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Common carp Cyprinus carpio is one of the most cultured fish in the world. In 2008, the world and the European production was 2Â 987 433 tons and 144Â 747 tons, respectively (FAO, 2011). Carp is also one of the most important farmed freshwater fish species in the central Europe. It is a well established cultured species with a well known production cycle and consumed as traditional food. Carp is an omnivorous species eating zooplankton and benthos (worms, insects, molluscs) as well as detritus in the natural conditions. Typical way of farming is using artificial shallow earthen ponds in which the production is based on natural food supplemented by cereals. The carp's digestive system is adapted to a diet including more carbohydrates compare to carnivorous species. The farming cycle in Europe usually takes 3-4 years
Common carp is divided in two subspecies, C. c. carpio from Europe and C. c. haematopterus from Asia. Productive populations were domesticated from both ancestral forms, as well as their hybrids and backcrosses followed by mass selection .
There is a large body of evidence that n-3 fatty acids, especially EPA and DHA are beneficial for human health. Today's western diet is deficient in omega 3 fatty acids and excessive in omega 6 resulting in a high n-6/n-3 ratio. Several studies suggest that human being evolved on a diet with the n-6/n-3 ratio close to 1 whereas in the western diet it exceeds 15:1 . This change is associated with pathogenesis of many diseases, including cardiovascular diseases, cancer, inflammatory and autoimmune diseases.
EPA and DHA influence the physical nature of cell membranes, membrane-protein-mediated responses, eicosanoid generation, cell signaling and gene expression in many different cell types . EPA and DHA have been shown to beneficially modify a range of cardiovascular risk factors, which result in primary cardiovascular prevention, reduction in total and cardiovascular mortality with an especially potent effect on sudden death .
Stable isotope studies indicate that conversion of ALA to EPA occurs but is limited in men and that further transformation to DHA is very low . Therefore it was proposed that EPA and DHA should be consumed directly to maintain optimal tissue functions.
conducted an extensive meta-study and concluded that the benefits of fish intake exceed the potential risks of possible harmful effect of pollutants.
The world capture fisheries have been relatively stable in the past decade and are predicted to be stable or slightly decline in the future. It is not possible to further increase the fish capture in world-wide scale otherwise there would be a problem with overfishing and potential depletion of resources. The aquaculture production is the only solution to meet the increasing consumer demand for fish. Aquaculture is the fastest growing animal-food-producing sector with a growth rate from 1970 of around 8.3 % per year and with 52.5 million tons (in 2008; 68.3 including aquatic plants) accounts for almost half of total food fish supply (SOFIA, 2010).
Fish oil and fish meal have been traditionally used as the basal ingredients in aqua-feeds for carnivorous fish culture. Fish oil has a high level of the n-3 highly unsaturated fatty acids (n-3 HUFA), EPA and DHA, which are healthy for fish as well as for humans. Since the aquaculture expands, fish meal and fish oil become more expensive. It consequently creates a high pressure on the aqua-feed producers to replace these ingredients with more sustainable alternatives . Generally, the vegetable sources of oil and protein are used as "The replacement". The vegetable oil can replace substantial amount of fish oil in the diets of many fish species without affecting growth of feed efficiency. However, the drawback of these alternatives is that they are lacking the n-3 HUFA and therefore are compromising the nutritive value of farmed fish for consumers. Several alternative oil sources, derived from unicellular algae, pelagic organisms or benthic invertebrates containing high amounts of n-3 HUFA have been identified and tested in aquafeeds. Nevertheless, their prices are still too high to be commonly used in aquafeeds (reviewed by .
Carps represent the largest group of cultured fish constituting around 70 % of freshwater aquaculture production. In Europe, the majority of carp production takes place in central Europe where it is produced in ponds using traditional semi-intensive techniques. There are two sources of n-3 HUFA in carp produced in ponds. First source is the natural feed, plankton and benthos, which is rich in n-3 HUFA and second is the n-3 HUFA synthesized by carp from ALA. Carps have been shown, in contrast to marine fish, to be able to bio-convert larger quantities of ALA to EPA and DHA citace pro carps . They have also relatively low requirements both for n-3 and n-6 fatty acids (0.5-1 %) which can be fulfilled by plant 18 carbon fatty acids . Inclusion of fish meal (5 %) and fish oil (0 %) in carp culture is also very low and therefore the substitution of fish meal and oil will be considerably easier than for carnivorous aquaculture.
