Effect of Inclusion of Tambaqui (Colossoma macropomum) Residual Oil in Diets for Commercial Hens on Productive Performance, Physiological Parameters and Egg Quality

Abstract

The increasing demand for sustainable and cost-effective animal feed alternatives has stimulated the use of agro-industrial by-products in poultry diets. This study evaluated the effects of tambaqui (Colossoma macropomum) residual oil (TRO), derived from aquaculture waste, on productive performance, physiological responses, and egg quality in commercial laying hens. A total of 144 Hisex Brown hens were assigned to diets containing 0%, 1.5%, 3.0%, or 4.5% TRO for 63 days. While TRO inclusion did not affect overall productivity, moderate levels (1.5% and 3.0%) improved egg weight, yolk pigmentation, and internal quality (Haugh unit). Hematological and biochemical parameters indicated metabolic adaptations, with increased cholesterol and decreased triglycerides in treated groups. The yolk fatty acid profile revealed higher omega-3 content with TRO inclusion, but lipid oxidation (TBARS) also increased, especially at higher levels. Sensory evaluation showed reduced aroma and flavor acceptability in eggs from hens fed 3.0% and 4.5% TRO. These findings suggest that moderate TRO inclusion can enhance egg nutritional value and support sustainable aquaculture waste reuse, though excessive levels may compromise product acceptability. Optimal inclusion levels should be further explored to balance metabolic benefits, oxidative stability, and consumer preferences.

1. Introduction

The increasing global demand for agricultural commodities for human consumption has intensified competition within the agricultural market, particularly in the area of animal nutrition, since many of these commodities are essential components of balanced livestock diets [1,2]. For decades, this has been one of the central issues guiding the animal production industry, as feed accounts for approximately 70% of total production costs, especially operational expenses [3]. This challenge becomes even more significant in regions with logistical difficulties, where geographic isolation leads to high costs for acquiring feedstuffs, making animal production more expensive [4,5].

In response, the agricultural sector has invested in research and the adoption of agro-industrial by-products as alternative feed resources [6]. These residues can partially or completely replace conventional feedstuffs or act as dietary additives to improve productivity, animal welfare, and product quality [6,7]. Beyond cost reduction, their use adds value to nutritionally rich residues while fostering sustainability through the reutilization of materials that would otherwise have low or no economic value [1,7]. This practice contributes directly to circular economy strategies and the development of environmentally responsible production systems [2].

Within this context, aquaculture stands out as one of the animal production sectors that generates the largest amount of residue with reuse potential, in addition to being one of the fastest-growing sectors globally [8]. Brazil occupies a leading position in this field, consolidating itself as one of the world’s top aquaculture producers [8,9,10]. Consequently, the country also generates a considerable amount of aquaculture residues [11,12]. Brazilian aquaculture is largely focused on native species, with tambaqui (Colossoma macropomum) playing a prominent role due to its high productivity, adaptability to tropical conditions, and strong consumer preference associated with the quality of its meat [9,13,14].

Technologies focused on the reutilization of aquaculture residues have been widely studied in order to harness their nutrient richness and make them viable for use in animal feeding, particularly in poultry diets aimed at enriching eggs and meat [15,16]. Research indicates that oils derived from fish processing residues can enrich poultry diets with polyunsaturated fatty acids (PUFA), notably omega-3 and omega-6. These compounds are essential for cell membrane stability, neural function, and disease prevention [17,18,19]. Additionally, they contribute to the improvement of the lipid profile of eggs and meat [18,19,20], enhancing their nutritional and functional value for human consumption, an important competitive advantage in markets that value foods with health-promoting properties [21].

Based on these premises, this study hypothesized that the inclusion of tambaqui residual oil (TRO) as an additive in commercial hens’ diets could enhance egg quality, optimize productive performance, and promote improvements in the birds’ physiological parameters [22,23,24,25]. This hypothesis is primarily supported by previous studies showing that tambaqui by-products can contain average concentrations of 21.23% omega-6 and 2.45% omega-3 fatty acids [26], as well as high levels of oleic acid (C18:1), palmitic acid (C16:0), and linoleic acid (C18:2) [27,28]. Additionally, redirecting these residues into animal feeding contributes to environmental sustainability by reducing waste and adding value to the aquaculture production chain [29,30]. Therefore, the aim of this study was to investigate the effects of increasing levels of TRO in diets for commercial hens on productive performance, physiological parameters, and the physical, chemical, and sensory quality of the eggs.

2. Materials and Methods

2.1. Processing of Tambaqui By-Products and Oil Production

TRO was obtained at the Animal Nutrition Laboratory of the Institute of Social Sciences, Education, and Animal Science at the UFAM, located in the municipality of Parintins (AM), Brazil, following the methodology used by Crexi et al. [31]. Tambaqui viscera, including liver, stomach, intestines, swim bladder and kidneys, as well as other minor visceral tissues discarded during commercial fish processing, were sourced from local commercial fish markets in Parintins and then transported in plastic containers, being immediately frozen upon arrival at the laboratory. In cases where some viscera were absent, the residual composition varied by less than 5% relative to the total mass collected. Approximately 120 kg of tambaqui viscera were collected for oil extraction. All viscera were obtained on the same day from a single batch of fish, ensuring homogeneity of the raw material and reducing potential oxidative degradation associated with prolonged storage.

To initiate the oil extraction process, the tambaqui viscera were thawed at room temperature and subsequently minced using a meat grinder (model PS-10, Skymsen, Brusque, SC, Brazil). The minced raw material was cooked at a temperature of 95–100 °C for 30 min. After cooking, the mixture was sieved using a Tyler No. 14 sieve to remove impurities. The fractions were then separated by centrifugation, and the oil fractions were collected and stored in amber bottles at −20 °C for further analysis. A sample of TRO was stored in a hermetically sealed, properly labeled container and sent to the commercial food analysis laboratory CBO^© (Campinas, São Paulo, Brazil) for the determination of fatty acid composition according to the procedures described by AOAC [32]. The results obtained are presented in

Table 1. Composition of the tambaqui residual oil.

Fatty Acids	Composition (%) 1
Butyric Acid (C4:0)	<0.0003 (LQ)
Caproic Acid (C6:0)	0.01
Caprylic Acid (C8:0)	0.01
Capric Acid (C10:0)	-
Undecanoic Acid (C11:0)	<0.0003
Lauric Acid (C12:0)	0.02
Tridecanoic Acid (C13:0)	<0.003
Myristic Acid (C14:0)	1.29
Myristoleic Acid (C14:1)	0.08
Pentadecanoic Acid (C15:0)	0.19
10-Pentadecenoic Acid (C15:1)	<0.003
Palmitic Acid (C16:0)	26.9
Palmitoleic Acid (C16:1n7)	2.63
Margaric Acid (C17:0)	0.46
Stearic Acid (C18:0)	15.31
Elaidic Acid (C18:1n9t)	0.22
Oleic Acid (C18:1n9c)	36.12
Linolelaidic Acid (C18:2n6c)	0.04
Linoleic Acid LA (C18:2n6c)	10.01
Gamma-Linolenic Acid GLA (C18:3n6)	0.10
Alpha-Linolenic Acid LNA (C18:3n3)	0.60
Arachidic Acid (C20:0)	0.20
Cis-11-Eicosenoic Acid (C20:1n9)	1.52
Heneicosanoic Acid (C21:0)	0.44
Cis-11,14-Eicosadienoic Acid (C20:2)	0.50
Cis-8,11,14-Eicosatrienoic Acid	0.45
Arachidonic Acid AA (C20:4n6)	0.31
Cis-11,14,17-Eicosatrienoic Acid	0.06
Behenic Acid (C22:0)	0.10
Erucic Acid (C22:1n9)	0.05
5,8,11,14,17-Eicosapentaenoic Acid EPA (C20:5n3)	0.06
Tricosanoic Acid (C23:0)	0.05
Cis-13,16-Docosadienoic Acid	0.02
Lignoceric Acid (C24:0)	0.06
Nervonic Acid (C24:1n9)	0.04
Docosahexaenoic Acid DHA (C22:6n3)	0.11
Monounsaturated Fat	40.61
Polyunsaturated Fat	12.31
Unsaturated Fat	52.92
Saturated Fat	45.04
Trans Fat	0.26
Omega-3	0.83
Omega-6	10.90
Omega-9	37.95
Ether Extract	97.96

¹ Fatty acid composition of tambaqui residual oil expressed as the relative percentage of each fatty acid in relation to the total identified fatty acids. Values below the limit of quantification of the analytical method are indicated as LQ (Limit of Quantification).

