Body composition and exercise in racing pigeons

Research in Veterinary Science 1990, 48. 321-326

Body composition and exercise in racing pigeons W. MULLIGAN, J. M. MACLEAN, Department oj Veterinary Physiology, The West Medical Building, University of Glasgow, GI2 8QQ, T. PRESTON, Scottish Universities Research and Reactor Centre, East Kilbride

Exercise-related changes in body protein 'turnover' and in the absolute amounts of body protein were studied in racing pigeons. Whole body radioactivity was followed in racing and control (limited exercise) birds after protein labelling by the injection of 75S e_ selenomethionine. Because of re-utilisation of the label this does not give a true picture of body protein turnover but the comparative data suggested an increased turnover in racing compared to control birds. Carcase analysis on a group of pigeons demonstrated a water content for lean body mass of 72· 7 per cent ± 3· 54. Lean body mass and exchangeable body potassium were used as indices of total body protein in a group of pigeons participating in an endurance race (15 + hours of flying). The results indicated that no body protein had been used as an energy source. These findings are compatible with the presence in' pigeons of a small labile pool or pools of protein. The presence and characteristics of such pools remains to be determined. THE objective of this work was to study changes in body composition in pigeons resulting from endurance nights and ultimately to use this information as a basis for a rational approach to pigeon nutrition. The traditional view of the body fuel consumed during exercise is to regard carbohydrate and fat as the normal materials with protein only becoming involved as a 'last resort' or emergency supply. There is a certain logic in this attitude if protein is regarded as essentially representing the structural and catalytic machinery of the body. Nevertheless, recent evidence to the contrary is emerging from studies on horses competing in endurance events (Rose et al 1977, Lucke and Hall 1980, Snow et al 1982), on marathon runners (Lemon et al 1983) and on laboratory animals exercised on treadmills (Dohm et al 1982). The results of these investigations suggest that in prolonged exercise protein can contribute a significant proportion of the energy requirement although the evidence, based mostly on elevation of blood urea, must be regarded as indirect. The present study was carried out on racing pigeons. In longer races these birds may spend up to 14 or more hours on the wing and will use up a much

higher proportion of their body energy stores than marathon runners or endurance event horses. They are, therefore, more likely to make inroads into body protein and are suitable subjects for direct observations of exercise-related changes in protein metabolism. The results are important in relation to the formulation of suitable diets for long distance racing pigeons, but also perhaps in the more general context of understanding the integration of fuel supplies in prolonged exercise in other species. The experiments described here represent attempts to measure changes in body protein 'turnover' and alterations to the absolute amounts of body protein in racing birds. L-selenomethionine, the analogue of the amino acid methionine is utilised by the body for protein synthesis (Awwad et al 1966). When labelled with 75Se which is a gamma emitter and thus suitable for whole body counting, it seemed to offer a convenient way of measuring total protein turnover (Waterlow et al 1969). However, there are doubts about its validity for such measurements, largely due to the considerable re-utilisation of selenomethionine and retention of the tracer itself with the resulting complexity in the kinetics involved (Waterlow et al 1969). Although absolute values for total protein turnover cannot be derived from analysis of whole body 75Se decay curves the method can provide valuable comparative information. This is borne out by the work of Yousef and Luick (1968) who used 75Se-selenomethionine turnover as an index of protein metabolism during cold exposure of mice. It did seem, therefore, a worthwhile technique to apply to the racing pigeon problem. If there is a significant utilisation of protein as an energy source it should be reflected not only in the turnover measurements but also in changes in the absolute amounts of body protein. In the experiments described here 'leah body mass' (LBM) and 'exchangeable body potassium' were used to assess changes in total body protein in pigeons taking part in 'endurance' races. Simple measurements of body mass in birds are complicated by the fact that the crop is a storage organ for food so that weighings must be carried out

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W. Mulligan, J. M. Maclean, T. Preston

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under conditions where variations in the amount of food in the crop are unlikely to occur. The authors have found that for healthy, fit pigeons the weight first thing in the morning before access to food or water is remarkably constant and this is referred to as 'standard body mass' (SBM). LBM (ie, the mass of the fat-free body) consists essentially of water, protein and minerals (the amount of carbohydrate in the body at any given time is quite small). Any changes in LBM as a result of exercise will reflect changes in body protein in that the gross mineral content of the body will be largely unaffected. Chemical analysis of a number of mammalian species has demonstrated a remarkable constancy in the water content of LBM, being very close to 73 per cent (Muldowney and Healy 1967). This enables LBM to be calculated from a measurement of total body water (TBW) as follows: LBM

(g) =

TBWg

x

n100 .

