Prophylactic action of lipoic acid on oxidative stress and growth performance in broilers at risk of developing ascites syndrome

Abstract

The objective of this study was to assess the effects of dietary supplementation with lipoic acid (LA) on broilers maintained at 2235 m above sea level with high risk to develop ascites syndrome (AS). A total of 2040 chicks were fed under commercial conditions with water and specific diets ad libitum during 7 weeks in two consecutive experiments. Mortality and indicators of performance and oxidative stress were compared weekly in broilers fed a basal diet plus 0, 10, 20, or 40 parts/10⁶ LA. The effects of LA at 40 parts/10⁶ were also studied during the initial 3 weeks or the last 4 weeks of the production cycle. Diets supplemented with 40 parts/10⁶ of LA during 7 weeks significantly improved feed conversion, decreased general mortality and mortality attributable to AS, and lowered thiobarbituric acid reactive substances and hydroxyl radicals in liver, and increased total glutathione pool. Smaller doses or shorter periods of exposure to LA were partially effective. In conclusion, LA under our experimental conditions has a prophylactic action in broilers with high risk to develop AS due to oxygen availability limitation.

1 Introduction

Ascites syndrome (AS) is well characterized from the epidemiological, clinical, and anatomopathological points of view. Accumulative evidence favors the idea that decreased oxygen tension or increased oxygen requirements causes AS in broiler chickens (Decuypere et al., 2000). If both conditions are present, risk of developing AS increases as more oxygen is required to sustain a high feed conversion rate and rapid growth, and oxygen tension decreases at altitudes>2000 m above sea level, as occurs on farms near Mexico City where mortality rates attributable to AS in this region can be as high as 29% (Arce et al., 1990). Thus, hypoxic conditions in tissues may result in a vicious circle in which reduction in pulmonary oxygen tension constricts arterioles, causing an increase in lung arterial pressure; this in turn results in right ventricle hypertrophy and gradual reduction in right ventricle output, which substantially increases venous pressure of the hepatic veins that results in edema in the lungs, the abdominal cavity, and the hydropericardium (Julian, 1993; Decuypere et al., 2000).

However, Bottje & Wideman (1995) and our research group (Díaz-Cruz et al., 1996) have shown that oxidative stress—that is, an important imbalance between production of reactive oxygen species (ROS) and reactive nitrogen species on the one hand, and antioxidant defenses on the other—is included in development of AS. To date, experiments are insufficient to define whether this oxidative stress is a cause or a consequence in the pathogenesis of AS. In any event, temporary reduction in feed intake (Suárez & Rubio, 1989) that in other systems limits ROS (Sohal & Weindruch, 1966), alternative lighting schedules (Buys et al., 1998), and dietary supplementation with antioxidants and free radical scavengers, as detailed in the Discussion of this work, have been attempted to decrease AS mortality.

The purpose of the study reported here was to evaluate whether dietary supplementation with lipoic acid (LA) would be a better alternative than the use of other prophylactic measures to ameliorate oxidative stress, growth performance, and cumulative mortality attributable to naturally developing AS in broiler flocks maintained under commercial conditions at an altitude of 2235 m above sea level (i.e. at high risk to develop AS). Indicators of oxidative stress in this work included thiobarbituric acid reactive substances (TBARS), total glutathione (TG), the presence of the hydroxyl radical (OH ), and the electrophoretic mobility of catalase, which is modified by the presence of singlet oxygen and gives rise to conformers with increased electrophoretic mobility (Lledias & Hansberg, 2000). Antioxidant properties of lipoic acid are well known, its use has some advantages over antioxidant vitamins, and its pharmacologic properties have been summarized and reported earlier (Packer et al., 1995; Biewenga et al., 1997).

