INTRODUCTION

The increasing cost and competition for conventional feed ingredients in pig production systems have intensified the search for alternative and locally available protein sources, particularly in tropical regions [1, 2]. In this context, the use of fast-growing forages with high nutritional potential represents a viable strategy to reduce dependence on soybean meal and improve feed autonomy for both smallholder and commercial producers [3]. Tithonia diversifolia (Hemsl.) A. Gray is a perennial shrub widely distributed in tropical ecosystems, characterized by rapid biomass accumulation, adaptability to low-fertility soils, and a notable crude protein and mineral content, which has supported its use in ruminant feeding systems [4].

However, despite increasing agronomic interest, the nutritional characterization of T. diversifolia for monogastric animals remains limited. Existing studies have primarily focused on ruminant systems or partial proximate analyses, with limited information available on amino acid balance, digestible and metabolizable energy (ME) estimation, and graded responses in digestibility under simulated monogastric conditions [5–7]. Furthermore, there is no standardized dataset describing how varying inclusion levels of T. diversifolia influence the nutritional and energetic profile of formulated pig diets, thereby restricting its practical application in swine nutrition [8]. To the best of our knowledge, this is the first report describing the amino acid profile and in vitro energy estimation of T. diversifolia meal specifically formulated for pig diets under Amazonian Ecuadorian conditions.

Despite the recognized agronomic and nutritional potential of T. diversifolia, its application in monogastric nutrition remains inadequately explored. Most existing studies have focused on ruminant feeding systems, where the high-fiber content is better tolerated, while investigations in pig nutrition are limited and often restricted to basic proximate composition. Critically, there is a lack of comprehensive data integrating amino acid profiling, energy estimation, and digestibility responses under controlled in vitro conditions that simulate monogastric digestion. Furthermore, previous studies have not systematically evaluated graded inclusion levels of T. diversifolia within complete diets, resulting in insufficient evidence regarding optimal inclusion thresholds and their impact on nutrient utilization. The absence of standardized datasets linking chemical composition with functional nutritional outcomes, particularly under tropical production conditions, represents a significant limitation. This knowledge gap constrains the practical incorporation of T. diversifolia into pig diets and highlights the need for a holistic evaluation that combines compositional, energetic, and digestibility parameters.

Therefore, the present study was designed to provide a comprehensive evaluation of T. diversifolia forage meal as a potential alternative ingredient in pig nutrition. Specifically, the objectives were to (i) characterize the proximate composition, mineral content, and amino acid profile of T. diversifolia meal; (ii) assess the effects of increasing inclusion levels (0%, 10%, 15%, 20%, and 25%) on the chemical composition and in vitro digestibility of formulated pig diets; and (iii) estimate digestible energy, ME for growing pigs, ME for finishing pigs, and net energy (NE) using established predictive models. By integrating compositional and functional analyses, this study aims to generate evidence-based insights into the nutritional value and feasible inclusion levels of T. diversifolia, thereby supporting its application as a sustainable feed resource in tropical pig production systems.

MATERIALS AND METHODS

Ethical approval

This study was conducted in accordance with internationally accepted ethical standards for research and reporting. All procedures complied with the ARRIVE 2.0 guidelines for transparent reporting of scientific studies and adhered to the principles outlined in the EU Directive 2010/63/EU on the protection of animals used for scientific purposes. Ethical clearance was obtained from the Ethical Committee of the Scientific Council, Faculty of Agricultural Sciences, Universidad Central “Marta Abreu” de Las Villas, Cuba (Protocol No. 57/2021; approved on March 14, 2021).

The experimental design involved exclusively in vitro analyses of plant-based feed materials, and therefore no live animals were subjected to experimental procedures, handling, or invasive sampling. Consequently, no animal welfare risks were identified. All laboratory procedures were conducted under standard biosafety conditions in accordance with institutional laboratory safety protocols.

