Energy values of different lipid sources with or without exogenous lipase for broiler chickens

Valores energéticos de diferentes fontes lipídicas com ou sem lipase exógena para frangos de corte

ABSTRACT

Lipid sources in broiler diets vary in digestion and absorption and may require exogenous lipase. This study evaluated different lipid sources, with or without lipase supplementation on dietary metabolizable energy, lipid digestibility, and blood biochemical parameters in broiler chickens. A total of 224 male broiler chickens, aged 14 days, were distributed in a completely randomized design in a 2 × 4 factorial arrangement, with two lipase levels (0 and 0.10 g/kg) and four lipid sources (degummed soybean oil, palm oil, hydrogenated soybean oil, and hydrogenated palm oil) added to a basal diet, totaling eight treatments with seven replicates of four birds per cage. Total excreta were collected between 19 and 23 days of age to determine the apparent metabolizable energy (AME), nitrogen-corrected metabolizable energy (AMEn), apparent coefficients of metabolization (ACM), ACM corrected for nitrogen balance (ACMn) and blood biochemical parameters. Lipase supplementation reduced AME, AMEn, ACM, and ACMn values in diets with hydrogenated soybean oil and hydrogenated palm oil. Soybean oil showed higher metabolization coefficients, regardless of lipase. Lipid source altered cholesterol, alanine aminotransferase, and aspartate aminotransferase, with higher values in diets with soybean oil and lower with hydrogenated palm oil. It is concluded that lipase supplementation does not improve lipid digestibility, energy utilization, or blood biochemical parameters in broiler chickens.

Index terms:
Blood parameters; hydrogenation; poultry

RESUMO

Fontes lipídicas em dietas de frangos de corte variam na digestão e absorção, podendo requerer lipase exógena. Este estudo avaliou diferentes fontes lipídicas, com ou sem suplementação de lipase, sobre a energia metabolizável da dieta, a digestibilidade de lipídios e os parâmetros bioquímicos sanguíneos em frangos de corte. Um total de 224 frangos de corte machos, com 14 dias de idade, foi distribuído em delineamento inteiramente casualizado em arranjo fatorial 2 × 4, com dois níveis de lipase (0 e 0,10 g/kg) e quatro fontes lipídicas (óleo de soja degomado, óleo de palma, óleo de soja hidrogenado e óleo de palma hidrogenado) adicionadas a uma dieta basal, totalizando oito tratamentos com sete repetições de quatro aves por gaiola. A excreta total foi coletada entre 19 e 23 dias de idade para determinação da energia metabolizável aparente (EMA), energia metabolizável aparente corrigida pelo nitrogênio (EMAn), coeficientes de metabolização aparente (CMA), CMA corrigido para o balanço de nitrogênio (CMAn) e parâmetros bioquímicos sanguíneos. A suplementação com lipase reduziu os valores de AME, AMEn, ACM e ACMn nas dietas com óleo de soja hidrogenado e óleo de palma hidrogenado. O óleo de soja apresentou maiores coeficientes de metabolização, independentemente da lipase. A fonte lipídica alterou o colesterol, alanina aminotransferase e aspartato aminotransferase, com maiores valores na dieta com óleo de soja e menores com óleo de palma hidrogenado. Conclui-se que a suplementação de lipase não melhora a digestibilidade de lipídios, o aproveitamento energético ou os parâmetros bioquímicos sanguíneos em frangos de corte.

Termos de indexação:
Parâmetros sanguíneos; hidrogenação; aves

Introduction

Broiler nutrition is a constantly evolving field, particularly in terms of the use of enzymes as additives and various oil sources to enhance feed efficiency and the productive performance of birds (Liu et al., 2023, Saleh et al., 2025). Among enzymatic additives, exogenous lipases have stood out for their ability to enhance lipid digestibility, thereby improving the energy availability of diets (Geng et al., 2022; Oketch et al., 2023). Lipases are enzymes that catalyze the hydrolysis of triacylglycerols into free fatty acids and monoacylglycerols, facilitating lipid absorption in the small intestine (Fernandes et al., 2025). This mechanism is particularly important for broilers, whose natural can be limited, especially in diets with high lipid content (Shoaib et al., 2021; Fernandes et al., 2023).

Lipids are crucial energy sources for broilers, and the choice of fat source can significantly impact the energy value of the diets (Rodriguez-Sanchez et al., 2019; Movagharnejad et al., 2020). Among the common lipid sources in poultry feed, notable lipid sources include degummed soybean oil, palm oil, hydrogenated soybean oil, and hydrogenated palm oil (Jimenez-Moya et al., 2021; Tenório et al., 2022). Each of these sources has distinct physicochemical characteristics that can affect their digestibility and utilization by broilers (Saleh et al., 2021; Shoaib et al., 2023).

