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 Animal Feed Science and Technology 170 (2011) 239–245

Contents lists available at SciVerse ScienceDirect

Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci

Control of ascites mortality and improved performance and meat

shelf-life in broilers using feed adjuncts with presumed antioxidant
activity
J. Rajani, M.A. Karimi Torshizi ∗ , Sh. Rahimi
Department of Poultry Science, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Four hundred 1-d old male broilers were assigned to five experimental groups, including:
Received 4 January 2011 1 – Control, 2 – Vitamin E (100 mg/kg), 3 – Aspirin (2000 mg/kg), 4 – Synthetic antioxidant
Received in revised form 23 August 2011 (125 mg/kg, Loxidan TD 30), 5 – Pomegranate peel (15,000 mg/kg). Ascites was induced
Accepted 1 September 2011
by providing broilers water containing 0.15 g/L NaCl from 6 to 12 d. Blood samples were
drawn from 12 birds per experimental group at the end of experiment (42 d). Malondialde-
hyde (MDA) concentration in meat samples was determined. Erythrocyte osmotic fragility
Keywords:
Antioxidant
(EOF) was determined in whole blood as a criterion of erythrocyte membrane fluidity. Right
Ascites syndrome ventricular weight ratio (RV/TV) was determined in all birds. Supplementation of diets by
Broiler antioxidant feed additives reduced the experimental induced ascites mortality in compar-
Performance ison to control group (P<0.05). The antioxidants were able to reduce the MDA occurrence
Lipid oxidation in meat of birds compared with control, where the pomegranate peel treatment being
the most effective (P<0.01). Erythrocyte osmotic fragility was reduced in aspirin fed birds
(P<0.01). Supplementation of antioxidants improved RV/TV of broilers in comparison to
controls with Loxidan TD 30 being the most effective group (P<0.01). In conclusion, the
results of the present study emphasized the efficacy of antioxidants’ role in reduction of
ascites incidence as a consequence of oxygen reactive substances’ scavenging activity. In
addition, these nutritional interventions also extended meat shelf-life.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Ascites is one of the most common metabolic diseases in broilers, which has caused great financial losses to the poultry
industry worldwide (Leeson, 2007). Many factors such as genetics, nutrition, environment, diseases and management play
a role in the etiology of ascites. Oxygen-derived free radicals play an important role in the genesis of tissue damage during
inflammatory reaction and ischaemia (Halliwell and Gutteridge, 1990). It was suggested that ascites syndrome might be
associated with oxidative stress and lipid peroxidation induced by reactive oxygen species (Bottje and Wideman, 1995;
Villar-Patino et al., 2002). The incidence of ascites may be due to in part to free radical generation in birds, with subsequent
depletion of tissue antioxidants (Bottje et al., 1995).
Several nutritional, medicinal and management programs are devised in order to alleviate economic loss from ascites.
The cornerstone of these strategies is reduction of oxygen needs of birds via controlling growth rate, provided that the

Abbreviations: MDA, Malondialdehyde; EOF, Erythrocyte osmotic fragility; RV/TV, Right ventricular weight ratio; PP, Pomegranate peel; PCV, Packed cell
volume; GAE, Gallic acid equivalent; PBS, Phosphate buffered saline; OD, Optical density; BHA, Butylated hydroxyanisole; BHT, Butylated hydroxytoluene.
∗ Corresponding author. Tel.: +98 21 48292356; fax: +98 21 48292200.
E-mail addresses: karimitm@modares.ac.ir, karimitm@yahoo.com (M.A. Karimi Torshizi).

0377-8401/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.anifeedsci.2011.09.001
240 J. Rajani et al. / Animal Feed Science and Technology 170 (2011) 239–245

