In animal production, two thirds of the total costs of meat, egg or milk production are due to feed expenses. The improvement of feed efficiency, i.e., the reduction of feed intake for a given level of production, has long been the main preoccupation of breeders and geneticists. In laying hens, an important part of the variation of feed consumption can be explained by the egg mass, body weight and its variation (Byerly et al. 1980, Fairfull and Chambers 1984). However, the remaining variation, referred to as residual feed intake or R criterion, may be used for direct selection of feed efficiency. The R is defined as the difference between the observed and the predicted feed intake. Two experimental lines have been divergently selected for high (R+)⁴ or low (R) residual feed intake (RFC), (Bordas and Mérat 1984, and Bordas et al. 1992). The observed feed intake differs by 40% for males of the same body weight and by 20% for females of the same body weight and egg production after 14 generations of selection. The higher energy intake of R+ birds should be offset by the following: 1) a lower digestion of ingested energy; 2) an enhanced energy expenditure (basal metabolic rate, physical activity or heat increment of feed, which is the heat production due to the increase in metabolic rate after feed ingestion, also referred as the thermic effect of food or diet-induced thermogenesis); or 3) changes in body composition. Selection for RFC did not modify the digestive use of energy intake of either cockerels or hens (Gabarrou and Géraert 1994, Géraert et al. 1991). The body composition of the two lines differs but, surprisingly, R+ birds are leaner than R birds (El Kazzi et al. 1995, Tixier et al. 1988, Zein-El-Dein 1985). Using indirect calorimetry, Géraert et al. (1991) and Gabarrou and Géraert (1994) demonstrated that differences in energy intake were balanced by enhanced heat production (HP) in the R+ compared with the R line. This difference in HP could result from an enhanced basal metabolic rate, physical activity or from diet-induced thermogenesis (DIT). When comparing White Leghorn hens of another RFC selection program, Luiting (1991) demonstrated that high RFC hens produce more heat than low RFC hens partly because of enhanced activity expenditure (+30-50%). The remaining 50-70% suggests higher DIT in high, compared with low RFC hens.

This DIT may be divided into an obligatory component related to digestion, absorption and processing of nutrients and into a regulatory or facultative component (Jéquier 1985). In rats, the hyperphagia induced by highly palatable diets is accompanied by a large increase in metabolic rate as a result of the regulatory DIT, which reduces weight gain and obesity. In mammals, this regulatory DIT allows body weight to be maintained in spite of a large increase in feed intake. DIT has been attributed mainly to sympathetic nervous stimulation of brown adipose tissue (BAT) thermogenesis after a meal (Rothwell and Stock 1979). BAT is involved in cold-induced thermogenesis in rodents, but regulatory DIT has not yet been demonstrated in birds, which have no BAT (Saarela et al. 1989). However, after prolonged cold exposure, ducklings exhibit nonshivering thermogenesis (NST) associated with loose-coupling of subsarcolemmal muscular mitochondria (Barré et al. 1985).

This experiment was thus performed to investigate the energy balance of R+ and R adult cockerels and in particular to clarify the origin of the difference in HP between the two lines by determining the basal metabolic rate, activity and DIT. The endocrinal and neuroendocrinological control of HP by thyroid hormones or by nervous sympathetic system was also investigated.

MATERIALS AND METHODS

Table 1. Diet composition [View Table]

Experiment 1. Twelve birds were transferred to individual respiratory chambers to measure their energy balance. They had free access to feed and water for 4 d and were deprived of feed for 2 d. The first day of each period (d 1 and 5) was considered as an adaptation period and was not taken into account for the calculations. Feed and water intakes were measured daily. Excreta were collected daily, weighed and freeze-dried before analysis. Endogenous energy and protein losses, and basal metabolic rate were determined during the second day without feed (d 6). True metabolizable energy (TME) was estimated by the difference between the gross energy ingested and excreted but taking into account the endogenous energy losses (Sibbald 1978); tube-feeding was not employed.