Soybean oil, extracted from soybeans (Glycine max) using organic solvents, is widely used due to its high content of unsaturated fatty acids, making it a highly digestible energy source (Faryadi et al., 2023; Calik et al., 2024). In contrast, palm oil, extracted from the fruits of the oil palm (Elaeis guineensis) through pressing the fruit pulp, is rich in saturated fatty acids and has intermediate digestibility (Chowdhury et al., 2018; González Sepúlveda et al., 2024; Tchablémane et al., 2024).

Additionally, the hydrogenation processes applied to soybean oil and palm oil result in products with higher saturated fatty acid content and, consequently, different physical and nutritional properties (Vienola et al., 2018; Cetıngul et al., 2022). Hydrogenation is a process in which unsaturated fatty acids are converted into saturated fatty acids through the addition of hydrogen in the presence of a catalyst (Torquato et al., 2021; Parmar et al., 2022) This process is performed to increase the oxidative stability of oils and alter their melting point, resulting in fat that is more solid at room temperature (Sizova & Ryazantseva, 2022). However, hydrogenation can also negatively impact the digestibility and efficiency of lipid utilization in birds (Ravindran et al., 2016). Therefore, the inclusion of hydrogenated oils in broiler diets requires detailed investigation to understand their nutritional implications (Kyselka et al., 2018; Thirumdas et al., 2023).

Given the potential impact of hydrogenation on lipid digestibility, supplementation with exogenous lipases may offer a promising solution (Rastello et al., 2025). Exogenous lipases have the ability to offset the disadvantages associated with hydrogenation by improving lipid digestibility and, consequently, the energy availability of diets (Ahmad et al., 2023). Studies have shown that lipase supplementation can improve feed efficiency and broiler performance (Hu et al., 2018; Arshad et al., 2021; Castro & Kim, 2021), but the interaction between lipases and hydrogenated oil sources has not been fully elucidated (Wang et al., 2024).

In addition to productive performance, it is essential to evaluate the effects of these nutritional strategies on the metabolic health of broilers (Fernandes et al., 2025). Blood biochemical parameters such as cholesterol, triglycerides, alanine aminotransferase, and aspartate aminotransferase are widely used to monitor lipid digestibility and liver integrity (Ai et al., 2025; Zálešáková et al., 2025). Cholesterol and triglyceride concentrations directly reflect the absorption and processing of dietary lipids, while alanine aminotransferase and aspartate aminotransferase are hepatic enzymes that are sensitive to changes in liver function, potentially indicating metabolic overload or adverse effects associated with the type of oil or the use of enzymes in the diet (Hu et al., 2018; Liu et al., 2025). The analysis of these indicators complements performance evaluation, providing a comprehensive view of the physiological impacts caused by different feeding strategies (Amer et al., 2020; Shoaib et al., 2021).

Despite the existence of studies addressing either exogenous lipase supplementation or different lipid sources individually, there is limited evidence on their combined effects, particularly when hydrogenated oils are involved and both energy metabolism and biochemical health indicators are considered (Rodriguez-Sanchez et al., 2021; Ahmad et al., 2023; Arshad et al., 2021). This study stands out for simultaneously assessing the interaction between lipase supplementation and hydrogenated or non-hydrogenated oils in young broilers, using precise measurements of metabolizable energy, digestibility coefficients, and blood biochemical changes. This integrative approach provides new insights into whether enzymatic supplementation can counteract the digestive limitations imposed by saturated fats, offering practical guidance for optimizing poultry diets.

Considering the reduced digestibility of hydrogenated fats and the potential of exogenous lipase to enhance lipid utilization, this study aimed to evaluate the apparent metabolizable energy (AME), apparent metabolizable energy corrected for nitrogen balance (AMEn), apparent coefficients of metabolization (ACM), ACM corrected for nitrogen balance (ACMn), and blood biochemical parameters of broilers fed diets containing with or without lipase supplementation. The hypothesis is that exogenous lipase improves lipid digestibility, energy utilization, and biochemical responses in broilers fed different lipid sources.

Material and Methods

Ethics committee

The procedures were approved by the Animal Use Ethics Committee of the National Council for Animal Control and Experimentation - UNIOESTE (Protocol No. 001/2024) and were previously sanctioned by the National Council for Animal Control and Experimentation in accordance with Normative No. 37 of February 15, 2018.

Broiler chickens

A total of 300 one-day-old male broiler chickens (Cobb 500), with an initial average weight of 47.8 g, were obtained from a commercial hatchery. The birds were raised in an experimental poultry house with a concrete floor covered with fresh pinewood shavings. The environment was equipped with semi-automatic feeders and nipple drinkers. During the first 13 days of age, broilers were fed a commercial corn- and soybean meal-based diet. Feed and water were provided ad libitum throughout the experimental period. The ambient temperature was maintained within the thermal comfort range recommended for the Cobb 500 strain, starting at 32 °C on day 1, 31 °C on day 2, 30 °C on day 3, 29 °C on day 4, 28 °C on day 5, 27 °C on days 6-7, 26 °C on days 8-10, and 25-26 °C on days 11-13. The ambient temperature was maintained within the thermal comfort range recommended for the strain, and a continuous 24-hour light was implemented.