. In addition, TRO samples were analyzed for heavy metals (Pb, Cd, Hg, As) following AOAC [32] recommendations, and no values above permissible limits were detected.

2.2. Facilities, Animals, Diets and Experimental Design

The aviary used was 17 m in length, 3.5 m in width, and a ceiling height of 3.25 m, with structural adaptations to improve bird welfare. The temperature and relative humidity were monitored using a digital thermohygrometer (Mylabor, São Paulo, SP, Brazil). The average temperature (29.1 °C) and relative humidity (63.4%) during the trial remained within the thermal comfort zone for commercial laying hens, and no clinical signs of heat stress were observed throughout the experimental period.

The experimental period of 63 days was divided into three consecutive 21-day subperiods to allow intermediate evaluations and trend analysis of the productive and physiological responses, in accordance with common recommendations for laying hen trials [14,16,22,23,24,25]. One hundred forty-four commercial hens of the Hisex Brown strain, 50 weeks old, previously subjected to a seven-day adaptation period to the diets and facilities, were used. The birds were weighed at the beginning of the experimental period to standardize the plots, with an average weight of 1.87 ± 0.213 kg. Hens were housed in galvanized wire cages (0.45 m in height, 0.40 m in width, and 1.00 m in length), accommodating 6 birds each suspended in a single line. It also had trough feeders and nipple drinkers. The birds were provided with 16 h of light per day (12 h of natural light + 4 h of artificial light) throughout the experimental period. The collection of eggs was carried out twice a day (9 a.m. and 3 p.m.), with a record of each daily occurrence (mortality, number of eggs, feed program, water supply checks, cleaning routines, and other management-related events).

The hens were distributed in a completely randomized design consisting of the control treatment (without inclusion) and three inclusion levels of TRO (1.5, 3.0, and 4.5%) in the diets, with six replicates of six birds each. The experimental diets (

Table 2. Experimental diet composition.

Feedstuffs	Tambaqui Residual Oil Levels (%)
	0	1.5	3.0	4.5
Corn (7.88%)	66.67	62.20	58.06	53.98
Soybean meal (46%)	20.98	23.99	26.51	28.79
TRO	0.00	1.50	3.00	4.50
Limestone	9.64	9.63	9.37	9.36
Dicalcium phosphate	1.66	1.65	2.03	2.34
Vit. min. supplement *	0.50	0.50	0.50	0.50
Salt	0.40	0.40	0.40	0.40
DL-methionine (99%)	0.15	0.13	0.13	0.13
Total	100.00	100.00	100.00	100.00
Nutrient
M.E., kcal.kg−1	2950.00	2950.00	2950.00	2950.00
Crude protein, %	16.50	16.50	16.50	16.50
Calcium, %	4.20	4.20	4.20	4.20
Available phosphorus, %	0.47	0.47	0.47	0.47
Crude fiber, %	2.54	2.63	2.72	2.76
Methionine + Cystine, %	0.66	0.66	0.68	0.70
Total methionine, %	0.40	0.39	0.40	0.41
Total lysine, %	0.75	0.82	0.88	0.94
Total threonine, %	0.59	0.63	0.66	0.69
Total tryptophan, %	0.18	0.19	0.21	0.22
Sodium, %	2.64	2.76	2.89	3.01

* Vitamin–mineral supplement: guaranteed levels per kilogram of supplement: Vitamin A 2,000,000 IU; Vitamin D₃ 400,000 IU; Vitamin E 2400 mg; Vitamin K₃ 400 mg; Vitamin B₁ 100 mg; Vitamin B₂ 760 mg; Vitamin B₆ 100 mg; Vitamin B₁₂ 2400 mcg; Niacin 5000 mg; Calcium pantothenate 2000 mg; Folic acid 50 mg; Coccidiostat 12,000 mg; Choline 50,000 mg; Copper 1200 mg; Iron 6000 mg; Manganese 14,000 mg; Zinc 10,000 mg; Iodine 100 mg; Selenium 40 mg; vehicle q.s.p. 1000 g. Values in parentheses indicate the crude protein content of the ingredients used (e.g., Corn: 7.88% CP; Soybean meal: 46% CP).

) were formulated to meet the nutritional requirements of the commercial hens using the reference values described by Rostagno et al. [33] and the values obtained for the TRO composition previously. All diets were prepared weekly by weighing ingredients on a precision balance, mixing them in a horizontal feed mixer (Nogueira Máquinas Agrícolas, Itapira, SP, Brazil) until homogeneous, and then storing the diets in hermetically sealed plastic bags under cool and dry conditions. A preliminary digestibility trial was conducted to determine the metabolizable energy value of TRO, obtaining a value of 8345.38 kcal/kg, which was considered for diet formulation.

2.3. Productive Performance and Egg Quality

The productive performance of the hens was evaluated according to the methodology described by Rufino et al. [34]. Every seven days, productive performance was evaluated for each replicate by recording feed intake, egg production, feed conversion ratios, and egg mass. Feed intake (g/bird/day) was calculated by weighing the total feed offered at the beginning of the week, subtracting the leftovers at the end of the period, and dividing the consumed amount by the number of birds and days. Egg production (%) was determined as the ratio between the total number of eggs produced and the number of hens, expressed as a percentage. Feed conversion ratio was expressed in two ways: as kilograms of feed consumed per kilogram of egg mass produced (kg/kg) and as kilograms of feed consumed per dozen eggs produced (kg/dz). Egg mass (g/bird/day) was obtained by multiplying the average egg weight by the egg production rate, expressed per bird per day. These measurements were performed weekly for each replicate, allowing the assessment of temporal variation as well as cumulative productive performance throughout the 63-day experimental period. On the last two days of each 21-day subperiod, 6 eggs per replicate (total of 36 eggs per treatment) were collected and separated, where 12 were randomly selected for physical quality evaluation, 8 eggs for chemical composition, 8 eggs for fatty acid profile and lipid oxidation, and 8 eggs for sensory evaluation.