overall growth performance not impaired. However, the majority of ascites controlling programs exert their role through
irreversible limitation of growth rate. It means that the maximum profitability may not be attained. It is of vast importance
to find strategies that effectively alleviate ascites while simultaneously supporting the optimum growth rate of broilers.
Role of reactive oxygen species (ROS) in ascites etiology was the spotlight of several studies (Bottje et al., 1995; Hassanzadeh
et al., 1997; Xiang et al., 2002).
The most common used synthetic antioxidants, butylated hydroxyanisole and butylated hydroxytoluene, have been
restricted recently, mainly because of their possible carcinogenicity causing liver swelling and changing liver enzyme activ-
ities (Martin and Gilbert, 1968). Aspirin has been shown to possess antioxidant and radical scavenging properties (Xianglin
et al., 1999). It has the capacity to directly protect the endothelium from the deleterious influences of oxidative stress
(Podohaisky et al., 1997). Vitamin E is an effective lipid-soluble free-radical scavenger (Morrissey et al., 1994). Its con-
sumption has been linked to the prevention of several metabolic disorders (Traber and Sies, 1996). Supplementation of
poultry diets with vitamin E could attain following objectives: prevention of nutritional deficiencies in poultry, improving
the oxidative stability of broiler meat and egg products and producing highly nutritional food source of vitamin E for the
human consumers (Sirri and Barroeta, 2007). However, use of vitamin E implants lead to the reduction of mortality rate
induced by ascites possibly by attenuating oxidative stress (Bottje et al., 1995).
Many herbs contain ingredients, which are active in three main areas: gut microflora, antioxidant properties, and liver
function (Lange, 1998). The pomegranate peel (PP) is a natural source of polyphenolic compounds such as ellagic tannins,
ellagic acid and gallic acid (Negi et al., 2003). The PP phenolics may act as antioxidants by donating electrons and reacting
with free radicals to convert them to more stable products and terminate free radical chain reactions (Negi and Jayaprakasha,
2003).
There are scant reports that test the preventive efficiency of commonly used antioxidants on ascites incidence in broilers
under farm conditions. These works were limited to vitamins (C and E), coenzyme Q10, l-carnitine, uric acid and selenium
(Bottje et al., 1995; Roch et al., 2000; Stinefelt, 2003; Geng et al., 2004a,b). The primary aim of the present study was
to assess the possible role of various antioxidants (synthetic and natural) in ascites control and meat stability of broiler
chickens. Although, antioxidative property of PP was reported already (Negi and Jayaprakasha, 2003; Naveena et al., 2008),
the effects of supplementation of PP into broiler diets are reported in the present work for the first time.

2. Materials and methods

2.1. Experimental birds and treatments

A Total of 400 1-d old male broilers (Ross 308) were assigned into five experimental groups, each was composed of 4 floor
pen replications of 20 birds including: 1 – Control group, 2 – Vitamin E (100 mg/kg, ␣-tocopherol acetate, Razak, Tehran,
Iran), 3 – Aspirin (2000 mg/kg, Pars Daru, Tehran, Iran), 4 – Synthetic antioxidant (125 mg/kg, Loxidan TD 30, Lohmann
Animal Health, Cuxhaven, Germany), 5 – Pomegranate peel (15,000 mg/kg, local market, Gorgan, Iran). All additives were
supplemented in mash form feed and the chosen supplementation levels for Loxidan TD, PP, aspirin and vitamin E were
adopted from previous published works by Wang et al. (1997), Brenes et al. (2008), Balog et al. (2000) and Konjufca et al.
(2004), respectively. The pomegranates were purchased from local market as fresh Iranian pomegranate (Punica granatum
L. Khoram Dizin Torshe Gorgan variety). The skins were manually removed and the peels were air dried under ambient
conditions and finely ground. The total polyphenolics content of PP was determined using the Folin–Ciocalteus assay as
gallic acid equivalent (GAE) (Escarpa and Gonzalez, 2001). The total polyphenolics concentration was 235.41 mg GAE/g PP.
Loxidan TD 30 is a mixture of three synthetic antioxidants including propylgallate, ethoxyquin and butylated hydroxy-
toluene, with synergistic effects.

2.2. Diet and husbandry

The animal use protocol was approved by the Institutional animal care of Tarbiat Modares University. Chicks had access
to maize-soybean based starter (1–14 d), grower (15–42 d) diets (Table 1) and water ad libitum. Birds were grown on floor
pens furnished with wheat straw litter for 42 d. Temperature was set at 32 ◦ C on the first day and gradually reduced to 24 ◦ C
by the third week, and maintained to the end of experiment at the same level. Light was provided according to the following
schedule 23 h light: 1 h dark. Birds were monitored for ascites symptoms (water belly) and mortality three times a day after
6 d.

2.3. Growth performance

Birds were weighed on pen basis every two weeks, and feed consumption was measured throughout the experimental
period to calculate feed conversion ratio (FCR). Performance data were corrected for dead birds during course of experiment.
 J. Rajani et al. / Animal Feed Science and Technology 170 (2011) 239–245 241

Table 1
Composition of the basal (control) diet (g/kg).

