The effects of short-term antioxidant supplementation on oxidative stress and flight performance in adult budgerigars Melopsittacus undulatus

Within all animals an oxidative balance exists between the production of reactive oxygen species (ROS) and the ability of endogenous and exogenous antioxidants to neutralise ROS. Oxidative stress is the term used to describe an imbalance whereby ROS production overpowers the detoxifying ability of the antioxidant systems. Oxidative stress can cause damage to proteins, lipids and DNA, and there is a growing understanding of the importance of oxidative stress in the development, health and ageing of animals(Knight, 1998). Levels of oxidative stress are therefore likely to be important fitness determinants,and identifying the factors influencing an individual's susceptibility to oxidative stress is of great interest. Physical activity is likely to be one factor influencing oxidative status because raising metabolism, as during exercise, will increase the production of ROS(Urso and Clarkson, 2003). Indeed, increased ROS production during exercise has been demonstrated in both humans and other animals (Kanter et al.,1988; O'Neil et al., 1996). An endogenous antioxidant system, well conserved across all taxa, controls and neutralises the ROS produced during exercise; however, prolonged strenuous exercise may overwhelm this antioxidant system (Sjodin et al., 1990). In this case dietary antioxidants may assist in removing free radicals and reducing oxidative stress. In birds, take-off is one of the most energetically demanding aspects of flight (Swaddle et al., 1999) and is likely to be associated with a burst of ROS production.

Since oxidative stress may cause damage to tissue(Bailey et al., 2007), there is growing interest in the possible role of dietary antioxidants in preventing exercise-induced oxidative stress, and improving performance(Powers et al., 2004). Much of the interest in this area has focused on the effects of antioxidant supplementation in human athletes, though the results are far from clear(Urso and Clarkson, 2003). Data from other, particularly non-model, taxa have the potential to improve our understanding of these links between dietary antioxidants and exercise. In birds high velocity initial vertical take-off, regardless of other escape strategies, is also vital in avoiding predation(Kenward, 1978). Although morphological traits such as wingtip shape(Swaddle and Lockwood, 2003)and fat load (Kullberg et al.,1996) have previously been demonstrated as important in determining flight performance in birds, it is possible that antioxidant status may also mediate flight performance by limiting oxidative damage associated with exercise. The extent to which flight exercise affects oxidative stress in birds, and how dietary antioxidants may ameliorate this,is unclear. Recently, Costantini and colleagues demonstrated that oxidative stress was increased with long flights in homing pigeons Columba livia, the first direct evidence of an oxidative cost to flight in birds(Costantini et al., 2008). In birds, evidence suggests that short flights tend to use glycogen stores for energy metabolism, whereas during long flights free fatty acids, produced by hydrolysis of adipose tissue, are oxidised by flight muscles(Jenni-Eiermann and Jenni,1991; Schwilch et al.,1996). Recent evidence suggests that anaerobic exercise may lead to increased ROS production through different pathways from aerobic exercise(Shi et al., 2007); thus take-off flight may have different oxidative costs than endurance flight. When fed on an antioxidant-rich diet, an individual should have a greater supply of available antioxidants and will be better equipped to remove free radicals associated with flight activity than when receiving a reduced antioxidant diet. Indeed, it has recently been demonstrated that male zebra finches Taeniopygia guttata fed supplementary carotenoids (nutrients displaying antioxidant properties) flew faster than controls, although oxidative stress was not measured (Blount and Matheson, 2006).

In this study, we fed individual budgerigars Melopsittacus undulatus (Shaw 1805) both an enhanced and a reduced quality diet. The main nutritional difference between these diets was in antioxidant concentration. In all cases, each bird received the two diets consecutively,and acted as its own control. After receiving each diet, we measured post-flight oxidative stress. We examined two indices of oxidative stress;malondialdehyde (MDA) level and comet assay. MDA is one of the major products of lipid peroxidation, after transition metal decomposition of lipid peroxides. High performance liquid chromatography (HPLC) can be used to examine the prevalence of MDA in its free form. The comet assay was developed by Collins and colleagues for measuring breaks in the DNA of lymphocytes and other single cells (Collins et al.,1997). We also calculated an average flight escape time across 4 days, in order to assess whether dietary antioxidants were capable of limiting flight performance. Our specific aims were to determine whether manipulating supplementary dietary antioxidants affected (1) post-flight oxidative stress levels and (2) take-off flight escape time.

