Nutritional and lipid profiles of the dorsal and ventral muscles of wild pirarucu

The objective of this work was to analyze the proximate and fatty acid composition of the dorsal and ventral muscles of wild pirarucu (Arapaima gigas) captured from a Brazilian Amazonian lake. Dorsal and ventral muscles were dissected out, freeze-dried, vacuum-packed, and had the proximate and fatty acid composition analyzed. Ash, total proteins, and lipids were inversely proportional to moisture and had higher levels in the ventral muscles. Twenty-seven fatty acids were quantified in both muscles without significant differences between them, except for the heneicosylic, palmitoleic, γ-linolenic, and dihomo-γ-linolenic acids. Saturated and monounsaturated fatty acids were predominant in both muscles. The eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids were quantitatively similar: 9.25 (dorsal) to 10.14 (ventral) and 8.50 (dorsal) to 10.63 (ventral) mg g-1 of total lipids, respectively. The EPA+DHA content of the dorsal and ventral muscles were 113.25 and 165.78 mg 100 g-1, respectively. The ratios of polyunsaturated/saturated (0.54 and 0.59 for the dorsal and ventral muscles, respectively), n-3/n-6 (0.20 and 0.21), and hypocholesterolemic/ hypercholesterolemic fatty acids (1.41 and 1.45) ratios, as well as the atherogenicity (0.59 and 0.53) and thrombogenicity (1.02 and 0.94) indices, indicate that pirarucu muscle is a good dietary source of EPA+DHA, and its nutritional lipid quality can be beneficial for human health.


Introduction
Pirarucu (Arapaima gigas) (Schinz, 1822) is the world's largest scaled freshwater fish, which is native to the Amazon basin and belongs to the most basal branch of extant Teleostei.This is a carnivorous fish reaching sizes of 3 m in length and weighting up to 200 kilograms (Fontenele, 1948).Arapaima gigas has an obligatory air-breathing system, moving to the water surface to breathe every 5 to 15 minutes (Fontenele, 1948), which makes it easy its identification and capture by fishermen.It is an emblematic species of the Amazonian ichthyofauna that has become very uncommon in the wild due to the overfishing in the last decades (Cavole et al., 2015), except in some natural reserves such as Pacaya-Samiria, in Peru, and Mamiraua, in Brazil (Núñez et al., 2011).Nowadays, A. gigas is in the Appendix II of the Convention on International Trade of Endangered Species of Wild Fauna and Flora, which means that its trade is controlled as a strategy to preserve the fish stocks (Cites, 2015).In Brazil, its fishing is regulated and only allowed under a community-based sustainable management system in protected areas during the dry season (Cavole et al., 2015).
Due to its large size, A. gigas represents a potential food resource not only for national trade in Amazonian regions, but also for international trade.In fact, this species has already been introduced in the United States (Hill & Lawson, 2015;Lawson et al., 2015), China, Cuba, Mexico, Philippines, Singapore and Thailand (FAO, 2012) as a promising alternative for food security.For local commercialization, Brazilian sellers have cut the fish and obtained the ventral and dorsal parts.These cuts have been sold at the market for different prices, US$ 3.75-5.00per kg for ventral muscle and US$ 6.25-7.00 per kg for dorsal muscle.Nevertheless, little is known about the flesh nutritional value of wild A. gigas, there is no scientific information regarding its fatty acid composition and whether there are differences between dorsal and ventral muscles.
Interest for studies regarding the nutritional and lipid value in foods has increased with particular focus on the highly unsaturated fatty acids (Hufa) of n-3 series, such as eicosapentaenoic (EPA; 20:5n-3) and docosahexaenoic (DHA; 22:6n-3) acids, due to their health benefits throughout human life (Inhamuns et al., 2009;Jabeen & Chaudhry, 2011;Swanson et al., 2012).EPA and DHA are associated with important physiological functions, such as the proper fetal development including neuronal, retinal, and immune functions, as well as the prevention of cardiovascular problems and Alzheimer's disease (Swanson et al., 2012).Since these fatty acids are primarily obtained by diet, the World Health Organization recommends the minimum consumption of 200 mg of EPA+DHA per day (WHO, 2015), and fish are considered to be one of the main natural sources of n-3 Hufa for human diets (Simopoulos, 2008;Inhamuns et al., 2009).
The objective of this work was to analyze the proximate and fatty acid composition of dorsal and ventral muscles of wild A. gigas, captured from a Brazilian Amazonian lake.