2 Materials and Methods

2.1 Experiment 1

Young (1-day-old) broilers (1032 chicks; Avian Farm x Arbor Acres strain) were used in this study. Broilers were weighed and randomly assigned to four dietary treatment groups (258 birds/group). Birds were housed for the duration of the 7-week study in six pens (43 birds/pen) per treatment at an altitude of approximately 2235 m above sea level. The chicks were brooded at 32°C during week 1; thereafter, the temperature was reduced gradually each week to reach 24°C at day 28. All chicks were fed the same starter diet (1–21 days) containing 3000 kcal/kg metabolizable energy (ME) and a finisher diet (22–49 days) with 3100 kcal/kg ME, both diets formulated to meet or exceed all requirements of the National Research Council (1994). Birds were randomly assigned to the control group or groups with LA. Control birds were fed the basal diet without additional LA, while birds with LA were fed the basal diet to which LA (10, 20, and 40 parts/10⁶) was added.

2.1.1 Thiobarbituric acid reactive substances

The concentration of TBARS was assessed by the method of Zentella de Piña et al. (1993). Briefly, homogenates of hepatic tissues were filtered through a cheesecloth, and an aliquot of the filtered homogenate was incubated with 0.1 ml of 0.15 M phosphate buffer (pH 7.4) for 30 min at 37°C. Then, 1.5 ml of 20% acetic acid (pH 2.5) and 1.5 ml of 0.8% thiobarbituric acid were added. The mixture was placed in boiling water for 45 min. Tubes were allowed to cool, and 1 ml of 2% KCl and 5 ml butanol:pyridine (15:1) solution was added to each tube. Tubes were mixed vigorously and the absorbance of the organic layer was measured by a spectrophotometer set at 532 nm. The concentration of TBARS in the samples was calculated with an extinction coefficient of 1.56 × 10⁵/M/cm (Wills, 1969). The protein concentration was determined by the method of Bradford (1976).

2.1.2 Total glutathione

The concentration of TG was determined by the enzymatic method of Akerboom & Sies (1981). Briefly, 1.0 ml of 0.1 M buffer phosphate, 100 μl sample containing 0.5 to 2 n moles of glutathione, 20 μl nicotinamide adenine dinucleotide phosphate reduced (NADPH), 20 μl 5,5′-dithiolbis(2-nitrobenzoic acid) (DTNB), and 20 μl glutathione reductase were placed in cuvette. After mixing the contents of the cuvette, the increase in absorbance at 412 nm was recorded. A blank assay without glutathione was run separately. For calibration, the procedure was repeated using oxidized glutathione instead of the sample. The temperature was controlled at 25°C.

2.2 Experiment 2

Broilers of 1 day old (1008 chicks, Ross x Ross strain) were fed the same starter and finisher ration as described in experiment 1 except for the ME (starter ration, 3100 kcal/kg; finisher ration, 3200 kcal/kg). Broilers were weighed and randomly assigned to four dietary treatment groups and were housed under the same conditions as in experiment 1. The dietary treatment groups were as follows; control basal diet group without LA throughout study; with LA (40 parts/10⁶) in basal diet for 1–21 days and with LA (40 parts/10⁶) in basal diet days for 22–49 only, and with LA (40 parts/10⁶) in basal diet throughout study.

In both experiments, feed and water were provide ad libitum. Feed consumption and body weight were recorded weekly. Liver samples were obtain weekly after euthanizing the birds by cervical dislocation; the liver was removed, blotted dry, and desiccated. Subsequently, the amounts of TBARS, TG, and OH in samples were determined. Birds were included in mortality by ascitic syndrome if they died with ascites without abdominal fluid accumulation but with obvious ascitic syndrome symptoms including right ventricular dilation, hydropericardium, and vascular congestion.

2.2.1 Concentration of hydroxyl radical

The concentration of hydroxyl radical (OH ) was assessed using the method described by Nash (1953). Briefly, 1 g hepatic tissue was homogenized in 5 ml of 20% trichloroacetic acid; an aliquot (500 μl) of homogenate was mixed in 1 ml Krebs–Ringer solution added with 0.1 mM FeCl₃, 0.2 mM ethylenediamine at pH 7.4 to avoid superoxide formation and 33 mM dimethylsulfoxide as the substrate for hydroxyl radicals in production of formaldehyde (Hallinan et al., 1991). Hydroxyl free radicals were measured as changes in absorbance at 412 nm in the supernate by the Hantzh reaction in which the formaldehyde formed, as an index of OH free radicals, was calculated using an extinction coefficient of 800 × 10³/M/nm.

