Effect of increasing lipid levels on liver metabolism, intestinal morphology and production performance of discus fish (Symphysodon aequifasciatus)

Efeito do aumento dos níveis lipídicos no metabolismo hepático, morfologia intestinal e desempenho produtivo de peixes discus (Symphysodon aequifasciatus)

Abstract

The objective of this study was to evaluate the effects of three levels of lipids in the diet on growth, metabolism, and intestinal and hepatic histopathology in discus fish (Symphysodon aequifasciatus). Fish were stocked in quadruplicate in aquarium (50L) and were fed experimental diets containing three levels (3%, 10%, and 14%) of dietary lipids (LP). At the end of the trial, no significant differences in growth performance were observed. Fish fed diets with 3% and 10% LP exhibited significantly higher protein efficiency rates compared to those fed the 14% LP diet. A significant increase in lipase and alkaline phosphatase activity was observed in fish fed 14% LP diets. The inclusion of 14% lipids in the diet led to a decrease in albumin activity and an increase in the height of the intestinal villi. Although no significant differences were observed in aspartate aminotransferase, alanine aminotransferase activities, and size of hepatocytes. The histopathology of the liver indicated reduced hepatocyte size, lipid vacuoles, and sinusoidal space in the group fed the 3% LP diet. In conclusion, the use of 10% LP diet was found to be more favorable for liver health and growth performance.

Keywords:
enzyme digestive; feed utilization; lipid level; morphology intestine

Resumo

O objetivo deste estudo foi avaliar os efeitos de três níveis de lipídios na dieta sobre o crescimento, metabolismo e histopatologia intestinal e hepática em peixes-disco (Symphysodon aequifasciatus). Os peixes foram estocados em quadruplicada em um aquário (50L) e alimentados com dietas experimentais contendo três níveis (3%, 10% e 14%) de lipídios dietéticos (LP). No final do experimento, não foram observadas diferenças significativas no desempenho do crescimento. Os peixes alimentados com dietas com 3% e 10% de LP apresentaram taxas de eficiência proteica significativamente mais altas em comparação com aqueles alimentados com a dieta de 14% de LP. Um aumento significativo na atividade da lipase e da fosfatase alcalina foi observado em peixes alimentados com dietas com 14% de LP. A inclusão de 14% de lipídios na dieta levou a uma diminuição na atividade da albumina e a um aumento na altura das vilosidades intestinais. Embora não tenham sido observadas diferenças significativas nas atividades de aspartato aminotransferase, alanina aminotransferase e no tamanho dos hepatócitos. A histopatologia do fígado indicou redução do tamanho dos hepatócitos, dos vacúolos lipídicos e do espaço sinusoidal no grupo alimentado com a dieta com 3% de LP. Em conclusão, o uso da dieta com 10% de LP foi considerado mais favorável à saúde do fígado e ao desempenho do crescimento.

Palavras-chave:
enzima digestiva; utilização de ração; nível de lipídios; morfologia intestinal

1. Introduction

The discus fish (Symphysodon aequifasciatus Pellegrin, 1904) is an ornamental species renowned as the “King of Aquarium Fishes” due to its disc-shaped body and bright (Livengood et al., 2009). Native to the Amazon River, this species has been successfully bred in various parts of the world including China, Malaysia, Korea, and Germany (Pirhonen et al., 2014).

This species holds significant importance within the realm of aquarium fish (Fu et al., 2020). Previous research has primarily focused on topics such as protein requirements (Chong et al., 2000), carotenoids usage (Abdollahi et al., 2019), the replacement of fishmeal with soybean meal (Chong et al., 2003) and the fruit meal (Honorato et al., 2022). While an earlier study indicated that a dietary lipid content of 12 to 17% is optimal for discus fish growth (Wang et al., 2009; Wen et al., 2017), the physiological and histological adaptations remain unaddressed.

