MATERIALS AND METHODS

Ethical approval

All experimental procedures involving broiler chickens were reviewed and approved by the Kasetsart University Institutional Animal Care and Use Committee, Bangkok, Thailand (Approval No. ACKU65-AGR-011). The study was conducted in accordance with the institutional guidelines for the care and use of animals in research and the principles of Good Agricultural Practices for broiler production. Before the experiment, all birds were inspected for health status and managed under controlled environmental conditions with appropriate stocking density, ventilation, temperature, lighting, vaccination, and ad libitum access to feed and water. Throughout the 35-day experimental period, birds were monitored daily for general health, behavior, morbidity, mortality, and signs of stress. Handling was minimized and performed by trained personnel to reduce discomfort. Birds selected for carcass evaluation were humanely euthanized by carbon dioxide inhalation in a controlled chamber, followed by exsanguination, ensuring rapid loss of consciousness and minimizing pain and distress. All efforts were made to safeguard animal welfare and use the minimum number of birds required to obtain reliable scientific data.

Study period and location

The study was conducted from January to April 2024 at the Poultry Research Center Farm, Department of Animal Science, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand.

Study design, experimental animals, and dietary treatments

A total of 750 one-day-old Ross 308 male broiler chicks were randomly allocated to five dietary treatments in a completely randomized design. Each treatment consisted of six replicates, with 25 birds per replicate pen.

Experimental diets were formulated for three feeding phases: starter (1–10 days), grower (11–24 days), and finisher (25–35 days). The dietary treatments consisted of: (T1) PC with standard ME; (T2) NC with ME reduced by 150 kcal/kg relative to the PC; (T3) NC supplemented with 250 g/ton emulsifier; (T4) NC supplemented with 200 g/ton emulsifier; and (T5) NC supplemented with 250 g/ton emulsifier during the starter and grower phases followed by 200 g/ton during the finisher phase. The ingredients and chemical composition of the experimental diets are presented in Table 1.

The PC diets were formulated to meet standard ME requirements for broilers, whereas the NC diets were formulated with a reduction of 150 kcal/kg ME. The calculated ME values for the PC and NC diets were 2,975 and 2,825 kcal/kg during the starter phase, 3,050 and 2,900 kcal/kg during the grower phase, and 3,100 and 2,950 kcal/kg during the finisher phase, respectively. The reduction in dietary energy was primarily achieved by decreasing the inclusion of energy-dense ingredients, such as corn, and increasing the inclusion of rice bran, thereby resulting in higher dietary fiber concentrations. The inclusion level of added fat remained relatively constant among treatments. Crude protein (CP) and essential amino acid concentrations (lysine, methionine, threonine, and valine) were maintained at similar levels across the PC and NC diets to minimize confounding effects of nutrient imbalance.

The nutrient values presented in Table 1 were calculated values. Analyzed nutrient composition and fatty acid profiles of the dietary fat source were not determined in the present study. The increased fiber concentration and reduced energy density of the NC diets were expected to negatively affect lipid digestion efficiency and therefore provided an appropriate nutritional model for evaluating the efficacy of emulsifier supplementation.

Birds were reared in floor pens within a controlled-environment poultry house. Each replicate pen contained 25 birds and provided 1.8 m² of floor space, corresponding to a stocking density of 13.89 birds/m². The poultry house was equipped with an evaporative cooling system, programmable artificial lighting, automated electric heating, and tunnel ventilation. The brooding temperature was initially maintained at 34°C and gradually reduced to 28°C during the first 3 weeks of the experiment. The lighting schedule consisted of 18 h light and 6 h darkness per 24-h cycle throughout days 10–35 of the experimental period. Feed and water were provided ad libitum, and birds were vaccinated in accordance with standard commercial management practices.

