Effects of days-fasting and refeeding on growth, biochemical and histometric liver parameters in pacu Piaractus mesopotamicus

Efeito da restrição alimentar e realimentação sobre o crescimento, parâmetros bioquímicos e histometria hepática em pacus Piaractus mesopotamicus

Abstract

This study evaluated the effects of days-fasting followed by days-refeeding on growth, biochemical, and hepatic parameters in pacu (Piaractus mesopotamicus). One hundred and twenty juveniles P. mesopotamicus with initial average weight and length of 47.7 ± 9.2 g and 13.4 ± 0.9 cm were randomly distributed into six experimental units (20 fish per unit) and subjected to treatments: 30 days-fasting followed by 50 days-refeeding, and control group, fed continuously throughout the period. During the fasting period, samples were collected at 10, 20, and 30 days, while during the refeeding period at 15 and 50 days. Animals in the control group were sampled at the same periods. Weight (g), relative condition factor (Kn), and hepatosomatic index (biometric parameters) were measured. Liver assessments were performed. Additionally, glucose, plasma biochemical parameters levels were measured. After 30 days of fasting, hepatocyte density (73.8 ± 1.09%), liver glycogen (14.9 ± 0.87%) and hepatocyte nuclear volume (27.3 ± 0.30 µm3) were lower compared to the control group (82.0 ± 0.67%, 19.4 ± 0.74% and 43.40 ± 0.48 µm3 respectively). The relative condition factor remained unchanged. Cholesterol values, blood vessels, and sinusoidal density increased significantly during fasting. After refeeding, parameters were restored to the control level. On the 50th day of refeeding, the hepatosomatic index was significantly higher than the control group. The results showed that fasting associated with refeeding did not affect fish growth. The period over 50 days of refeeding may influence the pacu's compensation compared to daily-fed animals. The effects of fasting and its relationship with the pacu's physiological response through nutritional status become useful in contributing to feeding practices in P. mesopotamicus fish farming.

Keywords:
aquaculture; food deprivation; hepatocyte; stereology

Resumo

Este estudo avaliou os efeitos de dias de jejum seguidos de dias de realimentação sobre o crescimento, parâmetros bioquímicos e hepáticos em pacu (Piaractus mesopotamicus). Cento e vinte juvenis de P. mesopotamicus com peso e comprimento médios iniciais de 47,7 ± 9,2 g e 13,4 ± 0,9 cm foram distribuídos aleatoriamente em seis unidades experimentais (20 peixes por unidade) e submetidos aos tratamentos: jejum de 30 dias seguido de 50 dias de realimentação e grupo controle, alimentado continuamente durante todo o período. Durante o período de jejum foram coletadas amostras aos 10, 20 e 30 dias, enquanto no período de realimentação aos 15 e 50 dias. Os animais do grupo controle foram amostrados nos mesmos períodos. Peso (g), fator de condição relativo (Kn) e índice hepatossomático (parâmetros biométricos) foram medidos. Avaliações hepáticas foram realizadas. Além disso, foram medidos os níveis bioquímicos do plasma. Após 30 dias de jejum, a densidade dos hepatócitos (73,8 ± 1,09%), o glicogênio hepático (14,9 ± 0,87%) e o volume nuclear dos hepatócitos (27,3 ± 0,30 µm3) foram menores em comparação ao grupo controle (82,0 ± 0,67%, 19,4 ± 0,74%). e 43,40 ± 0,48 µm3 respectivamente). O fator de condição relativo permaneceu inalterado. Os valores de colesterol, vasos sanguíneos e densidade sinusoidal aumentaram significativamente durante o jejum. Após a realimentação, os parâmetros foram restaurados ao nível de controle. No 50º dia de realimentação, o índice hepatossomático foi consideravelmente superior ao do grupo controle. Os resultados mostraram que o jejum associado à realimentação não afetou o crescimento dos peixes. O período superior a 50 dias de realimentação pode influenciar na compensação do pacu em relação aos animais alimentados diariamente. Os efeitos do jejum e sua relação com a resposta fisiológica do pacu por meio do estado nutricional tornam-se úteis para contribuir nas práticas alimentares na piscicultura de P. mesopotamicus.