At 14 days of age, 224 broilers with an average weight of 416 g were transferred to metabolic cages (50 cm² each) and allocated in a completely randomized design in a 2 × 4 factorial arrangement. The factors included two exogenous lipase levels (0 and 0.10 g/kg) and four lipid sources (degummed soybean oil, palm oil, hydrogenated soybean oil, and hydrogenated palm oil) added to a basal diet. This arrangement resulted in eight treatments, each with seven replicates of four birds per cage, for a duration of 10 days. The cages were equipped with external feeders and internal automatic drinkers. Throughout the experimental period, ambient temperature was maintained within the birds’ thermal comfort zone, with feed and water provided ad libitum, and a continuous 24-hour photoperiod implemented to stimulate feed intake.

Diets and treatments

All oils used in the study were sourced from the national market (Brazil) and are approved for use in both animal and human nutrition. The basal diet was formulated to meet the recommendations of Rostagno et al. (2017) for the initial phase, without the inclusion of oils (Table 1). The experimental diets were prepared using the substitution method proposed by Matterson, Potter and Stutz (1965). The treatments were prepared by blending the basal diet with the oil at a 9:1 weight-to-weight ratio, followed by thorough homogenization, as the oil was added after diet formulation to ensure uniform incorporation, in accordance with the substitution method described by Sakomura and Rostagno (2016).

Table 1:
Centesimal composition of the basal diet, without and with the addition of lipase.

Ingredients (g/kg) as fed	Basal diet
Corn grain	620.00
Soybean meal	340.00
Dicalcium phosphate	14.90
Limestone	11.10
Salt	4.50
Lysine sulfate	2.58
DL-Methionine	2.56
L-Threonine	0.36
Choline chloride	0.62
Salinomycin	0.55
Antioxidant	0.30
Avilamycin (10%)1	0.10
Premix Vitamin-Mineral2	2.00
Inert (kaolin)3	0.43
Calculated nutrient composition (g/kg) as fed
Metabolizable energy (kcal/kg)	2885
Crude protein	205.00
Ether extract	28.38
Total calcium	8.09
Total phosphorus	6.21
Available phosphorus	4.00
Sodium	1.98
Digestible Lysine	11.32
Digestible Methionine + Cysteine	8.15
Digestible Tryptophan	2.28
Digestible Threonine	7.36

• ¹The antibiotic used was avilamycin (10%), a commercial product containing 10 g of active ingredient per 100 g of product. ²Vitamin-Mineral Premix for broiler chickens, provided per kilogram of feed: Vitamin A (min.) 13,500.00 IU, vitamin D₃ (min.) 3,750.00 IU, vitamin E (min) 30.00 IU, vitamin K₃ (min) 3.75 mg, vitamin B₁ (min) 2.25 mg, vitamin B₂ (min) 9.00 mg, vitamin B₆ (min.) 4.50 mg, vitamin B₁₂ (min.) 18.00 mg. Pantothenic acid (min) 18.00 mg, niacin (min) 37.50 mg, folic acid (min) 1.20 mg, biotin (min) 0.09 mg, selenium (min) 0.38 mg, copper (min) 10.00 mg, iron (min) 50.00 mg, manganese (min) 80.00 mg, cobalt (min) 1.00 mg, iodine (min) 1.00 mg, zinc (min) 50.00 mg. Lipase was added to the basal diet at a level of 0.10 g/kg, using lipase preparation with an activity of 10,000 U/g. ³Inert (kaolin) The inert consisted of kaolin with the inclusion of lipase as a weight-for-weight substitution.

The treatments containing lipase were supplemented with 1000 U/kg of enzyme, replacing an inert ingredient in the basal diet to maintain the nutritional composition unchanged. The treatments containing lipase were supplemented with 1000 U/kg of enzyme, replacing an inert ingredient in the basal diet to maintain the nutritional composition unchanged. The inert consisted of kaolin with the inclusion of lipase as a weight-for-weight substitution. The enzyme used was a triacylglycerol hydrolase (Lipact®) with an activity of 10000 U/g of lipase, with each lipase unit (U/g) defined as the amount required to release 1 μmol of titratable aliphatic acid per minute.

To determine the gross energy values of the tested ingredients, combustion calorimetry was used as described by Silva and Queiroz (2006). In this method, the sample was placed in a sealed container known as a bomb calorimeter, where it was burned in the presence of oxygen. The heat release was measured and used to calculate the gross energy of the ingredient. Based on this analysis, the following gross energy values were obtained for the tested ingredients: degummed soybean oil (9621 kcal/kg), palm oil (9697 kcal/kg), hydrogenated soybean oil (8867 kcal/kg), and hydrogenated palm oil (7916 kcal/kg).

