Effects of Dietary Arsenical Inclusion on Lipid Metabolism and Liver Function in Mule Ducks

This study evaluated the effectiveness of different arsenical sources on inducing fatty liver, on changes in lipid metabolism and on liver function in mule ducks. Sixty twelve-week-old mule ducks were selected and randomly divided into five treatments, including the control group and four different arsenical sources; Roxarsone (300 mg/kg), arsanilic acid, As2O5 or As2O3, containing 85.2 mg/kg arsenic were included in the basal diet. The ducks were fed the medicated basal diet for 3 weeks followed by a one-week drug withdrawal. The results showed Roxarsone treatment decreased body weight, feed intake, liver weight and abdominal fat weight (p<0.05), while it increased the relative liver weight (p<0.05) during medication period (3 week). The As2O5 treatment decreased abdominal fat weight and relative abdominal fat weight when compared to the control (p<0.05). Only Roxarsone among the treatment groups increased feed intake, liver weight and relative liver weight, while the As2O3 group showed the lightest liver weight and relative liver weight among treatment groups during the withdrawal period (4 week). The Roxarsone group decreased (p<0.05) NADP-malic dehydrogenase (MDH) and acetyl-CoA carboxylase (ACC) activities and increased (p<0.05) cholesterol concentration during the medication period, and elevated the MDH and ACC activities during the withdrawal period. All four arsenical treatment groups showed lymphocytic infiltration in liver tissue, while the Roxarsone and As2O3 treatments showed an increase in aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) activities (p<0.05). During the withdrawal period, arsenical treatments resulted in liver vacuoles. However, the arsenicals differed in effectiveness and mechanisms of inducing fat vacuoles. (Asian-Aust. J. Anim. Sci. 2006. Vol 19, No. 3 : 412-417)


INTRODUCTION
Chronic arsenic toxicity has been discovered for more than a half century in some areas of the world.Arsenic in high dose generally decreases animal performance and causes abnormalities in the skin and hair (McDowell, 1992).However, organic arsenicals i.e., Roxarsone (3-nitro-4hydroxyphenylarsonic acid) and arsanilic acid, has been used as feed additive in poultry industries to improve production and control coccidiosis for decades (McDougald et al., 1992;Waldroup et al., 1995).
High dose (300 mg/kg) of Roxarsone inclusion in the diet induces fatty livers in laying hens, laying ducks and mule ducks (Chiou et al., 1997;Chen et al., 2000;Chen and Chiou, 2005;Wu et al., 2005).In mule ducks, the fatty liver probably is induced by arsenic or Roxarsone toxicity, which depresses feed intake during the treatment period.The large increase in feed intake after the arsenicals withdrawal causes an extra amount of lipid synthesis that cannot be metabolized efficiently (Chen and Chiou, 2001).Chen and Chiou (2005) indicated that the mechanism of fatty liver induced by Roxarsone is related to the structure of arsenic compound, and which is different from the restricted feeding.The degree of arsenic toxicity differs among the different arsenical structures.Inorganic arsenic is more toxic than the organic arsenic, while the trivalent arsenic is more toxic than the pentavalent arsenic (NRC, 1980).However, the mechanism on inducing fatty liver in different structural arsenic compounds is still not clear.
This study is therefore to evaluate the effectiveness of different arsenical sources on inducing fatty liver, changes in lipid metabolism and liver functions in mule ducks.

MATERIALS AND METHODS
Mule ducks were a three way cross bred from male Peking ducks crossed with female domestic Tsaiya ducks as described in Chen and Chiou (2001).Eleven-week-old healthy mule ducks with similar live weights were selected and placed into individual 40×30 by 38 cm high cages for one-week adaptation.After adaptation, sixty healthy 12week-old mule ducks (2,880±12 g) were selected and randomly allocated into five dietary treatments; a control without Arsenical inclusion and treatments with different arsenicals in the diet; Roxarsone, arsanilic acid, As 2 O 3 or As 2 O 5 , respectively, at the same arsenic level of 85.2 mg/kg.Feed and water were provided ad libitum.Trial was conducted for 4 weeks with 3-weeks medication and 1week withdrawal.The basal diet is presented in Table 1.
Feed intake and live weight were individually recorded weekly during the experimental period.Blood samples of the 3 rd and 4 th weeks were taken from the ulnar vein after 12-h feed withdrawal with water restriction.Ducks were sacrificed in the 3 rd or 4 th week in the trial, respectively.
The abdominal fats included the fat surrounding the gizzard and fat pad in the abdominal cavity were stripped off immediately after livers removed.Approximately 0.5 cm of the liver tissue samples were taken from the livers and preserved in neutral-buffered 10% formalin for tissue section.The tissues were then embedded in paraffin, sectioned at 6 µm, and stained with hematoxylin and eosin.These liver slides were prepared for pathological examination.
Approximately 5 g of liver samples were sampled and homogenized at low temperature for 2 min (12,000 rpm).The supernatant was taken after centrifugation at 10,000×g for 10 min under 4°C.After repeating the centrifugation procedure, the supernatant was centrifuged again at 105,000×g 4°C for 60 min for cell microsomes precipitation.The supernatant cytoplasm was taken for hepatic enzyme and protein concentration analysis.
Serum TG and CHOL concentrations were analyzed referred to Lien et al. (2005) using an automatic blood chemical analyzer with Roche testing kits (Roche COBAS MIRA, Switzerland).The AST, LDH and CK activities  were measured using kinetic methods as recommended by the Germen Society for Clinical Chemistry (1972) using an automatic blood chemical analyzer with Roche testing kits (Roche COBAS MIRA, Switzerland).Serum enzyme activities were expressed in international units (U) per liter of serum (Bergmeyer, 1983).The hepatic lipogenic enzyme activity of CCE, MDH, FAS and ACC were measured using the modified method of Takeda et al. (1963), Ochoa (1955), Kumar et al. (1970) and Numa (1969) respectively.The hepatic tissue protein content in the enzymatic activity calculation was measured according to Lowry et al. (1951) using bovine serum albumin as the standard protein.
A completely randomized design was applied to examine the effects of arsenic chemical forms.Analyses of variance were calculated using the general linear model procedure of the SAS (1984).Duncan's new multiple-range test was used to compare the means according to Steel and Torrie (1960).

