Research Note: Effect of fasting time on the fasting heat production, blood metabolites, and body components of layer-type pullets

Abstract

Fasting heat production (FHP) is used to assess the maintenance net energy requirement of animals. Herein, the FHP of layer-type pullets was estimated. In trial 1, 16 40-day-old Jingfen layer-type pullets were divided into 4 groups of 4 chickens and placed in 4 respiratory chambers. Pullets had free access to feed and water. After 4-d acclimatization, feed was withdrawn, and chickens were measured for FHP for 3 consecutive days. In trial 2, twenty-four 40-day-old pullets were placed in 4 respiratory calorimetry chambers, with 6 pullets per chamber. After 4-d acclimatization, one chamber was randomly selected and all pullets in the chamber was sampled at 5, 25, 50, or 65 h after feed withdrawal. The result showed that FHP declined with fasting time and reached the lowest level between 48 and 72 h. Respiratory quotient was decreased (P < 0.05) between 24 and 48 h compared with that in the first 24 h after fasting. The FHP in the light period showed a significant to decline with fasting time (P < 0.01), whereas the FHP in the dark period was decreased (P < 0.01) 24 h after fasting. Body weight, thigh mass, and abdominal fat decreased (P < 0.05) at 25 h after fasting. Serum glucose were increased (P < 0.01) and while triglycerides were significantly decreased (P < 0.01) at 50 h compared with that at 5 and 25 h time point. The result suggests that the adequate measuring period for FHP for layer-type pullets is from 24 to 48 h after fasting. The FHP of 7-wk-old layer-type pullets was 562.20 kJ/kg of BW^0.75/d under a 10-h light and 14-h dark lighting regime.

INTRODUCTION

Currently, the energy requirement of poultry is estimated with the metabolizable energy (ME) system. The ME system systematically underestimates the energy content of feed rich in starch or fat and overestimates the energy content of feed rich in protein or fiber (Noblet et al., 1994). Compared with the ME system, the net energy (NE) system can more precisely estimate the feed energy content to facilitate the improvement of feed efficiency and application of low-protein diet in poultry. Fasting heat production (FHP) is used for characterizing the basal metabolic rate of animals, their corresponding maintenance energy requirements, and the calculation of net energy value of feeds. Many factors can affect FHP measurement such as the diet consumed before fasting, fasting time, and light regime.

Herein, the adequate fasting time was evaluated with layer-type pullets. An open-circuit system, from which gases are extracted and analyzed for the concentration of O₂ and CO₂, was used to estimate the FHP. The blood metabolites and hormones and body composition were determined at different fasting time points.

MATERIALS AND METHODS

All procedures in the study were approved by the Animal Care Committee of Shandong Agricultural University and were performed in accordance with the guidelines for experimental animals of the Ministry of Science and Technology (Beijing, China).

Experimental Design

In trial 1, 16 40-day-old Jingfen layer-type pullets were reared in respiratory thermometry chambers for 7 d, including a 4-d acclimatization period and a 3-d fasting and thermometry period. The 16 pullets were randomly assigned to 4 respiratory calorimetry chambers with 4 pullets per chamber. The temperature in the calorimetry chamber was maintained at 23 (±1) °C. During the whole experimental period, the pullets was fed with a balanced corn-soybean based pullet diet (Metabolic energy, 2,864 Kcal/kg; 18.0% crude protein) that was formulated to meet the requirements for all nutrients. During the 4-d acclimatization period, the pullets had free access to feed and water. In the morning of d 5, feed was withdrawn at 4:30 pm and the pullets were weighed. The concentration of O₂ and CO₂ of respiratory chambers was continuously measured for 72 h for 3 d between 44 and 46 d of age. The light regime was 10-h light and 14-h dark period. Light was turned on at 10:00 am and turned off at 8:00 pm. Light intensity was 10 lux. The chamber window was covered with black shading film to avoid the interference of outside environment during the whole experimental period.

