferation, IL-2 and gamma interferon production thereby activating cellular immune response (Geetha et al., 2005). Quail are valuable research model due to its physiologic and morphologic similarities to chicken (Padget and Ivey, 1959) and are preferred over chicken because they have not been as intensively selected and bred by the poultry industry for traits of economic importance, such as eggs and production. Moreover, minimum requirements in terms of space, feed and mycotoxin make quail an excellent model for investigating toxicologic effects, disease resistance, and immune function (Sharma et al., 2008; Patial et al., 2013b). In previous study, we have reported the protective effect of SBT leaves against OTA induced nephrotoxicity (Patial et al., 2013b). Since liver is also one of the key organs in OTA toxicity in poultry (Dwivedi and Burns, 1986), the present work which is in continuation of our previous study (Patial et al., 2013b) demonstrates the protective effect of SBT leaves against the development of clinical disease with hepatic lesions in Japanese quail. 2. Materials and Methods 2.1 Experimental Birds and Their Management Day-old Japanese quail chicks were procured from Central Poultry Development Organization, Chandigarh, India. The quail chicks used in the present study were from the same breeding flock. The animal care and experimental protocol was approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Prior to arrival of chicks, the experimental room, cages, trays and cage stands were prepared as described in earlier study (Patial et al., 2013b). The waterers and feeders were washed daily with 5% potassium permanganate solution throughout the experiment. Excreta swabs taken from 20 randomly selected chicks on day 1 were streaked on brilliant green agar plates (BGA) and examined for bacterial growth after 48 hr of incubation at 37 o C. The excreta samples were found to be negative for Salmonella and Escherichia coli on bacteriologic examination. 2.2 Plant Material and Preparation of SBT- Leaf Extract Sea buckthorn (H. rhamnoides L.) leaves were collected from the orchard of Krishi Vigyan Kendra, CSK HPKV, Kukumseri, District Lahaul, Himachal Pradesh, India at an altitude of 8866 feet. The plant material was collected from the same source to rule out phytochemical variations caused due to regional and climatic conditions. Fresh leaves were dried under shade in a clean, dust free environment and crushed to fine powder using laboratory grinder. SBT-leaf powder extract was prepared using a Soxhlet extraction apparatus according to method described in earlier study (Patial et al., 2013b). 2.3 Ochratoxin-A and L-β-phenylalanine Ochratoxin-A was produced by growing the fungus Aspergillus ochraceous (NRRL-3174) on maize, and the culture material with known level of OTA was supplied by Dr. G. E. Rottinghaus, University of Missouri, Columbia, MO, USA. A pure compound of l-β-phenylalanine (Hi-Media, Mumbai, India) was used for the study. 2.4 Feeding Schedule The quail chicks were fed dietary treatments (Quail mash procured from Department of Animal Nutrition, COVAS, CSK HPKV, Palampur amended with the appropriate additives) from d 1 until the end of the experiment. Feed was autoclaved before feeding or mixing with OTA culture material(s) and SBT powder. Pre-boiled drinking water was provided during the 99

3 experiment. Feed and water were available for ad libitum consumption. 2.5 Experimental Design Two hundred and seventy, day-old Japanese quail chicks were randomly divided into 6 groups i.e. CX, SX, OX, OP, OS and OSS with 45 birds in each of the groups. The present study was conducted with three replicate pens of 15 quail each assigned to each of the six groups. The various treatments, starting from day 1 until 21 day in different groups, are presented in Table Clinical and Biochemical Studies Birds from all groups were closely observed, at least thrice daily, for the development of clinical signs and mortality. In order to evaluate the effect of OTA on body weight, 9 randomly selected quail (three quail per replicate) from each treatment group were weighed at 0, 7, 14, and 21 day post-feeding. After weighing, 2 to 3 ml of blood was collected via cardiac puncture from 6 randomly selected birds from each treatment group at 7, 14, and 21 day for estimation of alanine transaminase (ALT), cholesterol, total serum proteins (TSP), albumin and uric acid. All the biochemical estimations were done using diagnostic kits (Reckon Diagnostics Pvt. Ltd., Baroda, India) in a fully automatic Blood Chemistry Analyzer (RA-50 Auto Chemistry System) according to the manufacturer s recommendations. 