For egg quality analysis, this study followed the methodology described by Rufino et al. [34]. A total of 12 eggs per treatment were stored at room temperature for one hour and weighed using an electronic balance (model BL320H, Shimadzu, São Paulo, SP, Brazil) (0.01 g). They were then placed in wire baskets and immersed in buckets containing different concentrations of sodium chloride (NaCl) solutions with density variations from 1.075 to 1.100 g/cm³ (with 0.005 intervals) to determine specific gravity. Next, the eggs were placed on a flat glass plate to measure albumen and yolk height, as well as yolk diameter, using an electronic caliper (model 500-196-30, Mitutoyo, Suzano, SP, Brazil). Albumen and yolk were separated using a manual separator, placed in plastic cups, and weighed using an analytical balance. Eggshells were washed, dried in an oven at 50 °C (122 °F) for 48 h, and weighed. The dry eggshells were then used to determine shell thickness using a digital micrometer. The average eggshell thickness was analyzed considering three regions: basal, meridional, and apical. Yolk color was evaluated using a ROCHE^© colorimetric fan (Roche Diagnostics GmbH, Mannheim, Germany) with a scale ranging from 1 to 15. The Haugh unit was calculated using the formula [35]:

$${\mathrm{H}}_{\mathrm{u}\mathrm{n}\mathrm{i}\mathrm{t}}=100\ast \log \left(\mathrm{H}+7.57-1.7\ast {\mathrm{W}}^{0.37}\right)$$

(1)

where

• H = albumen height (mm)
• W = egg weight (g).

2.4. Physiological Parameters

Six hens per treatment were selected for blood collection and analysis of hematological, leukocyte differentiation and plasma biochemical parameters. One milliliter of blood was collected from the hens directly from the ulnar vein using disposable syringes containing heparin anticoagulant (5000 IU per sample). These samples were immediately centrifuged at 7000 rpm for 10 min to separate the red blood cells for hematological parameters evaluation, while the plasma was used for biochemical parameters analysis. The samples were identified and preserved at −80 °C (−122 °F) before starting the analysis.

For hematological analysis, the collected blood was used to count erythrocytes (M/mm³) using a Neubauer chamber after dilution in formaldehyde-citrate and toluidine blue, visualized under an optical microscope (Nikon Eclipse E-50i, DM3000, Tokyo, Japan) with a 40× objective lens. Hemoglobin concentration (g/dL) was determined by the cyanomethemoglobin method, while hematocrit (%) was measured using the microhematocrit method [36], with centrifugation of heparinized microcapillary tubes at 12,000 rpm for 5 min [37]. Based on these analyses, the mean corpuscular volume (MCV, μm³), mean corpuscular hemoglobin (MCH, pg/cell), and mean corpuscular hemoglobin concentration (MCHC, g/dL) were calculated according to Tavares-Dias and Moraes [38].

Leukocyte differentiation analysis was performed under an optical microscope (Nikon Eclipse E-50i, DM3000, Tokyo, Japan) with a 100× oil immersion objective lens. A total of 100 leukocytes per smear were counted and classified into heterophils, eosinophils, basophils, typical lymphocytes, atypical lymphocytes, and monocytes according to Tavares-Dias and Moraes [38].

For plasma biochemical analysis, the remaining plasma samples after centrifugation were subjected to commercial enzymatic-colorimetric assay kits (InVitro Diagnóstica, Itabira, MG, Brazil), following the manufacturer’s specific recommendations. The readings were taken using a mass spectrophotometer (model K37-UVVIS, Kasvi^©, São José dos Pinhais, Brazil) at a specific wavelength for each assay. The biochemical parameters analyzed included total protein, triglyceride, glucose, cholesterol, and albumin concentrations.

2.5. Chemical Composition of the Eggs

Eight eggs from each treatment were randomly selected and subjected to proximate composition analysis in order to determine moisture, mineral, fat, and protein contents. Moisture (%) was measured by oven-drying the samples at 105 °C until constant weight was achieved. Ash or mineral content (%) was obtained after incineration of the dried samples in a muffle furnace at 550 °C. Total fat (%) was determined by ether extraction using the Soxhlet method, and crude protein (%) was quantified by the Kjeldahl procedure, with nitrogen content converted to protein using the factor 6.25. All analyses were conducted in accordance with the official methods described by the AOAC [32].

2.6. Lipid Oxidation and Fatty Acid Profile of the Yolks

Eight eggs from each treatment were used to evaluate the potential lipid oxidation of the yolk (TBARS analysis). The eggs were broken, and the yolks were separated and frozen. Subsequently, the frozen yolks were subjected to lyophilization, where water and other solvents were removed by sublimation, bypassing the liquid state. The dehydrated yolks were then subjected to TBARS analysis to measure the degree of lipid oxidation using a modified version of the methodology described by Vyncke [39] and adapted by Ramanathan and Das [40]. Samples of same eight eggs were sent to the commercial food analysis laboratory CBO^© (Campinas, São Paulo, Brazil) for the determination of fatty acid profile of the yolks according to the procedures described by AOAC [32].

2.7. Sensory Characteristics of the Eggs

Eight eggs per treatment were selected for sensory analysis. For the sensory evaluation, 20 untrained judges of both genders were selected to assess the appearance, acidity, aroma, color, and taste of the eggs. A 9-point hedonic scale was used to measure these attributes, ranging from “liked extremely” (9) to “disliked extremely” (1), following the methodology described by Dutcosky [41]. Each judge was provided with a sample (half of a boiled egg, cooked in hot water for 10 min) from each treatment.

2.8. Statistical Analyses

The adopted statistical model was as follows:

$${\mathrm{Y}}_{\mathrm{i}\mathrm{k}}=\mathsf{\mu }+{\mathsf{\alpha }}_{\mathrm{i}}+{\mathsf{ϵ}}_{\mathrm{i}\mathrm{k}}$$

(2)

where

• Y_ik = Observed value for the variable under study;
• μ = Overall mean of the experiment;
• α_i = Effect of the TRO levels;
• ϵ_ik = Experimental error.

All data were analyzed by one-way ANOVA using the R software 4.1.3 (2021), following the guidelines outlined by Logan [42]. Significant variables (p < 0.05) were subjected to polynomial regression to analyze the influence of the independent variable (TRO levels) on dependent variables evaluated [42,43]. The mathematical model, linear (Y = a + bx) or quadratic (Y = c + bx + ax²), was chosen based on the influence of each independent variable on the dependent variable analyzed [43,44].

3. Results

3.1. Physical-Chemical Characteristics of TRO

The TRO analysis indicated that it was predominantly composed of monounsaturated fatty acids (40.61%) and saturated fatty acids (45.04%), while polyunsaturated fatty acids accounted for only 12.31% of the total (Table 1). Among the essential fatty acids, ω-3 and ω-6 levels were 0.83% and 10.90%, respectively, indicating a predominance of ω-6 (Table 1). The main fatty acid found was oleic acid (36.12%), followed by palmitic acid (26.9%) and stearic acid (15.31%) (Table 1). Moreover, heavy metal contents were below detection limits for Pb, Cd, Hg, and As, confirming the safety of the oil for dietary inclusion.

3.2. Productive Performance and Egg Quality

The inclusion of TRO in the diets did not significantly affect (p > 0.05) productive performance variables (Table 3). However, egg weight increased significantly with TRO inclusion (p ≤ 0.05), following a quadratic model, with maximum values observed in treatments with 1.5% and 3.0% TRO (Table 4). Yolk color was also influenced (p ≤ 0.05), showing a linear increase as TRO levels in the diet increased (Table 4).

Although eggshell thickness did not show significant differences among treatments (p > 0.05), specific gravity decreased linearly (p ≤ 0.05) with increasing TRO levels (Table 4). The Haugh unit followed a quadratic model, with better results (p ≤ 0.05) observed at intermediate TRO inclusion levels (Table 4). The pH values of the yolk and albumen were significantly affected (p ≤ 0.05), following linear and quadratic models, respectively.