Palavras-chave:
aquicultura; privação alimentar; hepatócitos; estereologia

1. Introduction

Brazilian aquaculture stands out worldwide due to the exotic species Nile tilapia Oreochromis niloticus (FAO, 2022). The main species of fish cultured are Nile tilapia (O. niloticus), tambaqui (C. macropomus) and its hybrids, species of carpa (C. carpio, C. Idella, H. molitrix, H. nobilis) and Pacu (Piaractus mesopotamicus) and its hybrids (Valenti et al., 2021). Nevertheless, native species such as the pacu Piaractus mesopotamicus have gained prominence in Brazilian fish farming, as it is considered a robust species, with excellent growth performance and adaptability to cultivation, in addition to excellent meat quality and marketability (Peixe Br, 2023). Meanwhile, production costs in fish farming are constant concerns and alternatives that allow reducing feed consumption are desirable in aquaculture research (Jensen et al., 2023; Kumar et al., 2023).

Among emerging strategies observed in fish farming, feeding management with feed deprivation or restriction followed by refeeding stands out. This purpose, in addition to reducing feeding costs, promotes better use of feed by fish, avoiding feed wastage and leftovers that deteriorate water quality, characterizing an economically improved system (Silva et al., 2013; Liu et al., 2019; Favero et al., 2019, Gabriel et al., 2023). Despite these advantages, evaluating the physiological responses triggered by this management is necessary, considering the period of partial or total feed deprivation with subsequent refeeding.

According to Souza et al. (2002), with the reduction in feed supply, the pacu showed impairments in growth and body weight. In the refeeding phase, it showed an increase in appetite and a consequent increase in feed intake, i.e., the pacu was able to restore growth performance through a compensatory gain. This means that fasting can cause an adaptation to starvation followed by improved utilization of feed after refeeding (Dobson and Holmes, 1984; Nicieza and Metcalfe, 1997; Xie et al., 2001).

On the other hand, the effects of starvation and subsequent refeeding caused metabolic and physiological responses such as hormonal variations correlated to the expression of metabolic enzymes in O. niloticus (Sakyi et al., 2020), oxidative damage, and lipid peroxidation in the intestine of juvenile Acipenser stellatus (Florescu et al., 2021), modify thermal imprinting at the egg stage in Dicentrarchus labrax (Mateus et al., 2023), and haemato-biochemical changes in juvenile Colossoma macropomum (Assis et al., 2020). In this respect, the practice could cause severe physiological disorders in the pacu.

According to Wieser et al. (1992), four physiological stages are identified during the period of fasting followed by refeeding, including stress (hyperactivity due to starvation), energy reduction (decrease in respiratory rate and glycemic activities, related to glycogen in muscles), adaptation (reduction in metabolic rate and use of protein in detriment of lipid to obtain energy) and recovery (increased growth rates and oxygen use). In this way, it is distinctly possible that fasting becomes a very challenging period for vital organs, mainly the liver.

The liver is a digestive gland responsible for several metabolic-nutritional functions (Liebl et al., 2022). After ingestion and absorption of food in the digestive system, hepatocytes begin the esterification and oxidation processes, which are responsible for synthesizing, storing, and releasing lipoproteins, cholesterol, and organic ions (Liebl et al., 2022). Additionally, the liver detoxifies and neutralizes toxins and maintains glycemia via gluconeogenesis and lipogenesis processes (Roberts and Ellis, 2012). For this reason, morphological and structural variations in the liver can be measured and analyzed morphometrically as direct responses to physiological adaptations to feed management and diet (Rodrigues et al., 2017; Owatari et al., 2018; Oliveira et al., 2024).