It is therefore of interest to understand and maximize the innate ability of carp to synthesize EPA and DHA from ALA in order to preserve the lipid quality of the fish as human food and for sustainable utilization of feed resources. Carp culture may be capable of becoming net producer of EPA and DHA by selecting fish with high enzyme activities in fatty acid elongation and desaturation.
PUFA biosynthetic pathway
Factors influencing lipid content and composition
Effect of nutrition
Freshwater fish are as well as marine fish an important source of n-3 fatty acids in human diet. Carp is traditionally reared in earthen ponds and its nutrition is based on natural food with cereal supplementation. Since cereals are rich in carbohydrates and have very low level of n-3 fatty acids the flesh of the farmed carps generally contain a high level of oleic acid and low level of favorable n-3 HUFA (Csengeri, 1996).
N-3 HUFA content in pond produced carp flesh can be increased by several means from which nutrition is the most important . Carp is omnivore and its natural food consists mainly of zooplankton, zoobenthos and detritus . Plankton (Domaizon et al, 2000) and benthos (Bell et al., 1994; Bogut et al., 2007) naturally contain high levels of n-3 FA, including EPA and DHA. Thus a proper pond management maintaining sufficient amount and appropriate structure of planktonic and benthic community is of great importance when improving carp fatty acid composition.
Carp was shown to have the ability for n-3 HUFA biosynthesis from its precursor 18:3 n-3, alpha-linolenic acid (ALA) . Thus supplemental feeding which is rich in ALA could be the alternative way to increase n-3 HUFA content in carp flesh. Feeds with a high level of ALA content, cheap and easily available, are rapeseed, linseed and hempseed. Rapeseed or rapeseed cake is becoming an important part of pellets for carp nutrition in the Czech Republic for its low price and availability. Rapeseed oil has a moderate amount of ALA (13 %) and favorable ratio n-6/n-3 around 2 and is commonly used in feed for salmonids as a replacement for fish oil (. However, there are no available data about impact of rapeseed oil on carp lipid content and composition when being used in pond production systems where many factors are influencing the muscle lipid composition, such as variations in amount and composition of natural food, fish density or environmental conditions as well as interactions among them.
It was shown that genes and LC-PUFA biosynthesis are up-regulated after plant oil feeding (Tocher et al., 2001; Zheng et al., 2005a; Leaver et al., 2008; Morais et al., 2009)
An alternative approach to influence muscle lipid composition might be the use of biologically active compounds which modulate the fish metabolism to synthesize or deposit more n-3 HUFA.
Such a potent compound could be sesamin. As a first study investigating sesamin effects in fish Trattner et al. (2008a) found that sesamin/episesamin supplementation increases the level of DHA up to 37 % in white muscle of rainbow trout (Oncorhynchus mykiss) fed with high ALA vegetable oil. An in vitro study with Atlantic salmon (Salmo salar) hepatocytes (Trattner et al., 2008b) showed that sesamin/episesamin exposure led to increased elongation and desaturation of 14C ALA to DHA indicating that sesamin has modulatory effects on lipid metabolism leading to increased levels of DHA and higher Î² oxidation activity. However, there are still many questions about the use of sesamin in fish feed, especially whether effects similar to those observed in salmonids can also be observed across different fish species, particularly cyprinids.
Another potential bioactive compound might be lipoic acid. Lipoic acid acts as an antioxidant both in the hydrophilic and hydrophobic phases (Navarri izo, 2002). It was shown to have several effects on lipid metabolism in chicken (Hamano 2002), humans (Jameel, 2006), rats and rabbits (Mythili 2006). Trattner et al. (2007) studied effect of lipoic acid on fatty acid composition in brain and muscle in South American pacu (Piaractus mesopotamicus) and found that lipoic acid increased level of EPA in muscle polar lipids.