Soybean oil, obtained from Glycine max seeds, was extracted through an industrial process using organic solvents, most likely hexane, following standard practices in the vegetable oil industry. Palm oil was extracted from the pulp of Elaeis guineensis fruits by mechanical pressing, followed by separation of the crude oil via decantation or filtration, in accordance with conventional industrial methods, without additional details on processing conditions.

For hydrogenation, both oils underwent a full hydrogenation process using hydrogen gas in the presence of a nickel catalyst, under conditions of 180 °C and 3 atm pressure, until complete saturation of fatty acids, according to industrial protocols described in the literature (Gunstone, 2011). No details were provided on catalyst concentration, reaction time, or reactor type. When applicable, complementary analyses, such as gas chromatography for fatty acid profile determination, were performed to characterize the oils, with experiments conducted in triplicate to ensure reproducibility.

Excreta collection and analysis

To avoid variations associated with partial excreta collection, which may lead to underestimation or overestimation of the obtained values, this study adopted the total excreta collection method, as also employed in previous studies (Khalil et al., 2021; Ellawidana et al., 2023; Liu et al., 2023). With this approach, it was possible to achieve greater accuracy in the quantification of apparent metabolizable energy (AME), apparent metabolizable energy corrected for nitrogen balance (AMEn), as well as apparent coefficients of metabolization (ACM) and ACM corrected for nitrogen balance (ACMn) (Lopes et al., 2024 ).

The broiler chickens at 14 days of age underwent a five-day adaptation period (14 to 18 days of age) in the metabolic cages, followed by total excreta collection for another five days (19 to 23 days of age), according to the methodology described by Sakomura and Rostagno (2016). The feed was weighed at the beginning of the experiment, and the leftovers were weighed at the end of the collection period to determine feed intake (Avila et al., 2006).

Excreta were collected from each experimental unit twice a day (at 9:00 AM and 6:00 PM) to prevent fermentation. After removing feathers, feed residues, and other sources of contamination, the excreta were transferred to labeled plastic bags, weighed, and stored in a freezer until the end of the collection period. Subsequently, the samples were thawed, pooled by replicate, and homogenized. Then, aliquots of 400 to 500 g were taken and placed in ventilated ovens at 55°C for 48 hours for drying and subsequent analysis.

After pre-drying the samples of excreta and basal diet (BD), and the test ingredients were ground and analyzed for dry matter (DM), gross energy (GE), and nitrogen (N). The ACM and ACMn values were determined according to the methodology of (Matterson, Potter & Stutz, 1965), using the following equations:

ACM (%) = (Gross Energy_consumed - Gross Energy_excreted) / Gross Energy_consumed × 100

ACMn (%) = (Gross Energy_consumed - Gross Energy_excreted - (8.22 × Nitrogen Balance)) / Gross Energy_consumed × 100

Endogenous excreted nitrogen was estimated using a constant value according to Lesson and Summers (2001), with the Kjeldahl method referenced for nitrogen determination as described by Silva and Queiroz (2006). A mean endogenous nitrogen excretion value of 250 mg N/kg body weight^0.75 per day was applied in the nitrogen balance equation:

NB = N_consumed - (N_excreted - N_exc.End​)

in which NB = nitrogen balance; N_consumed = nitrogen consumed; N_excreted = nitrogen excreted; Nexc.End = endogenous nitrogen excretion. This equation was used to calculate nitrogen-corrected apparent metabolizable energy (AMEn), and ACM corrected for nitrogen balance (ACMn).

The AME and AMEn values were determined according to the methodology of Lesson and Summers (2001), using the following equations:

AME_TD = GE_consumed - GE_excreted / DM_consumed

AME_BD = GE _consumed - GE_excreted / DM_consumed

AME_FEEDSTUFF = ME_BD + [(AME_TD - AME_BD) / g/g replacement]

AME_nTD = [(GE_consumed - GE_excreted) - 8.22 * NB] / DM_consumed

AME_nBD= [(GE_consumed - GE_excreted) - 8.22 * NB] / DM_consumed

AME_nFEEDSTUFF = [AME_nBD + (AME_nTD - AME_nBD)] / g/g replacement

in which AME_TD = apparent metabolizable energy of the test diet; AME_BD = apparent metabolizable energy of the basal diet; AME_FEEDSTUFF = apparent metabolizable energy of the feedstuff; AME_nTD = nitrogen corrected apparent metabolizable energy of the test diet; AME_nBD = nitrogen corrected apparent metabolizable energy of the reference diet; AME_nFEEDSTUFF = nitrogen corrected apparent metabolizable energy of the feedstuff; GE_consumed = gross energy consumed; GE_excreted = gross energy excreted; NB = nitrogen balance; DM_consumed = dry matter consumed; ME_BD = metabolizable energy of the basal diet; ME_TD = metabolizable energy of the test diet.