During metabolism, characterized by the influence of adrenal steroids insulin and glucagon, excess feed ingested is converted into energy reserves of glycogen, triglycerides, and cholesterol, which will be the primary sources of energy available during feed restriction, intensifying glycemic and lipid mobilization from the liver, and visceral lipids as a consequence of starvation (Nebo et al., 2018). Thus, the fatty acids released during lipolysis can be energy sources for cholesterol synthesis (Ashouri et al., 2020). Likewise, plasma biochemistry discloses information about the nutritional, metabolic and health status of fish, which may be related to the adaptive physiological responses produced by adverse situations such as feed deprivation (Furné et al., 2012; Assis et al., 2020; Mateus et al., 2023). Therefore, considering the above and the lack of information regarding the effects of fasting and refeeding on the cultivation of pacu P. mesopotamicus, the present study aimed to investigate a possible compensation in biometric parameters, and biochemical and hepatic-histological changes in P. mesopotamicus exposed to fasting and refeeding.

2. Material and Methods

All experimental procedures were approved by the Animal Use Ethics Committee (CEUA) of the Federal University of Mato Grosso do Sul – UFMS (protocol number 834/2017).

2.1. Experimental design

The experiment was conducted at the Experimental Pathology Laboratory - LAPEX at UFMS for 80 days. One hundred and twenty juveniles of pacu P. mesopotamicus, from the exact spawn obtained in the fish farming sector of the Aquidauana of the State University of Mato Grosso do Sul - UEMS, with an initial average weight and length of 47.7 ± 9.2 g and 13.4 ± 0.9 cm and relative condition factor (Kn) 1.0 ± 0.1.

The fish were acclimatized to the experimental conditions for 15 days, with controlled temperature (26~28°C), constant aeration, and physical and biological filtration. Water quality parameters (temperature, dissolved oxygen, pH, and electrical conductivity) were checked with a SANXIN SX751 portable meter twice daily. The observed values ​​were: temperature of 27.1 ±0.6ºC, dissolved oxygen of 4.8 ±0.8 mg L-1, pH 7.05 ±0.2 and electrical conductivity of 54.94 ±7.2 µS.

After the acclimatization period, weight (g) and standard length (cm) were recorded. The data were used to determine fish biomass (fish weight per experimental units) and the amount of feed (5% live weight divided into two portions) to be administered twice daily. Then, the fish were randomly distributed into six 140L-experimental units (20 fish per unit); where three were randomly selected for the control groups and three for the treated groups.

Two distinct experiments were conducted. The first involved a fasting experiment in which fish (n= 36) was subjected to fasting treatment for 10, 20 and 30 days. For each fasting treatment, 12 fish were sampled to compose the control groups.

The second experiment considered the remaining fish after the 30th day of fasting (n= 24). Here, fish fasting treatment were subjected to refeeding for 15 and 50 days. Control fish (n= 24) and refeeding groups were fed twice daily with an extruded commercial feed for omnivorous fish (Guabi-Pirá^® (4~6 mm), moisture 8%, crude protein 32%, ether extract 6.5%, crude fiber 7% and mineral matter 10%). Figure 1 shows the experimental design.

Figure 1
Experimental design showing the number of fish according to fasting and refeeding treatments. Refeeding period begins after the 30-day fasting period.

2.2. Fish growth and biochemical analyses

To determine the pacu's growth performance, at the end of each experimental period, the fish were sedated in a eugenol solution (175 mg/L) (Rotili et al., 2012). The parameters evaluated were final weight (g), final total length (cm), and relative condition factor (Kn) = (Wt/We), where Wt is the observed body weight, and We is the expected weight obtained by the weight-length ratio (Le Cren, 1951; Santos et al., 2012).

Blood was collected by caudal venipuncture for plasma biochemical analyses using syringes with 10% EDTA anticoagulant (Aride et al., 2016; Oliveira et al., 2016, 2017). Blood glucose (mg dL-1) was determined with a portable digital glucometer (Accu-Chek Active®). The blood samples were centrifuged at 3,000 rpm for five minutes (Aride et al., 2018, 2020). The plasma obtained was frozen at -20°C to determine cholesterol (mg/ dL) and triglyceride (mg/ dL) using the automated system Cobas® c111 analyzer® (Roche Instrument Center).

2.3. Liver collection, hepatosomatic index (HSI) and liver histology

For liver analyses, fish were euthanized with an overdose of eugenol anesthetic (450 mg/L). The liver was removed and weighed to determine the hepatosomatic index (HSI) (%) {[liver weight (g) / fish weight (g)] × 100}. Then, the organ was delicately sliced transversely and fixed in 10% buffered formalin (pH 7.2) for 24 h. Subsequently, the samples were washed and dehydrated in increasing series of ethyl alcohol, clarified in xylol, and embedded in paraffin at 60°C to be sliced into 4 μm sections and stained with haematoxylin-eosin (H & E).