Effect of genetics
Another important factor affecting lipid content and composition is genetic origin. It was shown that muscle lipid content is a highly heritable trait (>0.5) in common carp and that there is a relatively high positive genetic correlation between body size (standard length and body weight) and lipid content (0.71 and 0.59, respectively) (Kocour et al., 2007). Buchtova et al. (2007; 2008) studied lipid content in four hybrids of common carp. The lipid content correlated with body weight. There is evidence from mammals and birds that there is a heritable genetic component governing capacity to biosynthesize and/or deposit n-3 HUFA (De Smet et al., 2004; Karamichou et al., 2006; Khang et al., 2007).
Leaver et al (2011) analyzed flesh lipid parameters in 48 families of Atlantic salmon and showed that flesh nâˆ’3 HUFA composition is a highly heritable trait (h2=0.77Â±0.14). Eight families were further selected for transcriptomic analyses. They found that there were specific hepatic mRNA expressions patterns associated with high flesh nâˆ’3 HUFA, which indicate possible mechanisms for family-dependent deposition in flesh.
There are no relevant data available for carp about the effect of genetic origin on the lipid composition (Fauconneau et al., 1995). In the study with the four carp hybrids Buchtova et al. (2007; 2008) found that, the fatty acid composition was not affected to any great extent by the hybrid type.
Effect of sex
Another factor with a large effect on lipid content and composition in animals is sex (Nurnberg et al., 1998). In cattle, the order of leanness in young slaughter animals is normally bulls > steers > heifers. In slaughter pigs, it is boars > gilts > barrows (De Smet et al., 2004).
Kocour et al. (2007) reported that females of Hungarian synthetic mirror carp were fatter than males probably due to later maturation. In study with four common carp hybrids Buchtova et al. (2008) found only minor differences in lipid composition between males and females probably caused by different lipid content. Fajmonova et al. (2003) did not find any sexual dimorphism in lipid content and fatty acid composition in three-year-old carps.
Effect of body tissue
Fish fillet is highly heterogeneous and is composed from several different tissues (e.g.: white muscle, red muscle, adipose tissue and skin). The tissues differ greatly in lipid content and therefore the lipids are not equally distributed in the fillet.
Variation in lipid content has an effect on fatty acid composition, independent of species or breed and dietary factors. The content of SFA and MUFA increases faster with increasing fatness than does the content of PUFA in livestock (De Smeth et al., 2004). This was shown for bulls fed different diets (Raes et al., 2003). Simmilar findings were reported for pork (Riley et al., 2000) and sheep (Nurnberg et al., 1998). The effect of fatness on the fatty acid composition can be explained to a large extend by differences in the fatty acid composition of the major lipid fractions and the relative contribution of these fractions to total lipids. Phospholipids are particularly rich in PUFA, whereas triacylglycerols contain much lower amounts of PUFA (De Smet et al., 2004).
Decreasing level of PUFA with increasing fatness was also reported in several fish species: mapará (Hypopthalmus sp.)(Inhamuns et al., 2001), rainbow trout (Kiessling et al., 2001). However, with MUFA being the main fatty acid class correlated with the level of fatness (reviewed by Henderosn and Tocher, 1987; Kiessling and Kiessling, 1993).
Effect of starvation
Purging of fish before slaughtering or delivery to market is a common practise in aquaculture to remove possible off-flavours and eliminate undigested food from intestine (Lim and Webster, 2006). It is usually done by moving the fish to clean water and starving them from few days to many weeks (Masser et al., 1999). The purging can also improve nutritional quality of the farmed fish by reducing excessive fat and increase of n-3 HUFA percentage (Einen et al., 1998; Palmeri et al, 2008). Einen et al. (1998) studied effect of starvation prior to slaughter in Atlantic salmon. They found significant but rather marginal effects of starvation on fatty acid composition in muscle, belly flap and liver. However, the fish used in the study had quite high muscle lipid content (16 %) and therefore much bigger effects could be probably seen in fish with lower muscle lipid content.
