Effect of ambient temperature on dietary energy partitioning and utilization in broilers as measured by closed-circuit indirect calorimetry system

Abstract

This study investigated the effects of ambient temperatures on growth performance and energy partitioning in broilers using a computer controlled closed-circuit indirect calorimetry system. Sixty 21-day-old male Arbor Acres broilers (1,004 ± 11 g BW) were randomly allocated to four ambient temperature treatments (23°C, 25°C, 27°C, 29°C), with five indirect calorimetry chambers per treatments and three broilers per chamber. Broilers were fed the same test diet, and growth performance, gas exchange, dietary energy partitioning, and substrate oxidation were determined from d 25 to 28. Increasing ambient temperature from 23°C to 29°C resulted in a linear increase (P ≤ 0.036) in BW, metabolic BW, and ADG, whereas ADFI/ADG decreased linearly (P = 0.019). Daily O₂ consumption and CO₂ production also decreased linearly with increasing ambient temperature (P ≤ 0.003). Compared with broilers maintained at 23°C, those maintained at 29°C exhibited lower heat increment (HI) and HI/AME (P ≤ 0.017), but greater net energy (NE), retained energy (RE), NE/GE, NE/AME and RE/AME (P ≤ 0.047). Regression models incorporating NE or RE intake, together with CP and metabolizable CP (MCP) intake, produced higher R² for predicting ADG than the corresponding model based on AME, CP, and MCP intake. Ambient temperature had no significant effects on substrate oxidation or the contribution of carbohydrate, fat, and protein oxidation to total heat production. In conclusion, increasing ambient temperatures within the tested range improved dietary energy utilization efficiency, as reflected by higher NE and RE, lower HI, and a linear decrease in ADFI/ADG. The superior predictive performance of NE- and RE-based models further supports the use of NE and RE as more physiologically relevant indicators of dietary energy value than AME. These results demonstrate that closed-circuit indirect calorimetry is an effective approach for evaluating dietary energy partitioning under different thermal environments.

Introduction

Net energy (NE) is widely regarded as the most accurate estimate of available energy in feed for broilers (Liu et al., 2017; Noblet et al., 2010). It was calculated by subtracting heat increment (HI), defined as the difference between total heat production (THP) and fasting heat production (FHP) from AME (Zuidhof, 2019). Thus, fluctuations in HI may have a considerable impact on the overall efficiency of dietary energy utilization in broilers.

Previous study has shown that HI is influenced by ambient temperature when broilers were fed the same diet (Morillo et al., 2023). However, fluctuations in ambient temperature may affect THP, primarily by altering the energy required for thermoregulation, which constitutes a major component of the NE requirement for maintenance (Koh and Macleod, 1999a). Broilers expend energy for thermogenesis at low ambient temperature and for evaporative heat loss under hot conditions (Hossain et al., 2026; Pishnamazi et al., 2015). Consequently, broilers maintained outside their thermoneutral zone must adjust their metabolic activity to either increase THP or enhance heat dissipation. These thermoregulatory responses affect the efficiencies of energy utilization for protein and fat deposition, thereby altering body composition. Sakomura et al. (2005) reported that broilers reared at ambient temperatures of 13, 23, and 32°C from 25 to 28 days of age exhibited marked differences in body protein deposition (3.66, 2.53, and 2.62 g, respectively) and fat deposition (1.52, 2.32, and 1.52 g, respectively). These differences were attributed to increased feed intake and greater THP at low temperatures, whereas higher temperature reduced energy intake and THP, thereby changing the partitioning of dietary energy between THP and body energy retention (Belhadj Slimen et al., 2016; Liu et al., 2022; Sakomura et al., 2005). Consequently, ambient temperature plays a critical role in influencing energy loss as heat and dietary energy utilization in broilers (Liu et al., 2022; Reyes et al., 2012; Xu et al., 2024). However, quantitative information describing how ambient temperature affects the partitioning of dietary energy into THP, HI, NE, and retained energy (RE) remains limited.

The indirect calorimetry technique provides a reliable approach for quantifying THP by simultaneously measuring oxygen (O₂) consumption and carbon dioxide (CO₂) production. These measurements allow the partitioning of AME into THP, HI, NE, and RE as protein (RE_p) or fat (RE_f) to be determined (Barzegar et al., 2020; Gerrits and Labussière, 2015). By controlling ambient temperature within the indirect calorimetry chamber, differences in THP and the partitioning of AME can be evaluated under different thermal conditions. Therefore, this study used indirect calorimetry to investigate the effects of four ambient temperatures (23, 25, 27, and 29°C) on the growth performance, O₂ consumption, CO₂ production, dietary energy partitioning, and energy utilization efficiency in broilers. The results will improve our understanding of how ambient temperature influences energy utilization and energy partitioning in broilers.

Materials and methods

All experimental procedures related to the use of live broilers were approved by the animal care and welfare committee of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (Beijing, China). The code of ethical inspection was IAS 2024-61.