Following staining, analyses of liver morphometry and structural volumetric density were conducted, in addition to periodic acid-Schiff (PAS) staining to identify hepatocellular glycogen (Oliveira et al., 2021). For this purpose, six samples from each treatment were photographed with a 14-megapixel OptCam® camera (LOPT14003) coupled to a high-performance vertical optical microscope (Zeiss Primo Star®). Images were analyzed at 400x magnification to assess structural volumetric density, including the presence of glycogen in the liver, while 1000x magnification was employed for hepatocellular morphometry (Ferreira et al., 2016). The software ImageJ version 1.48v was utilized for the analysis.

2.4. Structural volumetric density and liver glycogen

Five randomized images of each cross-section from the liver fragment were captured. We overlaid a random offset grid of 252 intersections (quadratic lattice test system) to measure structural volumetric density to count points for each cross-section. The interpoint distance was 15 µm, according to Reid (1980). At each intersection, we tallied the number of hepatocytes, sinusoids, and blood vessels (arterial, venous, and capillaries). To calculate the final density for each structure, we used the formula SVD (%) = ([Ip x 100]/Tip): Ip represents the intersections counted for the structure, and Tip represents the total number of intersections in the image (Rodrigues et al., 2017). Analyses were conducted in the ImageJ version 1.48v.

2.5. Liver morphometry

Seven hepatocytes were randomly considered per image for liver morphometric analyses, totaling 420 hepatocytes per treatment. The hepatocyte area (Ha) (µm²), the hepatocyte nuclear area (Hna) (µm²), and the hepatocyte nuclear diameter (Hnd) (µm) were measured. The hepatocyte nuclear volume (Hnv) was obtained by the formula Hnv = (4/3 π.r³), where r is the nuclear radius. Hepatocyte volume was estimated indirectly by the formula Hv (μm³) = (Ha × Hnv) / Hna) (Rodrigues et al., 2017). Analyses were conducted using the Motic 2.0® (Motic Asia, Hong Kong) software.

2.6. Statistical analysis

The time-fasting and re-feeding effects between control and exposure groups were compared using a generalized linear mixed model of ANOVA, followed by pairwise comparisons using the least significant difference (LSD) test. SPSS 23.0 (IBM®) software was employed for these analyses. Box plots were created for the mean, 25^th, and 75^th quartiles, the minimum and maximum values, and the interquartile intervals. All analyses have a 5% significance level.

3. Results

3.1. Biometric values and biochemical analyses

Body weights and hepatosomatic index according the treatments are represented in the Figure 2. In the fasting fish both measures were reduced (p<0.05) compared to the control group, regardless of the sampling period being on the 10^th, 20^th, or 30^th days; however, there was no significant difference (p>0.05) between the sampling intervals in the fasting treatment. On the 15^th day after the start of refeeding, significant differences in pacu weight were observed between the refeeding group and the control group, i.e., the pacu weight in the refeeding group was significantly below (p<0.05) those observed in fish in the control group; however, on the 50^th day, no significant differences (p>0.05) in weight were observed between the groups, i.e., reestablishment of the pacu's weight was observed after 50 days of refeeding.

Figure 2
Body weights and hepatosomatic indexes (HSI) in pacu Piaractus mesopotamicus subjected to 10-20-30 days of fasting and 15-50 days of refeeding. (ab) represent significant differences among control pacu. (AB) represents significant differences among fasted pacu. (**) represents significant differences between control and fasted pacu; ns: not significant. All analyses have 5% significance level.

Hepatosomatic indexes (HSI) in fasted pacu were significantly reduced (p<0.05) compared to pacu in the control group. After the 15^th day of refeeding, HSI increased in pacu from the refeeding group; however, it did not differ from the control group, while on the 50^th day of refeeding, an increased HSI (p<0.05) was observed in pacu compared to pacu from the control group. The relative condition factor did not show a significant difference (p>0.05) in any period (Figure 2).

