Influence of protein-energy ratios and feed processing on the performance of juvenile pacu (Piaractus mesopotamicus): implications for sustainable aquacultureAbstract
The search for more efficient and environmentally sustainable aquaculture systems has driven the development of precision nutrition strategies. Growth performance and metabolism were investigated in juvenile pacu (Piaractus mesopotamicus) fed either extruded or pelleted diets formulated with three energy-to-protein (E:P) ratios (12, 14, and 16 MJ/kg Crude energy (CE)/Crude protein (CP), respectively). A total of 180 fish (10 per tank, triplicate tanks per treatment) were fed for 90 days. Key metabolites (glucose, lactate, free amino acids, ammonia, triglycerides) and enzyme activities (ALAT, ASAT, GDH, LDH, MDH) were analyzed in liver, white muscle, and plasma. Data were analyzed by one‑way ANOVA followed by Tukey’s test (p < 0.05). Fish fed pelleted diets showed no differences in weight gain (WG) among E:P ratios. In contrast, extruded diets promoted a peak in WG at E:P 14 (245 ± 12 g/kg). Fish fed high‑E:P pelleted diets exhibited increased hepatic ammonia (+40%) and triglyceride synthesis in liver and white muscle, indicating enhanced aerobic metabolism. Fish fed extruded diets showed reduced reliance on aerobic metabolism and no elevation in hepatic ammonia. Although pelleted E:P12 resulted in a favorable metabolic profile (lower ammonia, no aerobic stress), extruded E:P14 proved more efficient in terms of growth performance and is therefore recommended for pacu farming.
Keywords:
sustainability; fish nutrition; digestible protein; gluconeogenesis; metabolic efficiency
Resumo
A busca por sistemas aquícolas mais eficientes e ambientalmente sustentáveis tem impulsionado o desenvolvimento de estratégias de nutrição de precisão. O desempenho de crescimento e o metabolismo foram investigados em juvenis de pacu (Piaractus mesopotamicus) alimentados com rações extrusadas ou peletizadas formuladas com três relações energia-proteína (E:P) (12, 14 e 16 MJ/kg de Energia Bruta (EB)/Proteína Bruta (PB), respectivamente). Um total de 180 peixes (10 por tanque, tanques em triplicata por tratamento) foram alimentados por 90 dias. Metabólitos-chave (glicose, lactato, aminoácidos livres, amônia, triglicerídeos) e atividades enzimáticas (ALAT, ASAT, GDH, LDH, MDH) foram analisados no fígado, músculo branco e plasma. Os dados foram analisados por ANOVA one-way seguida pelo teste de Tukey (p < 0,05). Peixes alimentados com dietas peletizadas não apresentaram diferenças no ganho de peso (GP) entre as relações E:P. Em contraste, as dietas extrusadas promoveram um pico no GP na relação E:P 14 (245 ± 12 g/kg). Peixes alimentados com dietas peletizadas de alta relação E:P apresentaram aumento da amônia hepática (+40%) e da síntese de triglicerídeos no fígado e no músculo branco, indicando maior metabolismo aeróbico. Peixes alimentados com dietas extrusadas mostraram menor dependência do metabolismo aeróbico e nenhuma elevação da amônia hepática. Embora a dieta peletizada E:P12 tenha resultado em um perfil metabólico favorável (menor amônia, sem estresse aeróbico), a dieta extrusada E:P14 mostrou-se mais eficiente em termos de desempenho de crescimento e é, portanto, recomendada para a criação de pacu.
Palavras-chave:
sustentabilidade; nutrição de peixes; proteína digestível; gliconeogênese; eficiência metabólica
1. Introduction
Growth performance in fish is dependent on metabolic efficiency (Boonanuntanasarn et al., 2017). The metabolic use of dietary carbohydrates, mostly as energy sources, is a desirable condition in fish nutrition (Sandre et al., 2017). This condition is expected to be optimized to levels at which protein dietary intake can be spared to the maximum extent (Coutinho et al., 2018). During the growth process, this macronutrient is supposed to be mostly derived from diets for body mass. Both levels of protein and energy must be adjusted into proper ratios to prevent undesirable effects such as fat deposition in the tissues (Kennedy et al., 2007). Therefore, the search for ideal protein/energy ratios is frequently investigated in several fish species of commercial interest to improve growth and production.