2.7 Pathology Six randomly selected quail chicks (two per replicate) were euthanized from each treatment group on 7, 14, and 21 day and examined for gross lesions in the liver. Representative liver tissue samples after collection in 10% neutral-buffered formalin were processed for histopathological studies (Luna, 1968). Grossly, paleness and enlargement were roughly graded as mild (1), moderate (2) or severe (3) on the basis of extent of liver involvement. The microscopic lesions i.e. congestion, degenerative changes, necrosis, leukocyte infiltration, regenerative changes and bile duct hyperplasia were graded with intensity score of 1 = occasional/in few places (mild), 2 = fairly common/multifocal areas (moderate), 3 = diffuse (severe). The lesion score was determined for each single type of gross and microscopic lesion on each day of sacrifice by multiplying the intensity score by the number of birds showing that particular intensity of lesion. 2.8 Bacteriological Examination Liver swabs collected from the birds of different groups after sacrifice at weekly interval as well as from the mortality birds were streaked on brilliant green agar plates for the presence of bacteria, if any, after 48 hr incubation at 37 o C. 2.9 Statistical Analysis Data generated in the present study were expressed as mean ± standard error mean. The statistical analysis of weekly data on body weight and biochemical parameters was subjected to ANOVA and Duncan s Multiple Range Test for comparison of mean values among different groups at various intervals (Snedecor and Cochran, 1989). The histological total lesion score for each of above mentioned gross and microscopic lesions was subjected to non-parametric Kruskal-Wallis test and comparison of mean values employing Dunn s post-hoc test for comparison of mean lesion score among different groups (P 0.05). All levels of significance were based on the 95% level of probability. 3. Results 3.1 Clinical Signs, Growth Response and Mortality Feeding diets amended with 3 ppm OTA led to anorexia, dullness, diarrhoea, depression in body weight, poor feathering and incoordinated movements. Similar signs were observed in the combination groups, but were comparatively less severe in intensity. In group OX, diarrhoea was more severe from 7 to 21 days post feeding, with almost 60-70% of the birds were seen passing out watery faeces along with an increase in uric acid and mucus content in the droppings. In addition to this, nearly 5-10% of the birds in group OX showed clinical signs such as pressing of head on the ground, backward motion to gain balance, ataxia and sudden falls onto the ground from day 14 onwards. Reduction in body weight and stunted growth were observed in almost all of the birds fed OTA diets either alone or in combination from one week onward (Fig 1). Reduction in body weight in OX group birds was significant (P 0.05) in comparison to control groups at all interval i.e. on 7, 14 and 21 day. The body weight of birds from groups OS and OSS were slightly better than group OX on day 7 but the data was not found statistically significant. Mortality in different groups was observed only up to 14 days post feeding. The mortality was highest during the first week of experiment in group OX (40%) followed by 31% in group OP, 26% in group OSS and 24.4% in group OS. Overall mortality was found to be highest in group OX (51.1%) as compared to the group OS (31.1%), in which SBT-leaf powder was given in combination with OTA in the diet. The results also - 100