Experimental design

A total of sixty 21-day-old male Arbor Acres (AA) broilers with similar BW (1,004 ± 11 g) were selected and randomly allocated to four environmental temperature treatments (23, 25, 27 and 29°C), with five closed-circuit indirect calorimetry chambers per treatment to compare the dietary energy partitioning differences in broilers under varying temperature conditions. Each chamber contained one wire-mesh metabolic cage (70.5 cm length × 58.0 cm width × 50.0 cm height) equipped with an individual feeder and drinker. Each cage housed three broilers and served as one experimental unit.

Broilers management and experimental diets

The broilers were fed a starter diet from 0 to 21 days of age, and an experimental diet from 22 to 28 days of age, with free access to water. Broilers were acclimated to the calorimetry chambers from 21 to 24 days of age and were evaluated from 25 to 28 days of age at a stocking density of 7.36 kg/m² based on initial BW of broilers. Relative humidity and ventilation were managed according to the guidelines for Arbor Acres broilers (Aviagen, 2018). Experimental diets were formulated to meet or exceed nutrient recommendations for broilers in China (MOA, 2004) (Table 1). All ingredients were crushed through a 2 mm sieve, evenly mixed, and pelleted (3-mm diameter by 3-mm length pellets) using a laboratory non-steam press pellet mill (Model SKJ 150, Funong machine Co. Zhengzhou, Henan, China).

Table 1.

Ingredients and chemical composition of experimental diets (as-fed basis, %).

Items	Starter diet (days 1 to 21)	Experimental diet (days 22 to 28)
Corn	53.40	59.31
Soybean meal	35.38	29.67
Corn gluten meal	2.00	3.00
Soybean oil	4.51	4.08
Dicalcium phosphate	1.73	1.83
Sodium chloride	0.30	0.30
Limestone	1.07	0.74
Premix1	0.50	0.50
L-lysine HCl	0.50	0.28
DL-methionine	0.23	0.17
L-threonine	0.19	0.08
L-valine	0.14	0.02
Broiler complex enzyme2	0.03	0.00
Phytase3	0.02	0.02
Total, %	100	100
Nutrient content, %4	90.95	91.12
Gross energy, kcal/kg	4,168	4,257
CP	23.31	20.26
Acid detergent fiber	6.19	5.83
Calcium	0.94	0.82
Available phosphorus	0.44	0.46

¹Supplied per kilogram of diets: vitamin A, 10,000 IU, vitamin D₃, 4,000 IU, vitamin E, 55.0 IU, vitamin K₃, 3.20 mg, thiamin, 3.0 mg, riboflavin, 7.0 mg, vitamin B₆, 3.0 mg, vitamin B₁₂, 16.0 µg, pantothenic acid, 15.0 mg, nicotinic acid, 50.0 mg, folic acid, 1.8 mg, biotin, 0.22 mg, choline chloride, 1,500 mg, Cu (as copper sulfate), 16.0 mg, Fe (as ferrous sulfate), 20 mg, Mn (as manganese sulfate), 120 mg, Zn (as zinc sulfate), 120 mg, I (as calcium iodate), 1.25 mg, Se (as sodium selenite), 0.30 mg.

²Broiler complex enzyme (Beijing Challenge Bio-tech Co. Ltd., Beijing, China) provided 6,000 units/g of xylanase, 3,000 units/g of β-mannanase, 1,200 units/g of β-glucanase, and 100 units/g of cellulase.

³Phytase (Beijing Challenge Bio-tech Co. Ltd., Beijing, China) provided enzyme activity 10,000 units per g.

⁴Gross energy, CP and acid detergent fiber were measured value, whereas calcium and available phosphorus were calculated values (air-dry basis) according to the China Feed Database (IASCAAS, 2024).

Measurements of O₂ consumption and CO₂ production

The O₂ consumption and CO₂ production of broilers were measured using an automatic closed-circuit indirect calorimetry system (Model CRS-1), developed by the Institute of Animal Science, Chinese Academy of Agricultural Sciences (Beijing, China). The design, operating principles, and validation of this system were described by Zhao et al. (2022) and Liu et al. (2025). The indirect calorimetry system consisted of four subsystems: (1) an animal chamber system; (2) an environmental control system for regulating chamber temperature, humidity, and airflow; (3) an O₂ supply and consumption quantification system; and (4) an air filtration and CO₂ absorption system to purify incoming air and remove CO₂ from exhaust air. The procedure for quantifying O₂ consumption and CO₂ production was as follows: a 1-L bottle containing 0.70% (vol/vol) sulfuric acid solution, two 6-L bottles each containing 350 g/kg KOH solution, and a 1-L bottle of deionized water were connected in sequence by silicon tubing. The bottles were placed in a stainless-steel box (49.5 cm length × 43.5 cm width × 49.5 cm height) with a sealed door, which was connected to the air inlet and outlet of the box. Subsequently, an automatic airtightness self-test was initiated using the control software (Copyright 2024SR0019052).