At 23 days of age, after the birds were fasted for six hours, blood samples were collected via ulnar vein puncture and stored without anticoagulants. The samples were centrifuged at 1050 × g for ten minutes to obtain serum, which was stored at -20 °C. Blood serum cholesterol (CHO), triglycerides (TG), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) analysis were performed using a high-performance automatic spectrophotometer (Flexor EL 200, Elitech, Paris, France) with specific kits, calibrated with standards (Elical, Elitech).

To evaluate the apparent metabolizable energy coefficients (AME) and nitrogen-corrected apparent metabolizable energy coefficients (AMEn), the gross energy (GE) values of the analyzed lipid sources were used: degummed soybean oil, palm oil, hydrogenated soybean oil, and hydrogenated palm oil.

Fatty acid profile analysis

At the beginning of the digestibility trial, a sample of the degummed soybean oil used in the experimental diets was collected and analyzed by C.B.O. Análises Ltda. (Valinhos, SP, Brazil) to determine fatty acid composition according to AOAC method 996.06 and iodine value in accordance with AOCS method Cd 1-25. Due to experimental limitations, palm oil, hydrogenated soybean oil, and hydrogenated palm oil were not subjected to laboratory analyses of fatty acid profile and iodine value. For these lipid sources, the contents of saturated, monounsaturated, and polyunsaturated fatty acids (SFA, MUFA, and PUFA), as well as general physicochemical characteristics, were obtained from the literature and used to support the interpretation of lipid digestibility.

Statistical analyses

The data were subjected to analysis of variance (ANOVA) using a completely randomized design in a factorial arrangement, considering the different lipid sources (soybean oil, palm oil, hydrogenated soybean oil, and hydrogenated palm oil) and the inclusion of lipase (0.0 or 0.10 g/kg) as main factors. The normality of residuals was assessed using the Shapiro-Wilk test, and homogeneity of variances was evaluated using Levene’s test. When significant differences were detected (P < 0.05), comparisons between treatments were performed using Tukey’s test at a 5% significance level. Results were expressed as means with their respective standard error of the mean (SEM).

For the analyzed variables, the statistical model used was Y_ijk = m + F_i + E_j + FE_ij + ε_ijk, in which Y_ijk = average observation of the dependent variable in each plot, measured in the i-th lipid source, in the j-th lipase enzyme class and in the k-th replication; m = effect of the overall average; F_i = effect of class of lipid source, for i = (1, 2, 3 and 4); E_j = effect of lipase enzyme class, for j = (1 and 2); FE_ij = effect of interaction between the i-th class of lipid source and the j-th lipase enzyme class, and ε_ijk = random error of the plot associated with level i, class j, and replication k.

Results and Discussion

The reduction in AME, AMEn, ACM, and ACMn (Table 2) following lipase supplementation indicates that, under the conditions of this assay, the efficiency of lipid digestion was not limited by the triacylglycerol hydrolysis step, but rather by the subsequent processes of micellar solubilization and intestinal absorption (Geng et al., 2022; Oketch et al., 2023; Chan et al., 2025). Exogenous lipase anticipates the release of free fatty acids in the proximal lumen, which may exceed the immediate capacity for complexation by bile salts, particularly in lipid matrices with a high degree of saturation (Rodriguez-Sanchez et al., 2019; Movagharnejad et al., 2020; Shoaib et al., 2023). This imbalance compromises the formation and stability of mixed micelles, limits the transport of digestion products to the brush border, and consequently reduces the recovery of metabolizable energy, as reflected in the lower metabolization coefficients observed (Ravindran et al., 2016; Arshad et al., 2021).

Table 2:
Apparent metabolizable energy (AME), AME corrected for nitrogen balance (AME_n), apparent coefficients of metabolization (ACM), ACM corrected for nitrogen balance (ACM_n) for different lipid sources, as well as the interaction between lipid source and lipase on ACM values.