Plant material (T. diversifolia) was collected from agricultural land in Napo Province, Ecuador, with full compliance to national agricultural and environmental regulations. The collection did not involve protected species or restricted areas, and no specific permits were required under local legislation. Handling, processing, and disposal of plant materials followed established environmental safety guidelines to minimize ecological impact.

The study design, data handling, and reporting were conducted in accordance with principles of scientific integrity, reproducibility, and transparency. No human participants or personal data were involved in this study.

Study period and location

The study was conducted from March to July 2025 in Napo Province, Tena Canton, Misahuallí Parish (Santo Urku community), Ecuador (0°57′01″S; 77°51′46″W; 565 m a.s.l.). The region has a humid tropical climate (Af, Köppen classification), with an annual rainfall of approximately 3800 mm, a mean temperature of 24°C–26°C, and relative humidity of around 85%. Soils are sandy loam, acidic (pH 4.5–5.5), and of moderate fertility.

In vitro digestibility tests were performed at the Natural Products Laboratory, Universidad Regional Amazónica Ikiam (Tena, Ecuador). Harvesting was carried out at a cutting height of 50 cm from the ground [9], and the evaluation of green and dry matter production was conducted using the method proposed by Rodríguez and Torres [10].

Chemical composition

The bromatological characterization of the meal was conducted using 2 kg samples of green forage, analyzed in triplicate for each treatment. Samples were dried using a vertical solar dryer at 55°C ± 2°C for 72 h until reaching approximately 10% residual moisture. The dried material was ground using a Willey mill (Thomas Scientific, USA) with a 1 mm sieve, homogenized, and stored in airtight polyethylene bags for subsequent analyses.

Proximate analysis was performed according to U. Florida [11, 12], including dry matter (DM), organic matter (OM), crude protein (CP; Kjeldahl method, N × 6.25), ether extract (EE), and nitrogen-free extract (NFE). Fiber fractionation (neutral detergent fiber [NDF] and acid detergent fiber [ADF]) was determined according to Van Soest et al. [13]. Additionally, analyses followed AOAC International (2016) methods: DM (934.01), CP (976.05), EE (920.39), and ash (942.05). Lignin content was determined using permanganate oxidation.

Gross energy (GE), ME, and digestible energy (DE) were estimated using predictive equations based on nutritional composition (Table 1) [14, 15]. Mineral composition, including phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), copper (Cu), zinc (Zn), and manganese (Mn), was analyzed using atomic absorption spectrophotometry [16].

CP = Crude protein, EE = Ether extract, NDF = Neutral detergent fiber, ADF = Acid detergent fiber, NFE = Nitrogen-free extract.

Sixteen amino acids were quantified by high-performance liquid chromatography (HPLC; Agilent 1100 system, Ecuachemlab Cía. Ltda., Ecuador) following acid hydrolysis (6 N HCl, 110°C, 24 h). Tryptophan was analyzed separately using alkaline hydrolysis with fluorescence detection. Data were expressed as percentage of sample dry weight (% w/w).

In vitro digestibility of T. diversifolia

The study was conducted at the Natural Products Laboratory of Universidad Regional Amazónica Ikiam using five treatments based on formulated diets containing different inclusion levels of T. diversifolia meal (10%, 15%, 20%, and 25%), along with a control treatment (0%). Each treatment was evaluated with four replicates under a completely randomized design.

Proximate chemical composition parameters of all treatments (DM, EE, CP, and OM) were determined (Table 2). Based on in vitro OM digestibility values, DE and NE were estimated using predictive equations described by [17]. In addition, ME for growing pigs and finishing pigs was calculated according to the models proposed by previous studies [18, 19] (Table 3).

NDF = Neutral detergent fiber, ADF = Acid detergent fiber, EE = Ether extract, CP = Crude protein, St = Starch, dvMO = in vitro digestibility of organic matter, DE = Digestible energy.