Materials and Methods
This study has been approved by the ethical committee of animal experimentation and research of the Instituto Nacional de Pesquisas da Amazônia (Inpa), municipality of Manaus, state of Amazonas, Brazil (Protocol number 002/2015).
Six wild fish (48.80±14.34kg; 1.70±0.14m) were collected in the community of Ilha da Paciência (3°22'S, 60°12'W), located in municipality of Iranduba, state of Amazonas, Brazil.Fish were captured using a dragnet and immediately slaughtered by medullary transection.Dorsal and ventral muscles were dissected out and kept in ice during the boat-land transportation to Inpa facilities, and were immediately stored in a freezer at -80°C.Afterwards, samples were freezedried, vacuum packed and sent to the chemistry department of the Universidade Estadual de Maringá for proximate and fatty acid composition analyses.
Moisture, ash and total protein in dorsal and ventral muscles were determined according to the procedures described by Horwitz (2005), and total lipids were determined by the Bligh & Dyer (1959) method.
Fatty acid methyl esters were prepared by the method proposed by Santos Júnior et al. (2014).Methyl esters were separated by gas chromatography using a Thermo Scientific Trace Ultra Gas Chromatographer (Thermo Scientific, Waltham, MA, USA), fitted with a flame ionization detector (FID) and a fused-silica capillary column (100 m × 0.25 mm i.d., 0.25 µm cyanopropyl CP-7420 select Fame).The operation parameters were as follows: detector temperature, 240°C; injection port temperature, 230°C; column temperature, 165°C for 18 minutes, programmed to increase at 4°C min -1 up to 235°C, with final holding time of 14.5 minutes; carrier gas, hydrogen at 1.2 mL min -1 ; nitrogen was used as the makeup gas at 30 mL min -1 ; and there was split injection at 1/80 ratio.For identification, the retention times of the fatty acids were compared to those of standard methyl esters (Sigma-Aldrich, St. Louis, MO, USA).Retention times and peak area percentages were automatically computed by a Software Chronquest 5.0.Quantification of fatty acids (mg g -1 of total lipids) was performed using tricosanoic acid methyl ester (Sigma-Aldrich, USA) as an internal standard (23:0) (Joseph & Ackman, 1992).Theoretical FID correction factor values were used to obtain concentration values of fatty acids in mg g -1 of total lipids (Visentainer, 2012) by using the equation below: FA = [(AX × WIS × CFX)/ (AIS × CFAE × WX)]; in which FA is fatty acid in mg g -1 of total lipids, AX is the peak area (fatty acids), WIS is the weight (mg) of standard, CFX is the theoretical correction factor, AIS is the peak area standard (23:0), CFAE is the conversion factor necessary to express results as mg of fatty acid rather than as methyl ester, and WX is the sample weight (g).
For data analysis, proximate and fatty acid analyses of dorsal and ventral muscle were determined in triplicate for each fish.Proximate values were correlated between them by using Pearson's correlation at 5% probability.Means of proximate parameters, fatty acid composition and nutritional quality indexes of dorsal and ventral muscles were compared by student's t-test, at 5% probability.Data were processed using SigmaPlot 11.0 software.

