1. Introduction
Soybean meal (
SBM), with a well-balanced amino acid profile, is the primary protein feed used in the poultry industry. The available energy of SBM for broilers has been measured in previous studies [
1,
2,
3]. In recent years, expanded soybean meal (
ESBM) has been extensively used in the poultry industry. The expansion technology modified the physicochemical properties of SBM and inactivated anti-nutritional factors [
4]. Replacing SBM with ESBM increased nutrients and energy digestibility [
5] and improved growth performance of broilers [
6]. Hence, precisely determining the energy levels of ESBM for broilers could promote the application of ESBM in the poultry industry.
The metabolizable energy (
ME) system is widely used in poultry, mainly because feces and urinary losses of birds are voided together through the cloaca. However, the net energy (
NE) system provides a more accurate estimate of dietary energy and represents the actual energy demand for maintenance and production [
7]. NE values consider heat increment (HI) produced during the digestion and absorption process in the gastrointestinal tract [
8]. The heat increment of protein is higher than that of fat and carbohydrate in animals [
9]. Hence, the application of the NE system potentially reduces the addition ratio of protein feed and decreases nitrogen emissions, resulting in cost savings without detrimental effects on the performance parameters of animals [
10,
11]. Therefore, it is vital to measure the net energy of ingredients and promote the development of the NE system for broilers.
Factors associated with birds, including strain, age, and body weight, could affect the energy utilization of feeds [
12,
13]. As broilers grew older, the secretion of digestive enzymes, intestinal surface area, and digestive capacity, especially for SBM, increased [
14]. It is easy to illustrate that energy retained in the body improves with increasing age of broilers [
15]. AME values of feed ingredients, including corn, soybean, and bran, were reported to be higher in older broilers than in younger ones [
16,
17]. However, the age-dependent pattern of net energy values of ingredients for broilers is still unknown. A previous study reported that age affects HI due to changes in feed intake and intestinal digestive status of pigs [
18]. It is reasonable to speculate that the NE values of ingredients differ between birds at different ages. The objective of the study was to determine ME and NE values of ESBMs for broilers from 14 to 16 day and 28 to 30 day using the difference method. The results of this study could facilitate precise feed formulation.
2. Materials and Methods
The experiment was approved by the Institutional Animal Care and Use Committee of China Agricultural University (Beijing, China) for scientific purposes (AW51304202).
2.1. Expanded Soybean Meal and Diets
The ESBMs with different crude protein (
CP) contents were acquired from commercial plants (Wellhope Co., Ltd., Chengdu, China, New Hope Co., Ltd., Shenyang, China and Techlex Co., Ltd., Zhuozhou, China). The measured nutrient contents of three ESBM samples are shown in
Table 1. The basal diets were formulated to meet the energy and nutrient requirements for broilers in the grower and finisher phase (
Table 2). A corn–soybean meal basal diet was formulated, and three test diets, each containing an ESBM sample, were developed by replacing 25% of the energy-yielding ingredients in basal diets, including corn, soybean meal, corn gluten meal, distillers dried grains with solubles, peanut meal and amino acids (
Table 3).
2.2. Birds and Experiment Design
A total of ninety-six 10-day-old and forty-eight 24-day-old Arbor Acres broiler chickens with average body weight (BW) were sourced from Tieling city (Liaoning province, China) and housed in a climate-control shed at Jilin Academy of Agricultural Sciences (Gongzhuling, Jilin province, China). Broilers were given ad libitum access to feed and water. After dietary adaption, broilers were transferred from the shed to respiration chambers for 2 days of chamber adaption. A randomized design was used to evaluate four different diets in 12 calorimetry chambers (3 chambers per treatment) for 3 days. Each chamber represents a repeat run. The experiment was conducted in two batches, with each diet repeating six runs, with four (14 to 16 day) or two (28 to 30 day) birds per repeat.