Treatments	AME (kcal/kg)	AMEn (kcal/kg)	ACM (%)	ACMn (%)
Lipase (0.0 g/kg)	6145	5979	68.32a	66.50a
Lipase (0.10 g/kg)	5098	4983	54.42b	53.18b
Lipid source
Soybean oil	9746	9541	99.47a	97.39a
Palm oil	7559	7356	83.03b	80.80b
Hydrogenated soybean oil	1983	1899	20.00d	19.16d
Hydrogenated palm oil	3626	3538	45.43c	44.37c
SEM	1.92	1.88	4.74	4.65
P value
Lipase	-	-	0.001	0.001
Fat	-	-	0.001	0.001
Lipase vs. Lipid source	-	-	0.001	0.001
		ACM - Effect of lipase on apparent digestibility (%)
		Lipase (0.0 g/kg)	Lipase (0.10 g/kg)	P value
Lipid source	Soybean oil	98.99a	99.96a	0.294
	Palm oil	87.52bA	78.54bB	0.003
	Hydrogenated soybean oil	21.22d	18.78c	0.253
	Hydrogenated palm oil	68.30cA	18.76cB	0.001
	P value	0.0001	0.0001
		ACMn - ACM corrected for nitrogen balance (%)
		Lipase (0.0 g/kg)	Lipase (0.10 g/kg)	P value
Lipid source	Soybean oil	97.96a	96.81a	0.294
	Palm oil	84.66bA	76.94bB	0.006
	Hydrogenated soybean oil	20.29d	18.04c	0.294
	Hydrogenated palm oil	66.82cA	18.17cB	0.001
	P value	0.001	0.001

• SEM: standard error of means. ^{a, b, c}: within each lipase level (column), different lowercase letters indicate differences among lipid sources (P < 0.05). ^{A, B, C}: within each lipid source (row), different uppercase letters indicate differences between lipase levels (0.0 vs 0.10 g/kg) (P < 0.05).

The reduction in AME and AMEn may be attributed, in part, to the increased formation of insoluble salts in the gastrointestinal tract (Khalil et al., 2021; Ahmad et al., 2023). The calcium concentration in the basal diet (8.09 g/kg) associated with available phosphorus (4.00 g/kg) creates an environment conducive to the complexation of free saturated fatty acids with divalent cations, particularly in young birds with limited bile salt availability (Ravindran & Abdollahi, 2021; Vertiprakhov, Grozina, & Fisinin, 2024). Under these conditions, the premature release of free saturated fatty acids by exogenous lipase promotes the formation of calcium soaps and reduces the fraction of lipids available for micellar incorporation and energy recovery, which translates into lower metabolizable energy values (Geng et al., 2022; Oketch et al., 2023; Palomar et al., 2023).

The effect of lipid source on ACM and ACMn, regardless of lipase supplementation, is consistent with differences in fatty acid profile and degree of unsaturation described in Table 3. Soybean oil, with a high PUFA fraction (54.27%) and a high iodine value (125.40 g I₂/100 g), exhibits greater interfacial fluidity and a lower melting point, favoring emulsification and the formation of thermodynamically more stable mixed micelles (Elmore, Kerr, & Bobeck, 2023; Oketch et al., 2023; Ma et al., 2024). In addition, palm oil and, particularly, hydrogenated sources, with a higher proportion of SFA (>44%) and a lower iodine value (<46 g I₂/100 g), exhibit greater molecular ordering and lower micellar compatibility, which translates into the lower metabolization coefficients observed at both lipase levels (Kyselka et al., 2018; Song et al., 2022; Palomar et al., 2023).

Table 3:
Fatty acid profile and iodine value of lipid sources.

Lipid source	SFA (%)	MUFA (%)	PUFA (%)	Major fatty acids (approximate composition)	Iodine value (g I2/100 g)	Reference
Soybean oil	18.41	24.70	54.27	C16:0 (palmitic): 7-10% C18:0 (stearic): 2-5% C18:1 (oleic): 22-30% C18:2 (linoleic): 50-60% C18:3 (linolenic): 5-9%	125.40	-
Palm oil	46.60	43.60	9.80	C16:0 (palmitic): 38.8% C18:1 (oleic): 35.8% C18:2 (linoleic):14.8% C18:0 (stearic): 4.3% C14:0 (myristic): 1.1%	49.40	(Montoya et al., 2014)
Hydrogenated soybean oil	52.30	46.5	0.40	C16:0 (palmitic): 10.5% C18:0 (stearic): 41.8% C18:1 cis (oleic): 14.4% C18:1 trans (elaidic): 32.1% C18:2 (linoleic): 0.2% C18:3 (linolenic): 0.2%	40.70	(List et al., 2007)
Hydrogenated palm oil	51.06	46.18	2.67	C16:0 (palmitic): 41-43% C18:0 (stearic): 7%) C18:1 (oleic/elaidic), cis + trans : 46% C18:2 (linoleic/linolelaidic), cis + trans : 3%	46.00	(Musavi, Tekin, & Erinç, 2011)

• SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. Fatty acids are expressed as percentage of total fatty acids. The fatty acid profile of soybean oil was determined at the beginning of the experiment by an accredited external laboratory (C.B.O. Laboratory Analyses Ltda., Valinhos, SP, Brazil). Palm oil values were sourced from Montoya et al. (2014), hydrogenated soybean oil from List et al. (2007), and hydrogenated palm oil from Musavi, Tekin and Erinç (2011).