Results and Discussion
The content of ash, total proteins and lipids was inversely proportional to moisture content and higher in ventral muscles of wild A. gigas (Table 1).Regarding the total lipid content found in both muscles, A. gigas is considered a medium fat fish (4.00 to 8.00% body fat) in accordance with the classification of Ackman (1989).Moreover, this study reported higher lipid content in ventral muscles of wild A. gigas.In fact, it is known that ventral areas usually contain more lipids than the dorsal area in fishes (Thammapat et al., 2010).Despite significant differences between the lipid contents, the fatty acid compositions in both muscles were quantitatively similar, except for heneicosylic (21:0), palmitoleic (16:1), γ-linolenic (18:3n-6) and dihomo-γ-linolenic (20:3n-6) acids (Table 2).In these sense, 27 fatty acids were quantified in both muscles, with predominance of saturated (SFA) and monounsaturated (Mufa) fatty acids, in a range from 295.32±27.55 to 308.67±28.58(SFA), and from 242.24±28.49to 248.88±28.84(Mufa) (mg g -1 of total lipids) in dorsal and ventral muscles, respectively, without quantitative difference between them.Polyunsaturated fatty acids (Pufa) were lower than SFA and Mufa in the two analyzed muscles, varying from 158.22±11.93 to 183.18±16.29 (mg g -1 of total lipids) in dorsal and ventral muscles, respectively, being higher in the ventral part.A similar predominance pattern has been observed in some Amazonian fish, such as the tucunaré (Cichla sp.) in dry season (Inhamuns et al., 2009); mapará (Hypophthalmus sp.) (Inhamuns & Franco, 2001); and spotted sorubim (Pseudoplatystoma corruscans) (Tanamati et al., 2009); as well as in wild freshwater fish from other regions (Jabeen & Chaudhry, 2011).
All the quantified Pufa showed quite similar values in both dorsal and ventral muscles of wild A. gigas, except for linoleic acid, which was higher than the other Pufa and is considered one of the essential fatty acids for freshwater fish (NRC, 2011).Probably, the highest proportion of linoleic acid in both muscles comes from the fish diet, since A. gigas is a carnivorous fish showing a preference for fishes of the Loricariidae family, which are mainly herbivorous and planctophagic (Fontenele, 1948).Actually, the fatty acid composition is strongly influenced by the age, feeding, reproductive cycle, season, salinity, geographical location and biology of the fish species.Therefore, different results can be found even in the same species under different conditions (Inhamuns et al., 2009;Jabeen & Chaudhry, 2011).
The sum of EPA and DHA in A. gigas reached 113.25 mg and 165.78 mg in 100 g of dorsal and ventral muscles, respectively.It means that a person should eat at least 120.64 g of ventral muscle or 176.60 g of dorsal muscle to achieve the minimum EPA+DHA amount of 200 mg per day as recommended by the World Health Organization (WHO, 2015).That amount of flesh is relatively small in comparison to the size that A. gigas could reach and considering its availability in the Amazonian market.
The Pufa/SFA, n-3/n-6 and H/H ratios, as well as the AI and TI indexes, indicate that wild A. gigas flesh could be beneficial for human health.The Pufa/SFA ratio in A. gigas (Table 3) is higher than the value (0.45) recommended by the Department of Health and Social Security of the United Kingdom (DHSS, 1984), which considers diets with a Pufa/SFA ratio below 0.45 as not healthy due to their potential in inducing cholesterol increase in the blood.The n-6 series fatty acids showed higher proportions than the n-3 in both dorsal and ventral muscles, especially linoleic and arachidonic acids -20:4n-6; 14. 67±3.20 (arachidonic, dorsal) to 20.91±6.81(arachidonic, ventral) mg g -1 of total lipids.The n-3/n-6 ratios in A. gigas were not quantitatively different between muscles.However, these data are lower when compared with the ones of other Amazonian fish, such as the Cichla sp.(0.66-0.98) (Inhamuns et al., 2009), C. macropomum (0.41) (Almeida et al., 2008) and Hypophthalmus sp.(1.5-1.6)(Inhamuns & Franco, 2001).In those fish, all the n-3/n-6 ratios were affected by the high proportion of DHA in their muscles.Nevertheless, nutritionists believe that the n-3/n-6 ratio should be at least 0.20 (Simopoulos, 2008) to provide nutraceutical benefits for human health, and the A. gigas ratio was into that limit.

Conclusions
1. Dorsal and ventral muscles of wild Pirarucu are a good dietary source of eicosapentaenoic and docosahexaenoic acids (EPA+DHA), highly unsaturated fatty acids.
2. Both dorsal and ventral muscles show lower atherogenicity and thrombogenicity indexes, which indicate beneficial effects in preventing coronary artery diseases.
Results are shown as means ± standard deviations of triplicate analyses per fish (n = 6).

Table 1 .
Proximate composition (% in wet weight basis) of dorsal and ventral muscle of wild Arapaima gigas from Brazilian Amazon(1).
(1) Means followed by equal letters within rows, do not differ, by Student's t-test, at 5% probability.Results are shown as means±standard deviations of triplicate analyses per fish (n = 6).

Table 2 .
Fatty acid composition (mg g -1 of total lipids) of dorsal and ventral muscle of wild Arapaima gigas from Brazilian Amazon(1).

Table 3 .
Nutritional quality indexes of the lipid fraction of dorsal and ventral muscle of wild Arapaima gigas from Brazilian Amazon(1).