This experiment lasted 8weeks (2×4week blocks) and we employed a crossover design with each bird receiving either an enhanced quality (EQ) diet for 4 weeks followed by a reduced quality (RQ) diet for 4 weeks, or vice versa. In the fourth week of each dietary block, food intake and flight trials were performed, and all birds were blood sampled.

At the WALTHAM® Centre for Pet Nutrition, 12 male and 12 female green and yellow (wild-type colour) budgerigars were selected from stock aviaries. These domesticated budgerigars have been selected for large body size, so skeletally are approximately 80% bigger than wild budgerigars. Each bird was weighed, health checked and randomly caged with a member of the opposite sex before being placed together in cages measuring 1002 mm×545 mm×410 mm. These experimental cages were smaller than the stock aviaries, though they had multiple perches, allowing short flights between them. A previous observational study showed that birds did fly in these cages, with a mean frequency of 11.8±2.7 short flights per hour, when undisturbed (S.D.L.,unpublished observation). However, the cages did not allow long, uninterrupted flights, so the birds were relatively sedentary. Throughout this experiment birds were not breeding, and temperature and day length were held constant. Birds had ad libitum access to water and food throughout the experiment, except during intake trials. Grit was provided in a separate container. Birds were given ∼10g of seed each per day, which was weighed in and out of the cage on a daily basis. Water and cage lining were also changed daily.

In this experiment we manipulated the level of an antioxidant-rich diet supplement within a standard seed mix for adult budgerigars. Nutrivit® is a nutritional supplement in the form of a small seed-like grain that is mixed into the seed mix Trill® produced by Mars® (Mars, Csongrad, Hungary). The main nutritional effect of Nutrivit® is to provide a higher concentration of antioxidants than is present in seed alone. Thus, any differences between birds on the two diets are likely to be mediated by antioxidants. Every bird on the trial received a baseline diet consisting of seed mix (Trill®) with a standard inclusion of Nutrivit® for 4weeks prior to the start of the experiment. This was the same diet that the birds usually received in their home aviaries. This ensured the birds were used to the Nutrivit® supplement before beginning the experiment. Then, two experimental diets were made up using identical proportions of seeds, but differing in the percentage inclusion of Nutrivit®. The enhanced quality diet (EQ) contained a 10% inclusion (by mass) of Nutrivit®. The reduced quality diet (RQ) contained a 1% inclusion (by mass) of Nutrivit®. Concentrations of antioxidants in the EQ diet were: α-tocopherol 1675i.u.g^–1; retinol 220,000i.u.g^–1;β-carotene 1.14μgg^–1; lutein 41.7μgg^–1; zeaxanthin 14.75μgg^–1; and in the RQ diet were: α-tocopherol 174i.u.g^–1; retinol 22,000i.u.g^–1; β-carotene 0.654μgg^–1; lutein 40.5μgg^–1;zeaxanthin 13.65μgg^–1. The birds were retained in their caged pairs and were randomly assigned to one of two groups. Half the pairs received the EQ diet for the first experimental block, followed by the RQ diet for the second block. The other half of the birds received the RQ diet for the first experimental block, followed by the EQ diet for the second block.

Flight trials were conducted from days 23 to 27 of each experimental block. The flight apparatus and protocol were similar to those of a previous study(Veasey et al., 2001) with some modifications. The cage used was 2 m high by 70 cm wide by 70 cm deep. The front wall of the cage was made of transparent Perspex. Coloured masking tape was taped horizontally across the cage at 10 cm increments above the floor, on both the transparent front panel and the back wall. A hinged 20 cm³ holding box was used to release the birds. The lid of the box was transparent, and was attached to a string. When pulled sharply this collapsed the side panels, and stimulated escape from ground level to a perch situated at the top of the cage. The loud sound of the sides hitting the ground meant each bird exhibited a standardised vertical escape flight.