The airtightness of each respiration chamber was verified before the experiment using a pressure decay test. Each chamber was connected to a high-pressure oxygen cylinder (≥ 10 MPa) and pressurized with oxygen at 0.05 MPa. Chamber pressure was continuously monitored by a pressure sensor connected to the closed-circuit indirect calorimetry system. When the pressure differential between the chamber and ambient air reached 300 Pa, the oxygen supply was automatically shut off. The pressure differential was then recorded 3 min after valve closure (A) and again 30 min later (B). Chamber airtightness was considered acceptable when the pressure decrease over the 30-min period did not exceed 50 Pa (i.e., B ≥ A − 50 Pa). Under this condition, the system automatically passed the self-test; otherwise, the test failed and an alarm was triggered.

After the airtightness test, broilers were transferred to the chamber and provided ad libitum access to diet and water during a 3-day adaptation period. Throughout this period, the chamber door remained open while the environmental temperature was maintained at the preset level (23, 25, 27, or 29°C). Air was continuously circulated through each chamber by a computer-controlled air pump and solenoid valves without passing through the air filtration and CO₂ absorption system.

Experimental parameters, including chamber temperature, relative humidity, O₂ concentration, differential pressure, feeding schedule, and test duration, were preset in the control software. Before each run, the feeder was emptied, refilled with sufficient test diet for the 3-day experimental period, and weighed. The chamber door was then closed, and the bioassay was initiated through the control software. During the experiment, the closed-circuit indirect calorimetry system automatically monitored chamber temperature, relative humidity, O₂ concentration, CO₂ concentration, differential pressure in the chamber, BW, and O₂ consumption of the broilers. Oxygen consumption was calculated as the difference between the weight of the oxygen cylinder at the beginning and end of each run. Oxygen consumption (g) was converted to volume (L) using an O₂ density of 1.331 g/L under standard conditions (20°C and 101.325 kN/m²). All data were automatically recorded by the control software and used to calculate average BW, O₂ consumption, and ADG.

Upon completion of each bioassay, the residual feed was automatically discharged from the feeder by opening the feeder base plate. The air filtration and CO₂ absorption system were then deactivated, and the solenoid valve connected to the respiration chamber was opened to allow atmospheric air exchange. All excreta were collected into aluminum foil boxes and stored at −20°C for subsequent analysis. After three consecutive 24 h bioassays had been completed in each chamber, the CO₂-absorbing KOH solution was weighed and sampled to determine the total amount of CO₂ produced during the test period. The amount of CO₂ absorbed in the KOH solution was quantified using BaCl₂ precipitation method as described by Wu et al. (2019). The density of CO₂ at standard conditions (1.842 g/L, at 20°C and 101.325 kN/m²) was used to convert CO₂ weight (g) to volume (L).

Determination of AME, THP, and NE

The dietary AME was determined based on the total feed intake and total excreta collected from broilers aged 25 to 28 days over three consecutive 24 h calorimetry bioassays. Excreta from each experimental unit were pooled, dried in a forced-air oven at 65°C for 72 h, and analyzed for chemical composition and gross energy (GE). The O₂ consumption and CO₂ production were determined using computer-controlled closed-circuit indirect calorimetry throughout the three consecutive 24 h bioassays. The THP was calculated based on the volume of O₂ consumed and CO₂ produced. The respiratory quotient was calculated as the ratio of the volume of CO₂ production to the volume of O₂ consumption. The HI was calculated as the difference between THP and FHP. The dietary NE content (kcal/kg DM) was calculated by subtracting HI from AME.

Chemical analysis

Feed and excreta samples were finely ground using a laboratory mill (model BJ-150, Deqing Baijie Electrical Co., Ltd., Zhejiang, China) and passed through a 0.42 mm mesh screen prior to chemical analysis. The DM content was determined according to the AOAC (2007) method 925.10. GE was measured by adiabatic calorimeter (Parr 6400, Parr Instrument Co., Moline, IL), with benzoic acid as the calibration standard. The CP content was determined by the AOAC (2007) method, using a Kjeldahl nitrogen analyzer (model KDY-9820, Shandong Haineng Scientific Instruments Co., Ltd., Dezhou, China). The acid detergent fiber was determined by AOAC (2005) method 973.18. Uric acid content in excreta was determined according to the method of Marquardt (1983).

Calculation and statistical analysis

Dietary AME (kcal/kg DM) = (energy intake – energy output) / feed intake

THP (kcal) = 3.866 × O₂ consumed (L) + 1.200 × CO₂ exhaled (L) (Brouwer, 1965)

FHP (kcal/kg BW^0.70 per day) = 107.55 (Noblet et al., 2015)

HI (kcal/kg DM) = (THP – FHP × BW^0.70 × day) / feed intake in which THP is in kilocalories, O₂ is oxygen consumption in liters, CO₂ is carbon dioxide production in liters. A 107.55 kcal/kg BW^0.70 per day for broilers reported by Noblet et al. (2015) to calculate the FHP.