The efficiency in protein utilization is enhanced with appropriate amounts of dietary lipids (Sandre et al., 2017) and carbohydrates as well (Coutinho et al., 2018). However, despite an increase in nitrogen retention, an imbalance in protein/energy ratio may reduce growth rates and food conversion and increase body fat content (Kabir et al., 2018). Therefore, the rational inclusion of carbohydrates in fish feeding is crucial to optimize nutritional conditions (Sandre et al., 2017). An appropriate tool for estimating the optimal use of nutrients, in addition to the evaluation of growth performance, is gauging physiological conditions such as the metabolic profile.
The metabolic profile in fish is known to be altered according to the diet's nutrient content (Moro et al., 2010; Melo et al., 2016) and may reflect the availability of nutrients associated with adaptations in the digestive and absorption processes (Honorato et al., 2010). Thus, this use of nutrients can be accessed through the tissue's metabolic profile and the evaluation of the levels of key metabolites and their variation. For example, the improvement in protein catabolism resulting from increased dietary protein can be detected in the plasma concentrations of free metabolites such as amino acids (Corrêa et al., 2007; Melo et al., 2016).
Along with metabolite profiles, enzymes related to energy metabolism can be used as an index of nutritional status (De Almeida et al., 2011; Zhou et al., 2017). There are no reports on the physiological effects derived from diet processing methods in fish that have been reported (Venou et al., 2003; Ma et al., 2016). Extrusion is generally used in the manufacturing of diets for aquatic organisms (Mesquita et al., 2013; Dethlefsen et al., 2016; Cian et al., 2016) and is called a granulation process that differs from the granulation process without extrusion. Granulation is a common technique that has been widely used in recent years to produce dry foods in which nutrient mixtures are compressed under steam to produce expanded granules (Chen et al., 2018). According to Kumar et al. (2006) and Roychowdhury et al. (2020) extruded diets provide benefits in fish feeding due to the gelatinization of starch molecules that facilitates the digestive action of amylase.
Raw starch is a relatively poor source of energy due to its low digestibility (Enes et al., 2008; Honorato et al., 2010). Hence, extruded diets improve digestibility and, consequently, the availability of starch to the feeding organism. Some studies report improvements based on structural disarrangements in starch granules from different carbohydrate sources (Chen et al., 2018; Enes et al., 2008); therefore, structural changes must be considered in the dietary use of carbohydrates (Venou et al., 2003). Studies concerning such aspects are essential, particularly in the development of feeding for commercially relevant species. The freshwater omnivorous fish pacu (Piaractus mesopotamicus (Holmberg, 1887)) is a fruit-eating and warm-water species that presents high farming potential due to its rusticity, high growth rates, and high-quality filets. This species is easily adapted to artificial feeding, and therefore, improvements in the composition of commercial feed pellets have been extensively studied (Abimorad and Carneiro, 2007).
We now know that there is a difference in the digestibility of diets subjected to different cooking processes (Honorato et al., 2016), but we do not have information on the destination of these nutrients that are more available for the metabolism of fish.
Adequate protein-to-energy ratios in pacu diets also directly impact aquaculture sustainability. This is because balanced nutrition improves metabolic efficiency, reducing the excretion of nitrogen compounds (such as ammonia) and, consequently, greenhouse gas emissions associated with farming (Dawood, 2021; Ullah Khan et al., 2019). Thus, this strategy aligns with Brazil's goals of promoting carbon-neutral aquaculture, combining productivity with reduced environmental impact.