In the unfolding of the interaction for ACM and ACMn, the lower values observed for hydrogenated soybean oil and hydrogenated palm oil may be associated with structural alterations resulting from the hydrogenation process, including an increased fraction of saturated fatty acids and greater crystalline organization of triacylglycerols, which may compromise the efficiency of emulsification, incorporation into mixed micelles, and, consequently, intestinal lipid absorption (Rodriguez-Sanchez et al., 2021; Shoaib et al., 2021; Sizova & Ryazantseva, 2022). However, as the specific parameters of the hydrogenation process were not characterized in this study, this interpretation should be considered a hypothesis based on literature rather than direct experimental evidence.

In this context of the structural alterations associated with lipid hydrogenation, the reduction in the degree of unsaturation increases molecular packing and the phase transition temperature of the lipid matrix, making lipids less compatible with incorporation into mixed micelles (Movagharnejad et al., 2020; Song et al., 2022; Ahmad et al., 2023). In practical terms, this means that the lipid phase becomes more rigid and less flexible (Sizova & Ryazantseva, 2022; Wang et al., 2024). This greater structural rigidity may reduce the interaction of digestion products with fatty acid transport systems in the apical membrane of enterocytes, such as CD36 (fatty acid translocase) and FATP4 (fatty acid transport protein 4), thereby limiting intestinal uptake and, consequently, energy utilization and metabolization coefficients (Ahmadpour & Zarrin, 2024; Hu et al., 2024).

In this same unfolding of the interaction for ACM and ACMn, soybean oil exhibited high digestibility coefficients, regardless of lipase supplementation. This result is consistent with its high proportion of unsaturated fatty acids, particularly linoleic acid (C18:2), which favors emulsification and incorporation into mixed micelles (Rodriguez-Sanchez et al., 2019; Saleh et al., 2021). From a physiological perspective, these findings indicate that the addition of lipase tends not to promote additional gains when the lipid fraction of the diet is readily emulsifiable and efficiently absorbed (Ahmad et al., 2023; Fernandes et al., 2023).

Furthermore, when comparing lipase at 0.0 and 0.10 g/kg within palm oil and hydrogenated palm oil, the reduction in ACM and ACMn observed with lipase at 0.10 g/kg suggests that the increased release of fatty acids did not translate into greater energy utilization in these lipid sources (Oliveira et al., 2019; Saleh et al., 2025). A plausible interpretation is that the intensification of hydrolysis exposed a functional limit of subsequent digestive steps, such that part of the released products was not effectively incorporated into the absorption pathways (Tenório et al., 20202; Saleh et al., 2025). As a result, the fraction of recovered energy decreased, which was reflected in the lower metabolization coefficients (Rodriguez-Sanchez et al., 2019; Chan et al., 2025).

In this context, the results of this study indicate that supplementation with lipase, under the conditions evaluated, was not a determining factor for improving the energy utilization of dietary lipids. In more unsaturated sources, such as soybean oil, lipids are already easily emulsified and absorbed, which limits additional gains from enzymatic addition (Rodriguez-Sanchez et al., 2019; Oketch et al., 2023; Kerr et al., 2024). In more saturated or hydrogenated sources, the increase in fatty acid release promoted by lipase did not translate into greater absorption, since subsequent steps of digestion, such as micelle formation and interaction with minerals in the intestinal lumen, remained the main limiting factors (Ravindran & Abdollahi, 2021; Ravindran et al., 2016; Chan et al., 2025). Thus, the use of lipase should be interpreted as dependent on the characteristics of the lipid source and the intestinal environment, and not solely on the capacity for enzymatic hydrolysis.

There was no interaction effect between lipase supplementation and lipid sources on the evaluated blood biochemical parameters (P > 0.05; Table 4). However, a significant effect of lipid source was observed for cholesterol (P = 0.001), alanine aminotransferase (P = 0.001), and aspartate aminotransferase (P = 0.001).

Table 4:
Cholesterol (CHO), triglycerides (TG), alanine aminotransferase, and aspartate aminotransferase (AST) values in broiler blood fed different lipid sources with or without lipases.

Treatments	CHO (mg/dL)	TG (mg/dL)	ALT (UI/L)	AST (UI/L)
Lipase (0.0 g/kg)	141.26	28.34	11.91	168.23
Lipase (0.10 g/kg)	136.77	27.60	12.64	167.00
Lipid source
Soybean oil	148.47a	30.21	14.64a	179.79a
Palm oil	134.50b	26.14	12.07b	168.00ab
Hydrogenated soybean oil	139.81ab	27.93	13.84ab	161.79b
Hydrogenated palm oil	132.65b	27.50	8.61c	160.71b
SEM	1.63	0.70	0.47	2.04
P value
Lipase	0.149	0.672	0.154	0.812
Lipid source	0.001	0.225	0.001	0.001
Lipase vs. Lipid source	0.639	0.388	0.102	0.142

• SEM: standard error of means. ^{a, b, c}: Means followed by different lowercase letters in the columns differ (P < 0.05).