In Trial 2, twenty-four 40-day-old Jingfen layer-type pullets were selected and placed in 4 respiratory calorimetry chambers, with 6 pullets per chamber. Pullets were exposed to the same treatment as in Trial 1. After 4-d acclimatization, one chamber was randomly selected and all the chickens in the chamber were sampled at 5, 25, 50, or 65 h after feed withdrawal to match with the time points when respiratory quotient (RQ) descend from a peak to a trough (around 0.7) observed in trial 1. A blood sample was collected from a wing vein, and serum was obtained after centrifugation for 10 min at 3,000 rpm and 4°C and then stored at −80°C. After weighed, birds were euthanized with carbon dioxide and then the abdominal fat (including abdominal fat pad and fat around proventriculus and gizzard) and breast (including Pectoralis major and Pectoralis minor) and thigh muscles (the muscles adhering femur and tibia bone) were harvested and weighed.

Measurement of FHP

The “open-circuit negative pressure” respiratory thermometry device (Shandong Mingjun Ecological Agriculture Technology Co., Rizhao, Shandong, China) was used to determine the respiratory heat production. Briefly, the respiration chamber (800 L) was air conditioned to maintain a constant temperature and humidity using an air conditioner and a heater. Gas was continuously extracted from the respiration chamber by a vacuum pump at a rate of 20 L/min. CO₂ and CO₂ concentrations in each chamber were measured at 3-min intervals by gas analyzer. FHP and RQ were calculated with the following equations.

$$\mathrm{FHP}\left(\mathrm{kcal}\right)=3.866\times \mathrm{V}{\mathrm{O}}_2\left(\text{L}\right)+1.200\times {\text{VCO}}_2\left(\text{L}\right)$$

$$\text{RQ}={\text{VCO}}_2\left(\text{L}\right)/\mathrm{V}{\mathrm{O}}_2\left(\text{L}\right)$$

where VO₂ and VCO₂ are the volume of oxygen and carbon dioxide, respectively.

FHP during the light and dark phases and the whole period in 0-24, 24-48, and 48-72 h were calculated respectively.

Measurement of Serum Metabolites

The serum content of serum calcium (CA), inorganic phosphorus (IP), total protein (TP), serum glucose (GLU), and triglycerides (TG) were detected with commercial kits (Sichuan Mike Biotechnology Co., Ltd., China) with an automatic biochemical analyzer (7020 Clinical Analyzer: Hitachi High-Tech GLOBAL, Japan). The concentration of free triiodothyronine (FT3) and free tetraiodothyronine (FT4) in serum were determined using an enzyme-linked immunosorbent assay kit (ELISA; Enzyme-linked Biotechnology Co., Ltd. Shanghai, China). The concentration of nonesterified fatty acid (NEFA) in serum was determined using an assay kit (Jiancheng Bioengineering Institute, Nanjing, China). All the measurements were performed according to the manufacturer's instructions. All the samples were measured in duplicate. For the measurement of FT3 and FT4, the intra-assay variation was 10% and the lowest detectable concentration was 0.1 pmol/L and 0.1 pmol/L. All samples were included in the same assay to avoid interassay variability.

Statistical Analysis

Data were analyzed by one-way ANOVA model (SAS, Versioon8e, SAS Institute) with each chamber as replicate (n = 4) in trial 1 and each pullet as pullet as replicate (n = 6). Differences between the treatments were evaluated using Duncan's multiple comparisons tests. Data are expressed as mean ± standard error. Differences were considered as significant at P < 0.05.

RESULTS AND DISCUSSION

Effect of Fasting Time on FHP and RQ

In trial 1, FHP and RQ declined with fasting time (Figure 1A) and FHP was reduced by 14.75% and 23.48% (P < 0.01) from 24 to 48 h and from 48 to 72 h, respectively, compared with that in the first 24-h after fasting (P < 0.001, Figure 1B). Compared with that in the first 24-h fasting period of (RQ = 0.74), the RQ value decreased from 24 to 48 h (RQ = 0.70; P < 0.05) but remained unchanged from 48 to 72 h (RQ = 0.72; P > 0.05, Figure 1C). In previous study, FHP-measuring period for layer-type pullets was conducted from 24 to 48 h after fasting (Johnson and Farrell, 1984). Laying hens reportedly do not exhibit a difference in total heat production between 22 and 46 h fasting, suggesting that metabolic heat production from residual nutrients ingested after 22 h fasting was negligible (Li et al., 1991). In the present study, the FHP gradually decreased with fasting time and in the period from 48- to 72-h reached the lowest value. In this study, the FHP of 7-wk-old layer-type pullets was 562.20 kJ/kg of BW^0.75/d. The FHP of White Leghorn hens and cockerels (from 48 to 72 h of after fasting) was 404 to 464 and 223 to 349 kJ/kg BW^0.75/d (O'Neill et al., 1974). The FHP of broilers at 15 d of age was 462 kJ/kg of BW^0.75/d (Liu et al., 2017). Overall, the result suggests that FHP is species and age-dependent.