On day 22 all birds were allowed to acclimate to the flight apparatus in groups of six. On day 23 of each experimental block, the flight cage was positioned at a fixed location in an enclosed procedures room, and video recording apparatus was fixed on a tripod 3 m from the cage. Birds were then placed in the holding box and flown individually as described previously(Veasey et al., 2001);however, in this study we calculated escape time from 10 to 50 cm height. Following this, the birds were given two trial flights each day between day 23 and day 26, and three flights on day 27 (11 flights in total). Each bird had a 10 min break between flights. By watching videos back frame-by-frame we could calculate escape time in 0.04 s intervals. Because of the holding cup design,the bird could not be observed flying over the first few centimetres. Thus,the escape time measurement started when the head passed a line 10 cm from the ground, and finished when the head passed a line 50 cm high. We used a mean escape time across all 11 flights in our analysis.

We blood sampled every bird prior to the experimental phase, after 28 days of receiving their baseline diet, then on day 28 of each experimental block,around 24 h after the final exercise trial. Individual birds were weighed and tarsus, wing and mass measurements were taken. A small blood sample(∼250μl) was taken from the jugular vein via a syringe; 50μl of the whole blood was diluted in 1 ml PBS immediately in a sodium citrate tube for comet assay. The rest of the blood was transferred to 75μl capillary tubes. The capillary tubes of blood for antioxidant and MDA analysis were centrifuged and haematocrit readings were taken from each. Plasma was stored at –70°C, prior to antioxidant and MDA analysis.

At the University of Glasgow, we analysed levels of α-tocopherol,lutein, zeaxanthin and retinol in order to uncover any effect of feeding treatment or exercise on plasma antioxidant profile. To extract the antioxidants, 40μl of ethanol was added to 20μl of plasma and vortexed thoroughly; 50 μl of hexane was then added and vortexed before the hexane layer, containing the antioxidants was drawn off. This was repeated with 40μl of hexane before the hexane extract was placed in a SpeedVac model SPD 111V (Thermo Electron Corp., San Jose, CA, USA) for 20min. The final antioxidant extract was then dissolved in 20 ml of methanol.

A Spectra model 8800 HPLC pump system with a Phenomenex 250mm×2mm i.d. column (Phenomenex, Macclesfield, UK) was employed to determine the antioxidant composition of each sample. We used HPLC at a flow rate of 0.2mlmin^–1 with a mobile phase of water/acetonitrile(2.5:97.5), and water/ethyl acetate (2.5:97.5) in a gradient elution. Using a Diode array absorbance detector type Thermo SPECTRAsystem UV6000LP (Thermo Electron Corp.), we detected carotenoids by absorbance at 445nm,α-tocopherol at 295nm and retinol at 325 nm. Peaks were identified by comparison with chromatography and retention times of several standards(Sigma, Poole, UK; Fluka, Gillingham, UK).

The MDA method was based on that of Young and Trimble(Young and Trimble, 1991). Thiobarbituric acid (0.044 mol l^–1,100 μl) and phosphoric acid (1.22moll^–1, 100μl) were mixed together and added to 50 μl of plasma (per bird) in a test tube. An inert atmosphere was created by applying a nitrogen blanket, and the test tubes were sealed and vortexed prior to heating (60 min, 70–75°C). Samples were cooled in water,then 200 μl was transferred to a centrifuge tube containing sodium hydroxide (1 mol l^–1, 100 μl). Methanol (500 μl) was added and mixed. Samples were centrifuged (10 min, 12,000 g)and the supernatant analysed on a Summit HPLC system (Dionex, Idstein,Germany) using Chromeleon software (Dionex). An Acclaim 120 C18 5 μm 4.6 mm×250 mm column (Dionex) and guard were used with fluorescence detection (excitation 532 nm and emission 553 nm). The mobile phase was isocratic, 40:60 methanol:phosphate buffer (40 mmol l^–1, pH 6.5), with a flow rate of 1 ml min^–1, and a run time of 7 min. Samples were assayed against a standard of malonaldehyde bis(dimethyl acetal; Sigma Aldrich) that was simultaneously taken through the same procedure.