The highest CHO values were associated with diets containing soybean oil (148.47 mg/dL), whereas the lowest values were observed for palm oil (134.50 mg/dL) and hydrogenated palm oil (132.65 mg/dL). These levels remained within the reference range considered physiological for broiler chickens at the evaluated age, generally reported between 100 and 150 mg/dL (Cruz et al., 2018; Hu et al., 2018). This result is related to differences in the metabolic fate of absorbed lipids (Rodriguez-Sanchez et al., 2021; Oketch et al., 2023). More unsaturated lipid sources favor micelle formation and the intestinal absorption of fatty acids. Once absorbed, these lipids reach the liver, where they are reassembled into triacylglycerols and transported into the circulation in the form of lipid-rich lipoproteins (Cartoni et al., 2022; Izuddin et al., 2023). Thus, higher plasma CHO reflects a greater flux of lipids through systemic transport and distribution pathways, and not necessarily greater hepatic oxidation, remaining consistent with the higher coefficients of metabolization observed for soybean oil (Oketch et al., 2023; Zálešáková et al., 2025).

With respect to ALT, the highest values were observed in diets containing soybean oil (14.64 U/L), whereas the lowest values occurred with palm oil (12.07 U/L) and hydrogenated palm oil (8.61 U/L). These levels remained within the reference range considered physiological for broiler chickens at the evaluated age, generally reported below 15 to 25 U/L (Zálešáková et al., 2025). Unlike CHO, this response is more closely related to the intensity of hepatocellular metabolism than to plasma lipid transport (Ahmad et al., 2023). Sources with greater micellar compatibility tend to increase the supply of fatty acids to the liver, raising the demand for reesterification, oxidation, and integration of these substrates into energy pathways, which is reflected in higher cytosolic enzymatic activity (Hu et al., 2018; Oketch et al., 2023). In this way, the relative increase in ALT indicates a greater hepatic metabolic load associated with the processing of absorbed lipids, and not structural damage to hepatic tissue (Ahmad et al., 2023).

For AST, the highest values were associated with soybean oil (179.79 U/L), whereas the lowest values were observed for hydrogenated soybean oil (161.79 U/L) and hydrogenated palm oil (160.71 U/L). These levels remained within the reference range considered physiological for broiler chickens at the evaluated age, generally reported between 150 and 250 U/L (Zálešáková et al., 2025). Considering the broader tissue distribution of this enzyme, these results may reflect not only hepatic activity, but also greater systemic metabolic demand associated with diets showing higher efficiency of energy recovery, as evidenced by the higher values of AME, AMEn, ACM, and ACMn in the more unsaturated sources (Song et al., 2022; Ye et al., 2022).

This relationship between blood parameters and metabolization coefficients reinforces the interpretation that the differences observed in CHO, ALT, and AST are an indirect consequence of variations in the efficiency of digestion, micellar solubilization, and intestinal absorption of lipids (Song et al., 2022; Oketch et al., 2023). As demonstrated by the lower values of AME, AMEn, ACM, and ACMn in diets supplemented with lipase and in the more saturated or hydrogenated sources, the primary limitation of the process was not at the stage of triacylglycerol hydrolysis, but in the subsequent events of mixed micelle formation and transport of digestion products to the brush border of enterocytes (Ye et al., 2022; Wang et al., 2024).

In this sense, the lower values of CHO, ALT, and AST observed in the more saturated and hydrogenated sources can be interpreted as indicative of reduced direction of lipids toward systemic and hepatic metabolic pathways, reflecting a more restricted profile of energy utilization in these lipid matrices (Ravindran et al., 2016; Rodriguez-Sanchez et al., 2019; Kerr et al., 2024). Thus, the integration between blood biochemical parameters and the data on metabolizable energy and metabolization coefficients reinforces that the composition of the lipid source plays a central role in modulating the metabolic fate of absorbed lipids and the physiological response of the birds, regardless of lipase supplementation.

Conclusions

Lipase supplementation did not improve AME and AMEn values or the metabolization coefficients ACM and ACMn in broiler diets and was associated with reductions in these parameters in diets formulated with palm oil and hydrogenated palm oil. Soybean oil exhibited higher ACM and ACMn, whereas hydrogenated sources showed the lowest values, regardless of lipase inclusion. These results indicate that lipase inclusion did not enhance the efficiency of dietary energy utilization under the evaluated conditions.

Data Availability Statement

Data available upon request to authors.

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• Editor de seção:
  Renato Paiva
  0000-0001-5107-0285

Publication Dates

• Publication in this collection
  15 June 2026
• Date of issue
  2026

History

• Received
  17 Nov 2025
• Accepted
  26 Mar 2026