At the end of each period, the rectal and comb temperatures were measured using thermocouples (N93722 and N93713, Bioblock Scientific, Paris, France) previously calibrated against a mercury thermometer. At the same time, blood was also collected from a wing vein using a heparinized syringe. After centrifugation (1500 × g for 15 min) the plasma was separated and stored at 20°C until measurment of the triglyceride, uric acid, glucose, triiodothyronine and thyroxine concentrations.

Experiment 2. Twelve cockerels of each line were reared under the same conditions as described above. They had free access to water. The same diet was minced (diameter < 0.5 µm) and mixed with water (50:50, wt/wt). Birds were fed by crop intubation (Sibbald 1978). After an adaptation to tube-feeding for 3 d at 100% of their individual free feed intake, birds received on the fourth day either 60 or 100% of their individual free feed intake. Heat production was continuously monitored for 23 h/d.

Experiment 3. Feed intake patterns of 8 R+ and 8 R cockerels were measured as described by Picard et al. (1992). Feeder weights (± 0.01 g) and duration of any access to feeders (± 0.1 s) were recorded 6 times per second. Cockerels were adapted for 2 wk to the experimental device and the feed intake was recorded during a 24-h period. A meal was defined as a period of continuous eating not interrupted by more than 120 s (Savory 1979).

Experiment 4. Four cockerels of each line were fed 60 g of feed once a day by crop intubation. At the same time, they received a gelatin capsule orally that was either empty (control) or filled with DL-propranolol (5 mg/kg). Propranolol is a -antagonist that inhibits thermogenesis induced by -adrenergic activity. Birds had free access to water. Heat production was continuously monitored for 23 h.

The flow rate of each chamber was measured (± 0.5%) by means of a thermal-mass flow meter from 0 to 10 L/min (MKS 258B, MKS Instruments, Paris, France). Mass flow meters were calibrated every 6 mo from 1 to 10 L/min by weighing infused N₂. Two pairs of oxygen (OXYGOR 6N) and carbon dioxide (UNOR 6N, Maihak, Hamburg, Germany) gas analyzers continuously monitored O₂ and CO₂ concentration in the chambers. All analyzers operated with 1% ranges (O₂: 20-21%, CO₂: 0-1%). Day-to-day variation in the gas analyzers was corrected by calibration using two standard gas ranges (20.416% O₂, 0.570% CO₂ for standard 1; 20.346% O₂ and 0.708% CO₂ for standard 2). Chambers were calibrated yearly by alcohol combustion with a recovery ranging between 95 and 98%.

A Fluke data logger module (2286A, Fluke, les Ullis, France) was interfaced with the gas analyzers, flow rate controllers, temperature and relative humidity probes, Doppler-radar modules, strain gauges and switch valves. It collected data and switched gas samples for 90-s sampling periods over at least 23 h.

Gas samples were switched from respiratory chambers or from the reference-air line to the analysis system as follows: chambers 1-3 to analysis system 1 and chambers 4-6 to analysis system 2. Air was sampled from a chamber exhaust outlet and analyzed for 90 s. Values were averaged over the final 20 s before another gas sample was analyzed.

Oxygen consumption and CO₂ production rates were modeled, taking into account the accumulation rates of O₂ and CO₂ in chambers between two analyses as previously described by MacLean and Watts (1976).

Table 2. Body weight, feed intake, physical activity, energy balance, respiratory quotients, rectal and comb temperature of R+ and R adult cockerels1 (Experiment 1) [View Table]

Heat production (HP) was calculated as HP (kJ) = 16.18 O₂ (L) + 5.02 CO₂ (L) (Romijn and Lokhorst 1961). Heat production was measured continuously for 23 h (1 h was necessary for maintenance and calibration). Diet-induced thermogenesis (DIT) was estimated from the difference between heat production determined in feed-deprived and fed cockerels. Heat production in the inactive state was calculated by extrapolating the regression between heat production and physical activity to zero activity.