3.3. Physiological Parameters

In the results of hematological parameters (Table 5), hemoglobin concentration and hematocrit showed significant variations (p ≤ 0.05), with an initial reduction followed by an increase at higher levels of TRO inclusion. The erythrocyte count decreased linearly (p ≤ 0.05), while mean corpuscular volume increased in a quadratic manner (p ≤ 0.05). Conversely, mean corpuscular hemoglobin (p ≤ 0.05) and mean corpuscular hemoglobin concentration (p ≤ 0.05) decreased as TRO inclusion increased.

The inclusion of TRO in the diet of commercial hens significantly impacted several plasma biochemical parameters (Table 6). Total protein and albumin levels decreased (p ≤ 0.05) linearly with increasing TRO inclusion, reaching the lowest values in the diet with 4.5% TRO, suggesting a possible interference in protein synthesis or amino acid availability. On the other hand, cholesterol levels increased (p ≤ 0.05) linearly with TRO inclusion in the diets, possibly due to the lipid profile of TRO, which is rich in monounsaturated fatty acids. Lipid metabolism was also affected by a significant reduction in triglycerides (p ≤ 0.05), which dropped from 39.07 mg/dL to 19.58 mg/dL in the diet with the highest TRO inclusion, indicating a possible redirection of lipids to other metabolic processes. Meanwhile, glucose followed a quadratic trend (p ≤ 0.05), initially increasing in the diet with 1.5% TRO (239.29 mg/dL) but decreasing at higher inclusion levels (198.16 mg/dL with 4.5% TRO), which may reflect adjustments in the birds’ energy metabolism in response to fish oil inclusion.

The inclusion of TRO in the hens’ diet significantly altered the differential leukocyte count (Table 7), indicating possible immunomodulatory effects. The percentage of heterophils decreased in groups with 1.5% and 3.0% TRO but increased again in the diet with 4.5% TRO (p ≤ 0.05), which may be related to adjustments in the birds’ inflammatory response. Eosinophils decreased linearly with increasing TRO inclusion (p ≤ 0.05), suggesting a possible reduction in the immune response to parasitic and allergenic agents. Meanwhile, typical lymphocytes followed a quadratic pattern, increasing in the 1.5% TRO diet and decreasing at higher inclusion levels (p ≤ 0.05), which may indicate regulation of the adaptive immune response. The percentages of basophils and monocytes were not significantly affected by the treatments (p > 0.05), suggesting that TRO inclusion did not markedly alter these cellular components of the immune response. The reduction in heterophils and eosinophils, combined with the initial increase in typical lymphocytes, suggests that moderate TRO levels have a positive immunomodulatory effect.

3.4. Chemical Composition of the Eggs

The moisture content of the eggs decreased linearly (p ≤ 0.05) with the increasing levels of TRO in the diets (Table 8). The lipid percentage increased significantly (p ≤ 0.05), while protein content also followed a linear increase (p ≤ 0.05) as TRO levels in the diets increased (Table 8). However, the mineral content of the eggs was not significantly affected (p > 0.05) by the TRO levels in the diets (Table 8).

3.5. Lipid Oxidation and Fatty Acid Profile of the Yolks

The TBARS values followed a quadratic trend (p ≤ 0.05), with the highest results observed from the inclusion of 1.5% TRO in the diets (Table 9). The linoleic acid (C18:2n6c) content also followed a quadratic model (p ≤ 0.05), reaching maximum values with 3.0% TRO inclusion (Table 10). Alpha-linolenic acid (C18:3n3) increased linearly (p ≤ 0.05), while DHA (C22:6n3) also showed a significant linear increase (p ≤ 0.05) (Table 10).

3.6. Sensory Characteristics of the Eggs

The appearance and color of the eggs were significantly influenced (p ≤ 0.05) by the treatments, following a positive linear model, indicating increased acceptance of these characteristics as TRO inclusion in the diets increased (Table 11). However, aroma and taste exhibited a negative linear trend (p ≤ 0.05), suggesting greater rejection as TRO levels in the diets increased (Table 11). Egg texture was not significant (Table 11).

Table 3. Productive performance of commercial hens fed diets containing increasing levels of tambaqui residual oil (TRO).

Variables	TRO Levels, %				p-Value 1	CV 2 %	Model	R2
	0	1.5	3.0	4.5
Feed intake, g/bird/day	101.97	103.53	102.78	103.17	0.72	2.25	-	-
Egg production, %	89.55	91.49	91.20	89.50	0.60	3.51	-	-
Feed efficiency, kg/kg	1.92	1.93	1.95	1.96	0.48	6.89	-	-
Feed efficiency, kg/dz	1.36	1.35	1.44	1.38	0.12	4.89	-	-
Egg mass, g	53.15	53.78	55.90	52.47	0.40	6.68	-	-

¹ The means presented in the row differ significantly when p ≤ 0.05. ² CV = Coefficient of variation.

Table 3. Productive performance of commercial hens fed diets containing increasing levels of tambaqui residual oil (TRO).

Variables	TRO Levels, %				p-Value 1	CV 2 %	Model	R2
	0	1.5	3.0	4.5
Feed intake, g/bird/day	101.97	103.53	102.78	103.17	0.72	2.25	-	-
Egg production, %	89.55	91.49	91.20	89.50	0.60	3.51	-	-
Feed efficiency, kg/kg	1.92	1.93	1.95	1.96	0.48	6.89	-	-
Feed efficiency, kg/dz	1.36	1.35	1.44	1.38	0.12	4.89	-	-
Egg mass, g	53.15	53.78	55.90	52.47	0.40	6.68	-	-

¹ The means presented in the row differ significantly when p ≤ 0.05. ² CV = Coefficient of variation.

Table 4. Quality of eggs produced by commercial hens fed diets containing increasing levels of tambaqui residual oil (TRO).

Variables	TRO Levels, %				p-Value 1	CV 2 %	Model 3	R2
	0	1.5	3.0	4.5
Egg weight, g	53.70	55.93	55.96	55.04	0.05	7.75	Y = 53.763 + 1.845x − 0.35x2	0.97
Yolk, %	27.04	28.40	28.57	28.22	0.03	7.42	Y = 27.074 + 1.1023x − 0.19x2	0.98
Albumen, %	60.44	59.37	59.22	59.57	0.82	6.30	-	-
Eggshell, %	12.52	12.23	12.21	12.21	0.02	7.12	-	-
Yolk height, mm	18.10	18.29	18.56	18.21	0.39	4.12	-	-
Albumen height, mm	10.34	10.78	10.51	10.19	0.26	6.77	-	-
Yolk diameter, mm	40.12	40.06	39.94	39.97	0.62	3.14	-	-
Yolk color	5.18	5.26	5.32	5.54	<0.01	7.49	Y = 5.154 + 0.076x	0.91
Specific gravity, g/mL	1088.47	1087.43	1084.72	1084.37	<0.01	2.41	Y = 1088.50 − 1.0007x	0.92
Eggshell thickness, µm	0.44	0.43	0.43	0.43	0.06	5.88	-	-
Haugh unit	91.88	93.18	92.08	90.90	0.05	2.83	Y = 91.996 + 0.9707x − 0.2756x2	0.90
Yolk pH	6.06	5.92	5.92	5.88	<0.01	1.75	Y = 6.026 − 0.036x	0.78
Albumen pH	7.63	7.73	7.78	7.59	0.05	5.24	Y = 7.6205 + 0.1403x − 0.0322x2	0.92

¹ The means presented in the row differ significantly when p ≤ 0.05. ² CV = Coefficient of variation. ³ The mathematical model was adjusted to represent the relationship between the independent variable (TRO inclusion levels) and the corresponding dependent variable under evaluation.