Production performance
Table 2 presents the effect of different arsenical sources on mule duck growth performance and carcass characteristics.Roxarsone inclusion decreased (p<0.05)feed intake, live weight and liver weight and abdominal fat weight, and As 2 O 5 inclusion decreased abdominal fat weight (p<0.05).After a week of withdrawal, Roxarsone treatment increased (p<0.05)feed intake, liver and relative liver weight.However, these body weights were still lighter (p<0.05) as compared to the control.Czarnecki and Baker (1984) demonstrated that the trivalent arsenic depressed more prominently chick growth performance on As 2 O 3 versus As 2 O 5 feeding, but this phenomenon did not occurred in this trial.Chen and Chiou (2001) indicated that after one-week of force-fed on the respective arsenical (11.36 mg/day arsenic), the feed intake and body weight of mule ducks were decreased by the Roxarsone treatment only, and did not show any significant effect by the arsanilic acid, As 2 O 3 or As 2 O 5 treatment.These results agreed to the observation in the present trial that only Roxarsone group (p<0.05),but not the other treatment group decreased (p>0.05)growth performance.Apparently, Roxarsone was the most effective among arsenicals on the growth performance effect in mule ducks.
Roxarsone inclusion decreased feed intake and caused a decrease in body weight, abdominal fat weight and liver weight (p<0.05) but an increase in relative liver weight (p<0.05),reflecting that the toxic influence of Roxarsone on the hepatic lipid metabolism is less prominent than on live weight.The As 2 O 5 treatment did not affect (p>0.05) the feed intake but decreased (p<0.05) the abdominal fat weight and its relative weight, this implied that As 2 O 5 directly affect the abdominal fat storage.
During the withdrawal period, Roxarsone treatment significantly increased (p<0.05) the feed intake, hence increased liver and relative liver weight compared to the control, and which were consistent with previous studies (Chen and Chiou, 2001;2005).The highest liver weight and relative liver weight were found in Roxarsone group, followed by the order of As 2 O 5 , arsanilic acid and As 2 O 3 .