Plasma triiodothyronine (T3) and thyroxine (T4) were determined by RIA with the use of a commercial kit (CIS-ORIS Industries, Lyon, France). Plasma glucose was determined with a glucose analyzer based on spectrophotometry (model 2, Beckman Instruments, Palo Alto, CA). Plasma triglycerides, uric acid and nonesterified fatty acids (NEFA) were measured by enzymatic methods (Fossati et al 1980, Fossati and Prencipe 1982 and Okabe et al. 1980, respectively) by using the kits provided by BioMerieux SA (Charbonnières-les-Bains, France).

To determine liver deiodinase I activity, 12 cockerels of each line were slaughtered by cervical dislocation. Livers were removed and immediately weighed, frozen in liquid N₂ and stored at 80°C until analysis. Deiodinase I activity was determined as described by Darras et al. (1992). The animal protocols were approved by the French Ministry of Agriculture.

RESULTS

Regression equations were established for each line between HP and TMEi (HP = a × TMEi + b) (Table 3). The slope, a, which is an estimation of DIT expressed as the percentage of TME, was higher in the R+ line than in the R line, whether birds were tube-fed (+31%) or had free access to feed (+43 %). The extrapolation to zero feed intake HP, b, which corresponds to the basal metabolic rate, did not differ between lines whether birds were tube-fed or had free access to feed. The slope of the regression between retained energy and TMEi, representing the ability to retain energy, was lower in the R+ line. Moreover, the retained energy extrapolated to zero feed intake, representing the total energy losses when unfed was higher in the R+ line.

Table 3. Regression equations between heat production (HP) and true metabolizable energy intake (TMEi) of R+ and R cockerels1 [View Table]

Respiratory quotient, plasma uric acid and plasma glucose concentration did not significantly differ between lines (Table 4). Triglyceride concentration was lower in fed R+ birds and plasma NEFA concentration was higher in feed-deprived R+ birds than in their R counterparts. Plasma T3 concentration did not differ in fed birds but was lower in feed-deprived R+ birds. T4 and hepatic deiodinase I activity did not differ between lines. Rectal temperature did not differ significantly between lines but comb temperature was higher in fed R+ than R birds.

Table 4. Plasma triglyceride, uric acid, glucose, triiodothyronine (T3) and thyroxine (T4) concentrations in fed and unfed R+ and R adult cockerels, and hepatic deiodinase I activity1 (Experiment 1) [View Table]

Physical activity expressed in counts/min was 28% higher in the R+ than in the R line when birds had free access to feed (Table 2). Tube-feeding drastically reduced activity, particularly in the R line compared with the R+ line. The energetic cost of activity (the slope of regression, Table 5) was higher in the R line whether birds were tube-fed (+11%) or had free access to feed (+33%). HP extrapolated to zero activity (Table 5) was higher in the R+ line whether birds were tube-fed (+19%) or had free access to feed (+29%). As a consequence, the total energetic cost of activity was slightly but not significantly higher in the R+ line, 244 vs. 211 kJ/(kg^0.75·D), when cockerels had free access to feed. Tube feeding drastically reduced the total activity expenditure, which remained significantly higher in the R+ than in the R line, 91 versus 48 kJ/(kg^0.75·D).

Table 5. Regression equations between heat production (HP) and physical activity (PA) of R+ and R cockerels1 [View Table]

Table 6. Feed intake pattern in R+ and R male cockerels monitored for 24 h1 (Experiment 3) [View Table]

Table 7. An oral dose of DL-propranolol (5 mg/kg) reduces heat production of R+ but not R adult male cockerels1 (Experiment 4) [View Table]

DISCUSSION

Using a Doppler-radar system with White Leghorn hen lines selected for high or low RFC, Luiting (1991) found that as much as 50-70 % of the divergence in HP between lines could not be accounted for by changes in physical activity. In Experiment 1, the activity-related HP, which was higher in the R+ line (+16%), did not explain the total divergence in HP between lines. The difference due to activity [33 and 38 kJ/(kg^0.75·d), in Experiments 1 and 2, respectively) represented only 25 and 36%, respectively, of the difference in HP between lines. Because no difference was observed in basal metabolic rate, the remaining 75 or 64% of the difference in HP could thus be accounted for by DIT.

