The Institutional Animal Care and Use Committee of China Agricultural University (Beijing, China) approved all protocols used in this experiment.
Equipment for indirect calorimetry
Six open-circuit respiration chambers (1.4 × 2.7 × 2.1 m3) located in the FengNing Swine Research Unit of China Agricultural University (Academician Workstation in Chengdejiuyun Agricultural & Livestock Co., Ltd) were used to conduct the indirect calorimetry, which were described in details in previous studies [12, 13] with the exception of temperature settings. The relative humidity was controlled at 70–75%, and the air velocity was controlled at 0.1 m/s. Oxygen content was measured with a paramagnetic differential analyzer (Oxymat 6E, Siemens, Munich, Germany), whereas CO2 and CH4 contents were measured with infrared analyzers (Ultramat 6E, Siemens). The analyzers had a range of 19.5–21.0% for O2, 0–1% for CO2, and 0–0.1% for CH4, with a sensitivity of 0.2%. Gas concentrations in each chamber were measured at 5-min intervals. The ethanol combustion experiment was used to check the accuracy of the chamber in measuring gaseous exchange.
Animals, diets and experiment design
Two animal trials were conducted in this study. In Trial 1, 36 healthy growing barrows (Duroc × Large White × Landrace) with initial BW of 26.4 ± 1.9 kg were allotted to a 6 × 6 Latin Square Design in 6 consecutive periods. In each period, 6 pigs were moved into 6 open-circuit respiration chambers with ambient temperatures setting as 18 °C, 21 °C, 23 °C, 27 °C, 30 °C, and 32 °C, respectively. In Trial 2, 24 healthy growing barrows (Duroc × Large White × Landrace) with initial BW of 64.2 ± 3.1 kg were allotted to a 4 × 6 Youden Square Design in 6 consecutive periods. In each period, 4 pigs were moved into 4 open-circuit respiration chambers (only 4 chambers were available during Trial 2) with ambient temperatures setting as 18 °C, 23 °C, 27 °C, and 32 °C, respectively.
Two diets based on corn-soybean meal were formulated to meet or exceed the nutrient requirements for growing pigs at 25 kg and 65 kg according to the recommendations by NRC [14], and were applied in Trial 1 and 2, respectively. The analyzed chemical compositions of those two diets were shown in Table 1.
Table 1
Ingredient compositions and analyzed chemical components of the complete diets fed to barrows at different growth stages used in Exp.1 and Exp.2 (as-fed basis)
Items | Growth stage |
25 kg | 65 kg |
Ingredients, % | | |
Corn Soybean meal Dicalcium phosphate Limestone Sodium chloride | 77.13 20.00 0.90 0.75 0.35 | 80.39 17.00 0.80 0.65 0.35 |
Premix1 | 0.50 | 0.50 |
Lysine-HCl | 0.21 | 0.17 |
DL-Methionine | 0.11 | 0.09 |
L-Threonine | 0.02 | 0.02 |
L-Tryptophan | 0.03 | 0.03 |
Analyzed nutrient levels, % | | |
Dry matter | 89.50 | 89.60 |
Gross energy, MJ/kg | 16.38 | 16.67 |
Crude protein | 17.78 | 16.20 |
Ether extract | 2.89 | 2.57 |
Ash | 4.72 | 3.34 |
1Premix supplied the following quantities per kilogram of complete diet for pigs at 25 kg: vitamin A, 6,000 IU; vitamin D3, 1,500 IU; vitamin E, 15 IU; vitamin K3, 1.5 mg; vitamin B12, 10 µg; riboflavin, 3.0 mg; pantothenic acid, 9.5 mg; niacin, 18 mg; choline chloride, 350 mg; folacin, 0.4 mg; thiamine 0.9 mg; pyridoxine 1.5 mg; biotin, 25 µg; Mn, 17.5 mg (MnO); Fe, 80 mg (FeSO4·H2O); Zn, 65 mg (ZnO); Cu, 60 mg (CuSO4·5H2O); I, 0.3 mg (KI); Se, 0.2 mg (Na2SeO3), and supplied the following quantities per kilogram of complete diet for pigs at 65 kg: vitamin A, 5,000 IU; vitamin D3, 1,350 IU; vitamin E, 13.5 IU; vitamin K3, 1.45 mg; vitamin B12, 9 µg; riboflavin, 2.7 mg; pantothenic acid, 8.0 mg; niacin, 16 mg; choline chloride, 280 mg; folacin, 0.3 mg; thiamine 0.7 mg; pyridoxine 1.35 mg; biotin, 20 µg; Mn, 15.0 mg (MnO); Fe, 70 mg (FeSO4·H2O); Zn, 65 mg (ZnO); Cu, 25 mg (CuSO4·5H2O); I, 0.3 mg (KI); Se, 0.2 mg (Na2SeO3). |
