Fatty acid profile of Slovenian farmed rainbow trout

The present study was carried out to determine the fatty acid profile of rainbow trout (Oncorynchus mykiss) from three Slovenian fish farms; Zalog, Želimlje and Povodje. Fatty acids composition was determined on a gas chromatograph with a flame ionization detector (GC-FID). The results showed that farming conditions have a significant influence on the fatty acid composition of rainbow trout. The predominant saturated fatty acid (SFA) was palmitic acid, oleic acid was the main monounsaturated fatty acid (MUFA), while the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were the main long chain n-3 polyunsaturated fatty acids (n-3 PUFA). The percentage of DHA exceeded that of EPA in all rainbow trout samples studied. The n-6/n-3 ratio ranged from 0.89 to 1.54 and the PUFA/SFA ratio was between 1.81 and 2.36. In dorsal and ventral fillet parts, the content of most fatty acids was similar, exceptions were observed for some PUFAs; arachidonic acid, EPA and DHA.


Introduction
Fish meat is not only an important source of protein, but also contains nutritionally valuable lipids and fatty acids (FA). It is known as a rich source of long-chain n-3 polyunsaturated fatty acids (n-3 PUFA), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are essential for normal growth, development and reproduction in all vertebrates, including fish and humans (Lauritzen et al., 2001;Howe et al., 2005). Interest in the health benefits of longchain n-3 PUFA is increasing as numerous stud-ies report that a diet rich in fish protects against chronic diseases such as coronary heart disease. Consumption of fish meat is increasing today as consumers become aware of the positive health effects and numerous micronutrients such as vitamin A, vitamin B 12 , zinc, selenium and iodine (Guler et al., 2008).
Fats have a high energy density and are therefore an important source of energy in the diet of fish. They build cell membranes and are important for maintaining their functions. Fish, like all other animals, cannot synthesize n-3 and n-6 PUFAs themselves, so they must be supplied through the diet (Gonzalez et al., 2006). At least 1 % of the daily energy requirement must be provided by n-3 fatty acids. It is known that excessive levels of n-6 fatty acids can negatively affect fish growth, as it can inhibit the conversion of linolenic acid to polyunsaturated fatty acids (Ruxton et al., 2004). Vegetable oils such as soybean and canola oils are rich in linolenic acid and are used in trout nutrition. Fish oils have an even better effect on fish growth as they contain long chain PUFAs, namely EPA and DHA, in concentrations of over 30 %, while n-6 concentrations are much lower compared to vegetable oils. The trout diet should contain between 6 % and 14 % of the daily energy requirement from pure fat (Ruxton et al., 2004).
In fish, n-3 fatty acids are present in high concentrations in the phosphoglycerides of cell membranes. In farmed fish, diet has a major influence (McKenzie et al., 2000;Luzar, 2018). The lipid composition of farm fish is more constant than that in wild fish. Farmed fish have a higher fat content than wild fish and are therefore a better source of n-3 fatty acids (Ibrahim Haliloglu et al., 2004). Lipid distribution in fish muscle varies greatly, depending on species, type of muscle and sampling site; in rainbow trout, variations in lipid content were found both in dorsal and ventral part (Testi et al., 2006). Research and development in aquaculture has focused on feed ingredients, as the amount of lipids and fatty acids in the diet is of fundamental importance. However, in wild trout meat, the fatty acid profile varies greatly. It depends on the amount of food consumed, the season, the geographical area and the age of the fish. In farmed trout, there are approximately the same data between fish, which generates a positive response in the market. Farmed fish generally have higher total lipid levels than wild fish, 100 g of farmed fish can provide a higher amount of n-3 PUFAs (especially EPA and DHA) than 100 g of wild fish (Cahu et al., 2004). As farmed fish become an important part of the world's fish supply, it is important to maintain the high lipid nutrient quality of the product and continue to provide customers with large quantities of the n-3 PUFAs.
In Slovenia, rainbow trout or California trout (Oncorynchus mykiss) is widely distributed and represents an important freshwater fish from a nutritional and economic point of view. In pres-ent study we focused on the fatty acid composition of rainbow trout from selected fish farms and attempted to assess whether individual rainbow trout samples meet the required nutritional parameters, especially in terms of long chain n-3 PUFAs.