Table 4. Quality of eggs produced by commercial hens fed diets containing increasing levels of tambaqui residual oil (TRO).

Variables	TRO Levels, %				p-Value 1	CV 2 %	Model 3	R2
	0	1.5	3.0	4.5
Egg weight, g	53.70	55.93	55.96	55.04	0.05	7.75	Y = 53.763 + 1.845x − 0.35x2	0.97
Yolk, %	27.04	28.40	28.57	28.22	0.03	7.42	Y = 27.074 + 1.1023x − 0.19x2	0.98
Albumen, %	60.44	59.37	59.22	59.57	0.82	6.30	-	-
Eggshell, %	12.52	12.23	12.21	12.21	0.02	7.12	-	-
Yolk height, mm	18.10	18.29	18.56	18.21	0.39	4.12	-	-
Albumen height, mm	10.34	10.78	10.51	10.19	0.26	6.77	-	-
Yolk diameter, mm	40.12	40.06	39.94	39.97	0.62	3.14	-	-
Yolk color	5.18	5.26	5.32	5.54	<0.01	7.49	Y = 5.154 + 0.076x	0.91
Specific gravity, g/mL	1088.47	1087.43	1084.72	1084.37	<0.01	2.41	Y = 1088.50 − 1.0007x	0.92
Eggshell thickness, µm	0.44	0.43	0.43	0.43	0.06	5.88	-	-
Haugh unit	91.88	93.18	92.08	90.90	0.05	2.83	Y = 91.996 + 0.9707x − 0.2756x2	0.90
Yolk pH	6.06	5.92	5.92	5.88	<0.01	1.75	Y = 6.026 − 0.036x	0.78
Albumen pH	7.63	7.73	7.78	7.59	0.05	5.24	Y = 7.6205 + 0.1403x − 0.0322x2	0.92

¹ The means presented in the row differ significantly when p ≤ 0.05. ² CV = Coefficient of variation. ³ The mathematical model was adjusted to represent the relationship between the independent variable (TRO inclusion levels) and the corresponding dependent variable under evaluation.

Table 5. Hematological parameters of commercial hens fed diets containing increasing levels of tambaqui residual oil (TRO).

Variables 1	TRO Levels, %				p-Value 2	CV 3 %	Model 4	R2
	0	1.5	3.0	4.5
Hemoglobin, g/dL	13.20	10.36	10.47	11.53	0.05	10.66	Y = 13.1 − 2.2767x + 0.4333x2	0.96
Erythrocytes RBC, M/mm3	2.81	1.89	1.69	1.09	<0.01	17.71	Y = 2.674 − 0.3573x	0.94
Hematocrit, %	32.16	29.83	29.90	34.33	0.05	11.84	Y = 32.258 − 2.9413x + 0.7511x2	0.98
MCV, um3	24.60	51.99	41.31	41.50	0.05	18.77	Y = 27.047 + 16.268x − 3.0222x2	0.78
MCH, pg/cel	20.08	17.05	13.26	9.99	0.05	17.46	Y = 20.204 − 2.2707x	0.99
MCHC, g/dL	13.19	4.44	3.27	3.16	0.05	11.56	Y = 12.864 − 6.404x + 0.96x2	0.97

¹ MCV = Mean Corpuscular Volume. MCH = Mean Corpuscular Hemoglobin. MCHC = Mean Corpuscular Hemoglobin Concentration. ² The means presented in the row differ significantly when p ≤ 0.05. ³ CV = Coefficient of variation. ⁴ The mathematical model was adjusted to represent the relationship between the independent variable (TRO inclusion levels) and the corresponding dependent variable under evaluation.

Table 5. Hematological parameters of commercial hens fed diets containing increasing levels of tambaqui residual oil (TRO).

Variables 1	TRO Levels, %				p-Value 2	CV 3 %	Model 4	R2
	0	1.5	3.0	4.5
Hemoglobin, g/dL	13.20	10.36	10.47	11.53	0.05	10.66	Y = 13.1 − 2.2767x + 0.4333x2	0.96
Erythrocytes RBC, M/mm3	2.81	1.89	1.69	1.09	<0.01	17.71	Y = 2.674 − 0.3573x	0.94
Hematocrit, %	32.16	29.83	29.90	34.33	0.05	11.84	Y = 32.258 − 2.9413x + 0.7511x2	0.98
MCV, um3	24.60	51.99	41.31	41.50	0.05	18.77	Y = 27.047 + 16.268x − 3.0222x2	0.78
MCH, pg/cel	20.08	17.05	13.26	9.99	0.05	17.46	Y = 20.204 − 2.2707x	0.99
MCHC, g/dL	13.19	4.44	3.27	3.16	0.05	11.56	Y = 12.864 − 6.404x + 0.96x2	0.97

¹ MCV = Mean Corpuscular Volume. MCH = Mean Corpuscular Hemoglobin. MCHC = Mean Corpuscular Hemoglobin Concentration. ² The means presented in the row differ significantly when p ≤ 0.05. ³ CV = Coefficient of variation. ⁴ The mathematical model was adjusted to represent the relationship between the independent variable (TRO inclusion levels) and the corresponding dependent variable under evaluation.

Table 6. Plasma biochemical parameters of commercial hens fed diets containing increasing levels of tambaqui residual oil (TRO).

Variables	TRO Levels, %				p-Value 1	CV 2 %	Model 3	R2
	0	1.5	3.0	4.5
Total proteins, g/dL	1.87	0.53	0.60	0.59	0.05	14.91	Y = 1.463 − 0.2513x	0.76
Triglycerides, mg/dL	39.07	38.20	23.82	19.58	0.05	20.37	Y = 41.095 − 4.8567x	0.90
Glucose, mg/dL	214.72	239.29	211.51	198.16	0.05	12.75	Y = 218.06 + 13.796x − 4.2133x2	0.74
Cholesterol, mg/dL	28.71	28.82	37.66	78.64	0.05	17.31	Y = 19.663 + 10.575x	0.74
Albumin, mg/dL	3.49	3.19	2.49	2.39	0.05	18.61	Y = 3.49 − 0.2667x	0.93

¹ The means presented in the row differ significantly when p ≤ 0.05. ² CV = Coefficient of variation. ³ The mathematical model was adjusted to represent the relationship between the independent variable (TRO inclusion levels) and the corresponding dependent variable under evaluation.

Table 6. Plasma biochemical parameters of commercial hens fed diets containing increasing levels of tambaqui residual oil (TRO).

Variables	TRO Levels, %				p-Value 1	CV 2 %	Model 3	R2
	0	1.5	3.0	4.5
Total proteins, g/dL	1.87	0.53	0.60	0.59	0.05	14.91	Y = 1.463 − 0.2513x	0.76
Triglycerides, mg/dL	39.07	38.20	23.82	19.58	0.05	20.37	Y = 41.095 − 4.8567x	0.90
Glucose, mg/dL	214.72	239.29	211.51	198.16	0.05	12.75	Y = 218.06 + 13.796x − 4.2133x2	0.74
Cholesterol, mg/dL	28.71	28.82	37.66	78.64	0.05	17.31	Y = 19.663 + 10.575x	0.74
Albumin, mg/dL	3.49	3.19	2.49	2.39	0.05	18.61	Y = 3.49 − 0.2667x	0.93

¹ The means presented in the row differ significantly when p ≤ 0.05. ² CV = Coefficient of variation. ³ The mathematical model was adjusted to represent the relationship between the independent variable (TRO inclusion levels) and the corresponding dependent variable under evaluation.