This study evaluated the adaptive responses to dietary protein-energy ratios produced through two types of food processing (pelletizing and extrusion) in pacu (Piaractus mesopotamicus) through growth performance and metabolic parameters.
2. Material and Methods
The experiment was performed in the Aquatic Organism Nutrition Laboratory at the Aquaculture Center of São Paulo State University (UNESP) in Jaboticabal, São Paulo state, Brazil. The procedures applied were in accordance with the animal research ethical principles established by the Brazilian College of Experimentation (COBEA) and approved by the Animal Research Ethics Committee (CEUA), protocol 292/2010 (CEUA/UNIGRAN). Although the experiment was conducted at UNESP, the ethical approval was obtained from the committee of Centro Universitário da Grande Dourados (UNIGRAN), as per the institutional agreement between the universities.
Fish were held in 2,000 L tanks with water at 27 oC and pH 6.9, filtered through bio-beds, and bubbled with air to saturation. Before the experimental feeding, fish were fed over two weeks during acclimatization with commercial pellets. After this period, the fish were transported to the experimental conditions.
2.1. Experimental design
One hundred and eighty acclimated pacus (Piaractus mesopotamicus), averaging 11 ± 0.7 g and 8.21 ± 0.03 cm, were distributed in 18 fiberglass tanks (10 fish per tank) with a capacity of 180 L each; these tanks were filled with constantly recirculating and filtered water. The water temperature was kept at 27 ± 1 °C and pH 7.1 ± 0.1, with 1.64 ± 1.2 mg L-1 NH4+ and 3.8 ± 0.4 mg L-1 dissolved oxygen. Although below the ideal for some species, pacu can tolerate moderate hypoxia, and all groups were maintained under identical conditions. Fish were fed the experimental diets three times a day to satiety for 90 days (Table 1). Experiments included six treatments performed in triplicate.
At the end of the experimental period, feeding was discontinued for 24 h before sampling; 30 fish per treatment (10 fish per tank, three replicates) were randomly sampled. The initial body weight (IW) and final body weight (FW) were determined to express weight gain (WG) as WG% = (FW- IW). Blood samples were collected from the caudal vein with heparinized syringes, and plasma aliquots were separated by centrifugation at 13400 x g for 3 min (Lyndon et al., 1993). Fish were anesthetized with 30 g L-1 eugenol (Inoue et al., 2003), euthanized by cervical separation, and liver and white muscle samples were collected and stored at -20 °C for further analyses.
2.2. Experimental diets
The ingredients used to prepare the diets were previously crushed in a hammer mill using a 0.5-mm sieve. Food items and rations were assessed in terms of their percent composition (AOAC, 2000), and diets were formulated and mixed with water to 200 g kg-1 moisture. The protein and energy levels corresponded to each experimental formulation. Prepared feed was kept at 5 °C to ensure quality.
Six experimental isoproteic diets (27%) with three levels of Crude energy (16, 17, and 18 MJ·kg-1) were prepared using two processes (pelletization and extrusion) (Table 1). Extrusion was conducted using the Extrutec equipment with a simple spiral and a nominal capacity of 10 Kg h-1. Pelletization was performed in a laboratory pelletizer (California Pellet Mill). The pellets' diameter and length were 1.2-1.5 mm and 1.5-3.0 mm, respectively.
The diets presented three ratios of energy (E)/ protein (P): 12, 14, and 16.
2.3. Biochemical determinations
Tissue sub-samples were homogenized with a Teflon pestle motor-driven tissue cell disruptor (IKA- T10 basic- Ultra Turrax) into 0.02 M Tris / 0.01 M phosphate buffer, pH 7.0, plus glycerol 1/1 (v/v), at 4 °C under an ice bath and kept at a ratio 1 : 10 (tissue : buffer). Homogenates were centrifuged at 13400 x g for 3 min (Centrifuge Eppendorf 5415R), and the supernatants (crude homogenates) were preserved for analyses.