It was necessary to establish if a similar relationship held good in the case of the pigeon. Furthermore, the relationship between TBW and LBM applies only in circumstances where there is no disturbance of body fluid equilibrium. It was therefore essential to back up the LBM measurements with some suitable check on body fluid status. The most useful measurement for this purpose is 'exchangeable body sodium' (Na.} in that it reflects extracellular fluid volume and taken with the TBW measurement and plasma potassium concentration can be used to calculate 'exchangeable body potassium' (K e ) (Shizgal et al 1977). As intracellular potassium concentration is held within narrow limits K, bears a fairly constant relationship to intracellular water and to body nitrogen in both normal and pathological slates (Tal so et al 1960).

Materials and methods

Turnover studies (5 Se-selenomethionine) The birds involved in the study were from a genetically related stock. They were fed on a commercial grain mixture ad libitum. The control birds on limited exercise were housed in a 8 feet x 6 feet (2' 4 m X I . 8 m) stock loft with a small aviary from two weeks before the start and during the period of the experiment. On day 0 they were each injected intraperitoneally with II kBq of 75Se_ selenomethionine (I ·0 ml of a dilution in sterile saline of a stock solution containing 10 to 15 p,g ml : J of carrier selenomethionine) and whole body 75Se activity measured on that day and at frequent intervals throughout the experiments. The same amount of 75Se-selenomethionine was made up to

500 ml with saline in a polythene bottle of similar dimensions to a pigeon body for use as a calibration 'phantom' in the radioactivity measurements. The experimental groups had been exercised regularly and given frequent training flights of 20 to 40 miles. They were each injected on day 0 with II kBq 75Se-selenomethionine as in the case of the controls and whole body radioactivity measurements were carried out at regular intervals throughout the experimental period. The first experiment was carried out in 1986 (experiment I) and repeated in 1987 (experiment 2).

Tissue analyses To confirm the incorporation of 75Se-selenomethionine into body protein in these experiments, and to obtain information on its distribution, the five control birds in experiment 2 were killed on day 19 and subjected to carcase analysis. The pectoral and leg muscles were dissected out as completely as possible, liver and gut removed and these tissues homogenised in saline in a top drive macerator. It was found helpful in the case of the gut to subject it to freeze-drying before maceration. After making the homogenate to standard volume with saline, aliquots were removed to check the level of protein binding of the 75S e activity. Samples were treated with an equal volume of cold 20 per cent trichloracetic acid (TCA). After centrifugation at 2000 g for 15 minutes the supernatants were removed and the precipitated protein washed twice with 10 per cent TCA. The radioactivities of the precipitated proteins, supernatants and washings were measured in a well-type gamma counter and 'protein bound' activity expressed as a percentage of total activity. To examine the relative specific activities of proteins from the different tissues, TeA precipitates from four of the birds were washed several times with absolute alcohol followed by ether and finally oven dried. Samples of the dried protein were dissolved in dilute sodium hydroxide and assayed for 75Se-activity in a well-type gamma counter. This enabled the specific activity (counts per minute [cpm] mg- I protein) to be calculated. The remaining tissue homogenates in each case were diluted to 500 ml with saline in the standard polythene bottles and their radioactivity measured in the whole body counter. The activity of the carcase residue was likewise measured. TBW TBW was measured by the dilution technique using tritiated water (TOH). A 1·0 ml stock solution of TOH containing approximately 700 kBq of tritium was injected subcutaneously in the loose skin at the back of the neck. An equilibrium blood sample was