We performed the alkaline comet assay procedure according to Tice and colleagues at two different pH values for each bird(Tice et al., 2000):electrophoresis at low pH (0.03 mol l^–1 NaOH) to reveal single stranded breaks and electrophoresis at high pH (0.3 mol l^–1 NaOH), which converts alkali labile sites into single strand breaks. Slides were made and analysed on the same day as blood sampling. We used 50 μl SYBR Gold in each gel for visualisation of comets. Slides were viewed by epifluorescence microscopy using an Olympus BX-51(Olympus Optical Co., Tokyo, Japan) with a 460 nm UV filter for SYBR Green. Komet software (v.6, Kinetics Imaging, Nottingham, UK) was used for image analysis on 100 randomly selected cells for each bird and treatment. Cells were scored according to the percentage DNA in the comet head, as a measure of DNA intactness.

In order to ensure that the birds were eating the Nutrivit® supplement,and also to assess selection or otherwise of the high antioxidant food, we performed an intake trial during each experimental block. Each morning at 08:00 h on days 24, 25 and 26 of each experimental block, feeding dishes were removed from each cage for a period of 2 h to standardise hunger, and cages were cleaned. After 2 h without food, pairs were separated with the female on the left of the cage in all cases. Individual budgerigars were presented with a food bowl containing a prepared 10 g sample of their experimental diet containing very precisely weighed seed, and known numbers of Nutrivit®pieces. The dish and tray were removed after 2 h, along with any spilled seed. The contents of the dish and cage floor were then sorted and weighed, and the number of uneaten Nutrivit® particles was counted and subtracted from the original total.

Data from the EQ and RQ diet experiments were analysed using generalised linear mixed models (GLMM; Proc Mixed in SAS version 9; Cary, NC, USA) with a normal error distribution. Individual identity was entered as a random factor into the model to control for the non-independence of repeated measures from the same individual. The order in which each bird received the diets, the diet and sex were entered into the models as factors, and morphometric, escape time and blood measurements were entered as covariates. All models used a Satterthwaite correction, which can result in degrees of freedom that are not integers. Models were developed using backward elimination starting with the highest order interaction term. We tested for all two-way interactions between main effects and covariates, and removed non-significant factors stepwise from the full model beginning with the interaction terms. We used P<0.05 for statistical significance. All significance tests were based on the F distribution. Comet data were proportions and so were arcsine square-root transformed. Count data were square-root transformed prior to analysis. Means with standard errors (s.e.m.) are reported throughout the text. In some cases problems during blood sampling or repeated failure to fly in some birds resulted in a sample size unequal to 24 birds. One bird was omitted from all analyses because it was observed to exhibit abnormal behaviour throughout the experiment and subsequently had atypical results.

We found no effects of sex on any variable (P>0.1) so data from males and females were pooled.

When individuals were maintained on the RQ diet they had significantly higher levels of MDA than when on the EQ diet (GLMM F_1,37=8.37, P=0.0064, see Fig. 1A). Birds had a higher proportion of intact DNA, measured by high pH comet assay, which assesses strand breaks and alkali labile sites, on the enhanced antioxidant diet than on the reduced antioxidant diet (GLMM F_1,35=5.3, P=0.0273, see Fig. 1B). There was no effect of diet on the low pH comet(P>0.5, see Fig. 1C). There was no significant effect of diet order on any measure of oxidative stress, showing there were no carry-over effects between experimental blocks.

Mean baseline plasma oxidative stress levels prior to the start of the experiment, with a 3% Nutrivit® inclusion and no exercise, were: MDA,0.237±0.026 μmoll^–1; high pH comet,67.83±2.64%; low pH comet 79.81±2.62%.

There was no effect of diet on flight escape time (EQ, 0.58±0.1 s;RQ, 0.57±0.1 s). There was a significant, positive relationship between mass and flight escape time (GLMM F_1,32=7.39, P=0.011, see Fig. 2)but no interaction between diet and mass affecting escape time (GLMM F_1,17.7=2.81, P=0.112). There was no relationship between comet assay, plasma MDA or plasma antioxidant concentration and flight escape time. There was no effect of diet on blood plasma concentration ofα-tocopherol, lutein, zeaxanthin or retinol (P>0.1 in all cases), and no significant effect of diet order on plasma levels of the different antioxidants (P>0.1 in all cases), demonstrating that there were no carry-over effects of antioxidant supplementation (see Fig. 3).