Figure 1.

Effect of fasting time on fasting heat production (FHP) and respiratory quotient (RQ) of layer-type pullets in trail 1. (A) FHP and RQ at different time points; (B) FHP in the period of 0 to 24, 24 to 48, and 48 to 72 h after fasting; (C) RQ in the period of 0 to 24, 24 to 48, and 48 to 72 h after fasting; (D) FHP in light and dark periods; (E) RQ in light and dark periods. Data were presented as means ± SE (n = 4) *, P < 0.05; **, P < 0.01; ***, P < 0.001.

In the light period, FHP showed a tendency (P = 0.055) to decline with fasting time (Figure 1D), whereas FHP in the dark period was decreased by fasting time (P < 0.01) after 24-h fasting. The RQ was unchanged by fasting time in either the light or dark period (P > 0.05, Figure 1E). In previous studies, chickens used for the measurement of FHP were kept in a dark environment to mimic a resting state (Johnson and Farrell, 1984). In practice, however, chickens are reared under a light-dark cycle regime. Hens were subjected to a 14-h light and 10-h dark cycle for laying hens (Li et al., 1991). In the measurement of AME and NE of corn for laying hens, the birds received 16 h of light and 8 h of darkness in the respiratory chamber (Liu et al., 2020). In the present study, pullets were provided with a 10-h light and 14-h dark schedule. Although the FHP did not significantly differ between the light and periods (25.06 ± 1.38 vs. 22.63 ± 1.59 kJ/kg of BW^0.75/h), the relative lower value during dark period indicates the circadian rhythm in the FHP. Moreover, the FHP in the light and dark periods showed a tendency to decline and to decrease with time following the fasting period, suggesting that the FHP during the dark period plays an important role in the measurement of heat production for maintenance.

In addition, FHP measured in modern broiler chickens (0.5–3.0 kg) is suggested to be expressed as BW^0.70 per kg and that FHP values for broilers from should range from 410 to 460 kJ/kg of BW^0.70/d (Noblet et al., 2015). These results suggest that the estimated values of NEm are influenced by the type of animal (i.e., breed, age, sex), experimental environment, and measurement method. FHP may also be greater in growing animals than in mature animals probably because the amount of metabolic activity associated with energy deposition is greater in growing animals and remains at a higher level even during fasting.

Effect of Fasting on Organ Weight and Blood Metabolites

To refine the optimal measuring period for FHP, we determined the organ index and blood metabolite levels. The abdominal fat pad significantly decreased by fasting time (P < 0.001, Table 1) and underwent a large decline after 25-h fasting compared with that at 5 h after feed withdrawal, thereafter no further decrease occurred until 50 h, which decreased further by 65 h. Conversely, breast muscle mass was unchanged by fasting time, whereas the proportion to BW was increased with time (P < 0.01). For the thigh muscle, however, both the mass and proportion decreased by fasting time at 25 h (P < 0.001) but showed no further decline thereafter.

Table 1.

Effect of fasting time on organ index and blood metabolites of layer-type pullets in trial 2.