With regard to plasma biochemical parameters, notable alterations (p<0.05) were discerned during the fasting period. The fasting glucose levels were decreased (p<0.05) in comparison to the control group. However, on the 30^th day, the decrease was more pronounced than in the other sampling intervals. Following refeeding at the 15^th and 50^th day, the fishes exhibited a significant increase (p<0.05) in glucose levels, reestablishing values similar to those observed in the control group and differing significantly (p<0.05) from fish in the 30-day feed restriction treatment. A significant decrease (p<0.05) in triglycerides was observed in the fasted fish compared to the control group. However, on the 30^th day, a significant decrease was noted (p<0.05) when comparing to the control group and other fasting periods. When refeeding for 50 days, the pacu showed increased triglyceride levels without difference between control group, while it differed from the other refeeding period. Cholesterol levels increased significantly (p<0.05) in fasted pacu compared to pacu in the control group. With refeeding, the pacu showed reduced cholesterol levels but no significant difference (p>0.05) between the refeeding and control groups. However, the re-fed pacu differed (p<0.05) from those analyzed at 20 and 30 days, whose fish showed increased cholesterol levels (Figure 3).

Figure 3
Glucose, triglycerides and cholesterol biochemical parameters in pacu Piaractus mesopotamicus at 10-20-30 days of fasting and 15-50 days of refeeding. (ab) represent significant (p<0.05) differences among control pacu. (AB) represents significant (p<0.05) differences among fasted pacu. (**) represents significant differences between control and fasted pacu; ns: not significant. All analyses have 5% significance level.

3.2. Liver assessments

The liver parenchyma is constituted by polyhedral-shaped hepatocytes with centralized and peripheral nuclei (Figure 4). The hepatocytes of the fasted pacu exhibited a notable reduction (p<0.05) in volume, accompanied by an expansion of the sinusoids due to the increased blood circulation in response to vascular hyperemia (Figure 4b). In comparison to the fasting fish, the glycogen stores of the control fish were notably high (Figure 4c and 4d, respectively). Following a 30-day fast, the fish exhibited the most extensive sinusoidal network, while the hepatocytes displayed pale cytoplasm and scarcity of glycogen deposits (Figure 4d).

Figure 4
Histological evaluation of liver tissue from pacu Piaractus mesopotamicus after 30 days-fasting and control group. In (a) control group, normal liver parenchyma, sinusoids (s), bile duct (arrowhead), portal venous branch (✷). Highlighted: hepatocyte and centralized nucleus. In (b), 30 days-fasting group; marked cordonal appearance of the sinusoids largely filled by blood circulation; portal capillary (black arrow), and portal vein (✷). Highlighted: hepatocytes with reduced cytoplasm and nuclear contraction. In (c) control group, bile duct (arrowhead), sinusoids (s), and portal vein (✷). In highlight, glycogen-positive staining in the cytoplasm of hepatocytes. In (d) 30 days-fasting group, artery (arrowhead), portal venous branch (✷), and sinusoidal dilatation (black arrow). Highlighted: hepatocytes with reduced cytoplasmic glycogen deposits. (a) and (b) H&E staining; (c) and (d) Periodic Acid-Schiff (PAS) staining. In a, b, c and d bar scale = 50 µm; highlighted bar scale = 10 µm.

The fasting treatment modified (p<0.05) the structural volumetric density and liver glycogen deposit (Table 1). In the structural volumetric density, the percentage of hepatocytes was reduced considerably (p<0.05) only on the 30-day of fasting, while in the control group and the other fasting periods, they were increased. On the other hand, on the 50-day of refeeding, the hepatocytes density percentage was restored, not differing from the control group. The blood vessels increased at the 30-day of fasting, differing (p<0.05) from the control group. However, the other fasting sampling intervals did not differ. After refeeding, there was a reduction in blood vessels, but without significant difference either with the control group or between the refeeding times.

Table 1
Mean (± mean standard error) of structural volumetric density (SVD), hepatic glycogen and hepatocellular morphometry in Piaractus mesopotamicus according to the fasting time and refeeding (days).