Lipids, along with proteins and carbohydrates, are the primary macronutrients that contribute to energy production, tissue building, and maintenance of homeostasis in all vertebrates (Honorato et al., 2010; Almeida et al., 2011; Welengane et al., 2019). In carnivorous species, lipid-rich diets have been shown to enhance fish growth (Lin et al., 2018; Liang et al., 2022). These molecules also play a critical role in fish nutrition by providing energy. In lipid-deficient diets, costly amino acids are diverted to energy production rather than protein synthesis for growth (Favero et al., 2020; Ma et al., 2022; Song et al., 2023). In addition, low dietary fat levels can result in slow growth and symptoms associated with essential fatty acid deficiency in several fish species, such as red seabream (Pagrus major) (Takeuchi et al., 1990; Watanabe 1993). Conversely, too high a lipid content (above 12.1%) can lead to metabolic disorders and liver damage or dysfunction (Guo et al., 2019; Paulino et al., 2020). Furthermore, feeding fish a high lipid diet (up to 12.1%) has been associated with increased lipid accumulation in the body (Akpinar et al., 2012; Guo et al., 2019; Li et al., 2023).

The objective of this study was to assess the impact of three different lipid levels on the histopathology of the intestine and liver, as well as on the growth performance of discus fish S. aequifasciatus. Additionally, the enzymatic activities in both the liver and intestine were evaluated to provide insights into the effects of the lipid-feeding regimen on S. aequifasciatus at both the cellular and biochemical levels.

2. Material and Methods

The fish were purchased from Piscicultura Cascavel Fish Farm, located in Cascavel, Brazil. The experiment was conducted at the same location for 60 days. The experiment was conducted using a randomized design, involving three dietary treatment groups and four replicates. The 180 discus fish S. aequifasciatus (with an initial body weight of 2.15 ± 0.15 g and a body length of 3.81 ± 0.50 cm) were randomly placed into 12 individual 50 L glass aquaria, each containing 15 individuals. Throughout the experiment, S. aequifasciatus were provided with apparent satiation feeding at 08:00, 11:00, 15:00, and 19:00 daily for the entire 60-day period. The water temperature was maintained at 27.0 ± 0.5 °C, pH 7.2, and dissolved oxygen levels at 5.4 ± 0.42 mg L⁻¹. The aquaria were consistently supplied with flowing water.

The experiment was approved by the Ethics Committee for Animals Use of Universidade Federal da Grande Dourados (Protocol number 004/14).

2.1. Experimental diets

Three isonitrogenous commercial diets were utilized in this study (crude protein of 420 g kg⁻¹), and these diets featured varying lipid levels (3, 10, and 14% LP). The diets were sourced from a common manufacturer, Poytara, located in Araraquara-SP, Brazil. The formulation of these diets drew from a range of ingredients including acai (Euterpe oleracea) pulp, albumin, garlic, corn starch, oat, beet, canthaxanthin, carboxymethyl-cellulose, DL-Methionine, spinach, yeast extract, linseed meal, soybean meal, Schizochytrium sp., algae meal, artemia meal, squid meal, fish flour, salmon meal, passion fruit fiber, gelatin, dehydrated Gryllus assimilis, dehydrated Musca domestica, soy protein isolate, lycopene, L-Lysine, corn, linseed oil, salmon oil, paprika, beet pulp, broken rice, salt, spirulina, Tenebrio molitor, yucca extract, vitamin-mineral premix, antioxidant, mycotoxin absorbent, probiotic, prebiotic, and multienzyme supplement. The proximate composition of the experimental diets (Table 1), the composition analysis of the diets was conducted according to the methods outlined by the Association of Official Analytical Chemists (AOAC, 2000).

Table 1
Proximate composition of the experimental diets.

Centesimal Composition	Lipids inclusion
	3%	10%	14%
Dry matter (%)	97.88 ± 1.06	96.61 ± 1.05	95.70 ± 1.03
Crude protein (%)	42.68 ± 1.01	40.66 ± 0.97	42.08 ± 1.00
Crude lipid (%)	3.07 ± 0.05	9.82 ± 0.01	13.60 ± 0.06
Carbohydrate* (%)	49.70 ± 1.12	44.76 ± 0.41	38.40 ± 1.02
Ash (%)	2.43 ± 0.01	1.36 ± 0.06	1.61 ± 0.45
Gross energy (kcal kg−1)	4975.44 ± 43.61	5151.37 ± 58.21	5217.82 ± 57.84

• Analysis conducted in triplicate. Mean ± SD (Standard deviation). * Carbohydrate = dry matter - (crude protein + crude lipid + crude fiber + ash).