4 Table 1: Various treatments in different groups of quail chicks from day 1 to 21 Group Treatment Total level of culture material/sbt-leaf powder used Dosing level of culture material/sbtleaf powder/sbt--leaf extract achieved CX Chick mash alone 0 0 SX SBT-leaf powder 2% SBT-leaf powder 20 g/kg SBT in feed alone OX OTA alone 3.75% OTA culture material 3 ppm OTA in feed OP OTA + Phe 3.75% OTA culture material % Phe 3 ppm OTA + 25 ppm Phe in feed OS OTA + SBT-leaf 3.75% OTA culture material + 3 ppm OTA + 20 g/kg SBT in feed powder 2% SBT-leaf powder OSS OTA + SBT-leaf extract 3.75% OTA culture material + SBT-leaf powder in distilled water (10% w/v) for preparation of extract 3 ppm OTA in feed + SBT-leaf extract in drinking water (10% v/v) OTA = Ochratoxin-A; Phe = L-β-phenylalanine; SBT = Seabuckthorn Fig 1: Comparative body weights (Mean ± SEM) of Japanese quail from different experimental groups at various intervals indicated a decline in the mortality rate of birds in group OSS (35.5%) and in group OP (33.3%) in comparison to group OX (51.1%). 3.2 Serum Biochemistry Mean ALT activity on 7 days post feeding was higher in group OX and OP as compared to those in control groups i.e. CX and SX groups (Table 2) and the difference was statistically significant (P 0.05). Similarly on 14 days post feeding, the ALT activity was significantly higher in OX group in comparison to groups OP, OS and OSS in which the values were comparable to control groups (CX, SX). Estimation of serum cholesterol in group OX revealed significantly (P 0.05) lower values at 7 days post feeding in comparison to control groups (CX and SX), however, the values in the combination groups (OP, OS and OSS) were more or less comparable to control groups (CX and SX). At 14 day posts feeding the total serum cholesterol values were lower in all combination groups (OP, OS and OSS) but the difference was found to be statistically significant only in groups OX and OSS in comparison to groups CX and SX. At 21 days post feeding, all the treatment groups showed decrease- 101

5 Table 2: Effect of sea buckthorn on certain biochemical parameters in ochratoxin A fed Japanese quail Serum Biochemical Parameters ALT Cholesterol Total Protein Albumin Uric Acid Groups Interval (IU/L) (mg/dl) (g/dl) (g/dl) (mg/dl) CX 10.50±0.95 c 166±4.95 b 2.91±0.18 a 1.43±0.06 b 7.46±0.46 a SX 12.66±0.61 bc 193±13.2 a 2.90±0.05 a 1.65±0.06 a 7.28±1.26 a OX 23.16±4.48 a 94±9.90 c 1.26±0.15 c 0.60±0.05 d 8.10±0.31 a OP 7 DPF 19.16±2.75 ab 161±4.52 b 2.51±0.11 ab 1.20±0.07 c 8.01±0.61 a OS 10.50±0.42 c 210±5.50 a 2.70±0.11 ab 1.30±0.05 cb 7.58±0.22 a OSS 10.50±1.17 c 164±12.2 b 2.30±0.12 b 1.13±0.07 c 7.60±0.49 a CX 12.50±0.95 b 212±15.8 a 3.35±0.13 a 1.56±0.07 a 4.53±0.72 c SX 12.83±0.60 b 215±17.4 a 3.40±0.16 a 1.51±0.04 ab 5.66±1.27 bc OX 20.50±4.61 a 151±11.1 bc 2.78±0.29 a 1.10±0.09 c 9.15±1.60 a OP 14 DPF 13.83±0.94 b 191±20.6 ab 2.85±0.12 a 1.28±0.11 c 7.91±0.40 ab OS 13.50±0.99 b 172±7.36 abc 3.36±0.21 a 1.31±0.06 abc 5.35±0.27 bc OSS 13.16±0.79 b 158±12.5 bc 2.91±0.29 a 1.15±0.11 c 6.50±0.58 abc CX 8.83±0.94 a 128±9.74 a 3.36±0.18 a 1.46±0.04 a 5.12±0.62 c SX 7.00±0.57 a 149±8.86 a 3.46±0.08 a 1.43±0.06 ab 3.93±0.48 c OX 9.66±1.11 a 118±4.93 a 3.10±0.26 a 1.16±0.04 b 7.97±0.84 a OP 21 DPF 9.33±0.55 a 120±7.87 a 3.46±0.30 a 1.33±0.13 ab 7.32±0.57 ab OS 9.00±0.68 a 121±10.6 a 3.05±0.34 a 1.30±0.11 ab 6.45±0.99 ab OSS 9.00±1.15 a 121±6.45 a 3.35±0.20 a 1.30±0.06 ab 5.80±0.49 bc a-c Values within columns (between groups CX, SX, OX,OP, OS and OSS) with different superscripts are significantly different by ANOVA (P 0.05). Data are means ± SEM of three replicate pens of 2 quail each, Number of birds in each group = 45, DPF = day postfeeding, CX-control group, SX- sea buckthorn (SBT) group, OX= group kept on ochratoxin-a diet only, OP= group kept on L-βphenylalanine and ochratoxin-a, OS= group kept on SBT-leaf powder and ochratoxin-a, OSS = group kept on SBT-leaf extract and ochratoxin-a Fig 2: Liver paleness and swelling in OTA group (OX) in comparison to control group (CX) 102