Ingredients Starter Grower

Yellow maize 562.0 625.7

Soybean meal (440 g/kg protein) 330.0 295.0
Fish meal (650 g/kg protein) 40.0 25.0
Vegetable oil 37.8 26.0
Dicalcium phosphate 12.0 10.0
Calcium carbonate 10.0 10.0
Sodium chloride 2.0 2.3
Permixa 5.0 5.0
DL-Methionine 1.2 1.0

Calculated valuesb (as fed basis) (g/kg)

Metabolisable energy (MJ/kg) 12.97 12.55

Crude protein 220 200
Lysine 12.3 12.0
Methionine + Cystine 10.1 9.0
Threonine 9.0 8.1
Ca 10.0 9.0
Available phosphorus 5.0 4.5
a
Supplied per kg diet: Vit. A, 7040 IU; Vit. D3, 2000 IU; Vit. E, 8.8 IU; Vit. K3, 1.76 mg; Biotin, 0.12 mg; Thiamine, 1.2 mg; Riboflavin, 3.2 mg; Pantothenic
acid, 6.4 mg; Pyridoxine, 1.97 mg; Niacin, 28 mg; Vit. B12, 0.008 mg; Choline, 320 mg; Folic acid, 0.38 mg; Mn, 60 mg; Fe, 60 mg; Zn, 51.74 mg; Cu, 4.8 mg;
I, 0.69 mg; Se, 0.16 mg.
b
The values were calculated from NRC (1994).

2.4. Ascites induction

The experimental ascites was induced using sodium in drinking water model (Julian et al., 1992). Briefly, from 6 to 12 d,
NaCl in drinking water was provided at 1.5 g/L to all birds, and then returned to common tap water throughout the rest of
the experiment (days 13–42).

2.5. Malondialdehyde concentration in meat samples and ascites related parameters

Whole blood samples were drawn from twelve birds per experimental group at 42 d and then theses birds were slaugh-
tered, plucked and eviscerated. De-boned and skinless thigh and breast were ground, and apportioned into two sets of
sub-samples immediately after slaughter. The first set of samples was used to determine MDA concentration of fresh sam-
ples. The second set of samples was stored at −20 ◦ C for 30 d before MDA determination. The MDA content of meat samples
was determined as a measure of lipid oxidative susceptibility. The determination of MDA was based on colorimetric assay
of thiobarbituric acid reactive substances as described by Botsoglou et al. (1994).
Packed cell volume percentage was determined in whole blood samples by microhematocrit capillary tubes centrifuged
at 15,500 × g for 5 min (Jain, 1986).
Erythrocyte osmotic fragility was determined in whole blood samples as a criterion of red blood cell membrane fluidity
by Dacie’s method (Buffenstein et al., 2001) with minor modifications. Briefly, fresh blood (20 ␮L) was added to the tubes
containing 5 mL of phosphate buffered saline (PBS) solutions at 3 concentrations (0.1, 0.5, and 0.9% of NaCl w/v). The tubes
were mixed gently and incubated at room temperature (24 ◦ C) for 30 min. After mixing, the suspensions were centrifuged
at 430 × g for 5 min. The optical density (OD) of supernatant was measured at 540 nm with a spectrophotometer, against
blood in 0.9% PBS as a blank. Erythrocyte osmotic fragility was expressed as:
 OD at 0.5% PBS 
EOF (%) = × 100
OD at 0.1% PBS

All experimental birds were slaughtered at the end of experiment (42 d). Hearts were collected and pericardium, periph-
eral adipose tissues and atriums were removed. The left and right ventricles were separated and their individual weights
were measured. The RV/TV as a simple measure of ascites incidence was determined by the following formula (Arab et al.,
2006): RV/TV(%) = [Weight of right ventricle/(Weight of right ventricle + Weight of left ventricle)] × 100.

2.6. Statistical analysis

Mean values between treatments were compared by using analysis of variance followed by least significant difference
(LSD) test (SAS, 1990). Significant differences were accepted if P≤0.05. Ascites mortality data were analyzed by Chi-square
test.
242 J. Rajani et al. / Animal Feed Science and Technology 170 (2011) 239–245

Table 2
Effect of natural and synthetic antioxidants on performance of broilers.

Treatments Control Pomegranate Peel Vitamin E Aspirin Loxidan TD 30 SEM P value

Body weight at 42 d (g) 1957.2b 1864.5b 1947.7b 1877.7b 2223.0a 31.9 0.009
Body weight gain (g/bird/d) 45.64b 43.47b 45.43b 43.85b 51.98a 0.61 0.001
Feed intake (g/bird/d) 95.39ab 92.16bc 91.31c 93.40abc 96.68a 0.72 0.001
Feed conversion ratio 2.09a 2.12a 2.01ab 2.13a 1.86b 0.03 0.006
abc
Means with different superscripts in each row are significantly different (P<0.01).