Five experimental diets were formulated containing 0%, 10%, 15%, 20%, and 25% T. diversifolia meal as a partial replacement for soybean meal and fish meal. The control (basal) diet was formulated using corn meal, soybean meal, fish meal, wheat bran, and a vitamin–mineral premix (Table 4). All diets were designed to be iso-nitrogenous and iso-energetic, meeting the nutrient requirements for growing pigs according to NRC (National Research Council, 2012) [20].

Crude protein: 174 g/kg DM, Metabolizable energy: 14.2 MJ/kg DM, Ether extract: 35 g/kg DM, Crude fiber: 42 g/kg DM. DM = Dry matter.

Statistical analysis

Statistical analyses were performed using R software version 4.3.1 (R Core Team, 2023, Vienna, Austria) under a completely randomized design with one-way analysis of variance. Data are expressed as mean ± standard deviation, and significance was declared at p < 0.05 using Tukey’s post hoc test.

RESULTS

Chemical composition of T. diversifolia flour

T. diversifolia flour had a CP content of 318.127 (g/kg DM), with high levels of crude fiber (366.146 g/kg DM) and ash (176.471 g/kg DM). EE content was low (10.444 g/kg DM), while the neutral detergent fiber (NDF) and ADF contents were 534.214 (g/kg DM) and 533.013 (g/kg DM), respectively. Lignin reached 292.917 (g/kg DM), indicating a structurally complex fibrous fraction. In addition, the sample had appreciable levels of minerals such as calcium (32.413 g/kg DM), potassium (39.616 g/kg DM), and phosphorus (7.203 g/kg DM).

Amino acid profile

Chromatographic analysis revealed a total amino acid concentration of 12.38% (Figure 1). The most abundant amino acids were aspartic acid (3.37%), serine (1.46%), leucine (1.35%), valine (1.08%), and glycine (0.93%). Significant amounts of lysine (0.85%) were detected, whereas methionine was low (0.26%). Tryptophan was detected in minimal concentrations (0.0004%), and neither isoleucine nor glutamic acid were detected.

In vitro digestibility and chemical composition of experimental diets

The results of the chemical analysis (Table 5) of experimental diets with different levels of T. diversifolia flour inclusion showed no statistically significant differences (p > 0.05) in DM, OM, EE, or ADF contents among treatments. However, significant differences were observed in CP content (p = 0.03) and NDF content (p = 0.02).

The values refer only to treatments studied according to analysis of variance (analysis of variance). Values are expressed as mean (X) ± standard deviation (SD). Control: 100 % feed (finishing pig concentrate), T1: 90 % feed + 10 % T. diversifolia, T2: 85 % feed + 15 % T. diversifolia flour, T3: 80 % feed + 20 % T. diversifolia, T4: 75 % feed + 25 % T. diversifolia flour. Identical superscript letters within the same column did not differ statistically (p > 0.05) according to Tukey test. Different superscript letters within the same column differed statistically (p < 0.05) according to Tukey test.

Crude protein content showed a progressive increase from the control treatment (173.9 g/kg DM) to treatment T3 (20%), reaching a maximum value of 178.38 g/kg DM, followed by a decrease in T4 (171.68 g/kg DM). Post hoc analysis indicated that T3 differed significantly from the treatment with the highest inclusion level (T4), whereas treatments T1, T2, and the control exhibited intermediate values.

In terms of NDF, treatment T3 (20%) showed the highest value (201.23 g/kg DM), which was significantly higher than T2 (15%) with 188.78 g/kg DM. The remaining treatments did not show significant differences among them.

Starch, proximate stability, and energy trends

Total starch content (ST) ranged from 390.83 g/kg DM (T4) to 405.88 g/kg DM (T3), with no statistical differences (p = 0.4019). EE values remained constant (29.98–31.38 g/kg DM), with no evidence of treatment effects (p = 0.9033). DM and OM also remained stable among treatments (p > 0.4), indicating that the inclusion of T. diversifolia meal up to 25% does not significantly affect these fractions.

Energy trends derived from the in vitro digestibility assays (Figure 2) indicated a gradual numerical decline in DE, ME, and NE values as the inclusion level of T. diversifolia increased from 0% to 25%.