Table 7. Differential leukocyte count (white blood cell series) of commercial hens fed diets containing increasing levels of tambaqui residual oil (TRO).

Variables	TRO Levels, %				p-Value 1	CV 2 %	Model 3	R2
	0	1.5	3.0	4.5
Heterophils, %	62.88	52.52	56.22	58.38	0.05	17.14	Y = 75.6 − 16.63x + 3.13x2	0.78
Eosinophils, %	1.25	0.37	0.25	0.25	0.01	14.33	Y = 1.31 − 0.312x	0.70
Basophils, %	5.25	4.62	5.00	5.75	0.96	8.22	-	-
Typical Lymphocytes, %	28.75	40.62	37.12	33.75	0.05	19.23	Y = 13.135 + 20.2x − 3.81x2	0.84
Atypical Lymphocytes, %	0.00	0.00	0.00	0.00	-	-	-	-
Monocytes, %	1.87	1.87	1.41	1.87	0.70	7.27	-	-

¹ The means presented in the row differ significantly when p ≤ 0.05. ² CV = Coefficient of variation. ³ The mathematical model was adjusted to represent the relationship between the independent variable (TRO inclusion levels) and the corresponding dependent variable under evaluation.

Table 7. Differential leukocyte count (white blood cell series) of commercial hens fed diets containing increasing levels of tambaqui residual oil (TRO).

Variables	TRO Levels, %				p-Value 1	CV 2 %	Model 3	R2
	0	1.5	3.0	4.5
Heterophils, %	62.88	52.52	56.22	58.38	0.05	17.14	Y = 75.6 − 16.63x + 3.13x2	0.78
Eosinophils, %	1.25	0.37	0.25	0.25	0.01	14.33	Y = 1.31 − 0.312x	0.70
Basophils, %	5.25	4.62	5.00	5.75	0.96	8.22	-	-
Typical Lymphocytes, %	28.75	40.62	37.12	33.75	0.05	19.23	Y = 13.135 + 20.2x − 3.81x2	0.84
Atypical Lymphocytes, %	0.00	0.00	0.00	0.00	-	-	-	-
Monocytes, %	1.87	1.87	1.41	1.87	0.70	7.27	-	-

¹ The means presented in the row differ significantly when p ≤ 0.05. ² CV = Coefficient of variation. ³ The mathematical model was adjusted to represent the relationship between the independent variable (TRO inclusion levels) and the corresponding dependent variable under evaluation.

Table 8. Chemical composition of eggs produced by commercial hens fed diets containing increasing levels of tambaqui residual oil (TRO).

Variables	TRO Levels, %				p-Value 1	CV 2 %	Model 3	R2
	0	1.5	3.0	4.5
Moisture, %	77.30	76.88	76.09	75.43	0.05	0.57	Y = 77.385 − 0.4267x	0.99
Minerals, %	1.01	1.08	1.07	1.02	0.93	6.66	-	-
Fats, %	9.81	9.82	10.02	10.18	0.05	8.01	Y = 9.761 + 0.0873x	0.91
Proteins, %	11.88	12.22	12.82	13.37	0.03	6.77	Y = 11.812 + 0.338x	0.99

¹ The means presented in the row differ significantly when p ≤ 0.05. ² CV = Coefficient of variation. ³ The mathematical model was adjusted to represent the relationship between the independent variable (TRO inclusion levels) and the corresponding dependent variable under evaluation.

Table 8. Chemical composition of eggs produced by commercial hens fed diets containing increasing levels of tambaqui residual oil (TRO).

Variables	TRO Levels, %				p-Value 1	CV 2 %	Model 3	R2
	0	1.5	3.0	4.5
Moisture, %	77.30	76.88	76.09	75.43	0.05	0.57	Y = 77.385 − 0.4267x	0.99
Minerals, %	1.01	1.08	1.07	1.02	0.93	6.66	-	-
Fats, %	9.81	9.82	10.02	10.18	0.05	8.01	Y = 9.761 + 0.0873x	0.91
Proteins, %	11.88	12.22	12.82	13.37	0.03	6.77	Y = 11.812 + 0.338x	0.99

¹ The means presented in the row differ significantly when p ≤ 0.05. ² CV = Coefficient of variation. ³ The mathematical model was adjusted to represent the relationship between the independent variable (TRO inclusion levels) and the corresponding dependent variable under evaluation.

Table 9. Lipid oxidation of the yolks of eggs produced by commercial hens fed diets containing increasing levels of tambaqui residual oil (TRO).

Variables 1	TRO Levels, %				p-Value 2	CV 3 %	Model 4	R2
	0	1.5	3.0	4.5
TBARS values	0.46	1.38	0.68	0.44	0.03	6.22	Y = 0.564 + 0.5293x − 0.1289x2	0.72
Total peroxide values, %	4.37	4.54	4.03	3.98	0.02	10.59	Y = 4.427 + 0.002x − 0.0244x2	0.70

¹ TBARS values refer to the degree of lipid oxidation expressed in mg MDA/kg of lyophilized yolk samples. Total peroxide values indicate the degree of lipid oxidation expressed in meq O₂/kg of lyophilized yolk samples. ² The means presented in the row differ significantly when p ≤ 0.05. ³ CV = Coefficient of variation. ⁴ The mathematical model was adjusted to represent the relationship between the independent variable (TRO inclusion levels) and the corresponding dependent variable under evaluation.

Table 9. Lipid oxidation of the yolks of eggs produced by commercial hens fed diets containing increasing levels of tambaqui residual oil (TRO).

Variables 1	TRO Levels, %				p-Value 2	CV 3 %	Model 4	R2
	0	1.5	3.0	4.5
TBARS values	0.46	1.38	0.68	0.44	0.03	6.22	Y = 0.564 + 0.5293x − 0.1289x2	0.72
Total peroxide values, %	4.37	4.54	4.03	3.98	0.02	10.59	Y = 4.427 + 0.002x − 0.0244x2	0.70

¹ TBARS values refer to the degree of lipid oxidation expressed in mg MDA/kg of lyophilized yolk samples. Total peroxide values indicate the degree of lipid oxidation expressed in meq O₂/kg of lyophilized yolk samples. ² The means presented in the row differ significantly when p ≤ 0.05. ³ CV = Coefficient of variation. ⁴ The mathematical model was adjusted to represent the relationship between the independent variable (TRO inclusion levels) and the corresponding dependent variable under evaluation.

Table 10. Fatty acid profile of the yolks of eggs produced by commercial hens fed diets containing increasing levels of tambaqui residual oil (TRO).

FAP 1	Variables	TRO Levels, %				p-Value 2	CV 3 %	Model 4	R2
		0	1.5	3.0	4.5
SFA	Palmitic Acid (C16:0), %	15.75	15.01	15.16	15.69	0.48	4.35	-	-
	Stearic Acid (C18:0), %	4.16	4.18	4.04	4.05	0.91	6.51	-	-
	Myristic Acid (C14:0), %	0.20	0.18	0.21	0.22	0.11	11.36	-	-
MUFA	Palmitoleic Acid (C16:1n7), %	2.20	1.93	2.02	1.92	0.72	14.91	-	-
	Oleic Acid (C18:1n9c), %	25.71	25.31	26.83	26.91	0.15	4.09	-	-
PUFA	General Polyunsaturated Fatty Acids	39.05	38.82	38.28	38.94	0.33	6.59	-	-
PUFA ω6	Linoleic Acid LA (C18:2n6c), %	5.59	6.56	6.85	6.57	0.03	9.68	Y = 5.5955 + 0.8403x − 0.1389x2	0.99
	Arachidonic Acid AA (C20:4n6), %	1.02	1.07	1.02	1.12	0.16	5.91	-	-
PUFA ω3	LNA (C18:3n3), %	0.12	0.13	0.15	0.16	0.03	6.38	Y = 0.119 + 0.0093x	0.98
	EPA (C20:5n3), %	<0.01	<0.01	<0.01	<0.01	0.92	1.32	-	-
	DHA (C22:6n3), %	0.27	0.29	0.30	0.41	<0.01	7.87	Y = 0.253 + 0.0287x	0.78

¹ FAP = Fatty Acid Profile. SFA = Saturated Fatty Acids. MUFA = Monounsaturated Fatty Acids. PUFA = Polyunsaturated Fatty Acids. ² The means presented in the row differ significantly when p ≤ 0.05. ³ CV = Coefficient of variation. ⁴ The mathematical model was adjusted to represent the relationship between the independent variable (TRO inclusion levels) and the corresponding dependent variable under evaluation.