Protein-free acidic extracts were prepared from the homogenates by adding 1:10 of 20% trichloroacetic acid, followed by centrifugation at 21000 x g for 3 min. Neutral water extracts were prepared from tissue homogenates centrifuged at 21000 x g for 3 min. Metabolites from plasma or tissue extracts were colorimetrically determined. In the acidic protein-free extracts, it was quantified: total sugar (Dubois et al., 1956), pyruvate (Lu, 1939), lactate (Harrwer and Brown, 1972) and ammonia (Gentzkow and Masen, 1942). In the neutral extracts, it was determined the concentration of free amino acids (Copley, 1941) and free fatty acid (Norvák, 1965).
Glycogen was quantified in alcoholic precipitates after tissue alkaline disruption according to (Bidinotto et al., 1997). Triglycerides (TG) were estimated in total plasma at 510 nm with LABTEST enzymatic kit, Triglycerides Liquiform, Ref.: 87-2/100 (Labtest, 2026). Plasma and tissue protein concentrations were determined at 405 nm against 1 mg mL-1 of bovine serum albumin (Bradford, 1976). The metabolite concentrations are expressed in µmol-1 (mg of tissue); triglycerides in mg dL-1; glycogen in µmol of glycosyl-glucose units-1 (mg of tissue).
2.4. Enzymes assay
Tissue sub-samples were disrupted as described in 2.2. The homogenates from tissue disruption were centrifuged at 600 x g for 3 min, and the supernatants were centrifuged at 6000 x g for 8 min at 3 °C. Resultant supernatants were used as a crude enzyme source. The enzyme assay were done in the following reaction medium: Lactate dehydrogenase (LDH) – 5.0 mM pyruvate, 0.1 mM NADH and 42.5 mM Tris buffer, pH 7.5; Malate dehydrogenase (MDH) – 0.33 mM oxaloacetate, 0.2 mM NADH and 50.0 mM imidazole buffer, pH 7.0; Glutamate dehydrogenase (GDH) – 250 mM ammonium acetate, 5.0 mM α-ketoglutarate, 1.0 mM ADP and 50 mM imidazole buffer pH 7.0. The enzyme activities were determined following the NADH extinction at 340 nm (Hochachka et al., 1978). Alanine aminotransferase (ALAT) and Aspartate aminotransferase (ASAT) activities were determined by an end-point type assay and read at 430 nm. The reaction medium of both enzymes was 222 mM alanine (ALAT) or 44.4 mM aspartate (ASAT), 11.6 mM α-ketoglutarate, 0.22 mM arsenate, 0.27 mM pyridoxal phosphate, and 0.1 M phosphate buffer pH 7.0 (Reitman and Frankel, 1957). The enzyme activities are expressed in UI (g of protein-1).
Intermediary metabolites and enzyme assays were determined photometrically in a Beckman DU-520 spectrophotometer (Beckman Coulter Electronics Industria e Comercio, São Paulo, Brazil). All chemicals and reagents were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA) and Merck (Merck, Darmstadt, Germany).
2.5. Statistics
The experiments included three treatments (ratios of energy (E)/ protein (P) are 12, 14, and 16) in three replicates for pelletized diets and three treatments in three replicates for extruded diets. One-way ANOVA was used for each treatment to assess normality and homogeneity of variances among replicates. Data were tested for normality (Shapiro-Wilk) and homoscedasticity (Levene's). One-way ANOVA followed by Tukey's post-hoc test was used to compare treatments. Significance was set at p < 0.05. All values are reported as mean ± SD (n = 15). The data were analyzed using the Statistical Analyses System SAS® v.8 software package (SAS Institute Inc., Cary, NC, USA).
3. Results
No difference was observed in the WG in any of the E:P ratios in fish fed pelleted diets. Both extruded and pelleted diets affected the intermediary metabolism of pacus (Figure 1). This effect was observed through changes in metabolite profiles (Figures 2, 3 and 4) and the activity of enzymes (Figures 5 and 6) involved in intermediary metabolism.