6 in total serum cholesterol concentration, however, the difference was not found statistically significant in comparison to control groups (CX and SX). Amendment of diets with OTA had a significant effect on total serum protein (TSP) and the values were, in general, significantly lower in OTA treated groups compared to control groups (CX, SX) (Table 2). At 7 days post feeding there was a significant decrease (P 0.05) in TSP values in group OX in comparison to control groups (CX, SX), whereas the values in groups OS, OP and OSS were more or less comparable to controls groups (CX, SX). On 14 days post feeding, however, no significant difference was found in TSP values between group OS and control groups (CX, SX) whereas in groups OP and OSS the TSP values were more or less comparable to group OX. There was a significant decrease in albumin concentration in group OX when the corresponding values were compared with groups CX and SX at 7 and 14 days post feeding (Table 2). There was a dramatic reduction in serum albumin concentration at 7 days post feeding in group OX in comparison to groups OS, OP and OSS. The birds in group OX showed a significant decrease in serum albumin values on 14 days post feeding, however, the corresponding values at 14 and 21 days post feeding in group OS birds were more or less comparable to the control groups (CX, SX). No significant difference in mean uric acid concentration was observed at 7 days post feeding among the OTA and control group birds. At 14 days post feeding, a significant (P 0.05) increase in uric acid concentration was observed in group OX birds, however, in the combination groups the values were found less than group OX, even significantly less in group OS. At 21 day non significant numerical decrease was observed in the values of uric acid in combination groups as compared to group OX. Mean uric acid values were higher in OTA treated groups but in SBT and phenylalanine combination groups the increase was comparatively lower, indicative of partial protection. 3.3 Gross Pathology No gross lesion was observed in the liver tissues of control group (CX) (Fig 2, Table 3). In the liver of group OX, mild to severe enlargement and pale discoloration was observed at all the stages of experiment in comparison to phenylalanine (group OP) and SBT treated groups (OS, OSS) but the change was not significant. The liver was found to be significantly (P 0.05) enlarged and pale in group OX in comparison to the control group (CX) on 14 and 21 d, respectively (Table 3). The mean lesion score values for gross lesions were much lower in the SBT treated groups in comparison to group OX on 21 d though the difference was not statistically significant (Table 3). 3.4 Histopathology In the control groups (CX, SX), the liver sections revealed mild to moderate congestion and degenerative changes in the hepatocytes mainly comprising cytoplasmic vacuolations at various intervals of the experiment (Table 3) (Fig 2A). In group OX, the liver sections showed mild congestion and mild to moderate necrotic changes with leucocyte infiltration on 7 day. Prominent nuclei, karyomegaly, karyorrhexis, dissolution of nucleus and double nuclei were characteristic findings. Mild hepatocytes swelling with increased cytoplasmic granularity was consistent in all birds at all intervals. Individual cell necrosis, Kupffer cell hyperplasia and bile duct hyperplasia were also evident. Scattered heterophilic infiltration was occasionally observed in group OX birds. In addition, mild regenerative changes characterized by hepatocellular hyperplasia were observed in 50% birds on 7 day. At 14 and 21 day, mild to moderate hepatocellular hyperplasia, degenerative changes chiefly comprising mild to severe cellular swelling and distorted hepatic cords were predominant lesions. Hepatocytes appeared more eosinophilic with a marked increase in cytoplasmic granularity (Fig 2B). Focal to multifocal areas of necrosis accompanied with macrophages and few heterophils were consistently present in group OX on 14 and 21 day (Fig 2C). Presence of periportal granulocytic infiltrates along with mononuclear cells was interpreted to be the extramedullary haematopoiesis. Nuclear changes such as karyomegaly (Fig 2B), apoptotic nuclei and condensed nuclei were frequently observed. Mild to severe cellular swelling with multiple vacuoles in the cytoplasm of hepatocytes was evident until 21 day. Mean lesion score for each of the lesion in group OP was