Table 3
Effect of natural and synthetic antioxidants on ascites related parameters and MDA occurrence in meat of broilers.

Treatments Control Pomegranate Peel Vitamin E Aspirin Loxidan TD 30 SEM P value

Right ventricular weight ratio (%) 0.204a 0.195ab 0.187b 0.191b 0.183b 0.002 0.001
Ascites mortality† 7/80 (8.86)a 1/80 (1.25)bc 2/80 (2.50)b 0/80 (0.0)c 3/80 (3.75)b – 0.020
Erythrocyte osmotic fragility (%) 14.80a 15.31a 14.62a 10.43b 11.00b 0.49 0.001
Packed cell volume (%) 31.67b 29.56b 29.72b 30.61b 35.40a 0.54 0.001
Fresh meat malondialdehyde (␮g/g) 0.114 0.096 0.102 0.116 0.108 0.003 0.650
Stored meat malondialdehyde (␮g/g) 0.224a 0.176b 0.197b 0.196b 0.187b 0.004 0.001
abc
Means with different superscripts in each row are significantly different (P<0.05).
†
Number of dead birds/total no. of birds (%).

3. Results

Body weight and daily body weight gain (Table 2) in birds that received Loxidan TD 30 were higher (P<0.01) than control
and other groups, while other feed additives were not different (P>0.05) from control. Daily feed intake in Loxidan TD
30 supplemented birds was different (P<0.01) from birds provided vitamin E, PP and aspirin. Feed conversion ratio was
influenced (P<0.01) by supplementation of presumed antioxidant feed additives. Birds fed the Loxidan TD 30 diet had the
lowest FCR (P<0.01) which was not different from the vitamin E group. The FCR of other groups was similar to control.
The ascites index (RV/TV) was affected by supplementation of broiler diets with some of antioxidants (P<0.05). Broilers
fed diets supplemented with Loxidan TD 30, aspirin and vitamin E had significant reduction in RV/TV compared to control
(Table 3).
Antioxidant feed additives reduced the mortality cause by ascites induction during 6–12 d (P<0.05). The highest ascites’
related mortality (8.86%) was belonging to control group, and aspirin fed group had actually zero mortality.
Packed cell volume percentages were influenced by Loxidan TD 30 (P<0.05), however, other presumed feed additive
antioxidants were similar to control.
Erythrocyte osmotic fragility percentage was influenced by antioxidant feed additives (P<0.05). Aspirin and Loxidan TD
30 fed groups showed the lowest EOF values (10.43% and 11.00%, respectively) in comparison to control (14.80%).
The MDA levels of frozen stored meat samples (Table 3) were lower (P<0.05) in antioxidant fed groups (in the range of
0.176–0.197 ␮g/g for PP and vitamin E, respectively) compared to control (0.224 ␮g/g), while the fresh samples had similar
MDA concentrations (P>0.05).

4. Discussion

4.1. Performance

Birds fed Loxidan TD 30 had the best performance. Body weight, daily body weight gain and FCR of birds fed Loxidan TD
30 had considerable improvement compared to control (13.6 and 10%, respectively). Similar major increase in body weight
of broilers have been reported by Lin et al. (1989) and Wang et al. (1997) who investigated synthetic antioxidants BHA/BHT
and ethoxyquin, respectively. Although improvement in performances were generally reported in stress conditions (use
of highly unsaturated fatty acids, oxidized fats and high ambient temperature), our results showed that beneficial effects
of synthetic antioxidants on broiler performance could be exerted in normal condition with high salt model of inducing
ascites. The higher efficiency of some antioxidants may be attributed to their higher fat solubility, which would facilitate
their absorption from the intestinal lumen (Fellenberg and Speisky, 2006). However, this mechanism may not be relevant
to our results, as body weight was lower in vitamin E supplemented birds compared to Loxidan TD 30 group, despite of high
lipid solubility of vitamin E.
Although the metabolic cost of antioxidant defense mechanism has not been determined in broilers, it seems to be
significant due to the observed rapid growth rate in Loxidan TD 30, which means control of oxidative pressure (Ruiz-Feria,
2009), especially when taking into account the unsaturated nature of fat reserves of broilers (Labuza, 1971). Loxidan TD
30 may save nutrients, by increasing antioxidant potential through sparing endogenous antioxidants of birds. The saved
nutrients could be directed to growth of body. Authors wish to attract attentions to possible growth stimulating effect of
synthetic antioxidants (Loxidan TD 30) on broilers.
 J. Rajani et al. / Animal Feed Science and Technology 170 (2011) 239–245 243