NE (kcal/kg DM) = AME − HI

RE (kcal/kg DM) = AME – THP / FI

Retained nitrogen (RN) = (CP intake − CP excretion)/6.25 where CP intake (g/d) is the daily CP intake from the diet, and CP excretion (g/d) is the total of CP excreted in feces and urine. CP content in feed, feces and urine was determined by the Kjeldahl method (nitrogen content × 6.25).

RE as protein (RE_p, kcal/kg DM) = RN × 6.25 × 5.70

RE as fat (RE_f, kcal/kg DM) = RE – RE_p

Where RE is retained energy; RE_p is RE as protein; RE_f is RE as fat; 6.25 is the protein equivalent of 1 g nitrogen, and 5.70 is the energy equivalent of 1 g protein (kcal), as reported by Sharma et al. (2021).

Net substrate oxidation (g) was calculated from gas exchange measurements (L) and urinary nitrogen excretion (g) according to the equations described by Brouwer (1957, 1958).

Urinary nitrogen (g) = excreta (g) × uric acid (%) × 33.33%

Protein oxidation (g) = 6.25 × urinary nitrogen (g)

Carbohydrate oxidation (g) = – 2.965 × O₂ (L) + 4.170 × CO₂ (L) – 2.438 × urinary nitrogen (g)

Fat oxidation (g) = 1.718 × O₂ (L) – 1.718 × CO₂ (L) – 1.969 × urinary nitrogen (g)

Heat production from substrate oxidation was calculated using energy equivalents of 4.40, 9.50, and 4.19 kcal/g for protein, fat, and carbohydrate, respectively, as described by Gerrits and Labussière (2015).

The experimental unit was the group of broilers housed within each calorimetry chamber. Descriptive statistics for chamber environment variables (temperature, humidity, O₂ concentration, and CO₂ concentration), growth performance (BW and ADG), energy partition (daily O₂ consumption, daily CO₂ production, AME, THP, HI, RE, RE_p, and RE_f), and energy utilization indices (AME/GE, NE/GE, THP/AME, HI/AME, NE/AME, RE/AME, RE_p/AME, and RE_f/AME) were generated using JMP Pro 19.1 (SAS Institute Inc., Cary, NC). Data were analyzed by ANOVA using the Fit Model platform in JMP Pro 19.1. BW was included as a covariate to adjust the daily O₂ consumption and CO₂ production. The least squares means were compared using the Tukey honestly significant difference test. Polynomial orthogonal contrasts were applied to evaluate linear and quadratic responses to increasing ambient temperature during respiration calorimetry on all measured variables. Linear regression models were developed using the Fit Model platform in JMP Pro 19.1 to evaluate the relationships of ADG with AME, NE, or RE intake, together with CP and metabolizable CP (MCP) intake, and to evaluate the relationships of THP, HI, NE, RE, and RE_f with ambient temperature. The significance level was set at P < 0.05, whereas 0.05 ≤ P < 0.10 was considered a trend.

Results

Monitored chamber environment during indirect colorimetry system

The average ambient temperature for the four groups was 23.14, 25.11, 27.00, and 28.85°C, respectively (P < 0.001; Table 2). Throughout the 72-h measurement period, the actual chamber temperatures closely matched the target settings, with a maximum deviation of only 0.15°C. The temperature-humidity index (THI) increased linearly as the ambient temperature rose from 23°C to 29°C (P < 0.001). In contrast, relative humidity, O₂ concentration, or CO₂ concentration did not differ among treatments (P ≥ 0.083). The average relative humidity for the four groups were 52.17, 48.00, 48.71 and 48.73%, respectively. The average O₂ concentration remained stable between 20.84% and 20.89%, and CO₂ concentrations ranged from 2,831 to 3,614 ppm across the four treatment groups.

Table 2.

Monitored environmental conditions in closed-circuit respiratory chambers¹.

Items	Temperature				SEM	P-value2
	23°C	25°C	27°C	29°C
Temperature, °C	23.14d	25.11c	27.00b	28.85a	0.06	< 0.001
Relative humidity, %	52.17	48.00	48.71	48.73	1.41	0.192
THI3	69.53d	71.70c	74.22b	76.62a	0.12	< 0.001
O2 concentration, %	20.84	20.89	20.89	20.89	0.02	0.433
CO2 concentration, ppm	3,305	3,614	3,145	2,831	200	0.083

^a-dValues within a row with different superscripts differ significantly at P < 0.05.

¹Data are presented as least squares means of five observations per treatment.

²Means are separated using Tukey honest significant difference test.

³THI, temperature-humidity index, is equal to (1.8 × T + 32) - [(0.55 - 0.0055 × RH) × (1.8 × T - 26)], where T is the daily average temperature (°C) and RH is the daily average relative humidity (%).