2.3. Respiration Chambers
Twelve open-circuit respiration chambers (90 × 60 × 100 cm3 glass box with an automatic door on the top side) were used in our study. Briefly, chambers were equipped with a vacuum pump, as well as CO2 and O2 sensors. A zirconium oxide sensor (Model 65-4-20; Advanced Micro Instruments Inc., Huntington Beach, CA, USA) was used for O2 detection, and a non-dispersive infrared sensor (AGM 10; Sensors Europe GmbH, Erkrath, Germany) was used for CO2 detection. The real-time data of CO2 production and O2 consumption were collected at 3 min intervals and were expressed as L/min. The respiratory quotient (RQ) was automatically calculated. Moreover, the respiratory chamber was air-conditioned to maintain constant temperature (27 to 30 °C and 23 to 26 °C for broilers from 10 to 16 day and 24 to 30 day, respectively) and humidity (50% to 70%). The analyzer measured a range of 0% to 25% of O2 and 0% to 2.5% of CO2. In addition, it was suspended for about 1 h each day to replenish feed and collect excreta.
2.4. AME Measurement and Chemical Analysis
The AME values were measured by using the total collection method as described by Tillman and Waldroup [
19]. Feed intake (
FI) was measured and calculated daily. Excreta was collected every morning of the testing period. Then, the excreta of six replicates were mixed, oven-dried, weighted, and grounded until through a 1 mm
2 screen. Feed and excreta were analyzed on a dry matter basis. Gross energy (
GE) was determined using a bomb calorimeter (IKA-C3000, Bitterfeld-Wolfen, Germany). The CP was determined by using the Kjeldahl method (AOAC, 984.13) with Foss KT200 (Hilloerod, Denmark) [
20]. The neutral detergent fiber (
NDF) and acid detergent fiber (
ADF) were determined using an Ankom220 Fiber Analyzer (Ankom Technology, NY, USA) with filter bags as described by Van Soest et al. [
21]. AOAC methods were also used for the measurement of ether extract [
22] and ash (942.05) [
20]. The contents of sucrose, stachyose, and raffinose in ESBM samples were determined by using high-performance liquid chromatography (1260, Agilent Technologies Inc., Santa Clara, CA, USA) according to Kennedy et al. [
23]. Amino acids were determined using the acid hydrolysis method with an automatic Amino Acid Analyzer (ARACUS, Membrapure, Berlin, Germany). Briefly, the ESBM samples were hydrolyzed with 6 M HCl at 110 °C for 24 h. The samples were then equalized to a 50 mL volume, deacidified, dissolved in sample buffer, and analyzed.
2.5. Calculation
2.5.1. Respiratory Data
All respiratory data were corrected by BW
0.70 (
Table S1). The heat production (
HP) was calculated daily following the equation first proposed by Brouwer [
24].
The respiratory quotient (RQ) corresponds to the ratio between CO2 production and O2 consumption.
The fasting heat production (
FHP) value of 450 kJ/kg BW
0.70/day used in this study was measured by Noblet et al. [
25].
2.5.2. Energy Values of Diets
AME was corrected to zero nitrogen retention (
AMEn) using 34.41 kJ/g of nitrogen [
26].
AME intake was calculated as AME multiplied by FI.
2.5.3. Retained Nitrogen and Retained Energy
Retained energy (
RE, kJ/kg BW
0.70/day) = AME intake − HP [
2].
RE as protein (kJ/kg BW
0.70/day) was calculated as RN × 6.25 × 23.86, according to the previous study [
27], while RE as fat was calculated by subtracting RE as protein from total RE.
2.5.4. Energy Values of ESBMs
While R0 is the ratio of energy-yielding ingredients in the basal diet (96.76% and 97.30% for grower and finisher diets, respectively), R1 is the ratio of energy-yielding ingredients other than ESBM in test diets (71.76% and 72.30% for grower and finisher diets, respectively), R2 is the ratio of ESBM in test diet (25.00%).
The AME, AMEn, and NE values of ESBMs were then corrected by measured GE as follows:
2.6. Statistical Analyses
The chamber was considered an experiment unit. A one-way analysis of variance was conducted on growth performance, nitrogen balance, energy values, and energy balance using SPSS software 24.0 (SPSS Inc., Chicago, IL, USA). The “dietary treatment” was considered as a fixed variable, while the “chamber” and “batch” were considered as random variables. The results were displayed using the main effect of dietary treatment. Duncan’s method was used to make multiple comparisons. The energy values of ESBMs were analyzed using a two-way analysis of variance. “Age of birds” and “ESBMs” were considered as main effects. In addition, the variation of ESBMs was analyzed using the principal component analysis (PCA) procedure with “FactoMineR” and “factoextra” packages using R 4.3.3 software. Differences were considered significant at p < 0.05.