Aspirin fed birds had lower body weight compared to Loxidan TD 30 fed group and these broilers were 4.06% lighter
than control birds. Balog et al. (2000) also reported that aspirin depressed growth in broilers. Proudfoot and Hulan (1983)
observed similar reduction in body weight (4.93% compared to control) by using 1600 mg/kg at 49 d.
Supplementation of diet with 100 mg/kg of vitamin E did not improve body weight of chicks in comparison to control,
which is in agreement with results obtained by Konjufca et al. (2004). Pomegranate peel at used levels did not influenced
body weight of broilers compared to control.
The possible reason of observed growth improvement in Loxidan TD 30, could not be attributed solely to increase in feed
intake, because its feed intake was not different to control and aspirin-broiler groups. Therefore, the exact mechanism for
growth stimulating effects of Loxidan TD 30 remains unclear.
Interestingly, the lowest feed intake, which was found in vitamin E and PP groups did not concurrent to minimum FCR
of these birds, reflecting the lower body weight gain of these groups. The lowest FCR was belonging to Loxidan TD 30 group.
When taking into account the highest body weight and a feed consumption similar to control group, the better FCR of Loxidan
TD 30 group could be justified.
Antioxidants activity can occur outside of the body (e.g. in the feed or lumen of digestive tract) or inside the animal body
within extracellular or intracellular compartments. Some antioxidants are produced in protected form to increase their shelf-
life. For example, vitamin E, in form used in present study as ␣-tocopherol acetate is active only after undergoing hydrolyses
of protective chemical groups (acetate ester). Therefore, antioxidant activity of these products occurs after hydrolysis in the
gastrointestinal tract and protection of feed during storage and delivery is unexpected.
Synthetic antioxidants in the feed can be more effective than their natural counterparts due to, their relatively higher
effectiveness and lower cost. On the other hand, there are also growing concerns about widely using these synthetic antiox-
idants and its possible relation to cancer and mutation in consumers. However, the safety of natural antioxidants has not
guaranteed yet (Fellenberg and Speisky, 2006).

4.2. Ascites related parameters

Among several indicators for ascites assessment, the RV/TV proved the strongest relationship to ascites incidence (Lubritz
et al., 1995). The RV/TV values measured in our experiment (0.183–0.204%) are far less than the cutoff limits (about
0.25–0.30%) described for ascitic broilers (Julian, 1987; Cawthon et al., 2001). The reason for lower observed RV/TV values
(Table 2) could be attributed to lack of ascites predisposing factors: cold stress (Wideman and French, 1999), metabolism
stimulatory hormones (Hassanzadeh et al., 2000), sodium (Julian, 1987), hypobaric condition (Balog et al., 2000), and various
other factors. Also, because mortality occurred only during the administration of high NaCl in the waters, fully developed
ascites syndrome may not have been attained.
Control of growth rate, especially at early stages of broilers’ life gained promising prospective in alleviation of ascites
syndrome. However, this strategy should be adopted carefully, if there is not guarantee of catch up growth took placed
at the end of production period; the economic losses from low final body weight would out weigh any profit obtained by
reductions in ascites related mortality. Although adding Loxidan TD 30, vitamin E and aspirin to broiler diets significantly
reduced the RV/TV, these improvements in RV/TV in the aspirin and vitamin E groups was concurrent with lower final body
weight compared to Loxidan TD 30. The lowering effect of Loxidan TD 30 on RV/TV could be attributed to mechanisms
other than controlled growth rate as evidenced for aspirin and vitamin E. groups. The incidence of ascites is not the sole
consequence of systemic hypoxia, but the free radical generation would be encouraged under hypoxia (Bottje and Wideman,
1995). Therefore, enforcement of antioxidant statues could control the ascites incidence.
In the present study, ascites related mortality took place during 7 days of NaCl provision through drinking water. The
mortality ceased when normal drinking water was provided after day 12. The pattern of ascites mortality distribution
among experimental groups implies the effectiveness of short-term in-feed antioxidants on ascites mortality. Interestingly,
no case of mortality was observed in aspirin fed broilers while the control group had the maximum dead birds (8.86%). This
observation triggers the possible practical use of sodium stress in early life as a tool for diagnosis of susceptible individual
birds. The mean RV/TV for the dead broilers was 0.32 ± 0.03%, revealing ascites incidence.
Lipids are major constituents of cell membranes, conferring strength and fluidity properties to cells. Erythrocytes were
frequently investigated as indexes of cell membrane integrity (Xia et al., 1999; Geng et al., 2004a,b). Gutteridge et al. (1986)
reported that lower EOF reflects the protection of the structure and distensibility of the erythrocyte membrane and cell
geometry. Cell membranes are rich in unsaturated fatty acids; therefore, they are susceptible targets for oxidative attacks
in the absence of potent protective antioxidant mechanisms (Gutteridge et al., 1986).
Aspirin could lower EOF via two possible mechanisms; either by changing the lipid profile of the cell membrane (Steer
et al., 1997) and/or exerting antioxidant activity (Xianglin et al., 1999; Woollard et al., 1990; Aruoma and Halliwell, 1988).
Increases membrane cholesterol alters membrane surface area and decreases membrane fluidity. Thus, a decreased amount
of membrane cholesterol permits higher deformability (Cooper et al., 1975) and lower EOF. However, investigators have
demonstrated that an equilibrium exchange exists between the cholesterol in red cells and that in whole serum (Hagerman
and Gould, 1951). Inflammation (by definition) involves the production of toxic oxygen by human white blood cells. The toxic
oxygen may leak into nearby cells, damage the DNA, and provoke cancer (Ames et al., 1996). Aspirin as anti-inflammatory
drug could control the generation of toxic oxygen and prevent its detrimental consequences.
244 J. Rajani et al. / Animal Feed Science and Technology 170 (2011) 239–245