	0 h	5 h	25 h	50 h	65 h	P-value
Body weight, g	408.3 ± 13.2a	399.0 ± 15.2a	361.8 ± 12.0b	351.8 ± 5.7b	337.0 ± 12.9b	0.001
Abdominal fat, g	2.68 ± 0.37a	2.55 ± 0.41a	0.87 ± 0.22b	0.68 ± 0.13b	0.27 ± 0.13c	<0.001
%	0.65 ± 0.07a	0.63 ± 0.09a	0.25 ± 0.07b	0.19 ± 0.04b	0.08 ± 0.04c	<0.001
Breast muscle, g	39.0 ± 1.48	38.9 ± 1.56	38.2 ± 2.08	38.6 ± 0.61	36.3 ± 1.43	0.69
%	9.56 ± 0.21b	9.76 ± 0.20b	10.5 ± 0.27a	11.0 ± 0.14a	10.8 ± 0.17a	<0.001
Thigh muscle, g	49.5 ± 2.15a	49.3 ± 2.09a	40.9 ± 1.87b	38.8 ± 0.64b	38.0 ± 1.53b	<0.001
%	12.1 ± 0.23a	12.4 ± 0.13a	11.3 ± 0.20b	11.0 ± 0.17b	11.3 ± 0.11b	<0.001
Glucose, mmol/L	-	11.3 ± 0.24b	11.7 ± 0.38b	13.3 ± 0.48a	13.7 ± 0.48a	0.001
TG, mmol/L	-	0.56 ± 0.04a	0.51 ± 0.01ab	0.44 ± 0.01c	0.46 ± 0.01bc	0.002
NEFA, mmol/L	-	0.68 ± 0.06a	0.68 ± 0.04a	0.53 ± 0.03b	0.61 ± 0.04ab	0.051
TP, g/L	-	38.8 ± 1.07	38.7 ± 0.73	37.5 ± 0.44	39.1 ± 0.55	0.438
FT3, pmol/L	-	5.30 ± 0.06b	5.36 ± 0.03b	5.73 ± 0.08a	5.93 ± 0.09a	<0.001
FT4, pmol/L	-	53.2 ± 0.98b	54.6 ± 0.99ab	57.4 ± 1.63a	58.4 ± 1.73a	0.037

FT3, free triiodocarinine; FT4, free thyroxine; NEFA, Non-esterified fatty acid; TG, triglyceride; TP, total protein.

Data were presented as means ± SE (n = 6).

^abMeans in the same row with superscript differ significantly, P < 0.05.

Compared with 5- and 25-h time points, GLU concentration was increased at 50- and 65-h time points after fasting (P < 0.01, Table 1). In contrast, TG level was lowered at 50 h time point compared with 5 h and 25 h time points (P < 0.002). The concentration of NEFA showed a tendency to be decreased by fasting at 50 h time point (P = 0.051) compared to 5- and 25-h time points. TP level was not altered by fasting time (P > 0.05).

The decreased BW, thigh mass, and abdominal fat at 25 h after fasting indicated the mobilization of energy reserves. Indeed, fasting significantly increases the rate of fatty acid oxidation (Torchon et al., 2017). In this study, blood samples were obtained at time points when RQ decreased from peak point to the trough. The lack of change in blood concentrations of glucose, TG, and NEFA at 25-h time point, implies the energy supply is unaltered. Conversely, the significantly elevated concentration of glucose and decreased levels of TG and NEFA at 50 h after fasting indicated the reduced energy supply. Recently, the gastrointestinal tract emptying time was investigated to show that the emptying time of free-feeding broilers and adult roosters was 12 and 24 h, respectively (Wang et al., 2022). Overall, these results suggest that the period from 24 to 48 h is the adequate phase for FHP measurement.

Compared with 5 h time point, the concentration of FT3 was unchanged at 25 h but was increased at 50 h and 65 h (P < 0.001). The concentration of FT4 was elevated at 50 h and 65 h compared to 5 h (P < 0.05). During fasting, the increased T4 and decreased T3 was accompanied by decreased basal metabolic rate (Sechman et al., 1989). The relative high level of FT3 at 50 h and 65 h may be related to the metabolic fluctuations. In the present study the free T3 and T4 were measured and hence the result should be explained with caution.

In conclusion, the adequate measuring period for FHP of layer-type pullets was from 24 h to 48 h after fasting. The fasting heat production of 7-wk-old layer-type pullets was 562.20 kJ/kg of BW^0.75/d at 10-h light and 14-h dark lighting regime.