Weight gain (WG) expressed as g (Kg)-1 of Piaractus mesopotamicus after 90 days of feeding with pelletized and extruded pellets of diets at three ratios of digestible energy (DE): digestible protein (DP) ratios. Different superscript letters mean significant difference (p < 0.05). Diets with different energy-protein ratios.
Intermediary metabolites of Piaractus mesopotamicus fed with rations of pelletized pellets at ratios of digestible energy (DE)/ digestive protein (DP). Different superscript letters mean significant difference (p < 0.05).
Plasma metabolites of Piaractus mesopotamicus fed with pelletized and extruded pellets at ratios of digestible energy (DE)/ digestive protein (DP). Different superscript letters mean significant difference (p < 0.05).
Intermediary metabolites of Piaractus mesopotamicus fed with rations of extruded pellets at three ratios of digestive energy (DE)/ digestive protein (DP). Different superscript letters mean significant difference (p < 0.05).
Enzymatic activity of alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), and glutamate dehydrogenase (GDH) in the liver of Piaractus mesopotamicus fed with pelletized and extruded pellets of diets at three ratios of digestive energy (DE): digestive protein (DP) ratios. Different superscript letters mean significant difference (p < 0.05).
Enzymatic activity of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) in the liver of Piaractus mesopotamicus fed with pelletized and extruded pellets of diets at three ratios of digestive energy (DE): digestive protein (DP) ratios. Different superscript letters mean significant difference (p < 0.05).
Fish fed pelleted diets displayed the most significant metabolic changes in the white muscle (Figure 2). The glucose, lactate, free amino acids, and NH4+ concentrations were reduced with the increase in energy levels in the diets; similarly, the levels of pyruvate, free fatty acids, and triacylglycerol were enhanced. The liver glucose levels were augmented, and its lactate level was reduced. In the blood (Figure 3), the lactate level was reduced by 60%, while the glucose levels were enhanced by nearly 30%. Only free fatty acid concentrations were increased in the blood of fish fed extruded diets.
Different from fish fed pelleted diets, those fed extruded diets exhibited the most significant metabolite changes in the liver (Figure 4).
Their concentrations of glycogen, pyruvate, free amino acids, NH4+, triacylglycerol, and free fatty acids increased with increased dietary energy. In their white muscles, the concentration of free fatty acids was enhanced. In their white muscles, the concentrations of free fatty acids and free amino acids, and fish growth (WG) increased. Other metabolites did not show changes according to the dietary conditions (Tables 2 and 3).
Mean concentrations of intermediary metabolites in Piaractus mesopotamicus fed with pelletized diets.
Mean concentrations of intermediary metabolites in Piaractus mesopotamicus fed with extruded diets.
The activities of aminotransferases and GDH in the pacu liver were affected by the dietary energy levels. In fish fed pelleted diets, most of these enzymes’ activities were significantly reduced (Figure 5). The same pattern was observed in fish fed extruded diets, except for ASAT, which presented high activity in fish fed the E:P-14 diet.
The hepatic dehydrogenases of lactate and malate were also affected by the E:P ratios. Both LDH and MDH enzyme activities were decreased in fish fed pelleted diets with increased E:P ratios; they were increased in fish fed extruded diets with increased E:P ratios (Figure 6).
4. Discussion
The activity of metabolic pathways is associated with the concentration of cell metabolites, which are related to the level of nutrients from a diet. There is a complex and wide net of nutrient sensors spread over some tissues such as the hypothalamus, hindbrain, liver, Brockmann bodies, and intestine. These sensors control the nutrient intake and, consequently, their tissue levels (Efeyan et al., 2015; Ogunnowo-Bada et al., 2014). Variations in the concentration of metabolites in many body organs or compartments in sequential experimental moments, or distinct physiological conditions, allow observing the respective metabolic profile arising from those variations. In addition to available nutrients, the species’ metabolic preference plays a fundamental part in such adaptive processes, and these processes are directly linked to the organism or its genetic structure.