Items | S-PC | S-NC | G-PC | G-NC | F-PC | F-NC
Ingredients |  |  |  |  |  | 
Corn | 52.76 | 42.95 | 58.17 | 48.36 | 62.54 | 52.74
Palm oil | 2.50 | 2.50 | 2.50 | 2.50 | 2.50 | 2.50
Rice solvent bran | 5.92 | 17.92 | 4.86 | 16.87 | 6.32 | 18.33
Soybean meal (48% CP) | 34.93 | 32.76 | 31.55 | 29.38 | 26.11 | 23.94
L-Lysine | 0.28 | 0.30 | 0.21 | 0.23 | 0.25 | 0.27
DL-Methionine | 0.38 | 0.38 | 0.33 | 0.33 | 0.31 | 0.31
L-Threonine | 0.11 | 0.12 | 0.06 | 0.08 | 0.06 | 0.08
L-Valine | 0.07 | 0.07 | 0.04 | 0.04 | 0.06 | 0.05
Monodicalcium phosphate | 1.11 | 1.02 | 0.67 | 0.57 | 0.33 | 0.24
Calcium carbonate | 1.18 | 1.21 | 0.86 | 0.90 | 0.78 | 0.81
Salt | 0.30 | 0.30 | 0.30 | 0.30 | 0.30 | 0.30
Phytase + NSP enzyme (100 g/t; −70 kcal/kg) | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01
Choline chloride (60%) | 0.28 | 0.28 | 0.27 | 0.27 | 0.25 | 0.25
Premix | 0.18 | 0.18 | 0.18 | 0.18 | 0.18 | 0.18
Total | 100.00 | 100.00 | 100.00 | 100.00 | 100.00 | 100.00
Chemical composition |  |  |  |  |  | 
ME for poultry (kcal/kg)* | 2,975 | 2,825 | 3,050 | 2,900 | 3,100 | 2,950
Protein (%) | 23.00 | 23.00 | 21.50 | 21.50 | 19.50 | 19.50
Fat (%) | 4.76 | 4.50 | 4.91 | 4.65 | 5.03 | 4.77
Fiber (%) | 4.12 | 5.19 | 3.93 | 5.00 | 3.89 | 4.95
Calcium (%) | 0.95 | 0.95 | 0.75 | 0.75 | 0.65 | 0.65
Total phosphorus (%) | 0.88 | 1.00 | 0.76 | 0.88 | 0.69 | 0.81
Available phosphorus (%) | 0.50 | 0.50 | 0.42 | 0.42 | 0.36 | 0.36
Salt (%) | 0.34 | 0.34 | 0.33 | 0.33 | 0.32 | 0.32
Lysine (%) | 1.43 | 1.44 | 1.28 | 1.29 | 1.17 | 1.18
Methionine + cystine (%) | 1.08 | 1.08 | 0.99 | 0.99 | 0.93 | 0.93
Methionine (%) | 0.70 | 0.71 | 0.64 | 0.64 | 0.60 | 0.61
Threonine (%) | 0.98 | 0.98 | 0.88 | 0.88 | 0.80 | 0.80
Tryptophan (%) | 0.28 | 0.27 | 0.26 | 0.25 | 0.23 | 0.22
Valine (%) | 1.14 | 1.15 | 1.04 | 1.05 | 0.96 | 0.97
Choline (mg/kg) | 1,700 | 1,700 | 1,600 | 1,600 | 1,500 | 1,500
Lysine digestibility (%) | 1.32 | 1.32 | 1.18 | 1.18 | 1.08 | 1.08
Methionine digestibility (%) | 0.68 | 0.68 | 0.62 | 0.62 | 0.58 | 0.58
Methionine + cystine digestibility (%) | 1.00 | 1.00 | 0.92 | 0.92 | 0.86 | 0.86
Threonine digestibility (%) | 0.88 | 0.88 | 0.79 | 0.79 | 0.72 | 0.72
Tryptophan digestibility (%) | 0.26 | 0.26 | 0.24 | 0.24 | 0.21 | 0.21
Valine digestibility (%) | 1.00 | 1.00 | 0.91 | 0.91 | 0.84 | 0.84

Emulsifier product and specification

The emulsifier evaluated in this study was a commercial soy lysolecithin-based product, BergaFit 60 SO Lyso GMO (Berg+Schmidt Asia Pte. Ltd., Singapore, Singapore), formulated for use in feeds for poultry, swine, ruminants, aquaculture species, and companion animals. According to the manufacturer's specifications, the product contains soy lysolecithin on a carrier with acetone-insoluble matter ≥34%, ash ≤40%, and an acid value ≤42 mg KOH/g.