In vitro digestibility and energy estimation

The in vitro digestibility of DM and OM showed a numerical reduction with increasing levels of T. diversifolia inclusion (Table 6). However, no significant differences (p > 0.05) were observed among the 10%–25% inclusion treatments, indicating that the reduction pattern was not strictly progressive. The control diet (0%) exhibited slightly higher digestibility coefficients, followed by minor decreases in the experimental diets, although the overall variation remained within an acceptable range.

The values refer only to treatments studied according to analysis of variance (analysis of variance). Values are expressed as mean (X) ± standard deviation (SD). Control: 100 % feed (finishing pig concentrate), T1: 90 % feed + 10 % Tithonia diversifolia, T2: 85 % feed + 15 % T. diversifolia flour, T3: 80 % feed + 20 % T. diversifolia, T4: 75 % feed + 25 % T. diversifolia flour. Identical superscript letters within the same column did not differ statistically (p > 0.05) according to Tukey test. Different superscript letters within the same column differed statistically (p < 0.05) according to Tukey test.

Correspondingly, the estimated energy values (DE, ME, and NE) did not differ statistically (p > 0.05) among treatments, although a mild numerical downward trend was observed with increasing inclusion levels. These findings suggest that T. diversifolia meal can be incorporated up to 25% in pig diets without significantly compromising in vitro digestibility or energy content.

DISCUSION

Nutritional composition of T. diversifolia flour

T. diversifolia flour has a nutritional profile that stands out for its high CP content (318.127 g/kg DM), making it a promising alternative in animal diets, especially in production systems seeking accessible and sustainable protein sources. This value falls within the range reported by various authors, who have documented concentrations between 264.100 g/kg DM and 348.100 g/kg DM depending on factors such as the physiological age of the plant, cutting frequency, and agroecological conditions [21].

The crude fiber content was high (366.146 g/kg DM), as were the NDF (534.214 g/kg DM) and ADF (533.013 g/kg DM) values, reflecting a considerable structural fraction. These values suggest limited DM digestibility, especially for monogastric animals, although they could be functional as effective fiber in ruminants [4]. Lignin reached 292.917 (g/kg DM), a value that compromises the digestibility of the fibrous fraction by forming lignocellulosic complexes that are resistant to enzymatic action [22]. It has been observed that lignin levels above 180.100 (g/kg DM) can significantly reduce nutrient degradability in monogastric animals [23], and therefore this value must be carefully considered when formulating diets.

The low EE content (10.444 g/kg DM) is characteristic of shrubby forages, limiting their energy contribution via lipids. However, the energy balance can be maintained by including other energy-rich ingredients in the diet [24]. Ash accounted for 176.471 (g/kg DM), indicating significant mineral richness. This value is often associated with the presence of essential macro- and microminerals, as evidenced by the levels of calcium (32.413 g/kg DM), potassium (39.616 g/kg DM), and phosphorus (7.203 g/kg DM). The calcium content is particularly relevant for the diets of growing and lactating animals [25], while potassium and phosphorus play fundamental roles in osmotic regulation and energy metabolism [26].

The magnesium level (4.682 g/kg DM) and values for trace elements such as iron (0.267 g/kg DM), zinc (0.113 g/kg DM), manganese (0.068 g/kg DM), and copper (0.003 g/kg DM) are adequate and comparable to other tropical forage species [27]. The bioavailability of these elements must be evaluated based on their interaction with other dietary components, especially fiber and phytates [28]. Although the copper content was low, it can be supplemented with external sources according to the requirements of the animal species [29].

Overall, the nutritional profile of T. diversifolia flour indicates interesting potential as a protein–mineral supplement in diets for ruminants and, to a lesser extent, for monogastric animals, provided that its high-fiber and lignin content is considered. Its use could be particularly suitable in silvopastoral systems or as a component of multinutritional mixtures [30]. However, further in vivo or in vitro digestibility studies are recommended to confirm its effective nutritional value, as well as acceptance and palatability tests in the target species [31].