Strucutural volumetric density (%)			Fasting									Refeeding
			10d			20d			30d			15d			50d
Hepatocytes	Control		80.3	± 0.85a		82.7	± 0.86a		82.0	± 0.67a		78.8	± 0.91a		81.9	± 0.78a
	Exposed		78.9	± 0.96A		82.9	± 1.02A		73.8	± 1.09B*		76.0	± 0.98C*		82.2	± 0.86A
Sinusoids	Control		12.8	± 0.54a		9.8	± 0.65b		11.0	± 0.43b		13.8	± 0.55a		11.5	± 0.49b
	Exposed		12.8	± 0.65A		7.5	± 0.38A*		14.5	± 0.70A*		14.6	± 0.45A		11.2	± 0.53B
Blood vessels	Control		6.1	± 0.72a		7.4	± 0.93a		6.95	± 0.74a		7.38	± 0.99a		6.18	± 0.73a
	Exposed		7.9	± 0.87A		9.4	± 1.03A		11.6	± 1.08A*		8.95	± 1.01AB		6.36	± 0.91B
Hepatic glycogen	Control		18.6	± 1.05a		12.9	± 0.83b		19.4	± 0.74a		23.6	± 0.96b		25.8	± 1.45b
	Exposed		13.3	± 0.60A*		13.0	± 0.70A*		14.9	± 0.87A*		19.9	± 0.59B*		24.2	± 0.91C
Hepatocellular morphometry
Hepatocyte volume (µm3)	Control		351.6	± 6.57a		371.2	± 7.34a		449.0	± 6.40b		430.3	± 6.34a		411.3	± 6.07a
	Exposed		255.0	± 4.50aA*		282.1	± 6.47bA*		232.8	± 3.59bA*		457.4	± 7.16B		411.7	± 5.62B*
Nuclear volume (µm3)	Control		28.8	± 0.59a		44.1	± 0.87b		43.4	± 0.48b		44.7	± 0.54b		40.7	± 0.56b
	Exposed		25.6	± 0.37A*		33.5	± 0.73B*		27.3	± .030A*		44.1	± 0.52B		44.6	± 0.45B
Nucleocytoplasm rate (%)	Control		8.29	± 0.11a		12.6	± 0.17b		10.1	± 0.13c		10.8	± 0.12c		11.3	± 0.12c
	Exposed		10.2	± 0.15A*		12.4	± 0.14B		12.5	± 0.171B*		10.1	± 0.11A		11.9	± 0.13A

• ^ab
  represent significant (P < 0.05) differences among control specimens;
• ^AB
  represent significant (P < 0.05) differences among fasted;
• ^*
  represent significant (P < 0.05) differences between control and exposed.

The sinusoids were reduced (p<0.05) on the 20^th day of fasting compared to the control group; however, on the 30^th day, there was a significant increase (p<0.05), differing from the control group and the 20^th day of fasting. After the 50-day of refeeding, the sinusoids were reduced, and no significant differences were observed between treatments. Hepatic glycogen density remained stable during the different fasting periods; however, on the 10^th and 30^th days, there was a decrease in relation to the control group. At the 15-day of refeeding, the fish demonstrated a slight increase in glycogen density. However, they differed (p<0.05) from the control group, remaining inferior. Conversely, on the 50-day, the re-fed pacu showed an increase in glycogen density differing (p>0.05) from the previous period (i.e., 15-day of refeeding).

Regarding hepatocellular morphometry, the fasted pacu showed a decrease (p < 0.05) in the hepatocytes volume 10 days after exposition. After refeeding, the volume of hepatocytes increased, and there was no difference (p<0.05) neither between the control group nor between the refeeding intervals (p>0.05). Following the hepatocellular volume, the nuclear volume was significantly reduced (p<0.05) in fasted pacu. The refeeding pacu increased nuclear volume but did not differ from the control group or between the refeeding times.

4. Discussion

Programmed fasting or feed restriction, followed by refeeding, has been successfully investigated and experimentally applied in the feeding management of several aquacultured species, representing a favorable strategy for savings related to feed consumption without harm to fish health (Paz et al., 2018; Santos et al., 2018; Assis et al., 2020; Ashouri et al., 2020; Sakyi et al., 2020; Florescu et al., 2021; Mateus et al., 2023).