Changes in pigeon body composition collected after two hours, plasma removed and duplicate 100 j.tl samples delivered into preweighed counting bottles. Because of the small volumes involved the amounts of plasma for counting and the amounts of TOH injected were determined by weighing the measured volumes. A standard for the radioactivity measurements was prepared by diluting I . 0 ml of the TOH stock solution to 250 ml with distilled water. Triplicate 100 j.tl samples of this standard were delivered into counting bottles and weighed. Control pigeon plasma (100 j.tl)was added to each of the standard bottles and distilled water (100 j.tl) to each of the plasma samples. This ensured uniform composition of all the samples for radioassay. A scintillation cocktail (8 ml) was added to each bottle and after mixing radioactivity measurements were carried out in a liquid scintillation counter. Each sample was counted twice and quench corrections determined by the 'channels ratio' method (Rogers and Moran 1966). TBW was calculated as follows: TBW (g) =

Total tritium counts min - I injected counts rmn : I g - I of 2 h plasma water

Where repeat determinations of TBW were made a preinjection sample of plasma was collected and analysed to produce a background tritium count.

Exchangeable body sodium (Na] 24Na was obtained from the Scottish Universities' Research and Reactor Centre at East Kilbride. A preliminary experiment was carried out to study the equilibration of 24Na after subcutaneous injection in isotonic saline. Thereafter, the dilution technique was applied as in the measurement of TOH, ie, weighing of injected and plasma samples for analysis. A welltype gamma counter was used for the radioactivity measurements, and standards were measured at frequent intervals to aIlow for the rapid decay of 24 N a (ty, = 15'3 h). Calculations were as follows: Exchangeable sodium (Na.) mM Total 24Na counts min - I injected counts min I mM - I of 2 h plasma sodium

Simultaneous measurements of TRW Na, and K, An isotonic solution (1'0 ml) containing approximately 700 kBq of tritium and 500 kBq of 24Na was injected subcutaneously, an equilibrium blood sample collected after two hours and plasma separated. 100 j.tl samples of plasma were dispensed into counting bottles and weighed as before. Two standard solutions were prepared; I· 0 ml of injection mixture diluted to 250 ml for the tritium assay and 1'0 ml

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diluted to 100 ml for the 24Na measurements. The plasma samples and 24Na standard were counted first of all in the well-type gamma counter and then put aside for several days to allow the 24Na activity to decay. They were then assayed for tritium as described above. TBW and Na, were calculated as already described and K, from the relationship: K, =

TBW

([Na] + [K]) - Na,

(Shizgal et al 1977) where [Na] and [K] represent the concentrations of sodium and potassium in plasma water. Plasma concentrations of [Na] and [K] were measured by flame photometry.

Specific gravity and water content of pigeon plasma The specific gravity of pigeon plasma was taken at 1'015 based on a protein content of 3 g 100 ml" which is normal for the species. This corresponds to a water content of 952 g kg-I of plasma (Geigy Scientific Tables 1962).

Carcase analysis To determine the relationship between TBW and in pigeons a group of birds was killed, feathers removed and the carcases minced thoroughly. Weighed samples were subjected to Iyophylisation to determine the water content, and fat was measured by ether extraction of the dried residue. LBM

Results

Turnover studie~' Experiment 1. This trial involved five control and six experimental yearling male birds. On day 3 the experimental pigeons participated in a 120 mile race. Four returned in good time and the remaining two after intervals of two and four days. The four successful birds then took part in a 240 mile race on day 10 and again performed satisfactorily. The whole body 75Se activity (log scale) was plotted against time (linear scale) for each bird and a rate constant for the decline in activity calculated. The results are shown in Table I. Experiment 2. This was a repeat of the 1986 trial with some minor variations. Fiveexperimental birds successfully completed races of 150 miles (day 2),180 miles (day 9) and 240 miles (day 16). The results are summarised in Table I. They generally confirm the 1986 findings in terms of a more rapid decline in the whole body 75Se activity of the experimental compared to control birds. However, because of the greater scatter within each group the difference in the means is not significant at the 5 per cent level.