2.2. Growth performance analysis

At the end of the experimental period, fish were captured and placed in a container containing 100 mg L^-1 eugenol for anesthesia (Vidal et al., 2008). The fish were then manually immobilized and final biometric analyses, including measurements of weight, length, and number, were performed according to the Equations 1 to 5:

$$\begin{array}{l}WeightGain\left(WG\right)=\\ \left(finalbodyweight\left(g\right)-initialbodyweight\left(g\right)\right)\end{array}$$ (1)

$$\begin{array}{l}LengthGain\left(LG\right)=\\ \left(finalbodylength\left(cm\right)-initialbodylength\left(cm\right)\right)\end{array}$$ (2)

$$\begin{array}{l}SpecificGrowthRate\left(SGR\right)=100x\\ [\left(lnfinalbodyweight-lninitialbodyweight\right)/\\ numberofexperimentaldays\end{array}$$ (3)

$$\begin{array}{l}FeedConversionRatio\left(FCR\right)=\\ feedintake\left(g\right)/weightgain\left(g\right)\end{array}$$ (4)

$$\begin{array}{l}ProteinEfficiencyRatio\left(PER\right)=\\ weightgain\left(g\right)/crudeproteinintake\left(g\right)\end{array}$$ (5)

The Condition Factor (CF) of the fish was calculated by the allometric method, from the Expression 6:

$$K=W/Lb$$ (6)

where: W = total mass, L = standard length of the individuals, and b = regression coefficient (Lima-Junior et al., 2002).

The uniformity of weight was assessed using the Formula 7:

$$U=\left(N/N1\right)x100$$ (7)

where: U = uniformity (%); N = number of animals in the tank; N1 = total number of animals with weight or length 20% higher or lower than the average live weight in each experimental unit.

2.3. Enzymatic analysis

After completing the biometric evaluations, 20 fish per treatment were randomly selected for euthanasia using the methodology following CONCEA normative resolution no. 37, on 15 February 2018. This process involved sedation followed by an incision of the spinal cord once the fish exhibited no signs of movement, resulting in the euthanasia of 20 fish per treatment group. The digestive tract (100 mg) and liver (100 mg) were excised and homogenized individually to measure enzyme activity. Intestine homogenates were prepared using 0.02 M Tris (Sigma- Aldrich, USA) and 0.01 M di-potassium phosphate (Vetec Quimica, Brazil) buffer at pH 7.0 mixed with anhydrous glycerol (Vetec Quiımica, Brazil), in 1:1 ratio under ice bath, using a Potter-Elvehjem homogenizer (Sigma, UK). The homogenates were then centrifuged at 12000 × g for 3 minutes, and the resulting supernatants (crude homogenates) were collected for enzymes analysis. The following digestive enzymes were measured: nonspecific protease using casein as substrate (Walter 1984) lipase using p−nitrophenol acetate as substrate (Albro et al., 1985), amylase using starch as substrate (Bernfeld, 1955) and alkaline phosphatase which was analyzed using colorimetric methods (Alkaline phosphatase OSR6004). The analysis was conducted using a spectrophotometry (semi-automatic spectrophotometer Bio plus S-200). Liver assays included Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) following the procedures by (Reitman and Frankel, 1957). Protein concentrations in enzyme crude extracts were determined using the method described by (Bradford 1976), which measures the absorbance of the protein-dye complex at 450 nm. A standard solution of 1.0 mg mL⁻¹ of albumin was used for calibration.

2.4. Histopathological analysis

For the histopathological analysis, 20 fish per treatment were randomly selected for euthanasia using the methodology following CONCEA normative resolution no. 37, on 15 February 2018, then the liver samples (approximately 1.0 cm) and intestine segments (5.0 cm of midgut) were collected. These tissues were fixed in Bouin solution (Sigma-Aldrich, USA) for 24 hours before further processing. The processing procedure involved dehydration using a crescent series of ethanol, diaphanization in xylene, trimming, and embedding in paraffin wax (HistosecVR, Merck-lot K91225309; Merck KGaA, Jacarepagua, Rio de Janeiro, Brazil). The embedded tissues were microsectioned to a 5-µm thickness and placed on slides. The sections were then dewaxed using xylene before staining with Harris’s hematoxylin and eosin (H&E) (Anamol, Maharashtra, India) and by histochemical staining with periodic acid-Schiff hematoxylin (PAS-H). Three sections per replicate were prepared for the histological analysis.