Growth performance of broilers during indirect calorimetry system

Continuous monitoring of broiler BW showed different growth trajectories among the four ambient temperature treatments from 25 to 28 days of age (Fig. 1). The growth curve of broilers maintained at 25°C was consistently lower than those of broilers maintained at 27°C and 29°C groups, whereas broilers maintained at 23°C exhibited the lowest growth trajectory throughout the experimental period. No significant differences were observed among treatments in BW on d 25, metabolic BW (MBW), ADFI, ADG or feed conversion ratio (ADFI/ADG) during 25 to 28 days (P ≥ 0.102; Table 3). However, broilers maintained at 29°C tended to have a greater BW on d 28 than those maintained at 23°C (1,691 g vs. 1,609 g; P = 0.093). Increasing ambient temperature from 23°C to 29°C resulted in a linear increase in BW on d 28, MBW, and ADG from days 25 to 28 (P ≤ 0.036), whereas ADFI/ADG decreased linearly (P = 0.019).

Fig. 1.

Changes in BW of broilers housed in closed-circuit indirect calorimetry chambers during the 72-h experimental period (25 to 28 days of age) at four ambient temperatures (23, 25, 27, and 29°C). Each treatment consisted of five chambers, with three 25-day-old male Arbor Acres broilers per chamber. Time is expressed in hour.

Table 3.

Effect of ambient temperature on the growth performance of broilers housed in closed indirect calorimetry chambers from 25 to 28 days of age¹.

Items	Temperature				SEM	P-value2
	23°C	25°C	27°C	29°C		Anova	Linear	Quadratic
BW on d 25, g	1,262	1,289	1,304	1,297	16	0.325	0.121	0.321
BW on d 28, g	1,609	1,663	1,665	1,691	22	0.093	0.021	0.518
MBW, BW0.70/g	1,290	1,316	1,323	1,329	12	0.140	0.032	0.432
ADG, g/d	116	125	121	131	4	0.089	0.036	0.866
ADFI, g/d	148	153	147	153	4	0.670	0.684	0.912
ADFI/ADG, g/g	1.284	1.224	1.218	1.162	0.032	0.102	0.019	0.951

MBW, metabolic BW, is calculated as the mean BW raised to the 0.70 power.

¹Data are presented as least squares means of five observations per treatment.

²Means are separated using one-way ANOVA and subjected to polynomial orthogonal contrasts.

O₂ consumption, CO₂ production and respiratory quotient

The daily O₂ consumption per broiler was greater at 23 and 25°C than at 29°C from 25 to 28 days of age (P = 0.016; Table 4). Similarly, the daily O₂ consumption per kg of MBW, daily CO₂ production per broiler, and daily CO₂ production per kg of MBW were greater at 23°C than at 29°C (P ≤ 0.021). Increasing ambient temperature from 23°C to 29°C resulted in linear decreases (P ≤ 0.002) in daily O₂ consumption, from 60.43 to 54.24 L/bird per day and from 45.59 to 41.53 L/kg BW^0.70 per day, as well as daily CO₂ production, from 60.77 to 54.57 L/bird per day and from 45.59 to 41.92 L/kg BW^0.70 per day (P ≤ 0.003). Respiratory quotient was not affected by ambient temperature from 25 to 28 days of age (P = 0.624).

Table 4.

Effect of ambient temperature on O₂ consumption, CO₂ production, and respiratory quotient of broilers housed in closed-circuit indirect calorimetry chambers ¹.

Items	Temperature				SEM	P-value2
	23°C	25°C	27°C	29°C		Anova	Linear	Quadratic
Daily O2 consumption per broiler, L/d	60.43a	59.23a	55.85ab	54.24b	1.22	0.016	<0.001	0.851
Daily O2 consumption per kg of MBW, L/(kg BW0.70/d)	45.59a	45.05ab	42.64ab	41.53b	0.89	0.015	0.002	0.755
Daily CO2 production per broiler, L/d	60.77a	58.84ab	55.95bc	54.57c	1.07	0.002	<0.001	0.794
Daily CO2 production per kg of MBW, L/(kg BW0.70/d)	45.59a	44.76ab	42.80ab	41.92b	0.82	0.021	0.003	0.978
Respiratory quotient3	1.00	0.99	1.00	1.01	0.01	0.624	0.319	0.500

^a-cValues within a row with different superscripts differ significantly at P < 0.05.

MBW, metabolic BW, is equal to BW^0.70.

¹Data are presented as least squares means of five observations per treatment. BW was used as a covariate to adjust the daily O₂ consumption and CO₂ production.

²Means are separated using one-way ANOVA and subjected to polynomial orthogonal contrasts.

³Respiratory quotient = CO₂ production (L/d)/ O₂ consumption (L/d).