Csengery (1995) studied effect of starvation on lipid content and composition in common carp. He observed that there was a consistent decrease in the oleic acid levels both in muscle and liver and that PUFA were somehow protected. He also reported that the effect of starvation was dependent on the previous feeding. Vacha et al (2007) studied effect of long term starvation on fatty acid composition in common carp fed either on cereals or natural feed only. The carps supplemented by cereals had high lipid content (> 10 %) compared to the carps fed natural feed only (1.8 %). The biggest differences in fatty acid composition could be seen in the lean fish fed natural feed only, where mainly decreased levels of PUFA were observed.
Effect of processing and cooking
The last but not the least factor influencing lipid content and composition is processing and cooking. Especially, the quality of fats and oils added during processing has a very strong influence on lipid composition (Ansorena, 2004; Sampels et al., 2009. It was shown that lipid composition of fish products differ markedly from the raw fish (National Food administration, 2008). Sampels et al. (2009) found very high variation of n-6/n-3 ratio in fish products being up to 400 times higher than in the raw fish. They concluded that fat sources used during the processing and preparation has the largest impact on the food FA content and composition and proposed that it should be declared on the product label. The product lipid composition might further change when it is fried by the consumers (Ramirez 2005).
The overall aim of this work was to evaluate factors influencing lipid content and composition in common carp muscle. This work is a part of a bigger project focused on improvement of carp muscle lipid quality in order to be used as a local healthy product for prevention and treatment of cardiovascular diseases. The goal is to develop a technology of carp culture with using long term sustainable alternative feedstuffs.
Specific objectives were to:
Investigate lipid content, lipid classes and fatty acid composition in three parts of common carp fillets (Paper I).
Examine the effect of genetic origin on lipid content and fatty acid composition in common carp (Paper I).
Study the effects of sesamin on fatty acid composition in common carp (Paper II).
Study the effect of sesamin on global gene expression in vivo in common carp (Paper II).
Material and Methods
The chapter shortly describes the material and methods used in the studies included in the thesis. For a more detailed description of each method see Papers I-II. An overview of the material and methods used is shown in Table X
In Paper I, samples from four different crosses of common carp (Cyprinus carpio) reared in an earthen pond were sampled (Mean weight 2 kg). They were reared on the basis of natural food (plankton and benthos) with cereal supplementation (wheat). The sampled crosses were a pure line of Hungarian mirror carp (M2 x M2), a hybrid line of two Hungarian mirror carps (M2 x L15), a hybrid line of Hungarian mirror carp and Israeli mirror carp (M2 x Dor70), and a hybrid line of Hungarian mirror carp and Northern mirror carp (M2 x M72). Lipid content and total lipid fatty acid composition (in total lipids, phospholipids and triacylglycerols) of fillets of four fishes per cross were analyzed. The samples consisted of one slice dissected from the dorsal fin to the ventral line, including 50% of the red muscle tissue.
Other Six randomly selected individuals of the M2xM2 strain (mean weight 2 kg) were used to analyze differences among fillet parts. Three tissue samples, white dorsal muscle (WM), red muscle (RM), and abdominal wall with adipose tissue (AW), were dissected from each fillet of the six individuals. The samples were analyzed for lipid content, lipid classes composition and fatty acid composition (in total lipid, phospholipids and triacylglycerols).
Samples of plankton were collected from the pond in July, August and September and were analyzed for lipid content and total lipid fatty acid composition.