The specific experiment procedures were kept the same in both animal trials. In each period, pigs were kept individually in stainless-steel metabolism crates (1.2 × 0.5 × 0.6 m3) equipped with feeders and low-pressure nipple drinkers, and had a 7-d adaption period for crates and diets in the thermoneutrual-controlled room (24 ± 1 °C). On day 8, pigs were weighted and moved into the chambers for 5 days to measure the gases exchanges and calculate the total heat production (THP). Based on the feed intake during adaption, feed was supplied twice daily (08:30 and 15:30) ad libitum to pigs in chambers, and pigs had free access to water. For each pig, the total feces, urine output and wasted feed were collected twice daily from 08:00 to 08:30, and from 15:00 to 15:30, when the production of CO2, CH4 and O2 consumption were expelled in the calculation of daily HP, to determine the energy intakes of each day. On day 13, pigs were weighted in the morning and deprived of feed and fasted for 24 h, and the total urine output were collected during the fasting period to determine the fasting heat production (FHP), as an estimation of the energy used for maintenance [12]. On the morning of day 14, pigs were moved out the chambers and weighted again.
Sample collection
The daily urine output of each pig was collected into buckets with 50 mL 6 N HCl and sieved thereafter through multilayer gauze into plastic bottles and stored in -20 °C. The 5-day total urine collection and urine collection during the fasting period were quantified for each pig respectively, and a subsample of 50 mL for each pig was saved from the thoroughly mixed 5-day total collection for further analysis. The daily feces output of each pig was collected into plastic bags and immediately stored at -20 °C, then the 5-day total feces collection was thawed, mixed, and weighted, and a subsample of 350 g for each pig was oven-dried for 72 h at 65 °C to calculate the moisture content. Feed samples and subsamples of dried feces were finely ground through a 1 mm sieve prior to chemical analysis.
Blood samples of pigs in Trial 1 were collected from precaval vein into the heparinized tubes on day 13 morning during fasting, and then were centrifuged (Biofuge22R; Heraeus, Hanau, Germany) at 3,000 × g for 10 min at 4 °C. The supernatants were transferred to storage tubes, frozen in liquid nitrogen, and stored at -80 °C for further assays.
Chemical analysis and calculation
All chemical analysis of samples was conducted in duplicates and repeated if the duplicates differed by more than 5%. The gross energy (GE) in feed, feces, and urine samples was determined using an isoperibol calorimeter (Parr 6400 Calorimeter, Moline, IL, USA) with a standard reference of benzoic acid. For feed samples, the contents of dry matter (DM), crude protein (CP), ether extract (EE), and ash were determined following the methods of AOAC [15]. For fecal samples, the contents of DM and CP were determined. For urine samples, the nitrogen (N) content was measured according to Li et al. [13].
The dry matter intake (DMi) of each pig from day 8 to day 12 was calculated as the product of feed intake and the DM content of the diets. The GE intake was calculated as the product of actual DMi during the 5-day collection period and the GE values of the diets. The digestible energy (DE) intake and metabolizable energy intake (MEi) was calculated as the difference between GE intake and the energy loss in feces, and the difference between DE intake and the energy loss in urine and methane, respectively. Methane energy was calculated using the methane volume and a conversion factor of 39.4 kJ/L [16].