generally higher in comparison to other groups (OX, OS and OSS) on 7 day (Table 3). Focal areas of necrosis in the hepatic parenchyma infiltrated with heterophils and macrophages and individual cell necrosis (apoptosis) was evident as early as on 7 day in group OP (Fig 2D) in comparison to group OX and SBT treated groups (OS and OSS). In group OP, the regenerative activity of hepatocytes was slightly better in comparison to group OX on 7 and 14 day. The lesions in the SBT treated groups (OS, OSS) were similar in nature to those observed in group OX, however, these were comparatively less severe at all intervals of the experiment (Table 3). The regenerative changes comprising of hepatocellular hyperplasia, oval or binucleated hepatocytes and - 103

7 Fig 2A-F: A. Normal hepatocytes with round to oval nuclei in seabuckthorn fed control group (SX) at 14 days post feeding. H&E. 330x. B. Swollen and degenerating hepatocytes with increased granularity and vacuolations (v) in the cytoplasm and karyomegaly (arrows) in ochratoxin-a fed group (OX) at 14 days post feeding. H&E. 330x. C. Severe hepatocytic degeneration with a focal area of necrosis (N) largely infiltrated with mononuclear cells in ochratoxin-a fed group (OX) at 21 days post feeding. H&E. 66x. D. Presence of individual cell necrosis (apoptosis) indicated by arrows along with a focal area of necrosis (N) largely infiltrated with heterophils and mononuclear cells in ochratoxin-a plus L-β-phenylalanine fed group (OP) at 7 days post feeding. H&E. 66x. E. Regenerating oval hepatocytes (arrows) are accompanied with mitosis (arrowhead), single cell necrosis (asterisk) and karyomegaly in ochratoxin-a plus seabuckthorn-leaf powder fed group (OS) at 21 days post feeding. H&E. 132x. F. Cords of regenerating hepatocytes (arrows) in group OSS given ochratoxin-a in feed and seabuckthorn-leaf extract through drinking water at 14 days post feeding. H&E. 66x. mitotic activity were comparatively better in the SBT treated groups (OS, OSS) than in group OX throughout the study (Fig 2E, 2F), however, these were more or less comparable to those observed in group OP. Among the SBT treated groups (OS, OSS), the regenerative response was comparatively better in group OSS than 104

8 in group OS birds. At 21 day, degenerative changes such as cytoplasmic vacuolations and granularity were comparatively lower in the SBT treated groups (OS, OSS). Absence of necrotic areas and mild leucocytic infiltration in only one bird on 21 day in group OSS further suggested that SBT-leaf extract has promising potential in protection against liver damage due to OTA. 3.5 Bacteriological Examination None of samples collected from the liver tissues revealed either Salmonella or Escherichia coli on bacteriologic examination at any stage of the experiment in the sacrificed or mortality birds. 4. Discussion Results of the present study showed that feeding diets amended with 3 ppm OTA led to anorexia, diarrhoea, body weight depression, poor feathering and incoordinated movements in the quail chicks. Dwivedi and Burns (1984) found OTA to be extremely toxic to young growing broiler chicks (2 ppm and 4 ppm) and the severity of the clinical signs, behavioural changes, growth response and lesions were in a dose dependent manner. The lower intensity of clinical sign in combination groups (OS, OP and OSS) suggesting a protective effect of SBT and phenylalanine. The reduced appetite undoubtedly contributed to the depression in body weight OTA treated groups during the experiment. According to some workers, the growth depression in OTA treated chicks may be due to impaired protein synthesis caused by OTA (Mohiudin et al., 1993). The presence of diarrhoea may have been an additional factor contributing to the reduction in body weight in all groups fed OTA. In group SX, body weight was more or less similar to the control group (CX) indicating that addition of SBT-leaf powder to the feed had no adverse effect on feed consumption. The increased ALT levels agree with previous studies exposing broiler chicken to dietary OTA (Agawane and Lonkar 2004; Elaroussi et al., 2008). The elevated levels of serum ALT in the groups fed OTA could