2.2.2 Catalase

Catalase activity in polyacrylamide gels was analyzed by the method of Lledias & Hansberg (2000). Briefly, homogenates of liver tissues (1 g wet weight) were incubated with 5 ml of 20 mM 4-(2-hydroxy)-1-piperazineethanesulfonic acid (pH 7.2) containing 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 0.1 mM deferriferrioxamine B mesylate (Desferal). Samples of 18 μg protein in 10 μl were loaded into each lane. Gels were run at 150 V for 2.5 h; immediately after electrophoresis, these were stained for catalase activity, detected by incubating the gel for 5 min in 5% methanol and then rinsing three times with tap water for 10 min in 10 mM H₂O₂. The gel rinsed with tap water was incubated in a 1:1 mixture of freshly prepared 2% potassium ferric cyanide and 2% ferric chloride. A blue color developed in the gel except at zones in which H₂O₂ was decomposed by catalase.

2.2.3 Statistical analysis

One-way analysis of variance was used to compare differences in variables among and within experimental groups using statistical analysis calculated with SAS software (SAS Institute, 1995) with means comparisons using the Tukey test. A probability level of P<0.05 was considered statistically significant.

3 Results

3.1 Indicators of productivity

Data on feed consumption, growth rate, and feed conversion together with information on overall mortality and mortality attributable to AS are considered in this work as indicators of productivity. These indicators of productivity are reported first for experiment 1; that is, broilers fed with diets having lower ME and distributed in four experimental groups (without LA, and with three different levels of LA added to the diet). For better analysis of results, these were organized in two stages. The initial stage included the first 21 days of treatment with a diet having an ME of 3000 kcal/kg diet, while the final stage included days 22 to 49 of treatment with a diet having an ME of 3100 kcal/kg diet. Additionally, results of the entire 49-day study are presented (Table 1). During the initial stage, a statistically significant difference was found for general mortality by comparing number of birds fed with control diet with those fed with LA-enriched diet (10 parts/10⁶) (Table 1). Greater differences appeared in indicators of productivity of broilers during the final stage. Inclusion of LA in at least one diet resulted in the following statistically significant differences: decrease in feed consumption at 10, 20, and 40 parts/10⁶; better growth performance at 40 parts/10⁶; better feed conversion at 20 and 40 parts/10⁶; lower general mortality at 40 parts/10⁶, and much lower mortality attributable to AS at 40 parts/10⁶ (Table 1). For the entire experimental period, only feed conversion at 20 and 40 parts/10⁶, general mortality at 40 parts/10⁶, and mortality attributable to AS at 10, 20, 40 parts/10⁶ were improved by addition of LA to diets (Table 1). The effect (i.e. LA decreasing mortality attributable to AS) was increased as higher doses of antioxidant compounds were added to the diet (Table 1).

Indicators of productivity in broilers fed in stage 1 with a diet having a metabolizable energy content of 3000 kcal/kg and fed in stage 2 with a diet having a metabolizable energy content of 3100 kcal/kg: effect of lipoic acid