These sections were examined using a microscope at 40x magnification (model Precision P207), and pictures were captured with a digital camera (BEL model Eurekam 1.3), digital histomorphometry was prepared from three images captured randomly from histological sections. For image analysis, the Image J software was used. The height of intestinal villi and hepatocytes (Figure 1) were measured according to the method described by (Ota et al., 2019) which quantifies the number of all the cells per section.

Figure 1
Photomicrograph highlighting the the intestine showing epithelium region (EP), submucosa (s) e muscularis (m) of discus fish fed (A) 3% lipid diet, (B) photomicrograph showing villus height (continuous line) 10% lipid diet and (C) 14% lipid diet (H/E 20x).

2.5. Statistical analysis

The obtained data were grouped according to the treatments and subjected to a normality test using the Shapiro-Wilk test [[Q1: Q1]]). Data that showed normal distribution were then analyzed using analysis of variance (ANOVA). Significant differences (p< 0.05) were further examined using Tukey’s mean comparison test. All statistical analyses were performed using the R software (R Core Team, 2019).

3. Results

The performance of S. aequifasciatus fingerlings fed with diets containing varying percentages of lipids showed no statistical differences (p > 0.05) in terms of weight gain (WG), length gain (LG), feed conversion ratio (FCR) (Table 2), specific grow rate (SGR), and survival. However, fish fed a diet with 14% LP exhibited decreased protein efficiency compared to the other two groups (p < 0.05). Regarding protein efficiency rate, the highest values of 1.21 and 1.24 were obtained for diets containing 3% and 10% lipids, respectively. Additionally, the condition factor was three times greater in fish fed the 10% LP diet compared to those fed the 14% LP diet (Table 2). The uniformity of fish allotment was 85.5% for those fed the 10% LP diet and 78.3% for those fed the 14% LP diet.

Table 2
Growth, feed utilization efficiency, and homogeneity of juvenile S. aequifasciatus fed diets with increasing levels of dietary lipid.

Parameter	Lipids inclusion
	3%	10%	14%
Weight gain (WG) (g/fish)	1.97 ± 0.48	2.10 ± 0.51	2.07 ± 0.50
Length gain (LG) (cm/fish)	0.40 ± 0.14	0.53 ± 0.19	0.42 ± 0.15
Feed conversion ratio (FCR)	1.05 ± 0.20	1.11 ± 0.21	1.11 ± 0.21
Protein efficiency ratio (PER)	1.21 ± 0.13a	1.24 ± 0.13a	0.90 ± 0.09b
Specific growth rate (SGR) (%/day)	1.05 ± 0.337	1.70 ± 0.212	1.12 ± 0.264
Survival (%)	84.10 ± 9.40	90.90 ± 4.50	88.60 ± 5.10
Condition Factor	13.0	22.3	7.7
Uniformity (%)	80.6	88.8	78.3

• WG: weight gain; LG: length gain; FCR: feed conversion; PER: protein efficiency ratio; SGR: specific growth rate. Values are the mean ± SE of four replicates groups. Average initial body weight was 2.15 ± 0.15 g. Mean values within a given row with different superscripts are significantly different (P < 0.05).

In terms of enzymes activities, a significant increase in lipase (LIP) activity was observed in fish fed diets containing 10% and 14% lipids (128.40 and 127.70 UI mg⁻¹ protein; respectively) compared to those fed the 3% lipid diet (94.90 UI mg⁻¹ protein). As for alkalinity phosphatase (ALKP), it was significantly higher in S. aequifasciatus fed the 14% lipid diet (258.40 UI mg⁻¹ protein) compared to those fed 10% and 3% lipids (311.00 and 258.40 UI mg⁻¹ protein; respectively). Dietary lipid levels did not significantly affect amylase (AMYL), and alanine aminotransferase (ALT) activities. However, the lowest dietary lipid content (3%) resulted in the highest albumin (ALB) level (0.40 g dL⁻¹), and aspartate aminotransferase (AST) (541.00 UI mg⁻¹ protein) activity (Table 3).