Sampling and sample preparation
Rainbow trout or California trout (Oncorynchus mykiss) samples (n=12) were taken from three Slovenian fish farms (Povodje, Zalog and Želimlje), located in the Osrednjeslovenska statistical region, randomly from the pond where they were reared under specific farming conditions. The sampling was carried out between March and April, 2019. Trout samples were slaughtered in water and ice, packed in polystyrene cool boxes and transported to the laboratory where they were processed. The head and tail were removed, and the abdominal cavity was opened along a ventral midline incision. All visceral mass, skin and bones were removed and discarded. To separate both fillets from each carcass, an incision was made along the dorsal fin to the caudal fin and another incision was made behind the opercula, leaving out the lateral and ventral fins. Each fillet was cut along the insertion line of the ribs to obtain one dorsal and one ventral fillet. Two dorsal fillets from each fish were joined together. The same was done with the two ventral fillets. Sampling was done at 6 different locations; 3 on the dorsal and 3 on the ventral parts of the carcass. The samples were homogenized with a Grindomix homogeniser GM 200 (Retsch, Haan, Germany), vacuum packed and stored at a temperature of -20 °C until further analysis.

Fatty acid profile determination
Fatty acid composition of rainbow trout samples was determined according to the method of Park and Goins (1994), modified by Polak et al. (2008) The in situ transesterification (ISTE) was used to determine the individual fatty acid in the sample.

Statistical analysis
In the statistical analysis, the data were analyzed for normal distributions using the UNIVARIATE procedure (SAS/STAT, USA). The differences according to the fish farm or fish fillet part (dorsal, ventral) were analyzed through ANOVA procedure and Duncan test (SAS/STAT), with a 0.05 level of significance. The experiment was performed in three production replications.