		Diets supplemented with lipoic acid
Variable	Age (days)	0 (control diet)	10 parts/10^6	20 parts/10^6	40 parts/10^6
Feed consumption (g)	1 to 21	858±14^A	858±12^A	863±37^A	841±46^A
	22 to 49	3751±50^A	3450±30^B	3387±40^C	3438±40^B
	1 to 49	4609±21^A	4308±15^A	4250±48^A	4379±33^A
Growth performance (g body weight)	1 to 21	584±20^A	585±20^A	582±40^A	568±50^A
	22 to 49	1783±60^A	1678±70^A	1725±50^A	1832±50^B
	1 to 49	2367±57^A	2262±30^A	2308±18^A	2400±58^A
Feed conversion (g feed/g body weight gained)	1 to 21	1.46±0.02^A	1.46±0.03^A	1.48±0.07^A	1.48±0.08^A
	22 to 49	2.10±0.08^A	2.05±0.07^A	1.96±0.08^B	1.87±0.06^C
	1 to 49	1.95±0.02^A	1.89±0.02^A	1.84±0.01^B	1.82±0.02^B
General mortality (%)	1 to 21	3.52±0.81^A	0.78±0.03^B	1.17±0.07^AB	1.93±0.08^AB
	22 to 49	5.64±0.99^A	4.30±1.14^AB	3.51±1.03^AB	1.57±0.49^B
	1 to 49	9.16±1.43^A	5.08±1.16^AB	4.68±1.08^AB	3.50±0.93^B
Mortality attributable to ascites syndrome (%)	1 to 21	0.76±0.48^A	0.00±0.00^A	0.00±0.00^A	0.38±0.39^A
	22 to 49	3.92±0.88^A	2.31±0.84^AB	1.96±0.74^AB	0.76±0.48^B
	1 to 49	4.68±0.71^A	2.31±0.38^B	1.96±0.77^B	1.14±0.38^B
Values presented as mean (± standard error of the mean). Within each row, values with different superscript letters differ significantly (P<0.05).

Beneficial results due to LA-enhanced diet were evident during 22 to 49 days in experiment 1 as compared with scarce results reached during the initial stage of study (Table 1). Hence, a second experiment was carried out in which supplementation of LA (40 parts/10⁶) was offered to broilers in one or both stages. Control groups included broilers with an LA-free diet. To establish a higher risk in broilers to develop AS spontaneously, the ME content of diets was increased to 3100 kcal/kg during initial stages and to 3200 kcal/kg during the final stage. Additionally, a different chick strain was tested to validate LA preventing AS due to limitation in oxygen availability. Results with the same indicators of productivity as those presented in Table 1 are presented in Table 2 for the second experiment. An increase in the ME content of diets augmented feed consumption, growth performance, general mortality, and mortality attributable to AS, and decreased feed conversion in all groups (except one) of birds receiving 40 parts/10⁶ LA (comparison of data in Table 1 versus Table 2). Broilers included in the second experiment receiving diets with increased ME were subjected to a higher challenge to develop AS, as confirmed by an increase in mortality attributable to AS (see Table 2 versus Table 1). Despite this, supplementation with 40 parts/10⁶ LA during the entire 49 days of the study reproduced the benefits recorded for LA in the first experiment; that is, a significant increase in growth performance and decrease in both feed conversion and mortality attributable to AS (Table 2). It is of interest that the main effect of LA in preventing AS was reproduced with a different chick strain.

Indicators of productivity in broilers fed in stage 1 with a diet having a metabolizable energy content of 3100 kcal/kg and fed in stage 2 with a diet having a metabolizable energy content of 3200 kcal/kg: effect of lipoic acid, 40 parts/10⁶, during different experimental periods