Lipid synthesis and metabolism
Table 3 presents the effect of different arsenical sources on the hepatic lipogenic enzymes and blood lipid in mule ducks.Roxarsone was the only treatment group that decreased MDH and ACC activities and increased cholesterol concentration (p<0.05), and these changes were reversed i.e., an increase in the MDH and ACC activities (p<0.05) during the withdrawal period.
Restricted feeding and fasting decrease lipid synthesis in animals (Donaldson, 1990).Increased feed intake followed by restricted feeding or fasting usually increases lipid synthesis.The increase in lipogenic enzyme activities is highly related to the source and amount of substrate (Tanaka et al., 1975).In this trial, Roxarsone decreased (p<0.05)feed intake and hence decreased (p<0.05) the MDH and ACC activities during treatment period and this trend was significantly reversed (p<0.05) after one week of drug withdrawal.Hence, the Roxarsone treatment group showed the higher MDH, ACC and FAS activities in withdrawal (4 th week) than in medication period (3 rd week).Hasegawa et al. (1994) also observed the phenomenon demonstrated in this trial.ACC is a limiting enzyme in lipid synthesis.This activity decreases in fasting and increases in re-feeding (Tanaka et al., 1984).Converting malate to pyruvate that catalyzed by NADP-MDH provides a major NADPH source required for lipid synthesis.The FAS activity in the Roxarsone group was higher than in the As 2 O 3 group during the withdrawal period.This was implied that the mechanism of these two arsenicals were different, and resulted in higher liver and relative liver weight in the Roxarsone group than in the As 2 O 3 group (Table 2).
Lipids are synthesized mainly in the liver and these newly synthesized lipids are transported through lipoproteins to the body tissues in poultry (Hermier et al., 1985).The TG concentration in the Roxarsone group showed a decreased trend (p<0.1) and about 25% lower than the control during the medication period reflected that lipid was minor released (Table 2) and mostly cumulated in the liver, and resulted in a higher relative liver weight in the Roxarsone group (p<0.05).The blood TG concentration did not show differences between the Roxarsone and the control group (p<0.05)during withdrawal period.This may be attributed to the dramatically increased feed intake in both treatment groups, and induced higher hepatic lipogenic enzymes activities.But these TG content did not release from liver and were accumulated in the liver (p>0.05),resulted in the increased liver and relative liver weight (Table 2).The blood TG concentration increased about 21% more in the As 2 O 3 treatment than the control (p<0.1)reflected more lipids was released from liver, hence the lowest liver and relative liver weight in the As 2 O 3 group.The cholesterol concentration was higher in the Roxarsone group than control (p<0.05), and this agreed with Chen et al. (2000) who fed 300 mg Roxarsone to laying ducks; the cholesterol concentration was increased after 3-week Roxarsone treatment, but recovered to normal after withdrawal (Chen et al., 2000).Lien et al. (1999) also found that the cholesterol concentration in ducks increased after 3day fasting, and was attributed to the body lipid lipolysis.

Liver function
Table 4 presents effects of different sources of arsenicals on blood chemistry and hepatic histology in mule ducks.During medication period, the CK and AST activities were higher in the Roxarsone group and the AST and LDH activities were higher in the As 2 O 3 group.All arsenical treatment groups were demonstrated a liver lymphocytic infiltration from the histological observation.All hepatic enzyme activities returned (p>0.05) to the normal physiological range in all treatment groups after drug withdrawal, except the As 2 O 5 treatment, which showed higher (p<0.05)CK activity compared to the other treatment groups.All the arsenical treatment groups showed the abnormal lymphocytosis and vacuoles in the liver cells from the microscope observation.
AST and LDH are widely distributed in the heart, liver, kidney and muscle cells in poultry and pigeons.CK is the specific enzyme for muscle cells.Increase in these enzyme activities without an increase in CK activity represents a damaged liver or kidney (Lumeij ad Wolfwinkel, 1988;Wang, 1992).The Roxarsone group showed an increase (p<0.05) in CK activity during medication and was agreed with our previous trials using 3-weeks Roxarsone inclusion (300 mg) in the diet in laying hens (Chiou et al., 1997) and laying Tsaiya ducks (Chen et al., 2000) and reflected that damaged muscle cells in poultry by Roxarsone treatment.The elevated CK activity in the As 2 O 5 group means the damaged muscle tissue after withdrawn.From histological observation, all arsenical treatments caused abnormal lymphocytosis in the liver cell and Roxarsone treatment caused the bile duct proliferation in the liver cells in 50% ducks.These results proved that the over-dose arsenical had adverse effects on the liver function.These liver cell abnormalities were also observed in our previous trials using 300 mg Roxarsone inclusion in the diet on laying Tsaiya ducks (Chen et al., 2000) and mule ducks (Chen and Chiou, 2005).Arsanilic acid or As 2 O 5 treatments did not increase AST and LDH activities.This reflected that liver was only slightly damaged and still had not reached the threshold to induce the increase in these enzyme activities.

CONCLUSION
During withdrawal period, the Roxarsone treatment but not the As 2 O 3 , As 2 O 5 and arsanilic acid treatments showed the higher liver weight and relative liver weight than the control.All arsenical treatments would result in the liver vacuoles from histological observations, which actually defined as fatty liver, and would have adverse effects on animals.However, the arsenicals were different in effectiveness and in mechanisms in inducing the fat vacuoles.

Table 2 .
Effects of different sources of arsenicals on growth performance and carcass traits in mule ducks a, b Means in the same row with different superscripts are significantly different (p<0.05).x,y Means in the same column with different superscripts are significantly different (p<0.05).

Table 3 .
Effects of different sources of arsenical on hepatic lipogenic enzymes and blood lipid in mule ducks a, b Means in the same row with different superscripts are significantly different (p<0.05).x,yMeans in the same column with different superscripts are significantly different (p<0.05).*The enzyme activities were measured NADH at 340 nm and expressed as amount of NADH consumed or produced per min.1 unit = 1 n mole/mg protein.** p<0.1 compared with control.

Table 4 .
Effects of different sources of arsenicals on blood chemistry and liver histology in mule ducks Means in the same row with different superscripts are significantly different (p<0.05).x,y Means in the same column with different superscripts are significantly different (p<0.05).