Table 10. Fatty acid profile of the yolks of eggs produced by commercial hens fed diets containing increasing levels of tambaqui residual oil (TRO).

FAP 1	Variables	TRO Levels, %				p-Value 2	CV 3 %	Model 4	R2
		0	1.5	3.0	4.5
SFA	Palmitic Acid (C16:0), %	15.75	15.01	15.16	15.69	0.48	4.35	-	-
	Stearic Acid (C18:0), %	4.16	4.18	4.04	4.05	0.91	6.51	-	-
	Myristic Acid (C14:0), %	0.20	0.18	0.21	0.22	0.11	11.36	-	-
MUFA	Palmitoleic Acid (C16:1n7), %	2.20	1.93	2.02	1.92	0.72	14.91	-	-
	Oleic Acid (C18:1n9c), %	25.71	25.31	26.83	26.91	0.15	4.09	-	-
PUFA	General Polyunsaturated Fatty Acids	39.05	38.82	38.28	38.94	0.33	6.59	-	-
PUFA ω6	Linoleic Acid LA (C18:2n6c), %	5.59	6.56	6.85	6.57	0.03	9.68	Y = 5.5955 + 0.8403x − 0.1389x2	0.99
	Arachidonic Acid AA (C20:4n6), %	1.02	1.07	1.02	1.12	0.16	5.91	-	-
PUFA ω3	LNA (C18:3n3), %	0.12	0.13	0.15	0.16	0.03	6.38	Y = 0.119 + 0.0093x	0.98
	EPA (C20:5n3), %	<0.01	<0.01	<0.01	<0.01	0.92	1.32	-	-
	DHA (C22:6n3), %	0.27	0.29	0.30	0.41	<0.01	7.87	Y = 0.253 + 0.0287x	0.78

¹ FAP = Fatty Acid Profile. SFA = Saturated Fatty Acids. MUFA = Monounsaturated Fatty Acids. PUFA = Polyunsaturated Fatty Acids. ² The means presented in the row differ significantly when p ≤ 0.05. ³ CV = Coefficient of variation. ⁴ The mathematical model was adjusted to represent the relationship between the independent variable (TRO inclusion levels) and the corresponding dependent variable under evaluation.

Table 11. Sensory characteristics of eggs produced by commercial hens fed diets containing increasing levels of tambaqui residual oil (TRO).

Variables	TRO Levels, %				p-Value 1	CV 2 %	Model 3	R2
	0	1.5	3.0	4.5
Appearance	5.40	5.91	6.02	6.48	0.05	3 > 53	Y = 5.45 + 0.2233x	0.95
Texture	6.68	6.42	6.34	6.60	0.86	2.85	-	-
Aroma	6.28	6.14	6.11	5.82	0.05	2.72	Y = 6.299 − 0.094x	0.88
Color	5.71	6.28	6.37	6.88	0.05	2.92	Y = 5.77 + 0.24x	0.94
Taste	7.00	6.51	6.34	6.22	0.05	3.10	Y = 6.894 − 0.1673x	0.89

¹ The means presented in the row differ significantly when p ≤ 0.05. ² CV = Coefficient of variation. ³ The mathematical model was adjusted to represent the relationship between the independent variable (TRO inclusion levels) and the corresponding dependent variable under evaluation.

Table 11. Sensory characteristics of eggs produced by commercial hens fed diets containing increasing levels of tambaqui residual oil (TRO).

Variables	TRO Levels, %				p-Value 1	CV 2 %	Model 3	R2
	0	1.5	3.0	4.5
Appearance	5.40	5.91	6.02	6.48	0.05	3 > 53	Y = 5.45 + 0.2233x	0.95
Texture	6.68	6.42	6.34	6.60	0.86	2.85	-	-
Aroma	6.28	6.14	6.11	5.82	0.05	2.72	Y = 6.299 − 0.094x	0.88
Color	5.71	6.28	6.37	6.88	0.05	2.92	Y = 5.77 + 0.24x	0.94
Taste	7.00	6.51	6.34	6.22	0.05	3.10	Y = 6.894 − 0.1673x	0.89

¹ The means presented in the row differ significantly when p ≤ 0.05. ² CV = Coefficient of variation. ³ The mathematical model was adjusted to represent the relationship between the independent variable (TRO inclusion levels) and the corresponding dependent variable under evaluation.

4. Discussion

The inclusion of TRO in the diet of commercial hens, by not significantly affecting feed intake, feed conversion, and egg production, may indicate that the birds maintained their energy intake and feed efficiency regardless of the TRO level in the diet, similar to results reported by Lelis et al. [45] and Ceylan et al. [46]. However, egg weight increased significantly in diets containing 1.5% and 3.0% TRO, and this effect may be associated with the supply of essential fatty acids present in TRO, such as oleic acid and linoleic acid, which may have favored lipid deposition in the yolk [22,46,47]. However, when the inclusion level reached 4.5%, this improvement in egg weight was not maintained, possibly due to an imbalance in lipid metabolism or the utilization of available nutrients [27,48,49].

Other egg quality variables were also influenced by TRO inclusion, especially parameters such as yolk color, Haugh unit, and specific gravity, indicating that TRO may, even subtly, affect different physical parts of the eggs. The yolk color, which increased linearly with TRO inclusion, may be associated with the possible presence of liposoluble pigments deposited in the yolk from TRO, as observed in other oils when incorporated into poultry diets [22,50,51]. The results for the Haugh unit, which measures the internal quality of the egg, indicated improvement at intermediate TRO levels (1.5% and 3.0%) but a slight reduction at the highest level (4.5%), possibly related to changes in albumen composition, potentially due to the impact of TRO on water retention and egg white viscosity [49,52].

Regarding egg specific gravity, its linear decrease with increasing TRO in the diet suggests reduced mineral deposition in the eggshell, which may be attributed to a possible interference of lipids from TRO in the absorption or metabolism of calcium and phosphorus [53]. This phenomenon is also commonly observed when higher levels of oils are incorporated into poultry diets [22,51,54]. Although eggshell thickness did not show significant changes between treatments, the reduction in specific gravity may indicate a subtle impact on shell structure. In this regard, the stability of yolk and albumen pH reinforces the idea that TRO primarily influenced the lipid fraction of the egg, without causing major changes in the protein stability of the albumen [22,49]. Thus, these results suggest that moderate TRO levels (1.5% to 3.0%) may be beneficial for egg quality by increasing yolk weight and color, while higher levels may compromise mineral deposition in the shell and internal egg quality.