		Days on which 40 parts/10^6 lipoic acid was included in the diet
Variable	Age (days)	Day 0 (control diet)	Days 1 to 21	Days 22 to 49	Days 1 to 49
Feed consumption (g)	1 to 21	949±6.97^A	924±11.78^A	944±9.85^A	923±8.14^A
	22 to 49	4109±12.27^A	4041±27.20^A	4029±24.03^A	4044±40.68^A
	1 to 49	5058±12.85^A	4966±22.02^A	4970±30.64^A	4967±24.51^A
Growth performance (g body weight)	1 to 21	649±5.45^A	655±2.25^A	659±3.51^A	664±4.75^A
	22 to 49	2160±27.34^A	2205±25.75^AB	2219±21.38^AB	2269±26.65^B
	1 to 49	2809±17.77^A	2873±19.82^AB	2877±24.56^AB	2933±25.70^B
Feed conversion (g feed/g body weight gained)	1 to 21	1.46±0.02^A	1.41±0.02^AB	1.43±0.02^AB	1.39±0.01^B
	22 to 49	1.90±0.01^A	1.83±0.02^B	1.82±0.02^B	1.78±0.01^B
	1 to 49	1.80±0.01^A	1.73±0.01^B	1.73±0.01^B	1.69±0.01^B
General mortality (%)	1 to 21	4.37±2.16^A	3.57±1.46^A	1.59±0.48^A	3.57±0.53^A
	22 to 49	12.69±1.70^A	11.11±2.00^A	11.90±1.87^A	9.52±1.94^A
	1 to 49	17.06±2.97^A	14.68±2.16^A	16.66±1.62^A	13.09±2.01^A
Mortality attributable to ascites syndrome (%)	1 to 21	0.40±0.39	0.00±0.00	0.00±0.00	0.00±0.00
	22 to 49	9.12±1.67^A	5.55±1.00^AB	6.74 ±1.29^AB	3.96±1.00^B
	1 to 49	9.52±1.62^A	5.55±1.00^AB	6.74±1.29^AB	3.96±1.00^B
Values presented as mean (± standard error of the mean). Within each row, values with different superscript letters differ significantly (P<0.05).

3.2 Markers of oxidative stress

For the first experiment, the TBARS and TG content in hepatic tissues of broilers euthanized weekly throughout the study were measured. Dietary supplementation with three increasing doses of LA resulted in significantly lower TBARS values from days 21 to 42 with a single exception; that is, day 35 in birds supplemented with 10 parts/10⁶ LA compared with values in the control group (Table 3 and Fig. 1). Although there were no differences in hepatic TG during the first 14 days of the experiment in the four groups, higher hepatic TG values were observed later in broilers fed with supplemented diets as doses of LA were increased (Table 4 and Fig. 2). Using 40 parts/10⁶ LA diet, an increase in TG as compared with the control diet was statistically significant from days 21 to 35.

Mean (± standard error of the mean) values of thiobarbituric acid reactive substances (nmol/mg protein) in liver of broilers being fed a basal diet supplemented with lipoic acid

	Lipoic acid
Age (days)	0 (control)	10 parts/10^6	20 parts/10^6	40 parts/10^6
1	0.416±0.208^A	0.316±0.168^A	0.229±0.155^A	0.338±0.168^A
7	0.168±0.037^A	0.082±0.035^A	0.051±0.048^A	0.093±0.066^A
14	0.384±0.140^A	0.195±0.097^A	0.172±0.068^A	0.211±0.092 ^A
21	0.939±0.244^A	0.467±0.103^B	0.353±0.850^B	0.336±0.134^B
28	0.897±0.137^A	0.235±0.085^C	0.521±0.114^B	0.294±0.095^C
35	0.650±0.182^A	0.579±0.097^A	0.285±0.111^B	0.307±0.178^B
42	0.487±0.102^A	0.206±0.045^B	0.196±0.056^B	0.186±0.088^B
Means with different superscript letters differ significantly (P<0.05, n=6) against the control.

Means (± standard error of the mean) values of total glutathione (μmol/g ww) in liver of broilers being fed a basal diet supplemented with lipoic acid

	Lipoic acid
Age (days)	0 (control)	10 parts/10^6	20 parts/10^6	40 parts/10^6
1	2.40±0.66^A	2.53±0.47^A	3.40±0.63^A	2.33±0.55^A
7	2.16±0.41^A	2.78±0.31^A	2.56±0.33^A	3.22±0.79^A
14	2.40±0.72^A	2.73±0.21^A	2.06±0.46^A	3.07±1.10^A
21	1.32±0.39^B	1.00±0.26^B	2.19±0.58^A	2.93±0.43^A
28	2.03±0.38^B	1.04±0.58 ^C	2.66±0.42^B	3.32±0.40^A
35	0.89±0.59^B	3.22±0.53^C	2.01±0.42^A	2.54±0.46^A
42	2.07±0.26^A	2.50±0.62^AB	3.19±0.71^B	2.48±0.39^AB
Means with different superscript letters differ significantly (P<0.05, n=6) against the control.