AME, THP, HI, NE, RE and energy utilization efficiency

No differences were observed among treatments in dietary AME or RE_P in broilers from 25 to 28 days of age (P ≥ 0.298; Table 5). Dietary THP tended to be greater at 23°C than at 29°C (2,023 kcal/kg DM vs. 1,839 kcal/kg DM; P = 0.073), whereas HI was higher at 23°C and 25°C than at 29°C (P = 0.017). Increasing ambient temperature from 23°C to 29°C resulted in linear decreases in THP, from 2,023 to 1,839 kcal/kg DM, and HI, from 1,081 to 900 kcal/kg DM (P ≤ 0.014). Regression analysis further indicated that each 1°C increase in ambient temperature reduced THP and HI by 28.28 and 29.94 kcal/kg DM, respectively (Fig. 2a-b). In contrast, dietary NE was higher at 29°C than at 23°C (2,771 kcal/kg DM vs. 2,579 kcal/kg DM; P = 0.008). Similarly, the RE was greater at 29°C than at 23°C (1,831 kcal/kg DM vs. 1,636 kcal/kg DM; P = 0.035), whereas RE_f tended to be greater at 29°C than at 23°C (1,030 kcal/kg DM vs. 841 kcal/kg DM; P = 0.058). Increasing ambient temperature from 23°C to 29°C resulted in linear increase in NE (from 2,579 to 2,771 kcal/kg DM), RE (from 1,636 to 1,831 kcal/kg DM) and RE_f (from 841 to 1,030 kcal/kg DM) (P ≤ 0.012). Regression analysis showed that each 1°C increase in ambient temperature increased NE, RE, and RE_f by 30.77, 29.07, and 28.07 kcal/kg DM, respectively (Fig. 2c-e).

Table 5.

Effect of ambient temperature on dietary energy partitioning and utilization in broilers housed in closed-circuit indirect calorimetry chambers ¹.

Items	Temperature				SEM	P-value2
	23°C	25°C	27°C	29°C		Anova	Linear	Quadratic
Energy partitioning, kcal/kg DM
AME	3,659	3,701	3,683	3,671	15	0.298	0.820	0.100
THP	2,023	1,961	1,947	1,839	46	0.073	0.014	0.621
HI	1,081a	1,035ab	978ab	900b	36	0.017	0.002	0.661
NE	2,579b	2,666ab	2,705ab	2,771a	34	0.008	<0.001	0.757
RE	1,636b	1,740ab	1,735ab	1,831a	42	0.035	0.007	0.927
REp	795	798	800	801	7	0.947	0.563	0.922
REf	841	942	935	1,030	44	0.058	0.012	0.949
Energy utilization, %
AME/GE	78.36	79.26	78.87	78.61	0.33	0.297	0.819	0.101
NE/GE	55.22b	57.11ab	57.92ab	59.34a	0.73	0.008	<0.001	0.755
THP/AME	55.27a	52.98ab	52.88ab	50.11b	1.16	0.047	0.009	0.839
HI/AME	29.52a	27.96ab	26.55ab	24.51b	0.95	0.012	0.001	0.805
NE/AME	70.48b	72.04ab	73.45ab	75.49a	0.95	0.012	0.001	0.805
RE/AME	44.73b	47.02ab	47.12ab	49.89a	1.16	0.047	0.009	0.839
REp/AME	21.73	21.56	21.72	21.82	0.16	0.713	0.556	0.405
REf/AME	23.00	25.45	25.40	28.07	1.22	0.069	0.014	0.932

^a-bValues within a row with different superscripts differ significantly at P < 0.05.

NE, net energy; THP, total heat production; HI, heat increment; RE, retained energy; RE_f, retained energy as fat; RE_p, retained energy as protein.

¹Data are presented as least squares means of five observations per treatment.

²Means are separated using one-way ANOVA and subjected to polynomial orthogonal contrasts.

Fig. 2.

Linear regression of effect energy on ambient temperature. Regression analyses were performed using data from four ambient temperature treatments (23, 25, 27, and 29°C). Each treatment consisted of five closed-circuit indirect calorimetry chambers, with three 25-day-old male Arbor Acres broilers per chamber. THP, total heat production (kcal/kg); HI, heat increment (kcal/kg); NE, net energy (kcal/kg); RE, retained energy (kcal/kg); RE_f, retained energy as fat (kcal/kg); T, temperature (°C).

No differences were observed among treatments in AME/GE or RE_p/AME, which ranged from 78.36% to 79.26% and from 21.56% to 21.82%, respectively (P ≥ 0.297; Table 5). In contrast, the NE/GE and NE/AME values were greater at 29°C than at 23°C (P ≤ 0.012). Similarly, the RE/AME value was greater at 29°C than at 23°C (P = 0.047), whereas, the RE_f/AME value tended to be greater at 29°C (P = 0.069). Increasing ambient temperature from 23°C to 29°C resulted in a linear increase in NE/GE (from 55.22% to 59.34%), NE/AME (from 70.48% to 75.49%), RE/AME (from 44.73% to 49.89%), and RE_f/AME (from 23.00% to 28.07%) (P ≤ 0.014). In contrast, THP/AME and HI/AME were greater at 23°C than at 29°C (P ≤ 0.047), and both decreased linearly with increasing ambient temperature, from 55.27% to 50.11% and from 29.52% to 24.51%, respectively (P ≤ 0.009).

The linear regressions of ADG on effective energy (AME and NE) intake, together with protein (CP and MCP) intake, are presented in Fig. 3. The models incorporating NE, CP and MCP, or RE, CP, and MCP intake, yielded higher R² values than the corresponding model incorporating AME, CP, and MCP intake.

Fig. 3.