Birds consumed significantly more Nutrivit® on the EQ diet(1.07±0.23 pieces per 2 h) than on the RQ diet (0.08±0.039 pieces per 2 h; GLMM F_1,44=19.32, P<0.0001). However, there were no differences in the mass of seed eaten.

Dietary antioxidant availability had an impact on individual levels of oxidative stress. We found a significant effect of diet on both comet assay and MDA analysis. First, using the high pH comet assay we found a higher proportion of intact DNA on the EQ diet than on the RQ diet. In addition,birds had significantly higher levels of plasma MDA after receiving the RQ diet than after the EQ diet. Strenuous exercise seemed to impose an oxidative burden on the birds, which was eased by the consumption of a nutritional supplement rich in antioxidants. Oxidative stress levels appeared to be lower during the pre-experimental phase, when birds were not exercising, than during the experimental blocks. It appears that the flight exercise imposed did lead to increased oxidative stress, which is in accordance with other studies of DNA damage and exercise in humans and animals(Hartmann et al., 1995; Aniagu et al., 2006). Increases in MDA after exercise have also been shown in other taxa (for a review, see Vollaard et al., 2005). The high pH comet assay is thought to reveal alkali labile sites in DNA,representing several different forms of oxidative damage, rather than just single stranded breaks as found in low pH comet(Rojas et al., 1999). Although we found no difference between EQ and RQ diet in our low pH comet assay, the higher abundance of alkali labile sites on a reduced antioxidant diet suggests that dietary antioxidants prevented some exercise-induced oxidative damage of DNA.

Although in a short-term study it is difficult to assess the biological importance of exercise-induced damage, we suggest that increases in DNA damage may have long-term consequences. Lymphocytes are the most prevalent cells used in comet assays of avian blood (C.A.T., unpublished data). Avian lymphocytes are only produced early in development, so can be repaired but not replaced during adulthood, and circulate for a high proportion of an animal's life(Glick, 1979). Increases in DNA damage, including alkali labile sites, can eventually lead to apoptosis(Monti et al., 1992). Long-term DNA damage may induce a reduction in lymphocyte number and therefore immunocompromise an individual. The preventative action of dietary antioxidants on exercise-induced oxidative stress may thus have important effects besides those pertaining to exercise performance. Hartmann and colleagues previously reported that dietary vitamin E prevented oxidative damage of DNA in human subjects after treadmill running(Hartmann et al., 1995), though to our knowledge, ours is the first study to report dietary antioxidants preventing exercise-induced DNA damage in birds.

Increased oxidative stress was predicted to be one of the factors involved in limiting strenuous exercise in animals, where there may be a trade-off between minimising damage to tissue, yet maximising the chances of escape from a predator. Over 4 days we calculated average take-off escape time, an important aspect of fitness, for each bird when on EQ and RQ diets. We found that there was no effect of dietary antioxidant intake on escape time. In contrast, Blount and Matheson reported that zebra finches Taeniopygia guttata, a small passerine bird, receiving a carotenoid-enhanced diet flew faster than controls (Blount and Matheson, 2006). In their study oxidative stress was not measured,and it is possible that the difference was due to an effect of carotenoids aside from antioxidative protection. Indeed, carotenoids are useful in their role in immune signalling and as precursors for retinoids(Hartley and Kennedy, 2004). Of course, the difference may also represent a species-specific difference in behaviour.

In our experiment it is possible, though hard to prove, that the birds always flew as fast as possible, but they paid a price for this through increased oxidative stress. The consequences of this increased oxidative stress are unclear from a short-term experiment; although there is some evidence that oxidative stress can cause muscle damage(Bailey et al., 2007), other studies have found no effect of oxidative stress on muscle performance. Indeed, there is some evidence that increasing ROS production is necessary to optimise muscle performance (Reid,2001). Measuring flight speed repeatedly over a longer course of time on each diet may have revealed differences in performance, and it is possible that oxidative damage accumulating in muscle tissue is more important in affecting muscle stamina, rather than burst exercise. For example, one recent study showed that the length of flight in homing pigeons Columba livia was directly related to oxidative stress after flight(Costantini et al., 2008). In our study, although we expect the birds exerted maximal effort, the flight procedure was quick, and birds were rested between each flight. It has been shown that energy metabolism in short flights is quite different from that during long flights (Schwilch et al.,1996). Thus, flying birds for long periods, perhaps using a wind tunnel, or flying birds more often without rest, would reveal further differences in oxidative stress and flight performance.