Results and Discussion
The fatty acid profile (as a percentage of total fatty acid) of the rainbow trout species is presented in Table 1. The analysis showed that there were significant differences in the fatty acid profile between the rainbow trout samples from all tested fish farms.
The highest total level of SFAs was found in trout fillets from Želimlje fish farm (21.70 %), compared to trout fillets from Zalog (17.49 %) and trout fillets from Povodje (15.39 %). Among SFAs, myristic acid (C14:0), palmitic acid (C16:0) and stearic acid (C18:0) were found most frequently. Palmitic acid was the predominant fatty acid in trout fillets, with significant differences among farms (15.42 % of the total FA for Želimlje, 11.81 % of the total fatty acid for Zalog and 10.06 % of the total fatty acid for Povodje). When we compare the data obtained for total SFA with data from the study of Blanchet et al. (2005), who analyzed the fatty acid composition of farmed rainbow trout, the total SFA content was higher (26.9 %) compared to our results. In the present study, MUFA content in rainbow fillets was higher than SFA content in all three fish farms, which is in agreement with the results of Celik et al. (2008).
The major MUFA was oleic acid (C18:1) and differed significantly between fish farms. Rainbow trout from Povodje showed the highest percentage (40.33 %), followed by Zalog (35.37 %) and the lowest for Želimlje (28.83 %). Various vegetable oils are added to fish feed as a source of energy. The most common is rapeseed oil, which contains erucic acid. The presence of erucic acid (C22:1) in the trout samples studied confirmed its origin from the feed for the farmed specimens (Barbara et al., 2003).
In general, the total content of PUFAs ranged from 36.25 % (Povodje), 38.48 % (Zalog) to 39.21 % of the total FA (Želimlje). Compared to the data of Blanchet et al. (2005), PUFAs content in farmed rainbow trout samples was 40.60 % and in wild trout samples 58.60 %, where the total lipid content was three times higher in farmed trout samples. In contrast, in the study by Renko (2012), the total PUFAs content in farmed European bass fish (Dicentrarchus labrax) was 35.00 %, which was higher than wild European bass (26.95 % of total fatty acids). Significant differences in the content of long-chain n-3 and n-6 fatty acids, referred to as PUFAs, were found in our study. The percentage of total n-3 PUFAs tended to be higher in trout samples from Želimlje (20.17 %) than in rainbow trout samples from Povodje (15.29 %) and samples from Zalog (14.80 %), while the content of n-6 PUFAs were highest in trout samples from Zalog (22.84 %) and lowest in trout samples from Želimlje (18.01 %). Among the long chain n-3 PUFAs, it is worth mentioning EPA and DHA, which are essential nutrients as they are main components of cell membranes and have an important role in nutrition for human health. EPA and DHA possess extremely beneficial properties for the prevention of human coronary artery disease and are precursors for prostaglandins, leukotrienes, and tromboxanes of n-3 family. Therefore, fish diet is recommended as an important component for human health (Simopoulos, 2002). The percentage of DHA exceeds the percentage of EPA in all studied trout samples, the highest value was observed in Želimlje fish farm (13.52 %), followed by Povodje (9.54 %) and Zalog (8.23 %). Dietary recommendations for EPA and DHA based on cardiovascular risk considerations are between 250 and 500 mg/ day for European adults (EFSA, 2012). On average, with consumption of 100 g rainbow trout from our study daily recommendations for EPA and DHA are reached or even exceeded.
About 90 % of total n-6 PUFAs belonged to linoleic acid (C18:2), which is an essential fatty acid and precursor of arachidonic acid with elongation and saturation. This fatty acid is found in vegetable oil, which is present in the diet of farmed fish (Grigorakis et al., 2002). In the present study, trout samples from all three fish farms contained arachidonic acid, which is a precursor for prostaglandins, leukotrienes, and tromboxanes of n-6 family (Gurr, 2000). Arachidonic acid also plays a role in brain, retinal and infant growth. In the study of Blanchet et al. (2005), linoleic acid, alpfa-linolenic acid, and arachidonic acid were higher in farmed trout than in wild trout samples. This could be explained by the use of vegetable oils in fish feed. The n-6/n-3 ratio ranged from 0.89 (Želimlje) to 1.54 (Zalog). Data were comparable with study of Luzar (2018), where the average n-6/n-3 ratio of rainbow trout from Slovenian fish farms was 0.98. All rainbow trout samples tested had the n-6/n-3 within the recommended ratio. Suggested ratio n-6/n-3 (5:1) constitute a healthy human diet (Simopoulos, 2002). Moreover, the P/S ratio was much lower in the trout samples from Želimlje (1.81) due to the abundance of SFAs, especially palmitic acid. The n-6/n-3 is important because n-6 fatty acid derived eicosanoids are more inflammatory than n-3 fatty acid derived eicosanoids (Connor, 2000). As a result, n-3 fatty acids are considered anti-inflammatory because replacing the more inflammatory n-6 fatty acid derived eicosanoids with n-3 fatty acid derived eicosanoids will decrease inflammation.
The fatty acid profile of the dorsal and

Conclusion
Despite significant differences in the fatty acid profile of rainbow trout samples between farms, the results showed that consumption of all rainbow trout tested provided high levels of beneficial long-chain n-3 PUFAs, especially in terms of cardiovascular disease prevention. Considering the data from our recent research (Luzar, 2018), where the total fat content of farmed rainbow trout was determined (2.52 g/100g), the recommended daily intake of EPA + DHA is achieved with the consumption of 100 g of rainbow trout meat. The data obtained are important for our country, where farmed fish raised in appropriate conditions represent a large part of nutritionally desirable fatty acids.