Table 3
Activity of digestive enzymes and liver metabolic enzymes of S. aequifasciatus fed diets with increasing levels of dietary lipid.

Enzymes (UI mg−1 protein)	Lipids inclusion
	3%	10%	14%
Nonspecific Protease1	34.30 ± 1.70	32.20 ± 1.60	34.9 ± 1.73
Lipase (LIP)1	94.90 ± 10.10b	128.40 ± 13.66a	127.70 ± 13.59a
Amylase (AMY)1	1932.30 ± 306.70	1674.30 ± 265.79	1259.80 ± 199.99
Alkaline phosphatase (ALKP)1	258.40 ± 37.80b	311.0 ± 45.50b	478.50 ± 70.50a
Alanine Aminotransferase (ALT)2	75.00 ± 6.90	73.98 ± 6.81	67.9 ± 6.43
Aspartate Aminotransferase (AST)2	541.00 ± 67.70a	421.80 ± 52.70ab	346.6 ± 43.37b
Albumin (ALB) (g dL−1)2	0.85 ± 0.10a	0.75 ± 0.09a	0.40 ± 0.05b

• ¹
  Digestive enzymes;
• ²
  Metabolic enzymes. Values are the mean ± SE. Mean values within a given row with different superscripts are significantly different (P < 0.05).

Regarding intestinal morphometry (Figure 1A-C), it is noteworthy that fish subjected to the diets containing 14% and 10% lipids content (37.50 and 23.25 µm) exhibited significantly increased villus height compared to those fed with a 3% LP content diet, which displayed the lowest values (19.90 µm) (Figure 2). The histopathological analysis of the liver revealed differences among the treatments (Figure 3), particularly between the 3% and 10% lipid content diets compared to the 14% (p < 0.05) lipid content diet as depicted in Figure 2. fish subjected to a 3% LP diet exhibited reduced hepatocyte size, characterized by small lipid vacuoles (160.67 µm; Figure 3) and a diminished sinusoidal space. In contrast, the liver tissue of the 10% LP group demonstrated hepatocytes with a centralized nucleus and cordonal arrangement. Fish fed diet containing 14% LP diet exhibited vacuolized hepatocytes, with the nucleus displaced toward the periphery of the cell and increased sinusoidal space. Furthermore, the presence of hepatocellular necrosis, steatosis, a discreet inflammatory reaction, and septal fibrosis was evident in fish fed with the highest level of lipid content.

Figure 2
Intestinal villi height and hepatocyte size of S. aequifasciatus fed diets with increasing levels of dietary lipid. Different superscript letters are significantly different (P < 0.05). LP = lipid.

Figure 3
Histological appearance of the liver tissue (A, C) in liver of fish fed 3% lipid, and preserved cordonal architecture (fine trace) in fish fed 10% lipid diet, liver fed 14% lipid diet. Stained with PAS 400x.

4. Discussion

A previous study conducted by Wang et al. (2009) revealed that a dietary lipid content ranging from 12 to 17% is considered ideal for promoting the growth of S. aequifasciatus. However, in the current study, it was observed that the 14% lipid diet led to a reduction in PER and had a negative impact on fish performance. There were no significant differences among treatments for FCR, despite a significant decrease in PER in fish fed the 14% LP diet compared to those on the 3% and 10% LP treatments. These findings suggest that higher dietary lipid levels have a passive influence on nutrient utilization by fish, a phenomenon consistent with the observations made in a study by Fan et al. (2021) that assessed the effects of excess lipids on common carp (Cyprinus carpio). The results of the present study and that of Fan et al. (2021), may indicate that the protein-sparing effect is not as effectively achieved with excessive lipid supplementation in the diet.