In this study, resolution of different flight escape times was achieved using a standard video camera. This meant flight duration could only be measured to the nearest 0.04 s. Given the short flight distance, any difference in within-individual flight performance may have required a more sensitive filming technique. In order to properly assess flight take-off, a high speed camera would attain a more effective measure of performance (e.g. Williams and Swaddle, 2003). This is especially true over the first few centimetres, potentially those most important in predator evasion (Kenward,1978). However, the flight protocol employed here was certainly suitable to induce exercise, and there were differences in oxidative stress apparently mediated by the flight procedure. In this context the design was successful, and similar apparatus has been used previously to assess differences in vertical flight escape times(Veasey et al., 1998; Veasey et al., 2001; Blount and Matheson, 2005).

In spite of the effect of our dietary treatment on oxidative stress, we found that there was no significant difference in plasma levels of lutein,zeaxanthin, α-tocopherol or retinol after each 4week block. However, we do not believe this indicates a failure in the dietary treatment. We found that during the 2 h intake trials, intake of Nutrivit®, but not seed, was significantly increased on the EQ diet compared with the RQ diet. It is possible that the EQ birds had greater levels of antioxidants in other organs,though not in blood plasma. For example, it has been shown repeatedly (e.g. Surai et al., 2002; Karadas et al., 2005) that birds are capable of storing antioxidants in their liver at levels far greater than those seen in plasma. However, our experimental measurements indicated oxidative damage was increased after the birds received the RQ diet,regardless of the diet order. If antioxidants from the EQ diet were stored, we may have expected a lesser effect of exercise on oxidative stress after the RQ diet in birds that received the RQ diet in the second experimental block,which was not the case. Indeed, there was no effect of diet order on any plasma antioxidant we measured. Other recent studies of antioxidants in birds have found no effect of increased dietary antioxidants on plasma antioxidant levels (e.g. Biard et al.,2006). They did, however, find effects of the supplementation on other parameters, supporting the idea that although the antioxidants are not evident in plasma, they may have been used in other physiological systems. While retinol and α-tocopherol are increased in Nutrivit® to relatively high concentrations, other antioxidants, which we have not sampled,are possibly more important in this species. By assessing other important antioxidants perhaps we would attain a more effective estimate of antioxidant status.

Given the apparent beneficial effects of antioxidants, it could be expected that animals may selectively choose antioxidant-rich food items. We found no evidence that birds were selectively eating the antioxidant supplement. On the RQ diet Nutrivit® was seldom eaten at all, whereas on the EQ diet the average number of pieces eaten was around one during the 2 h palatability trial. This suggests that rather than selecting Nutrivit®, the birds ate it by chance when it was mixed into their seeds. However, after a 2 h deprivation it is conceivable that the birds selected foods based on their calorific content, rather than their antioxidant status.

In our study, we found evidence for exercise-induced lipid peroxidation and DNA damage. We also found that dietary supplementation with an antioxidant supplement was capable of countering this increased oxidative stress. Although we found no evidence of dietary antioxidants modifying exercise performance,longer term studies will be crucial in elucidating the roles of both oxidative stress and dietary antioxidants in determining physical capabilities in animals. DNA damage may have implications for immune function, as well as exercise performance. Thus access to dietary antioxidants may be an important fitness determinant, through various physiological pathways.

We would like to thank Russell Newnham and Julie Jones for help with bird care and logistics, Alistair Mellon for scoring the comet assay, and Bill Mullen for expert help with HPLC. Neil Metcalfe provided useful comments on the manuscript. S.D.L. was funded by a BBSRC Industrial CASE studentship and K.E.A. by a Royal Society University Research Fellowship. This work was carried out under licence from the UK Home Office and was subject to an ethical review by WALTHAM's ethical committee.