The emulsifier consisted of a blend of sorbitan esters, lysolecithin, and de-oiled lecithin and was incorporated into experimental diets at 250 g/ton, 200 g/ton, or under a phase-specific step-down supplementation strategy as described previously.

Productive performance and carcass yield

Individual body weight (BW) was recorded on day 1 and at the end of each feeding phase (days 10, 24, and 35). Feed consumption was recorded on a pen basis at the end of each respective growth phase.

Productive performance variables, including BW, average daily gain (ADG), FI, FCR, corrected FCR (cFCR), feed cost per gain (FCG), and mortality, were evaluated during four periods: starter phase (1–10 days), grower phase (11–24 days), finisher phase (25–35 days), and the overall experimental period (1–35 days) [18, 19]. The cFCR was calculated by adjusting FCR for mortality.

At 35 days of age, two birds per replicate were randomly selected for carcass evaluation. Selection was restricted to birds with BW values close to the treatment mean. Live BW was recorded before slaughter. Birds were humanely euthanized by carbon dioxide (CO₂) inhalation in a controlled chamber followed by exsanguination. Carcasses were defeathered and eviscerated, and internal organs were removed to obtain eviscerated carcass weight. Abdominal fat was excised and weighed separately.

The eviscerated carcasses were subsequently dissected into commercial cuts, including wings, inner breast (Pectoralis minor), outer breast (Pectoralis major), thighs, and drumsticks. Individual carcass components were weighed separately. Carcass yield was calculated as the percentage of eviscerated carcass weight relative to live BW. The relative weights of wings, inner breast, outer breast, thighs, drumsticks, and abdominal fat were expressed as percentages of eviscerated carcass weight [20].

Feed digestibility, AME, and fecal score evaluation

On day 30, two birds per replicate (12 birds per treatment) were selected for digestibility assessment. Birds were transferred to metabolic cages (two birds per cage) and fed experimental diets containing 0.5% chromium oxide (Cr₂O₃) and 1% Celite as external markers.

The interval between marker administration and the appearance of green-colored feces was recorded as gastrointestinal retention time. Excreta were partially collected over a 6-h period for subsequent analyses [21]. Fecal consistency was visually scored using a 4-point scale, where 0 = normal, 1 = slightly wet, 2 = wet, and 3 = very wet [22].

A 100-g subsample of fresh excreta was weighed immediately and dried to determine dry matter content. Dried excreta samples were ground through a 1-mm screen, whereas feed samples were ground through a 0.5-mm screen before laboratory analyses.

Feed and excreta samples were analyzed for OM using standard proximate analytical procedures based on moisture and ash determination. AME corrected for nitrogen was determined by analyzing GE using an adiabatic oxygen bomb calorimeter and nitrogen concentration using the Kjeldahl method [23].

AME was calculated using the following equation:

AME = GEdiet − (GEexcreta × (Markerdiet/Markerexcreta))

where GE represents gross energy. Marker concentrations in the diet and excreta were used to quantify digesta flow and nutrient retention, with Cr₂O₃ as an indigestible marker.

Statistical analysis

Data were analyzed using a one-way analysis of variance using SAS software [24]. When significant treatment effects were detected, means were separated using Duncan's multiple range test. Statistical significance was declared at p < 0.05. Results are presented as mean ± standard deviation.

The experimental unit was the replicate pen. The sample size (n = 6 replicates per treatment) was considered sufficient to detect biologically meaningful differences in broiler growth performance based on previous studies conducted under similar experimental conditions.