3. Results
The nutrient contents of ESBMs are presented in
Table 1. The concentrations of CP and EE in 3 ESBMs ranged from 43.46% to 46.31% and 0.80% to 0.98%, respectively (as fed basis). The percentage of GE was greater in ESBM2 (17.46 MJ/kg) compared with ESBM1 and ESBM3 (17.15 MJ/kg and 17.26 MJ/kg, respectively), while the contents of NDF and ADF were higher in ESBM1 (11.66% and 8.94%) compared with ESBM2 (11.03% and 6.50%) and ESBM3 (9.60% and 5.98%). In addition, the contents of most amino acids in the 3 ESBMs were similar.
Effects of dietary characteristics on growth performance, nitrogen balance, energy utilization, and energy balance of broilers from 14 to 16 day and 28 to 30 day are shown in
Table 4 and
Table 5, respectively. BW, FI, AME intake, and AME intake/BW gain of broilers were not affected by different diets regardless of age (
p > 0.05). Compared with basal diets, test diets increased nitrogen intake and nitrogen excreta of broilers irrespective of bird age (
p < 0.01). As a result, a reduction of RN was observed in birds fed basal diet from 28 to 30 day (
p < 0.05); however, the RN was not influenced by test diets from 14 to 16 day (
p > 0.05). Compared with test diets, the AME, AMEn, and AME/GE were significantly higher in basal diet for broilers from 14 to 16 day (
p < 0.001). Specifically, substituting ESBM1 and ESBM2 increased RE as a protein in broilers from 14 to 16 day compared with basal diet (
p < 0.05). As expected, substituting ESBMs increased broilers’ RE as protein from 28 to 30 day (
p < 0.05). Correspondingly, the RE as fat was significantly higher in broilers fed with basal diet from 28 to 30 day compared with those fed ESBM1 and ESBM3 diets (
p < 0.05). Although dietary AME values were similar from 28 to 30 day (
p > 0.05), test diets decreased AMEn values compared with basal diet (
p < 0.05). Additionally, the NE, RQ, HP, and HI were not affected by different diets irrespective of bird ages (
p > 0.05).
As shown in
Table 6, no treatment interaction was observed (
p > 0.05) between ESBMs and ages for energy values and energy utilization of ESBMs. The AME, AMEn, and NE values of ESBMs for broilers from 14 to 16 day varied from 9.79 to 10.88 MJ/kg, 8.24 to 9.29 MJ/kg, and 6.61 to 7.29 MJ/kg, respectively (DM basis). The AME, AMEn, and NE values of ESBMs for broilers from 28 to 30 day varied from 11.33 to 12.84 MJ/kg, 9.88 to 11.36 MJ/kg, and 5.89 to 6.77 MJ/kg, respectively (DM basis). The AME/GE, AMEn/GE. NE/AME and NE/AMEn ranged from 0.50 to 0.66, 0.42 to 0.58, 0.51 to 0.68, and 0.59 to 0.80, respectively. Interestingly, the average AMEn of ESBMs in 28-day-old birds was significantly higher than those in 14-day-old birds (10.42 vs. 8.93 MJ/kg), while the average NE/AME of ESBMs was significantly higher in 14-day-old birds compared with those in 28-day-old birds (
p < 0.05). Correspondingly, the AME (
p = 0.095), AME/GE (
p = 0.092), and AMEn/GE (
p = 0.052) of ESBMs were numerically higher in broilers from 28 to 30 day compared with those from 14 to 16 day. As age transitioned from 14 to 28 day, the NE (
p = 0.062) and NE/AMEn (
p = 0.084) of ESBMs were numerically decreased.
Variables of PCA showing correlations between energy values and nutrient contents of ESBMs (
Figure 1). An acute angle among energy values and nutrient contents indicates a positive correlation, while an obtuse angle indicates a negative correlation. The energy values (AME and NE) of ESBMs in birds at different ages were positively correlated. As expected, the CP and GE contents were positively related to energy values, while the NDF content was negatively related to energy values. However, the EE content tended to be negatively correlated with energy values. There were no relationships between energy values and raffinose, sucrose, stachyose, ash, and ADF. Yellow colors represent a high parameter contribution to the variation of ESBMs, while blue colors represent a low contribution. Specifically, the contribution of dry matter content to the variation of ESBMs is relatively small, while the variation of ESBMs is highly dependent on energy values and contents of CP, GE, NDF, and ADF.