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W. Mulligan, J. M. Maclean, T. Preston TABLE 1: Whole body 75Se activity of pigeons injected with 75Se-selenomethionine: first order tracer loss rates: mean ± SE Rate constant Id - ,)

1986 experiment

1987 experiment

Control In = 51 Experimental In = 4)

0·01086 ± 0·00056 0·01880 ± 0'00053

Significance (t test)

P<0·001

NS

In=51 In=51

0·01178 ± 0·00091 0·01541 ± 0·00203 NS

Not significant

Details of the tissue analyses are summarised in Table 2. The good agreement between the sum of the radioactivities of the individual tissues and the whole body activity confirms the validity of the analytical procedures, in particular, the use of the 500 ml polythene bottle as a sample container for the whole body counting. In all cases the protein bound activity of the tissue samples was found to be in excess of 96 per cent.

Relationship between

TBW and LBM

in racing pigeons

A group of 11 pigeons covering an age range of six weeks to two years and hence a range of fat content were killed and subjected to carcase analysis as described above. The water content of LBM was uniform (72' 2 per cent ± 1· 07) agreeing very closely with the corresponding figure for mammalian species. In nine of these birds, TBW had also been determined by TOH dilution. The agreement was generally good (74' 5 ± 3· 54) with a tendency for TOH dilution to overestimate TBW and a slightly greater degree of scatter around the mean.

Equilibration of subcutaneously injected 24 Na A group of six normal pigeons were each injected subcutaneously with 1·0 ml of an isotonic solution containing 500 kBq of 24Na. Blood samples were collected at intervals up to six hours after injection and plasma assayed for 24Na activity. The results were expressed as a percentage of the two hour plasma level where the activity appeared to have levelled off. On the basis of this curve it was decided to measure a two hour exchangeable sodium in the exercise experiment.

Exercise-related changes in body composition Choosing a race that represents a true endurance

performance of many hours on the wing can be difficult. This is influenced by two main factors distance and prevailing weather conditions: strong winds and, or, poor visibility can turn middle distance races (200 to 300 miles) into true endurance events. If conditions are too difficult and birds become lost for a day or two they are unsuitable subjects for study because exercise-related changes in body composition wilt be complicated by some degree of starvation. A suitable race occurred on June 18 1988 from Dorchester (366 miles). Birds were liberated at 07.00, weather conditions en route were not good and none had arrived home by nightfall. Five birds returned the next morning at intervals from 07.00 onwards. The minimum flying time in each case was about IS hours and it was obvious from handling alone that they had lost a significant amount of bodyweight. The first measurements of body composition were carried out on June 21, three days after the date of liberation. The authors have found that even in short races (four to five hours flying) there is a drop in SUM with a rapid 'bounce back' to near prerace values within a day or two. The time interval is probably too short for this to represent significant replenishment of body fuel stores and almost certainly largely corresponds to rehydration and restoration of gut contents to 'standard' conditions. The changes in body composition resulting from the endurance race and the gradual return to prerace values are summarised in Table 3. The prerace data apart from SBM are from a group of five birds from the same racing team, matched for weight and following the same programme up to the Dorchester race. The mean drop in SUM (prerace to three days after the race) was about 14 per cent but the true loss of body substance as measured by the fall in total body solids was more than 30 per cent. LUM and K, remained steady throughout suggesting that the weight loss could be accounted for entirely by the utilisation of body fat.

TABLE 2: Organ distribution of 75Se in five control birds 19 days after injection of 75Se_ selenomethionine (as % whole body activity)

Mean ± SE

Pectoral muscle

Leg muscle

Liver

Gut

27·3 ± 1'02

4'78 ±0·28

3·85 ±0·35

7·32 ± 1·02

Residue

Total

56·8 100·0 ±0·37 ± 1·47

Changes in pigeon body composition

325

TABLE 3: Changes in body composition of racing pigeons resulting from an endurance flight (mean ± SE)

SBM Igi Prerace Iday Post race Iday Iday 101 Iday 171 Significance (t Day - 2 v day Day - 2 v day

SBM TBW TBS LBM NS

21 31 test)

3 17

502 433 471 492

± 8·0 ± 11·6 ± 7·1 ± 9·8

P<0·002 NS

TBW Ig)