Likewise, Favero et al. (2020) subjected juvenile pacu P. mesopotamicus to 30 days of fasting followed by 30 days-refeeding and noticed that the refeeding period promoted hyperphagia and total growth compensation in fasted fish, which achieved weight gain and specific growth rate like those of fish fed continuously. In the Siberian sturgeon Acipenser baerii significantly decreased weight during the fasting phase and did not exhibit compensatory growth after 1, 2, or 3 weeks of fasting followed by four weeks of refeeding, indicating that a more extended period of refeeding would be necessary (Ashouri et al., 2020). Here, the fish reached compensatory weight at 50 days post-refeeding. However, despite this, HSI, glucose, and triglyceride levels demonstrated typical values at 15 days post-refeeding, indicating an early resumption of basal metabolism without irreversible damage to the digestive tissues. Two metabolic pathways facilitate the regeneration of blood glucose: glycogenolysis, which is based on glycogen stores, and gluconeogenesis, which allows glucose formation from non-carbohydrate compounds (Polakof et al., 2012). Furthermore, a lipostat model proposed by Jobling and Johansen (1999) suggests that hyperphagia and the rapid recovery of lipids modulate the compensatory growth rate. This provides an influx of energy and metabolic substrates that are then allocated to somatic growth by resumption of insulin-like growth factor (IGF) signaling, resulting in hyperanabolism and more pronounced growth (Won and Borski, 2013). On the other hand, Florescu et al. (2021) observed that stellate sturgeon A. stellatus was able to adapt to the regime of 7 days-fasting followed by 21 days-refeeding, while Sakyi et al. (2020) investigated the effects of 21 days of starvation and subsequent 21 days of feeding in Nile tilapia O. niloticus. They observed that starvation and refeeding can modulate the diversity of intestinal microbial communities and the metabolic responses of Nile tilapia, which could favor nutrient absorption. Such information indicates that starvation strategies are diversely related to fasting and feeding periods and should be adjusted according to the target species. In contrast, other species, such as pacu, can regulate growth within a few days of refeeding.

In the present study, fasted pacu showed a progressive reduction in the HSI, demonstrating the physiological capacity of the pacu to gradually mobilize hepatic glycogenic and lipid reserves to withstand the very challenging period. Unlike the fasting strategy in the present study, Favero et al. (2019) fed P. mesopotamicus with different diets to obtain lean and fat conditions and subjected the fish to 15 days of fasting and five days of breastfeeding. At the time, the researchers noted that fasting reduced blood glucose, triglycerides, and liver glycogen, while just a five-day-refeeding increased plasma glucose and liver lipids in lean fish, indicating that the species is efficient in utilizing feed. On the other hand, Favero et al. (2020) observed that the 30 days-fasting period followed by 30 days-refeeding decreased serum glucose and triglycerides and increased serum cholesterol concentrations, as well as decreased HSI, glycogen, and total lipid concentrations in the liver. These responses suggest an intense physiological adaptation of the pacu based on the conditioning and mobilization of energy reserves to establish new homeostatic patterns of organic vital processes (Pérez-Jiménez et al., 2007; Li et al., 2018).

Refeeding P. mesopotamicus stimulates the adrenal hormones insulin and glucagon, causing excess energy such as glycogen and lipids to be replenished, leading to an increase in liver weight and body weight, increasing the hepatosomatic index. Nebo et al. (2018) subjected O. niloticus to 1, 2, or 3 weeks of fasting and found recovery and increased HSI when refeeding the fish for 10 days. In contrast, Favero et al. (2019) did not observe the same responses in L. alexandri after five days-refeeding, while Favero et al. (2020) observed an increase in HSI during pacu refeeding.