The average daily THP was calculated according to the following equation based on the gas exchanges (O2 consumption and productions of CO2 and CH4) during day 8 to day 12 recorded at 5-min intervals and then averaged and extrapolated to a 24-h period:
HP (kJ) = 16.18 × O2 (L) + 5.02 × CO2 (L) − 2.17 × CH4 (L) − 5.99 × urinary N (g) [16].
The FHP was calculated using the same equation, but the 24-h FHP was predicted from the 8-h HP after feed deprivation from 22:00 to 06:00 during the last day of each period, which was then extrapolated to the 24-h period [13]. The RE was calculated as the difference between MEi and THP, while the RE as protein (REP) was calculated as N retention (g/d) × 6.25 × 23.86 (kJ/g), and the RE as lipid (PEL) was calculated as the difference between RE and REP. The net energy (NE) intake was calculated as the sum of RE and maintenance energy estimated by FHP [17]. Moreover, the oxidation of carbohydrates (OXCHO) and fat (OXF) were calculated by the method described by Chwalibog et al. according to the following equations [18]:
OXCHO (kJ/d) = [-2.968 × O2 (L) + 4.147 × CO2 (L) − 1.761 × CH4 (L) − 2.446 × Urinary N (g)] × 17.58.
OXF (kJ/d) = [1.719 × O2 (L) − 1.719 × CO2 (L) − 1.719 × CH4 (L) − 1.963 × Urinary N (g)] × 39.76.
The rates of DE:GE, ME:DE and NE:ME were then calculated based on the calculations of GE, DE, ME, and NE intakes. The respiratory quotient (RQ) was calculated as the ration between CO2 production and O2 consumption. All the energy balance indexes were present on the metabolic body weight (per kg BW0.60) basis.
Hormone and biochemical marker assays in serum
The concentrations of cortisol, insulin, triiodothyronine (T3) and thyroxine (T4) in serum were analyzed using radioimmunoassay, while the activities of growth hormone (GH) and glucagon in serum were analyzed using commercial enzyme-linked immunosorbent assay (ELISA) kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s guides. The levels of albumin, glutamic-pyruvic transaminase (ALT), glutamic oxalacetic transaminase (AST), globulin, high density lipoprotein (HDL), low density lipoprotein (LDL), total cholesterol (TC), triglyceride (TG), total protein and urea in serum were determined using the automatic biochemical analyzer (7170, Hitachi Corp., Tokyo, Japan) with corresponding commercial kits (Nanjing Jiancheng Bioengineering Institute) following the manufacturer’s guides.
Non-target metabolomics profiling in plasma and data analysis
Plasma samples from pigs kept under the ambient temperatures of 18 °C, 23 °C and 32 °C (defined as low, neutral and high ambient temperature group, respectively) were used for non-target metabolomics profiling assay, followed the procedures described by Liu et al. with some modifications [19].
Briefly, 100 µL plasma samples were added into 400 µL ice-cold extraction mix (methanol: acetonitrile = 1:1, v: v). After vortexing for 10 s, the mixture was centrifuged (Eppendorf, Germany) at 15,000 × g for 10 min at 4 °C to remove protein, and 400 µL supernatant was collected and evaporated to dryness using a vacuum concentrator (Concentrator PLUS, Eppendorf). The resulting dry residues were re-suspended in 200 µL recovery solution (water: methanol = 4:1), vortexed and centrifuged again at 15,000 × g for 10 min at 4 °C. The supernatant was filtered through a 0.22 µm membrane and transferred to sampler vials to be analyzed on a UPLC-MS system (UPLC, ACQUITY UPLC H-Class PLUS Bio System, Waters Corporation, MA, USA; MS, Q-Exactive, Termo Scientific, NJ, USA) equipped with a heated electrospray ionization (HESI) source. The UPLC separation was operated on a BEH C18 column (2.1 × 100 mm, 1.7 µm, Waters Corporation), with mobile phase comprised of 0.1% formic acid water solution (A) and 0.1% formic acid acetonitrile solution (B), flow rate set as 0.3 mL/min, column temperature set at 35 °C, and injection volume set as 5 µL. The MS analysis was performed in an electrospray ionization positive mode, and data were acquired with full scan using a mass resolution of 70,000 and a scan range of 50 to 750 m/z. For MS/MS analysis, an isolation window of 2.0 m/z and a mass resolution of 17,500 were selected.