289 284 294 296

± ± ± ± NS NS

4·9 9·4 7·1 8·9

TBS Igi

213±2'7 149 ± 6·7 177 ± 8·9 196 ± 8·6 P
LBM Igi

396 391 403 406

Fat Ig)

± 6·7 ± 11·6 ± 8·9 ± 11·6

105 43 67 86

± ± ± ±

NS NS

P
2·3 6· 7 4·5 8·5

Na e ImMI

17·1 17· 1 17'4 18'3

± ± ± ± NS NS

O· 70 0·58 0·24 0·16

x, ImMI 25·7 ± 1 ·05 26· 5 ± 1· 13 27·4 ± 1 ·57 26·5±1·46 NS NS

Standard body mass Total body water Total body solids Lean body mass Not significant

Discussion 75Se tissue analysis data showed that the tracer remained protein-bound and was well dispersed throughout the body, for example, in skeletal muscle as well as in liver (Table 2). This, however, was the situation on day 19 and the pattern would be expected to be different in the earlier stages because of the variations in the turnover rate of protein in different tissues. A proper insight into the kinetics of incorporation and turnover and how these are influenced by exercise requires frequent measurements of specific activity of proteins in gut, liver, skin, muscle, etc in limited exercise and racing birds. The most obvious interpretation of the whole body activity data is that there was an increase in protein breakdown in the racing pigeons compared to the 'limited exercise' controls. This is by no means the only explanation and the picture may be complicated by possible differences between racing and control birds in their protein intake and a greater potential washout of label from the exercising pectoral muscles. As already emphasised, because of its recycling 75Se-selenomethionine seriously underestimate~ absolute rates of whole body protein breakdown. By analogy with work with chicks using 3H-Iabelled phenylalanine (Muramatsu and Okumura 1985) a much more rapid breakdown for the limited exercise birds than the 75Se data presented here might be expected. However, the recycling problem applies to both control and experimental birds and we recognise the limitations of 75Se-methodology in comparative studies of whole body protein turnover. It is difficult to imagine that the differences in whole body protein turnover rates reflect a significant use of body protein as a fuel in the experimental birds bearing in mind that none of the races is likely to have exhausted fuel reserves as represented by glycogen and fat. This is supported by the findings in the endurance race. The results shown in Table 3 are clear cut: LBM and K, were quite unaffected by flying times of' 15 + hours, indicating no significant utilisation of body

protein as an energy source in what could truly be described as an endurance event. The constancy of the relationship between Na, and TBW throughout suggests no disturbances in body fluid distribution which might have compromised the validity of the LBM measurement. The possibility of the involvement in exercise of a small labile pool of protein whose rapid turnover would explain the 75Se-labelled protein results and which might be significantly replaced by day 3 when the first post race measurements were made cannot be overlooked. Acute protein catabolic phases during exercise followed by increased post exercise anabolism has been shown to occur in human athletes (Rennie et al 1981). This interpretation would support the concept of 'labile protein stores' which are available to replace a protein deficit and as an energy source (Munro 1964). Of particular relevance is the possible existence of a labile store in th.e non-contractile proteins of the pectoral muscles of the pigeon. Such a labile protein pool does exist and plays a crucial role in egg production in some wild bird species (Kendall et aI1973). Its occurrence in the pigeon and possible role in exercise remains to be examined. Clarification of this and other aspects of exercise-related changes in protein metabolism in the pigeon will require further turnover studies using nitrogen or carbon labelled amino acids, combined with measurements of total body protein. Bearing in mind Griminger's (1983) comments on this aspect of pigeon physiology: 'very little has been published on the actual nutritional requirements or on the metabolism of nutrients in this species' , it is clear that the present study represents but an early step along the road to adequate understanding. Acknowledgements This work was supported by grants from the Wellcome and Leverhulme Trusts. Carcase analyses were kindly carried out by the Department of Animal Husbandry, Glasgow Veterinary School.

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W. Mulligan, J. M. Maclean, T. Preston

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Received March 13, 1989 Accepted September 6, 1989