As observed previously in cockerels (MacLeod 1991), tube-feeding drastically reduced physical activity (Table 3) in both lines (0.8 counts/min), suggesting a similar level of activity related to feed intake in the R+ and R lines. Both lines spent the same time eating, which reinforces such a hypothesis. However, the energetic cost of activity (slope) appeared lower in the R+ line (Table 3). The difference in activity between lines may therefore be due to a difference in behavior such as higher water intake to enhance heat dissipation, as observed previously (Gabarrou and Géraert 1994).

The results suggest the existence of a regulatory thermogenesis, at least in the R+ line, as described previously by Jéquier (1985) in humans as a facultative DIT. For 100 kJ TME ingested, R+ birds dissipated 27.5 kJ as DIT compared with 20.9 kJ in R birds. Regressions between HP and feed intake (Table 3) indeed revealed higher coefficients in the R+ cockerels than in the R cockerels, i.e., 0.261 versus 0.182 with free access to feed and 0.230 versus 0.176 when tube-fed. Moreover, DL-propranolol decreased HP only in the R+ line. Such results suggest that the R+ line dissipated more heat than required for obligatory thermogenesis induced by feed ingestion through a facultative or regulatory thermogenesis under sympathetic control (Jéquier 1985). Such regulatory thermogenesis has also been observed previously in rats fed highly palatable diets (Rothwell and Stock 1979). In this rat model, the increase in energy intake induced by highly palatable diets is partially eliminated by enhanced thermogenesis through -adrenergic stimulation of the BAT (Rothwell and Stock 1980).

Thyroid control of thermogenesis did not seem to differ significantly in fed R+ and R birds, which generally exhibited similar plasma T3 and T4 concentrations and hepatic deiodinase activity. Danforth and Burger (1989) were also unable to show any difference in plasma T3 and T4 when increased energy intake was balanced by enhanced HP, but an increase in plasma norepinephrine concentration was observed. However, the lower plasma T3 concentration observed in feed-deprived R+ birds might indicate higher T3 turnover in this line. T3 regulation of thermogenesis should be further investigated in these lines.

The metabolic pathways involved in regulatory thermogenesis remain unclear. Indeed, there was no difference in the respiratory quotient or plasma glucose concentration in either fed or feed-deprived R+ and R birds, suggesting similar metabolic pathways in both lines. However, fed R+ birds exhibited a lower triglyceridemia (as previously observed by El Kazzi et al. 1995), which could contribute to the lower adiposity of R+ birds (El Kazzi et al. 1995, Tixier et al. 1988, Zein-El- Dein 1985). When feed deprived, R+ birds showed a higher NEFA concentration than R birds. This has also been reported in rats fed highly palatable diets (Rothwell and Stock 1979). In the hyperphagic rat model, enhanced thermogenesis results from a -adrenergic and T3 stimulation of the BAT (Rothwell and Stock 1979) and of the liver mitochondria (Liverini et al. 1994). Fatty acids are indeed the main fuel of the enhanced thermogenesis of the BAT. But because birds have no BAT (Saarela 1989), other tissues could be involved. However, DIT and NST may have a common origin (Rothwell and Stock 1980). Barré et al. (1985) showed that NST of cold-acclimated ducklings was controlled by glucagon and involved the loose-coupled oxidative phosphorylation of subsarcolemmal muscle mitochondria. Liver, through its very important metabolic role in birds, might also be involved in the enhanced thermogenesis of R+ birds as suggested in rats fed highly palatable diets (Liverini et al. 1994). The higher plasma NEFA level might also suggest that fatty acids can be used as fuel or even enhance the uncoupling of mitochondria (Barré et al. 1985).

The R+ and R lines are an interesting model for the study of the genetic control of DIT. The enhanced HP due to a higher DIT allows R+ birds to eliminate their excessive energy intake. Metabolic pathways such as the uncoupling effect of NEFA and their tissue localization (liver or muscles) should be futher investigated in relation to the endocrine and/or neuroendocrine control. Although -adrenergic regulation appears to be involved, thyroid control remains unclear. The investigation of thyroid hormone turnover could clarify its role in the enhanced thermogenesis of the R+ birds.

ACKNOWLEDGMENTS