Amino acid profile

Chromatographic analysis of T. diversifolia flour revealed a total amino acid content of 12.38%, confirming its potential as a protein source for animal feed systems in tropical areas. This value significantly exceeds the contribution of conventional tropical grasses (Cenchrus clandestinum: 8–10% CP) and is comparable to the range reported in forage legumes such as Leucaena leucocephala (18%–25%) [5].

Among essential amino acids, leucine and valine predominated, with values of 1.35% and 1.08%, respectively. These amino acids are essential for muscle metabolism and protein synthesis, particularly in growing animals [32]. The concentration of lysine (0.85%) is also noteworthy, as it is commonly a limiting amino acid in plant-based diets, highlighting the potential of T. diversifolia as a complementary ingredient in monogastric diets [33].

However, low concentrations of methionine (0.26%) and an almost negligible presence of tryptophan (0.0004%) were observed, along with the absence of isoleucine and glutamic acid, which represents a nutritional limitation. The deficiency of methionine, an essential sulfur-containing amino acid, can compromise protein utilization efficiency [34]. Therefore, it is advisable to formulate diets that compensate for this deficiency, either through methionine-rich ingredients such as treated soybean meal or through synthetic supplementation [35].

Among non-essential amino acids, aspartic acid (3.37%), serine (1.46%), and glycine (0.93%) were predominant. These compounds play key roles as metabolic precursors and in the synthesis of nucleotides, collagen, and other structural components [36–38].

Overall, the amino acid profile suggests that T. diversifolia is a valuable resource for diet formulation; however, its use must be carefully balanced to address deficiencies in essential amino acids. Integration of this ingredient into animal diets should be supported by evaluations of true protein digestibility and productive performance, particularly in species sensitive to amino acid imbalances [39].

In vitro digestibility and nutritional implications

The results of the chemical analysis (Table 5) indicate that the progressive inclusion of T. diversifolia flour up to 25% did not significantly affect the DM, OM, EE, or ADF contents (p > 0.05), which is consistent with previous findings [6]. This behavior suggests that the incorporation of this forage does not substantially alter the structural or non-DE fractions of the diet [40]. However, significant effects were observed for CP (p = 0.03) and NDF (p = 0.02), which are critical parameters in diet formulation.

CP content increased up to treatment T3 (20%), reaching 178.38 g/kg DM, followed by a decrease in T4 (25%). This pattern suggests that moderate inclusion levels may enhance protein intake, possibly due to the high concentration of soluble nitrogen in the leaves of this species. The reduction observed in T4 may be attributed to protein dilution associated with increased fiber content, a phenomenon also reported in diets with high inclusion of fibrous forages [41]. Post hoc analysis confirmed a significant difference between T3 and T4, indicating that 20% may represent an optimal inclusion level for maximizing protein content.

NDF content was highest in T3 (201.23 g/kg DM), significantly exceeding that of T2 (188.78 g/kg DM). This increase in effective fiber may contribute to satiety in monogastric animals and stimulate rumination in ruminants [42], although it may also reduce digestibility. Despite variations in CP and NDF, total starch content remained stable (p = 0.40), suggesting that non-structural carbohydrate fractions were not affected by T. diversifolia inclusion [43]. EE content also remained constant (p = 0.90), confirming the limited lipid contribution of this forage.

In vitro DM digestibility showed a significant decrease (p = 0.0128) with increasing inclusion levels, particularly in treatment T3 (31.68%) compared with the control (42.88%). Although no significant differences were observed in in vitro digestibility of OM or in estimated energy values (DE, MEcr, MEfc, and NE), a general numerical decline (p > 0.1) was evident as inclusion levels increased. This reduction may be associated with increased structural fiber and lignified fractions, which limit enzymatic degradation and nutrient availability [44]. According to Weimer [45], elevated lignin and hemicellulose levels reduce cell wall degradability and negatively impact digestive efficiency.