The digestion and absorption of some types of carbohydrates represent metabolic preferences that vary among species at different rates due to distinct digestive traits (Boonanuntanasarn et al., 2017; Coutinho et al., 2018; Kamalam et al., 2013). This is consequently reflected in metabolic responses (Almeida et al., 2011; Ma et al., 2018) or biochemical profiles in different body compartments. Likewise, the utilization of lipids and carbohydrates dietary can allow other nutrients to have roles other than energetic ones. Consequently, the balance of dietary energetic components is fundamental to adjust fish demands for best growth with minimal protein expenditure as an energetic source, driving amino acids to anabolic paths (Ma et al., 2018) in most tissues (Campeche et al., 2018; Melo et al., 2016). In addition, the use of carbohydrates in diet formulation accomplishes energetic dietary aspects, contributes to reducing costs, and prevents nitrogen excretion into the aquatic environment (Bombardelli et al., 2017; Mohseni et al., 2013).
In the present study, two main contrasts were observed between diets: i) the increased levels of lipids in D2 diets, and the increase of lipids plus carbohydrates in D3 diets when comparing both with E:P12 diets; and, ii) the manufacturing of pellets: pelleting or extrusion. The similar growth performance observed among fish fed pelleted diets can be a consequence of two possibilities: a) partial availability of some dietary nutrients in the pellets, making the distinction between their composition ineffective, and b) the impaired action of the digestive juice because of the physical characteristics of pellets, resulting in different compositions among them being ineffective. Otherwise, fish fed extruded diets presented different WG depending on the energy level in the diet. The effect of temperature on the structure of dietary nutrients, leading to cross-linking in some sulfur amino acids, dispersion of lipid drops, and gelatinization of polysaccharides, is well reported as enhancing the digestive processes in several cases (Sørensen, 2012).
However, the best WG observed in fish fed extruded diets did not surpass the average values observed in fish fed pelleted diets. This fact calls special attention to the E:Pratio when diets will be manufactured by pelletization processes. Weight performances and observed metabolic responses were different when pacus were fed pelleted or extruded diets. The growth increase in response to the dietary carbohydrate is dependent on organoleptic properties (Jobling et al., 2001). In fish, food intake is a complex process regulated by many factors, including an abundance of energy and nutrients, metabolic regulation, cell glucose availability, and others (Li et al., 2018; Ma et al., 2018).
The most relevant responses of pacus fed pelleted diets were observed in the white muscle; the liver was the most relevant physiological compartment in fish fed extruded diets. The increase in energy from the pelleted diets promoted an increase in glucose in white muscle, becoming readily available and resulting in increased TG in white muscle. In other fish species fed high-carbohydrate diets, high triglyceride and plasma cholesterol are also reported (Boonanuntanasarn et al., 2017). The consequence of the enhanced supply of free fatty acids for muscle metabolic demands was an aerobic preference, and the fermentative path of glucose was lessened by nearly 25%, followed by a reduction in liver lactate concentrations. The source of carbohydrates, their level of inclusion, and the balance between macronutrients in the diet stimulate the secretion and action of some hormones such as insulin and glucagon, influencing glucose metabolism, especially in omnivorous species such as pacu (Abimorad and Carneiro, 2007).
The role of insulin versus amino acid levels in the control of their flux and metabolism is widely known, which makes the glucose and amino acid cell concentrations closely related (van de Pol et al., 2017). The increase in muscle glucose is consistent with diminished amino acid levels. The enhancement of muscle glucose dropped the levels of amino acids, nitrogenous catabolism, and ammonia. A second possibility concerning the decrease of amino acids is their export to the plasma. Even with the plasma amino acids concentration remaining stable, their hepatic levels, followed by hepatic ammonia, were very suggestive of enhanced nitrogenous catabolism in the liver.