Statistical Analysis
For the metabolomics data, the software SIEVE 2.1 (Thermo Scientific) and Progenesis QI (Nonlinear Dynamics, Waters Corporation) were used for raw data processing, including background subtraction, peak alignment, identification, and normalization with Pareto scaling to get the standardized relative abundance of the metabolites in different samples. Then principal components analysis (PCA) was conducted using SIMCA-P 13 software (Umetrics, Umea, Sweden), and one-way ANOVA was conducted using JMP Pro 14.3.0 (SAS Institute Inc., Carry, NC, USA) to compare the abundance of metabolites among different treatments. The metabolites with fold change > 1.5, CV < 20% and P < 0.05 were selected for further identification by comparison of the ion features in the experimental samples to the chemical standard entries in reference libraries (METLIN, HMDB and KEGG) that included retention time, molecular weight (m/z) as well as their associated MS/MS spectra. Boxplot was plotted using the ggplot2 package in R software (http://cran.r-project.org/, version 4.0.2) to illustrate the concentrations of the compounds that identified with significantly difference among the three treatments. Those compounds were then imported into the module of pathway analysis in Metaboanalyst 3.0 (https://www.metaboanalyst.ca/MetaboAnalyst/upload/PathUploadView.xhtml) to generate the pathway topology analysis. The metabolic pathway with impact value greater than 0.1 was characterized as the significantly relevant pathway [19].
For the other data, normality and homogeneity of variance were checked using the normal probability plot and residual plot from JMP Pro 14.3.0, and outliers were removed before further analysis. For N and energy balance data and serumal hormone and biochemical markers data collected in Trial 1, a statistical model including ambient temperature as the only fixed effect and chamber as the random effect was fitted using the Fit Model function in JMP Pro 14.3.0. Tukey’s HSD test was used to separate the least square means among treatments with significantly different effects. Moreover, linear and quadratic effects of increased ambient temperature on responses were tested by polynomial contrast using the Fit Y by X function in JMP Pro 14.3.0. To better illustrate the comprehensive effects of ambient temperature and pig BW, the N and energy balance data collected in Trial 2 were combined with some corresponding data (those under the same temperature settings) in Trial 1 for analysis. A statistical model included the main effects of ambient temperature (18 °C, 23 °C, 27 °C, and 32 °C) and BW (25 kg and 65 kg), and their interaction effect as fixed effects, and included the chamber as the random effect. The Fit Model function in JMP Pro 14.3.0 was used to conduct the two-way ANOVA, and Tukey’s HSD test was used to separate the least square means among treatments with significantly different main effects only when the interaction effect was not significant. Moreover, equations were developed to predict the voluntary feed intake (VFI), MEi, REP and REL using metabolic body weight (BW0.6) and ambient temperature as predictors based on some modifications of the equations from Quiniou et al. [20]:
VFI or MEi = a + b × BW0.6 + c × (BW0.6)2 + d × T + e × T2 + f × BW0.6 × T,
REP or REL = a + b × BW0.6 + c × (BW0.6)2 + d × T + e × T2 + f × BW0.6 × T + g × MEi.
All the data obtained in Trial 1 and 2 were combined and used for model development. Covariance analysis was used to further confirm the quadratic effects of BW and T on response variables, and coefficients were estimated by Gauss-Newton analysis using the Nonlinear Modelling function in JMP Pro 14.3.0. The predicted vs. actual plot and predicted vs. residuals plot were used to check the goodness of fit of the models. For all the analysis, P < 0.05 was considered as significantly different.