Despite this trend, treatments T1 (10%) and T2 (15%) maintained moderate energy losses and acceptable digestibility coefficients (>36%) (Table 6), indicating that low to moderate inclusion levels may be viable without significantly compromising nutritional value [46]. This finding is relevant for practical applications, as it supports the use of T. diversifolia as a functional and economical feed component without adversely affecting animal performance [47]. Although DE and NE values did not differ significantly (p > 0.45), the control diet maintained the highest values (16.83 kcal/kg and 9.44 kcal/kg, respectively), likely reflecting the higher efficiency of conventional diets lacking high-fiber components [48].

CONCLUSION

The present study demonstrated that T. diversifolia forage meal possesses a nutritionally valuable profile characterized by high CP content (318.127 g/kg DM) and appreciable mineral levels, alongside a moderate amino acid composition dominated by leucine, valine, and lysine. However, the high-fiber fractions, particularly NDF (534.214 g/kg DM), ADF (533.013 g/kg DM), and lignin (292.917 g/kg DM), were identified as critical limiting factors affecting nutrient utilization. The in vitro evaluation revealed that increasing inclusion levels of T. diversifolia resulted in a significant reduction in DM digestibility (p < 0.05), while OM digestibility and estimated energy values (DE, ME, and NE) remained statistically unaffected (p > 0.05), despite a consistent numerical decline.

From a practical perspective, inclusion levels of 10%–15% appear to represent an optimal balance between nutritional contribution and digestibility, whereas higher inclusion levels (20%–25%) may compromise feed efficiency due to increased structural fiber and lignification. These findings suggest that T. diversifolia can be effectively utilized as a complementary protein–mineral source in pig diets, particularly in tropical production systems where conventional feed resources are limited or costly. Its incorporation may contribute to improved feed sustainability and reduced dependence on soybean-based ingredients.

A key strength of this study lies in its integrated approach combining chemical composition, amino acid profiling, and in vitro digestibility with energy estimation under controlled conditions. This provides a more comprehensive understanding of the functional nutritional value of T. diversifolia compared with studies limited to proximate analysis. Additionally, the evaluation of graded inclusion levels offers practical insights for feed formulation.

However, the study is limited by its reliance on in vitro methodologies, which may not fully replicate in vivo digestive and metabolic responses in pigs. Furthermore, factors such as palatability, anti-nutritional compounds, and amino acid bioavailability were not assessed, which may influence the practical applicability of the results.

Future research should focus on in vivo feeding trials to validate digestibility, growth performance, and health outcomes in pigs. Investigations into processing methods to reduce fiber and lignin content, as well as strategies to correct amino acid imbalances, are also recommended. Additionally, economic feasibility and long-term sustainability assessments will be essential to support large-scale application.

In conclusion, T. diversifolia forage meal represents a promising alternative feed resource with significant potential for sustainable pig production, provided that its inclusion is optimized to balance nutritional benefits and digestibility constraints.

DATA AVAILABILITY

The data generated and analyzed during this study are included in this published article. Additional data are available from the corresponding author upon reasonable request.

AUTHORS’ CONTRIBUTIONS

JADLTM: Conceptualization, study design, supervision, data analysis, and writing–original draft preparation, RLO: Study design, data interpretation, and critical revision of the manuscript, VCAY: Experimental design, data collection, and manuscript editing, JEDL: Sample processing, data collection, and manuscript revision, MAML: Laboratory analyses, data collection, and manuscript editing. All authors have read and approved the final manuscript.

COMPETING INTERESTS

The authors declare that they have no competing interests.

PUBLISHER’S NOTE

Veterinary World remains neutral with regard to jurisdictional claims in the published institutional affiliations.

ACKNOWLEDGMENTS

The authors express their sincere gratitude to the Universidad Regional Amazónica Ikiam for providing laboratory facilities and logistical support. Special thanks are extended to the technical staff of the Natural Products Laboratory for their collaboration during sample processing and analysis.

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