The increase in hepatic ammonia and free amino acids concentrations suggests a breakdown of proteins, which, considering the steady values of WG in every feeding condition, were likely driven to gluconeogenesis, as also suggested by Boonanuntanasarn et al. (2017). These hepatic increases in ammonia and free amino acids, followed by decreases in ALAT and ASAT, are apparently controversial. However, the reduction of both transaminases in fish fed the D3 diet would increase the level of free amino acids, as observed, under a regular input of amino acids. Considering enhanced nitrogenous catabolism, as proposed above, the increasing levels of ammonia were expected under the observed steady activity levels of GDH. Decreasing activities in hepatic LDH are suggestive that the enhanced plasma glucose came more from the nitrogenous source than from lactate. From the metabolic frame discussed in fish fed pelleted diets, it is possible to infer the following points: i) the aerobic preference resulting from the increase in energy in fish fed pelleted diets facilitates an oxidative stressing condition, ii) growing levels in hepatic ammonia with the increase of dietary energy would be detrimental to fish, iii) the synthesis of TG in white muscles can result in possible undesirable changes in the quality of fillets, and finally iv) a possible export of free amino acids from white muscle risks reducing their plentiful dispose toward growth processes.
Investigations on the composition of extruded diets demand more precision and accuracy. In addition to differences between pelleted and extruded diets concerning the growth performance parameter of WG, the differences in metabolic profiles among fish were remarkable, which has also been reported elsewhere (Honorato et al., 2010). Hence, the metabolic changes in the liver of pacus fed extruded diets were more evident than those observed in white muscle. The increase in energy in extruded diets enhanced glycogen bulks in the liver. This metabolic event can be described either as glycogenesis from dietary glucose or gluconeogenesis from amino acids, as proposed for Micropterus salmoides (Ma et al., 2018). Both assumptions seem to have the same relevance because the increase in pyruvate concentrations, or the remarkable fall in the Lipid/Protein ratio, allows accepting a gluconeogenic process as observed in Wuchang bream (Megalobrama amblycephala), black carp (Mylopharyngodon piceus) (Zhou et al., 2017), and Micropterus salmoides (Ma et al., 2018). Gluconeogenesis from protein breakdown should be more substantial than that from other intermediates. In fact, the increase in NH4+ and free amino acids sustains the assertion that the mobilization of liver amino acids was remarkable in fish fed the D2 and D3 extruded diets. However, the free amino acids would be driven to other processes than gluconeogenesis; for instance, the synthesis of muscle mass. This proposal is particularly pertinent to fish fed the D2 diet. However, fish fed extruded D3 diets showed distinct readings in metabolic outline. The plasma free amino acids concentration followed the same profile as the liver, suggesting a direct relationship between both compartments, and, therefore, the uptake of plasma free amino acids by the liver and peripheral tissues. This set of data concerning protein metabolism shows a clear interrelationship between liver, plasma, and white muscle, portraying a conversion of free amino acids into body mass in pacus fed the extruded E:P14 diet in the present experimental condition. The increase in energy in the rations resulted in metabolic alterations such as an increase in hepatic adjustments, which reflected higher bioavailability of proteins in extruded diets, precisely as reported before (Ma et al., 2018; Venou et al., 2003).
TG and free fatty acids in the liver also increased with the dietary energy; in addition, the white muscle free fatty acids concentration grew in parallel to that observed in the liver. This profile as a whole is highly suggestive that extruded diets make macronutrients easily available to metabolic processes. A more accurate adjustment of nutrient levels, as partially discussed above, must be implemented in such diets to achieve maximum output. Metabolic differences in fish fed diets of distinct compositions are primarily related to the digestibility of pellets as a consequence of the type of processing method used. The best use of glucose by fish, for example, is attributed to the rapid uptake of monosaccharides compared to polysaccharides from extruded diets (Cian et al., 2016; Ma et al., 2016).