The activity of digestive enzymes plays a pivotal role in assessing a fish’s capacity to utilize dietary nutrients effectively. In this study, we observed a notable increase in lipase concentration in the intestine tissue as the lipid content in the diet increased. This finding underscores a positive correlation between substrate concentration and enzyme activity. This result aligns with previous research, such as a study on Japanese sea bass (Lateolabrax japonicus) by Luo et al. (2010) and juvenile northern snakehead fish (Channa argus) by Sagada et al. (2017), both of which reported increased lipase activity with higher lipid levels in the diet. However, it’s important to note that some studies show that have indicated that elevated lipid levels can adversely affect the function of proteases, suggesting that the heightened production of lipase due to a high lipid diet may lead to reduced protease production (Luo et al., 2010; Fan et al., 2021; Lee et al., 2021; Ma et al., 2022; Li et al., 2023). This phenomenon is noteworthy, as diets with high fat content of the diet can reduce protein efficiency impacting PER and reducing overall food consumption. In such cases, animals may reach their energy requirements and become satiated, subsequently decreasing the absorption of dietary protein.

ALP plays a crucial role in the absorption of nutrients, including lipids, glucose, calcium, and inorganic phosphate (Tengjaroenkul et al., 2000). It is noteworthy that several studies have demonstrated a direct relationship between high dietary lipid levels and increased in alkaline phosphatase activity (Gawlicka et al., 1995; Grewal et al., 2020). Furthermore, several studies (Narisawa et al., 2003; Hansen et al., 2007) have suggested that alkaline phosphatase is implicated in the mobilization of lipid droplets following dietary intake. The activity of this enzyme is also associated with hepatic lipidosis showing increased alkaline phosphatase activity and rising dietary lipid levels (Fernandez and Kidney, 2007), align with the accumulation of lipids in the liver (Figure 3).

In animals, including fish, AST and ALT are vital transaminases primarily located in hepatocytes. The activities of these enzymes can significantly increase in response to liver damage or inflammation (Zhou et al., 2020; Ma et al., 2022). However, in the present study, no significant differences were observed in the activities of AST and ALT. Therefore, it becomes essential to consider other variables, such as digestive enzymes and albumin activity, to draw conclusions regarding the incorporation of lipids into the diet of S. aequifasciatus.

Albumin has been employed as a standard indicator in numerous liver function studies (Cheng et al., 2019; França et al., 2022), and is regarded as a standard for evaluating the structural and functional integrity of the liver sinusoidal unit (Rodrigues et al., 2017). With regard to albumin activity, a group fed a 14% lipid diet exhibited significantly lower albumin activity, suggesting the diminished albumin activity, observed in fish fed high-lipid diets, indicates impaired liver function and an elevated risk of liver damage due to excessive lipid intake (Wang et al., 2021; Ziemniczak et al., 2021).

The liver is a multifaceted organ responsible for various functions, including metabolism, detoxification, digestion, and excretion (Ota et al., 2019; Silva et al., 2021; Santos et al., 2021). The utilization of high-energy diets has a direct impact on fat deposition in the liver, a condition with significant health implications in fish (Ma et al., 2020; Yin et al., 2021; Paul et al., 2022). Hence, the examination of hepatic micrographs becomes pivotal in assessing the liver’s condition. In our study, the structural analysis of the liver revealed an excessive accumulation of fat in the cytoplasm (Figure 3).

Notably, the displacement of the nucleus within a hepatocyte is a frequent observation when describing the accumulation of lipid droplets (Ziemniczak et al., 2021). Pathological lipid accumulation is suggested when the nucleus deviates from its central position within the cell (Honorato et al., 2014). Furthermore, the development of a fatty liver is closely associated with compromised zootechnical performance. While discus fish in our study maintain their growth levels, alterations in albumin levels and liver structure indicate that this nutritional condition might not be sustainable over extended periods. Conversely, discus fish have exhibited plasticity in adapting their enzymatic and morphological profiles of the digestive tract to diets with moderate lipid levels, thereby enhancing their overall productive efficiency.

5. Conclusion

Excessive dietary lipids (14%) did not significantly affect growth performance but did show evidence of disrupting lipid homeostasis, reducing protein efficiency ratio, and affecting intestinal villus height. The results of the current study suggest that among the three different levels of lipids in the diet, the 10% lipid showed the most favorable outcomes for discus fish.

Acknowledgements

We would like to thank CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). The Sr. Henrique Momo Ziemniczak contribution to the histology manufacturer.

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Publication Dates

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

History

• Received
  20 Mar 2024
• Accepted
  25 July 2024