Linear regression of ADG on dietary nutrient intake. Regression analyses were performed using pooled data from the four ambient temperature treatments (23, 25, 27, and 29°C). Each treatment consisted of five closed-circuit indirect calorimetry chambers, with three 25-day-old male Arbor Acres broilers per chamber. AMEI, AME intake (kcal/d); CPI, CP intake (g/d); MCPI, metabolizable CP intake (g/d); NEI, net energy intake (kcal/d); REI, retained energy intake (kcal/d).

Substrate oxidation and its contribution to THP

No differences were observed among treatments in the oxidation of protein, fat, and carbohydrate, or in the contribution of each substrate to THP on a dietary intake basis (P ≥ 0.199; Table 6). Furthermore, no linear or quadratic responses were detected in substrate oxidation or substrate-specific contribution to THP as ambient temperature increased from 23°C to 29°C (P ≥ 0.100).

Table 6.

Effect of ambient temperature on the substrate oxidation and the corresponding energy yield per kg of feed consumed by broilers¹.

Items	Temperature				SEM	P-value2
	23°C	25°C	27°C	29°C		Anova	Linear	Quadratic
Uric acid nitrogen excretion, g/kg	7.3	6.6	7.0	7.1	0.3	0.540	0.781	0.247
Protein oxidation, g/kg	45.5	41.3	43.5	44.0	2.0	0.533	0.804	0.254
Fat oxidation, g/kg	−13.6	−8.7	−16.0	−20.1	5.6	0.544	0.294	0.427
Carbohydrate oxidation, g/kg	461.8	440.6	451.4	434.5	9.2	0.199	0.101	0.815
THP derived from protein, kcal/kg	200	182	191	193	9	0.541	0.783	0.255
THP derived from fat, kcal/kg	−129	−83	−152	−191	53	0.545	0.296	0.426
THP derived from carbohydrate, kcal/kg	1,939	1,850	1,895	1,824	38	0.199	0.100	0.816

THP, total heat production.

¹Data are presented as least squares means of five observations per treatment.

²Means are separated using one-way ANOVA and subjected to polynomial orthogonal contrasts.

Discussion

Accurate measurement of O₂ consumption, CO₂ production, and THP by indirect calorimetry depends on control of the chamber environment, particularly temperature, relative humidity, and gas concentrations (O₂ and CO₂). Previous research has demonstrated that ambient temperature markedly influences the logarithmic value of THP, highlighting the importance of maintaining stable environmental conditions during calorimetric measurements (Brown-Brandl et al., 2004). In the present study, the actual chamber temperature closely matched the preset values throughout the 72-h measurement period, with a maximum deviation of only 0.15°C. This level of precision is particularly important in broiler studies because minor changes in ambient temperature can influence feed-to-gain ratio (Bottje and Harrison, 1985). Relative humidity was also maintained within a narrow range (48.00%−52.17%) across treatments. Excessive humidity, particularly when combined with ambient temperature, has been reported to impair feed intake, carbohydrate metabolism, protein synthesis, fat deposition, and oxidative status (Kpomasse et al., 2021). The stable humidity observed in the present study reflects the ability of the closed-circuit indirect calorimetry system to automatically regulate both temperature and humidity, thereby avoiding environmental conditions that could compromise calorimetric measurements. Stable regulation of temperature and humidity also ensured reliable measurement of respiratory gases. Throughout the experiment, O₂ concentration remained constant at 20.84% to 20.89%, minimizing potential errors associated with changes in chamber during closed-circuit operation. Furthermore, the CO₂ concentration remained below 3,614 ppm, well below the 5,000 ppm limit recommended by the healthy farming technical specification for multi-layer caged white broiler (T/SDAA 0041-2021). Collectively, these findings indicate that this closed-circuit indirect calorimetry system provided a stable and well-controlled environmental suitable for accurately evaluating energy metabolism in broilers.

Accurate BW measurements are essential in indirect calorimetry because O₂ consumption and CO₂ production are commonly expressed relative to per kg of MBW when comparing energy metabolism among treatments (Ning et al., 2022; Tay-Zar et al., 2024). Moreover, FHP is required for NE calculations, is estimated from MBW using the value of 450 kJ/kg BW^0.70 proposed by Noblet et al. (2015). Consequently, abnormal growth during indirect calorimetry may bias measurements of O₂ consumption, CO₂ production, THP, and NE. Previous respiratory calorimetry studies have generally reported only the initial and final BW of broilers (Tay-Zar et al., 2024), whereas continuous BW changes during the measurement period have rarely been monitored. In the current study, real-time BW was continuously recorded throughout the 72-h calorimetry period, allowing automatic calculation of O₂ consumption per kg of MBW. The resulting growth curves showed that broilers maintained at 27°C and 29°C exhibited similar growth trajectories, whereas those maintained at 25°C grew more slowly and those at 23°C showed the lowest growth trajectory. Accordingly, BW, MBW, and ADG increased linearly, whereas ADFI/ADG decreased linearly from days 25 to 28 as ambient temperature increased from 23°C to 29°C. Ambient temperature directly influences heat exchange between the broiler and the environment, thereby affecting nutrient utilization and growth performance (Kim et al., 2021). The improved growth performance observed at higher ambient temperatures in the present study likely reflects a reduction in the energy required for thermoregulation, allowing a greater proportion of dietary energy to be directed toward tissue deposition and body weight gain.