Fig. 1 Summary of thiobarbituric acid reactive substances (TBARS)±standard error of the mean recorded in liver samples from broilers being fed with a basal diet without lipoic acid (LA), or this basal diet supplemented with LA, during 42 days. Each bar represents the mathematical sum of each value of each column in Table 3 divided by 7; that is, the number of weekly experiments (each with six birds) performed throughout the 42 days. Means with different letters differ significantly (P<0.05) against the control.

Fig. 2 Total gluthatione±standard error of the mean recorded in liver samples from broilers being fed with a basal diet without lipoic acid (LA), or this basal diet supplemented with LA during 42 days. Each bar represents the mathematical sum of each value of each column in Table 4 divided by 7; that is, the number of weekly experiments (each with six birds) performed throughout the 42 days.

In the second experiment, indicators of ROS were monitored measuring OH radicals and electrophoretic mobility of catalase in liver. Results included in Fig. 3 show the value results of OH recorded in birds receiving LA during the first 21 days of the experiment; the content of hepatic OH remained reduced despite the fact that the supplement was withdrawn in these animals during days 22 to 49. In the group receiving LA from days 22 to 49, values of OH during the first stage were similar to control values: once LA was added to the diet beginning on day 22, the hepatic value of OH was statistically lower than in the liver of broilers with a control-fed diet except at day 49 (Fig. 3). Changes in the electrophoretic mobility of catalase were not observed in liver samples of broilers whether fed with or without LA; that is, no evidence of singlet oxygen generation was observed (results not shown).

Fig. 3 Mean±standard error of the mean values for OH free radical pool in liver tissues obtained every 7 days from broilers fed with the following diets: basal diet without lipoic acid (LA) (solid circle); with 40 parts/10⁶ LA during 49 days (open circle); with 40 parts/10⁶ LA during the first 21 days of the experiment (solid triangle); or with 40 parts/10⁶ LA on days 22 to 49 of the experiment (open triangle). The assay was performed with four birds in each group at each time point.

4 Discussion

The main objective of our research work was to prevent the incidence of AS in broilers maintained under strict commercial conditions at 2235 m above sea level. Our reasoning was that a 10% decrease in atmospheric oxygen availability at an altitude of 2235 m above sea level is sufficient to lower oxygen tension in the inspired air of broilers; this, together with increased oxygen requirements to sustain rapid growth rates and high feed efficiencies, created hypoxic conditions in tissues and favored onset of pulmonary hypertension syndrome, whose ultimate consequence is AS (Decuypere et al., 2000). Hypoxic conditions also facilitated progressive establishment of oxidative stress, which played an important role in genesis of tissue damage (McCord, 1985).

The incidence of AS has been experimentally enhanced by decreasing ventilation and temperature in environmental chambers (Enkvetchakul et al., 1993) or by dietary administration of 1.5 parts/10⁶ triidothyronine (Decuypere et al., 1994). Furthermore, in broiler flocks maintained at an altitude of 2235 m the risk to develop AS increased (Arce et al., 1990; Díaz-Cruz et al., 1996). In these cases, cumulative mortality attributable to AS was near 30% and evidence of tissue oxidative stress as described later was documented in at least two of these studies. In general, the lung and liver concentrations of ascorbic acid, tocopherol, and TG were lower in birds maintained in the low ventilation chamber compared with control birds (Enkvetchakul et al., 1993). In the model of spontaneous AS by high altitude, TBARS values in the liver and heart were 2.4-fold and 2.8-fold higher in birds with clinical signs of AS compared with control birds, respectively (Díaz-Cruz et al., 1996).

Although temporary reduction in feed intake (Suárez & Rubio, 1989) and alternative lighting schedules (Buys et al., 1998) have proved successful in reducing the incidence of AS, the search for alternative possibilities including generalized use of feed supplemented with specific anti-oxidants continues. Diets supplemented with α-tocopherol acetate exerted a dose-dependent response on hepatic and pulmonary α-tocopherol concentrations but had no effect on growth performance or mortality attributable to pulmonary hypertension syndrome provoked by reducing ventilation and decreasing temperature (Bottje et al., 1995). In the model of high risk of AS at an altitude of 2235 m, vitamin E-supplemented diets resulted in better growth performance, lower rates of feed conversion, and lower TBARS content; nonetheless, the vitamin did not modify mortality attributable to AS (Villar-Patiño et al., 2002).