An increase in EOF could decrease erythrocytes deformability, which could lower oxygen transport and increase blood
viscosity. The resulting hypoxia induced by high EOF values could contribute in facilitating of ascites incidence (Julian, 1993).
In our experiment, reduction in EOF compared to control was in accordance to decrease in RV/TV observed in aspirin group.
Elevation of PCV was regarded as a manifestation of ascites, because erythropoises activation occurs in response to
hypoxia (Leeson et al., 1995). As severe ascitic birds died during NaCl administration (7–12 d), it may conclude that only
more resistant birds against ascites were the survivors at the time when PCV was determined (42 d). Therefore, the elevated
PCV in Loxidan TD 30 fed broilers compared to other groups at 42 d, could not reflex the ascites condition. Scheele et al. (2003)
showed limited practical value of high PCV in juvenile chickens as an ascites predictor. Higher PCV may be a compensatory
response of birds to supply more oxygen to tissues. Katzung, 1998, reported the PCV lowering effect of apsirin.

4.3. Malondialdehyde of meat

Malondialdehyde occurrence in meat is a popular assessment of lipid oxidation (Botsoglou et al., 1994). Antioxidants used
in the present experiment maintained MDA level of boilers’ meat 9–21% lower than the control group. Lipid peroxidation
within cell membranes caused by reactive oxygen species is believed to play a significant role in the etiology of ascites (Bottje
et al., 1995). Thus, inadequate antioxidant protection or higher free radical generation from various sites in the cell including
mitochondria may be key factors in the pathophysiology of ascites. Antioxidants like vitamins C, E and glutathione (GSH)
were diminished in tissues of severe ascitic birds, while the levels of lipid peroxides were increased compared to control
(Enkvetchakul et al., 1993; Bottje et al., 1995).
Although there was no study examining the antioxidative effect of dietary PP on stored meat, Swamy et al. (2011) reported
the lower MDA in fresh liver samples of rats fed PP extract (25 mg/d polyphenols equivalent). PP ability to lower MDA levels
could be explained by antioxidant compounds including, (a) ellagitannins, a precursor of ellagic acid, that has been found
to have antioxidative properties (Osawa et al., 1987; Mass et al., 1991), and (b) punicalagin which has the strongest lipid
peroxidation inhibitory and free radical scavenging activities (Kulkarni et al., 2004). Alpha-tocopherol and ␥-tocopherol are
present in PP could also contribute to its antioxidant property.

5. Conclusions

In summary, dietary antioxidants used in the present study were effective in reducing RV/TV, mortality, and MDA of
stored meat. These benefits were accompanied by higher body weight for synthetic antioxidant (Loxidan TD 30), while
vitamin E, PP and aspirin had no improvement in body weight of broilers. As only single dosage levels were tested in the
present study for each antioxidant, investigation of different dose levels is warranted for future studies.

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