Respiratory calorimetry quantifies substrate oxidation by measuring O₂ consumption and CO₂ production, making these variables, together with the respiratory quotient, reliable indicators of nutrient utilization in broilers. Both ambient temperature and feed intake have been shown to influence daily THP (Apalowo et al., 2024; Yunianto et al., 1997). Previous study has reported that increased feed intake and lower ambient temperature are associated with greater daily THP (Koh and Macleod, 1999b). In the present study, broilers maintained at 23°C exhibited greater daily O₂ consumption per broiler, daily O₂ consumption per kg of MBW, daily CO₂ production per broiler, and daily CO₂ production per kg of MBW than those maintained at 29°C. These findings indicate that decreasing ambient temperature increased metabolic activity and oxygen demand, reflecting greater energy expenditure for thermoregulation. Consequently, more dietary energy was dissipated as heat at lower temperatures, which is consistent with the greater THP and HI observed in the 23°C group. This response agrees with the established concept that broilers increase metabolic heat production under cooler conditions to maintain body temperature (Pishnamazi et al., 2015).

Energy utilization efficiency in broiler diet is determined by the partitioning of ME between THP and energy retention. Ambient temperature influences this partitioning by altering metabolic rate and the allocation of energy to protein and fat deposition (Sakomura et al., 2005). Previous study reported that the ME required for fat and protein deposition differs with ambient temperature, indicating that the efficiency of utilizing dietary energy for growth is temperature dependent (Sakomura et al., 2005). In the present study, broilers maintained at 23°C exhibited greater HI and THP but lower RE than those maintained at 29°C, resulting in a lower RE/AME. These findings indicate that a greater proportion of dietary ME was dissipated as heat under cooler conditions, whereas less energy was retained for tissue deposition. In contrast, the lower thermoregulatory demand at 29°C group reduced HI and favored energy retention, particularly as fat deposition, leading to greater RE and RE/AME. These findings are consistent with those of Zuidhof (2019), who reported that ambient temperature is a major determinant of the proportion of dietary energy retained in body tissues, and with Sakomura et al. (2005), who demonstrated that the energy cost of tissue deposition varies with ambient temperature. Furthermore, regression analyses showed that replacing AME intake with either NE intake or RE intake in models including CP and MCP intake progressively increased the R² for predicting ADG. This finding suggests that NE or RE more accurately represent the biologically available energy supporting growth than AME. Together with the observed changes in energy partitioning across ambient temperatures, these results demonstrate that the closed-circuit indirect calorimetry system provides a robust approach for evaluating dietary energy utilization in broilers under different thermal environments and further support the use of NE and RE as more physiologically relevant indicators of dietary energy value than AME.

The oxidation of carbohydrates, fat, and protein provides the energy released as THP in farm animals (Chwalibog, 2002). Although indirect calorimetry has been widely used to estimate the THP from respiratory gas exchange, relatively few studies have applied this approach to quantify individual substrate oxidation in broilers (Chwalibog, 2004; Liu et al., 2025). In the present study, substrate oxidation was estimated from O₂ consumption, CO₂ production, and uric acid nitrogen excretion, allowing the contributions of carbohydrate, fat, and protein oxidation to THP to be evaluated. The results showed that ambient temperature did not significantly affect uric acid nitrogen excretion, oxidation of carbohydrates, fat, and protein, or the contribution of individual substrates to THP. Carbohydrate oxidation accounted for the largest proportion of THP, followed by protein oxidation, whereas calculated fat oxidation was negative across all treatments. These findings agree with those of Chwalibog (2004), who reported that carbohydrate oxidation is the primary contributor to heat production in broilers and that negative fat oxidation values are commonly observed when carbohydrate is converted into lipid. Thus, the negative fat oxidation values observed in the present study likely reflect net lipogenesis rather than fat catabolism.

Conclusion

In summary, increasing ambient temperature from 23 to 29°C reduced THP and HI while increasing NE and RE values, thereby improving dietary energy utilization efficiency in broilers. The superior performance of NE- and RE-based models for predicting growth further supports the use of NE and RE as more physiologically relevant indicators of dietary energy value than AME. These results demonstrate that the closed-circuit indirect calorimetry system provides a reliable approach for evaluating dietary energy partitioning under different thermal environments and offers a useful tool for improving dietary energy evaluation and environmental management in broiler production.

CRediT authorship contribution statement

Hansuo Liu: Writing – original draft, Methodology, Investigation, Conceptualization. Changlin Guo: Validation, Investigation. Xiaomeng Ye: Methodology, Investigation. Jingjing Xie: Software, Methodology. Yuming Wang: Software, Methodology. Bing Lei: Validation. Feng Zhao: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

Disclosures

The authors declare no conflict of interest in this study.