be due to the hepatocellular disease. In the present study, the reduced ALT level in SBT combination groups was supported by the study of Geetha et al. (2008) in which that SBT leaf extract showed protective effect against CCl 4 induced hepatotoxicity in rats by lowering serum ALT values. OTA lowered the serum cholesterol levels, might be due to impairment of cholesterol synthesis or transport (Kubena et al., 1989). Cholesterol is synthesized primarily in the liver, and OTA has been reported to decrease the synthesis of sterol carrier protein (Ritter and Dempsey, 1971) and competitively inhibit mitochondrial transport carrier protein (Meisner and Chan, 1974), which could result in decreased energy for cholesterol synthesis. The increased cholesterol levels in SBT treated groups in the present study suggested that SBT might have reduced the OTA induced mitochondrial damage and thus restored the energy deficit for the cholesterol biosynthesis (Patial et al., 2013b; Solcan et al., 2011). OTA inhibits the protein synthesis through competitive inhibition of phenylalanyl-t-rna synthetase with phenylalanine (Bunge et al., 1978). The TSP values in groups OS, OP and OSS suggest the protective effects of SBT and phenylalanine against the OTA toxicity as the serum protein concentration did not change appreciably in relation to both control groups (CX, SX). Similarly, improvement in the serum uric acid levels in SBT and phenylalanine treated groups was also in accordance with earlier reports (Stoev et al., 2002a; 2002b). The liver enlargement found in the present study was in accordance with the earlier trials conducted on chicken (Elaroussi et al., 2008) and the effect was found to be dose-dependent. The liver enlargement in group OX was mainly attributed to more degenerative changes such as cellular swelling and vacuolation hepatocytes (Stoev et al., 2000). The restored liver size in SBT treated groups was in consistent with earlier findings where SBT treatment found to reduce the relative liver weight in chicken intoxicated with AFB 1 (Solcan et al., 2013) and mice poisoned with CCl 4 (Hsu et al., 2009). The slight liver enlargement observed in OS and OSS groups might be attributed to the regenerative changes including hepatocellular hyperplasia in comparison to group OX. The histopathological findings indicated that the supplementation of SBT-leaf powder at 2% level in feed has no harmful effect on the liver (Patial et al., 2013a; 2013b). The degenerative changes such as swollen and eosinophilic hepatocytes along with an increase in the cytoplasmic granularity and vacuolations in group OX might be due to route of elimination of OTA through the liver (owing to enterohepatic recirculation and the hepatobiliary route of excretion of OTA), and to a direct toxic action of OTA on these cells (Stoev et al., 2000). In the present study, phenylalanine was found to potentiate the hepatotoxic effect of OTA at early stages in quail. This might probably be due to an increase in the absorption of OTA from the stomach and intestine as well as gastrointestinal transit of OTA in the presence of phenylalanine, which could also increase its elimination through the liver and the kidneys (Roth et al., 1988; Stoev et al., 2002a). The partial hepatoprotection shown by SBT leaves in the present study is in accordance with an earlier study in which ethanolic extract of SBT leaves - 105

9 Table 3: Gross and microscopic leison scores (Mean±SE) in the liver of Japanese quail at various intervals Parameter CX SX OX OP OS OSS Mean lesion score in the liver of different groups 7 DPF Gross Lesions Paleness 0.0±0.0 a 0.6±0.3 a 0.8±0.4 a 0.8±0.3 a 1.1±0.3 a 1.3±0.4 a Enlargement 0.0±0.0 a 0.5±0.3 a 0.3±0.2 a 0.5±0.2 a 0.3±0.2 a 0.0±0.0 a Microscopic lesions Congestion 0.3±0.2 b 0.5±0.2 b 1.1±0.1 ab 1.6±0.2 a 0.6±0.2 ab 1.1±0.1 ab Degenerative changes 0.6±0.3 a 0.6±0.4 a 1.0±0.2 a 0.6±0.4 a 0.6±0.3 a 0.8±0.1 a Leucocyte infiltration 0.1±0.1 b 0.3±0.2 b 1.3±0.2 ab 2.3±0.4 a 0.6±0.3 ab 1.0±0.0 ab Necrosis 0.0±0.0 b 0.3±0.2 b 1.0±0.2 ab 2.3±0.4 a 0.8±0.4 ab 1.0±0.0 ab Regenerative changes 0.0±0.0 b 0.0±0.0 b 0.5±0.2 ab 1.0±0.0 ab 0.6±0.2 ab 1.8±0.3 a Bile duct