Administration of triiodothyronine (T₃) to broilers increased susceptibility for AS; in this model, food supplementation with vitamin C significantly reduced ascites mortality while displaying no effect on performance parameters (Hassanzadeh et al., 1997). In broilers maintained at 2235 m altitude, vitamin C-supplemented diets resulted in significantly lower feed consumption and lower rates of feed conversion; nevertheless, supplementation had no effect on mortality attributable to AS (Villar-Patiño et al., 2002). In the same model in birds grown at an altitude of 2235 m, diets supplemented with piroxicam, a non-steroidal anti-inflammatory drug that prevented hepatic increase of triacylglycerols and TBARS as well as decrease in TG levels resulting from acute ethanol intoxication in rats (Zentella de Piña et al., 1992), lowered the TBARS pool in the lung, liver, and heart of broilers with high risk to develop AS. However, the compound did not change the growth rate, feed consumption, nor feed conversion and did not modify cumulative mortality caused by AS (Valle et al., 2001).

In this work, further information reinforced findings that (1) oxidative stress was the molecular basis of AS and that (2) supplementation of diets with LA improved molecular indicators of oxidative stress, performance indexes, and cumulative mortality attributable to AS. LA (40 parts/10⁶) administered to broiler flocks maintained under commercial conditions at 2235 m altitude resulted in a lower TBARS content, a lower pool of OH radicals, and higher levels in TG (Figs. 1–3 ) in liver of birds with high risk to develop AS. Supplementation also improved feed consumption, feed conversion, growth performance and, above all, decreased cumulative morality attributable to AS (Tables 1 and 2 ). These results were observed in two different chick strains, even after increasing dietary ME to levels higher than in average diets commonly used at this altitude, which would increase broiler acceptability to develop AS. To the best of our knowledge, diet supplementation with LA is advantageous over other supplementation to prevent AS, at least in the model in which birds were maintained at high altitude.

Fig. 4 includes a possible mechanism (modified from Biewenga et al. (1997) and Lodge & Packer (2000)) to explain the benefical action of LA in decreasing oxidative stress previous to onset of AS. Cytosolic oxidation of suitable substrates (glucose) generates dehydrolipoate from feed lipoate; dehydrolipoate has a central role in regenerating reduced glutathione (GSH), reduced thioredoxine, ascorbate, and ubiquinol; the former two regenerate α-tocopherol. Some of these reduced regenerated compounds are useful to inactivate ROS and other pro-oxidant molecules such as H₂O₂, and in this way decrease cellular oxidative stress.

Fig. 4 Pivotal role of lipoic acid (LA), which used reduced coenzymes generated by cytosolic glucose oxidation to recycle oxidized antioxidants. The reaction of an antioxidant (vitamin E, vitamin C, reduced glutathione (GSH)) and a reactive oxygen species (ROS) (or H₂O₂) eliminates ROS (or H₂O₂), but the antioxidant is converted into a product no longer able to function. This oxidized product is regenerated to its native form to function again via the dehydro LA/LA redox couple. OS, oxygen species: GSSG, oxidized glutathione; NAD, nicotinamide aderine dinuclcotide (oxidized); NADH, nicotinamide adenine dinucleotide (reduced); NADP, nicotinamide adenine dinucleotide phosphate (oxidized); NADPH, nicotinamide adenine dinucleotide phosphate (reduced).

Acknowledgments

The authors are grateful to Dr Miguel Beltrán for his suggestions and comments on the method of catalase, to Margaret Ellen Reynolds Adler for helpful suggestions and critical reading the manuscript, and to Ms. Alejandra Palomares for secretarial assistance. This work was partially supported by grant 262577B from CONACyT, México.