From a physiological perspective, the inclusion of TRO in the diet of commercial hens significantly influenced hematological parameters, indicating possible physiological adjustments in the birds’ metabolism as TRO inclusion levels increased, even though this increase did not alter the dietary energy density [24,49]. The linear reduction in the number of erythrocytes with increasing TRO levels suggests a possible effect on erythropoiesis or the longevity of red blood cells [55,56]. Conversely, hemoglobin concentration and hematocrit followed a quadratic model, with an initial decline followed by recovery at the highest TRO level (4.5%). This pattern may be related to the adaptation of the birds’ metabolism to a diet with a more diverse lipid content [55,57], which can alter the availability of nutrients for blood cell synthesis.

Biochemical parameters were also affected, particularly total protein, albumin, cholesterol, and triglyceride levels. Total protein and albumin levels decreased linearly as TRO levels increased, which may indicate reduced hepatic protein synthesis or a redistribution of nitrogen metabolism toward other physiological functions [24,55]. Cholesterol levels, on the other hand, increased linearly, reaching significantly higher values in the diet containing 4.5% TRO. This effect is likely related to the lipid composition of the oil, especially its high content of monounsaturated fatty acids, which are known to modulate hepatic cholesterol metabolism [56,58]. Meanwhile, triglyceride levels significantly decreased, suggesting increased lipid utilization as an energy source or a shift in lipid metabolism toward yolk formation [59].

Regarding the differential leukocyte count, TRO inclusion appeared to have an immunomodulatory effect on the birds. The percentage of heterophils decreased in groups receiving 1.5% and 3.0% TRO but increased again in the 4.5% TRO diet, suggesting that intermediate levels may have a positive effect on the birds’ inflammatory response [60,61]. The linear reduction in eosinophils may be associated with a lower allergic or parasitic response, while the initial increase followed by a decline in typical lymphocytes suggests a regulation of the adaptive immune system [61,62]. Overall, these results indicate that TRO can influence the hematological and metabolic homeostasis of birds, with its impact varying depending on the inclusion level in the diet [63], potentially providing benefits at moderate levels while posing physiological challenges at higher levels.

As a consequence of these physiological effects, the chemical composition of the eggs was significantly influenced by the inclusion of TRO in the diet of commercial hens, reflecting modifications in the moisture, lipid, and protein content of the yolk. The moisture content of the eggs, which showed a linear reduction as TRO levels in the diet increased, suggests a possible replacement of the aqueous fraction with a greater deposition of macronutrients, primarily lipids [22,30], as well as a reduction in water retention within the egg content [49,54]. Similarly, the lipid content of the eggs increased significantly as TRO levels rose, not only corroborating the previous result but also indicating a potential transfer of TRO’s lipid profile to the egg yolk, given that TRO is rich in monounsaturated and polyunsaturated fatty acids [27,46]. This deposition of essential fatty acids in the yolk enhances the egg’s nutritional value, potentially making it a more beneficial dietary source for human consumption [64]. Furthermore, the increase in the lipid fraction of the egg may also be related to the metabolic adjustments previously observed in the birds, which redirected more dietary lipids toward yolk formation, especially at higher TRO levels, a result similar to that reported in other studies using oils in poultry diets [48,51,52].

The protein content also exhibited a linear increase with TRO inclusion, suggesting that the oil may have played a positive role in protein or other solid particles retention in the egg [65]. This effect can be explained by the improved energy availability provided by TRO, allowing for a greater allocation of amino acids toward protein deposition in the eggs [66,67]. However, the mineral content of the eggs was not significantly affected by the TRO levels in the diet, even though structural or physical changes in the eggshell may have occurred, as previously discussed. These results reinforce that the use of TRO can be advantageous in enhancing the nutritional value of eggs by increasing their lipid and protein fractions without compromising the mineral balance of the final product.

As observed in the chemical composition, the results of lipid oxidation and the fatty acid profile of the yolks, which were significantly influenced by the inclusion of TRO in the diet of commercial hens, reflect changes in the oxidative stability and chemical composition of the eggs. The TBARS values, which increased with the inclusion of 1.5% TRO and then decreased at higher levels (3.0% and 4.5%), can be explained by the higher content of unsaturated fatty acids in the yolk, which are more susceptible to peroxidation [27,68,69]. However, this effect may also be attributed to the presence of natural antioxidant compounds in TRO, such as tocopherols and other polyphenols, which may have mitigated the oxidative process at higher inclusion levels [69].

More specifically, TRO inclusion in the diet significantly affected the fatty acid profile of the yolk, increasing the concentration of polyunsaturated fatty acids, particularly those of the omega-3 and omega-6 series. These changes result from the direct addition of TRO to the diets and the subsequent transfer of its properties to the eggs. This modification in the lipid profile of eggs due to TRO inclusion may provide nutritional benefits for consumers, as omega fatty acids, especially omega-3, are associated with positive effects on cardiovascular and brain health [21,70]. Meanwhile, saturated and monounsaturated fatty acids showed less pronounced variations with TRO inclusion, indicating that the oil’s impact was more concentrated in the polyunsaturated fraction of the yolk [54,68]. This increase in essential fatty acids reinforces the feasibility of TRO as a functional ingredient in hen diets, enabling the production of nutritionally enriched eggs [64]. However, the effects on lipid oxidation suggest that an optimal balance in inclusion levels is necessary, as excessively high concentrations may require additional strategies to maintain the oxidative stability of the final product.

Finally, the sensory characteristics of the eggs, a key indicator of commercial acceptance, were significantly influenced by the inclusion of TRO in the diet of commercial hens. The visual appeal of the eggs increased linearly with TRO levels, possibly due to the intensification of yolk coloration, which became more vibrant with oil inclusion—an effect previously observed with the addition of other oils in hens’ diets [22,70,71]. This change is generally attributed to the presence of liposoluble pigments in the oil, such as carotenoids and xanthophylls, which are directly incorporated into the yolk and enhance its hue, increasing consumer attractiveness [72,73].

However, despite this positive effect on appearance and color, the aroma and flavor attributes declined as TRO levels in the diet increased. This sensory rejection may be related to the presence of volatile compounds derived from lipid oxidation or secondary metabolites of fatty acids present in tambaqui by-products, a phenomenon also reported in other studies using fish oils in hens’ diets [22,73]. The increased concentration of polyunsaturated fatty acids in the yolk, particularly those of the omega-3 series, may have led to the formation of aldehydes and ketones, which are responsible for undesirable odors and flavors [72,74]. This explains why eggs from hens fed higher TRO levels (≥3.0%) had lower sensory acceptance, especially regarding taste. Finally, the texture of the eggs did not show significant variations between treatments, suggesting that TRO did not negatively alter the protein structure of the egg white and yolk.

5. Conclusions

The results of this study demonstrate that TRO can be used as a sustainable ingredient in the diets of commercial laying hens, promoting both egg enrichment and the valorization of aquaculture by-products. While productive performance was not affected, moderate inclusion levels (1.5–3.0%) improved egg weight, yolk pigmentation, and internal quality (Haugh unit), in addition to inducing favorable metabolic adjustments without compromising bird health. Conversely, high inclusion (4.5%) impaired oxidative stability, negatively altered hematological parameters, and reduced sensory acceptance, especially in terms of aroma and flavor.

These findings indicate that TRO can be strategically incorporated into laying hen diets to enhance egg nutritional quality and contribute to circular production systems, provided that inclusion levels are carefully balanced to avoid adverse effects. Future research should aim to define optimal levels of TRO inclusion and explore complementary strategies, such as antioxidant supplementation, to mitigate lipid oxidation and improve the sensory acceptance of TRO-enriched eggs.