The extrusion process efficiently delivers carbohydrates (Akpinar et al., 2012; Cheng and Hardy, 2003), leading to faster absorption of glucose that results in the protein-sparing effect, and likely the increase of free fatty acids and TG. The activity of assayed enzymes in the liver of fish fed extruded diets seems to attest to the discussion above on the metabolism of pacus in such a condition. It is interesting to emphasize the MDH activities. Different from that observed in fish fed pelleted diets, the increase in such activity suggests that the aerobic processes were less effective. Several studies report on the relationship between metabolic enzymes, protein metabolism, and nutritional status in fish (Campeche et al., 2018; Ma et al., 2016). From the metabolic frame discussed in fish fed extruded diets, it is also possible to infer: i) the aerobic preference observed in fish fed pelleted diets seems to fade in fish fed extruded diets, ii) this fact would hinder an oxidative stressing condition; however, iii) the growing levels of hepatic ammonia with the increase in dietary energy is likely less relevant because the maximum level observed in fish fed the extruded E:P 16 diet is very close to that of fish fed pelleted E:P 13 diets, and less than 50% the levels observed in fish fed pelleted E:P 16 diets. Finally, iv) the inconvenience of increased synthesis of TG in white muscles was not observed in fish fed extruded diets.
Aiming for low-carbon aquaculture, which aims to minimize greenhouse gas emissions and mitigate the environmental impacts associated with aquaculture production, a promising strategy in this context is the formulation of diets with an adequate balance of protein and energy. Poorly balanced diets, especially those with excess protein, lead to the deamination of excess amino acids, resulting in increased nitrogen excretion in the form of ammonia (NH3/NH4+), which can compromise water quality and increase indirect emissions of nitrogen oxides. By optimizing the protein-energy ratio, it is possible to improve nutrient utilization efficiency, reducing the excretion of nitrogen compounds and, consequently, the pollutant load in farming systems. This approach not only favors the environmental sustainability of the activity but also contributes to the bioeconomic efficiency of production, maximizing biomass gain per unit of feed supplied. In which extruded diets with an intermediate P:E ratio showed better feed conversion and lower nitrogen excretion, suggesting greater metabolic efficiency and potential reduction of the environmental footprint.
5. Conclusions
This study evaluated the adaptive responses of juvenile pacu to different protein-energy ratios (12, 14, 16) in pelleted and extruded diets. We conclude that pelleted diets led to aerobic preference, increased hepatic ammonia and triglyceride synthesis in white muscle – effects that may be undesirable for growth and fillet quality. Extruded diets, particularly at E:P 14, promoted better growth performance, reduced hepatic ammonia accumulation, and avoided muscle triglyceride deposition. Therefore, extruded diets with an E:P ratio of 14 are recommended for pacu farming, combining metabolic efficiency with reduced environmental impact. These findings support the use of precision nutrition and appropriate feed processing to enhance sustainability in aquaculture. Regenerative aquaculture aims to improve nutrient utilization efficiency by integrating formulated diets with high bioavailability into technologically optimized production systems. This approach seeks to promote the closure of biogeochemical cycles, reducing the need for external inputs and minimizing effluent emissions and waste generation, thus contributing to mitigating environmental impacts and increasing the resilience of aquaculture systems. It is concluded that the application of precision nutrition principles, combined with the appropriate choice of feed processing, can optimize productive performance and promote more sustainable practices in pacu fish farming.
Acknowledgements
This work was supported by Coordination for the Improvement of Higher Education Personnel - CAPES (Scholarship and PROAP); the CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and Foundation for the Support of Education, Science, and Technology Development in the State of Mato Grosso do Sul [Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul]—FUNDECT. Foundation for the Support of Education, Science, and Technology Development in the State of Mato Grosso do Sul [Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul]—FUNDECT (Process Nº 83/035.027/2024, FUNDECT Project Nº 274/2024, SIAFIC Nº 1481), São Paulo State Research Foundation (FAPESP 04/07081-7), and Brazilian National Research Council (CNPq).
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Ethics and consent
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