hyperplasia 0.0±0.0 a 0.0±0.0 a 0.6±0.3 a 0.6±0.2 a 0.8±0.4 a 0.5±0.2 a 14 DPF Gross lesions Paleness 0.0±0.0 a 0.5±0.3 a 0.5±0.3 a 0.8±0.4 a 1.1±0.4 a 1.3±0.6 a Enlargement 0.0±0.0 b 0.3±0.2 ab 1.8±0.4 a 1.0±0.3 ab 1.1±0.3 ab 1.3±0.3 ab Microscopic lesions Congestion 0.3±0.2 b 1.1±0.1 ab 1.5±0.2 a 1.1±0.3 ab 1.0±0.2 ab 1.1±0.1 ab Degenerative changes 0.0±0.0 b 1.1±0.3 ab 2.0±0.3 ab 0.8±0.5 ab 1.5±0.4 ab 2.1±0.3 a Leucocyte infiltration 0.3±0.2 ab 0.0±0.0 b 1.1±0.4 ab 1.1±0.1 a 0.5±0.2 ab 1.0±0.2 ab Necrosis 0.3±0.2 ab 0.0±0.0 b 1.1±0.4 ab 1.1±0.1 a 0.1±0.1 ab 1.0±0.2 ab Regenerative changes 0.0±0.0 b 0.0±0.0 b 0.8±0.3 ab 1.1±0.1 a 1.0±0.2 ab 1.6±0.3 a Bile duct hyperplasia 0.0±0.0 b 0.0±0.0 b 1.3±0.5 ab 1.3±0.2 a 0.1±0.1 ab 1.3±0.4 ab 21DPF Gross lesions Paleness 0.0±0.0 b 0.5±0.2 ab 1.6±0.3 a 1.3±0.3 ab 0.3±0.3 ab 0.3±0.2 ab Enlargement 0.0±0.0 a 0.5±0.2 a 1.5±0.5 a 0.1±0.1 a 0.6±0.3 a 0.1±0.1 a Microscopic lesions Congestion 0.3±0.2 b 0.8±0.3 ab 1.8±0.1 a 1.3±0.2 ab 1.1±0.3 ab 1.3±0.2 ab Degenerative changes 0.8±0.4 a 1.1±0.4 a 2.0±0.4 a 1.6±0.4 a 0.6±0.4 a 0.8±0.4 a Leucocyte infiltration 0.1±0.1 ab 0.0±0.0 b 1.1±0.1 a 1.0±0.0 a 1.1±0.4 ab 0.1±0.1 ab Necrosis 0.0±0.0 a 0.0±0.0 a 0.8±0.3 a 0.6±0.2 a 1.0±0.4 a 0.0±0.0 a Regenerative changes 0.0±0.0 b 0.0±0.0 b 1.0±0.2 ab 1.0±0.2 ab 1.3±0.2 a 0.1±0.1 ab Bile duct hyperplasia 0.0±0.0 b 0.0±0.0 b 1.1±0.3 a 0.8±0.3a ab 0.8±0.3 ab 0.0±0.0 b a-b Values within rows (between groups CX, SX, OX, OP, OS and OSS) at various intervals with different superscripts are significantly different (P 0.05) by non-parametric Kruskal-Wallis test followed by Dunn's multiple comaparison post-hoc analysis, Data are means ± SE of three replicate pens of 2 quail each, Number of birds in each group = 45, DPF = day postfeeding, CX-control group, SX- seabuckthorn (SBT) group, OX= group kept on ochratoxin-a diet only, OP= group kept on L-βphenylalanine and ochratoxin-a, OS= group kept on SBT-leaf powder and ochratoxin-a, OSS = group kept on SBT-leaf extract and ochratoxin-a (100mg/kg bw) was reported to protect the liver from CCl 4 induced liver damage in rats revealed by the hepatic cells with well-preserved cytoplasm and a marked decrease in inflammatory cells (Geetha et al., 2008). Similarly, Solcan et al. (2013) reported less intensity of hepatocytic swelling, degeneration, necrosis and mononuclear cell infiltration in chicken given oil from SBT berries along with AFB 1. SBT seed oil in mice (Hsu et al., 2009) and SBT leaves in rats also (Geetha et al., 2008; Maheshwari et al., 2011) reported to have significant hepatoprotective effects due its antioxidant activity against CCl 4 induced liver damage. The hepatoprotective effect of SBT leaves or its extract against OTA induced liver damage in the present study as evident by histopathologic findings was also supported by the improved levels of alanine transaminase, total protein, albumin and cholesterol in the serum. The combined treatment with glucomannan at dietary level of 1 g/kg of feed and 50% aquamethanolic SBT leaves extract at 10,000 ppm has also been found to protect against OTA induced immunosuppression in Japanese quail (Jain et al., 2013). 106

10 Oxidative stress is one of the major mechanisms for OTA induced toxicity. So the hepatoprotective role of SBT might be associated to free radical scavenging potential (Geetha et al., 2008) due to its antioxidant constituents such as selenium, carotene, tocopherol, phenolic compounds and vitamin E and C, working individually or in synergy. The SBT has been shown to be effective against free radical-induced cellular transformation, and it can inhibit cytochrome P450- mediated reactions involved in the formation of reactive metabolites of the hepatotoxins (Solcan et al., 2013). SBT leaves however, have also been found to have high amount of tannins and other bioactive principles. So the protective effect of SBT leaves as a result of increased diuresis with rapid excretion of toxin metabolites from the body before the full manifestation of its toxic effect in the tissues would have actually been attained cannot be ruled out and needs further studies. 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