Glucose is one of the primary metabolic sources responsible for distributing energy to the body (Polakof et al., 2012). The reduction in blood glucose and triglycerides in the present study is related to the fasting suffered by the specimens, considering that such energy sources available and stored in the liver were mobilized to carry out vital processes. The body's glucose mobilization occurs with energy sources during feed deprivation. Hepatic glycogen is catabolized, leading to the release of glucose (glycogenolysis) for body maintenance. With the depletion of liver glycogen, glucose is synthesized and distributed throughout the bloodstream through gluconeogenesis from non-glycosidic substrates (lactate, glycerol, pyruvate, and some amino acids) (Polakof et al., 2012; Campos et al., 2017; Li et al., 2022). During feed restriction, lipids are also mobilized to obtain energy, with triglycerides being degraded to release free fatty acids, and finally, lipolysis occurs and the release of lipids as an energy source (Shimeno et al., 1990; Lehninger et al., 2006; Kim et al., 2014).

In the present study, the production of circulating cholesterol was regulated by stimulating the concentration and synthesis of intracellular (endogenous) cholesterol and by the glucagon and insulin hormones during restriction. This means that during refeeding, endogenous cholesterol production is reduced due to the increase in exogenous cholesterol obtained from the food supply (Berg et al., 2002; Lehninger et al., 2006; Kumari, 2023).

As observed in pacu, Ashouri et al. (2020) found that after one week of fasting, total cholesterol levels increased in juvenile Siberian sturgeon A. baerii compared to the fish in the control group, while after four weeks-refeeding, cholesterol levels were restored in fish plasma. Similarly, Godavarthy et al. (2012) found that after a long period of feed restriction (60 days), climbing perch Anabas testudineus presented high cholesterol levels conditioned by a decrease in insulin levels, causing the synthesis of cholesterol, a precursor of stress hormone synthesis (glucocorticoids). This increase is related to the production of gluconeogenesis and adrenocorticoids, in addition to compounds such as ketones, acetate, acetyl-CoA, and glucose, which help combat stress due to the energy provided.

The reduction in liver morphometry is related to the mobilization and consumption of energy stores (catabolism), leading to the depletion of reserves (Strüssmann and Takashima, 1990; Souza et al., 2001; Poursaeid and Falahatkar, 2022). The increase in hepatocyte nuclei could be related to anabolism (glycogen reserve) in the liver, characterized as an energy reserve obtained through feeding (Souza et al., 2001; Adhami et al., 2021).

Generally, fish can overcome long periods without feeding, considering that the amount of endogenous energy reserves will be sufficient to ensure the metabolic demands imposed during fasting (Navarro and Gutiérrez, 1995; Favero et al., 2020). Furthermore, the effects of fasting on metabolism depend on multiple factors, including the health of organ tissues such as the liver, which stores essential reserves of carbohydrates such as glycogen (Navarro and Gutiérrez, 1995). Indeed, in the present study, we observed critical changes in the hepatic indexes, structural volumetric density, glycogen density, and hepatocellular morphometry in the fasted pacu, which, upon being received, were physiologically reconfigured to the primary state of normality. However, periods of fasting can drastically affect liver function, which could significantly modify the concentrations of plasma metabolites such as glucose, cholesterol, and triglycerides, as observed in the present study.

5. Conclusion

In the present study, it was found that different fasting periods (10, 20, or 30 days) caused zootechnical impairments; however, after the refeeding period (15 or 50 days), the pacu P. mesopotamicus was able to recover the lost weight completely. The fasting strategy for up to 30 days induced reversible changes in biometric, biochemical, and histological parameters, with compensatory gains after 50 days of refeeding. Even with physiological impairments, the pacu can resist and overcome such events until periodic refeeding, demonstrating its remarkable resilience, which makes it extensively regarded in Brazilian aquaculture. This management measure can be an alternative to reduce costs in production systems.

Acknowledgements

The authors thank Taynara Leão, Mayara S. Siqueira, Maria Eduarda C. Garcia and Brenda de O. Martins for their support in laboratory and fieldworks. This study was funded in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior — Brasil (CAPES) — Finance Code 001. C.E.F. has been continuously supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico [grant 309358/2023-0]. A doctoral fellowship from CAPES supported K.N.N.F.

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Publication Dates

• Publication in this collection
  31 Jan 2025
• Date of issue
  2024

History

• Received
  06 June 2024
• Accepted
  28 Oct 2024