Table . Overview of study design for Papers I-II
Type of culture
Cages with recirculation system
Natural food + cereals
Natural food + cereals
Pelleted feed +/- sesamin
Total cytochrome P450
Global gene expression profiling
In Paper II, two-year-old common carp (Cyprinus carpio) individuals (mean weight 830 g) were reared in six 1 m3 tanks (6 fish per tank) connected to a recirculation system. The fish were fed diets with or without sesamin addition (0.58 g/100 g feed) for 9 weeks. Survival, specific growth rate and feed conversion ratio were calculated for each treatment. Samples of white dorsal muscle and hepatopancreas were taken from all fish. The white muscle samples were analysed for lipid content and fatty acid composition (in total lipids, phospholipids and triacylglycerols). The samples from hepatopancreas were analyzed for total content of cytochome P450, EROD activity and global gene expression profiling.
Lipid extraction and fatty acid composition analyses
Lipid analyses were performed as already described by . Lipids from tissues, diets and plankton were extracted by hexane-isopropanol method . Total lipids were fractionated by thin layer chromatography for separation of lipid classes (Pickova et al., 1997). Fatty acids were methylated (Appelqvist 1968) and analyzed with a Varian CP3800 gas chromatograph (Stockholm, Sweden) equipped with flame ionization detector, split injector and fitted with a 50 m length x 0.22 mm i.d. x 0.25 Âµm film thickness BPX 70 fused-silica capillary column (SGE, Austin, TX, USA) (Fredriksson-Eriksson and Pickova, 2007). Fatty acids were identified by comparison with the standard mixture GLC-461 (Nu-check Prep, Elysian, MN, USA) using retention time. Peak areas were integrated by means of Star chromatography workstation software version 5.5 (Varian AB, Stockholm, Sweden). Fatty acids were quantified using the internal standard 15-methylheptadecanoate (Larodan Fine Chemicals AB, Malmo, Sweden).
Lipid classes composition
Analyses of lipid classes composition was performed according to Olsen and Henderson (1989) with minor modifications. Extracted lipid samples were applied by a Camag ATS 4 automatic TLC sampler on the pre-developed and activated TLC plates. The lipid classes were separated with hexane-diethyl ether-acetic acid (85:15:1, v/v) and detected by spraying with copper acetate-phosphoric acid solution. Quantitative analyses of the separated lipid classes were performed densitometrically by use of the Camag TLC scanner 3. The lipid classes were identified by comparing with an external standard (TLC 18-4A; Nu-Check Prep, Elysian, Minnesota, USA).
Sesamin was analyses form extracted lipid samples with HPLC according to Moazzami and Kamal-Eldin (2006). Separation was done on a silica column using hexane/1,4-dioxane (94:4, v/v) as mobile phase. Detection was done by a fluorescence detector (excitation wavelength 296 nm and emission wavelength 324 nm). External standards were used for identification and quantification.
Total content of cytochrom P450 and ethoxyresorufin O-deethylation
Microsomal fraction was prepared from the hepatopancreatic homogenate using Ca-aggregation method as described by Zamaratskaia et al., (2009). The total cytochrome P450 content was determined using the spectrophotometric method of Omura and Sato (1964), measuring the differences in the spectra (dithionite+carbon monoxide) - dithionite. The activities of 7-ethoxyresorufin O-deethylase were estimated using an HPLC-based method according to Zamaratskaia and Zlabek (2009).
Global gene expression profiling
The global gene expression analysis was performed using c DNA common carp microarray with 26K gene probes (carp ARRAY ver 5. Williams et al., 2008). Total RNA was extracted from hepatopancreas using the TRIzol Plus RNA Purification Kit (Invitrogen 12183-555, Paisley, UK). Total RNA was quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The RNA was reversly transcribed to cDNA and labeled using the SuperScript Plus Indirect cDNA Labeling System (Invitrogen L1014-04, 05 and -06). The labeled cDNA was hybridized on the microarray using the Maui hybridization system (BioMicro Systems, Salt Lake City, UT, USA). The microarrays were scanned using the Agilent DNA microarray scanner and the obtained data were processed by BLUEFUSE software (BlueGnome, Great Shelford, Cambridge, UK).
The data were processed using data analysis software STATISTICA CZ, v. 8, StatSoft, Inc. All the values were expressed as mean Â± standard deviation. For statistical analyses one way ANOVA and Tukey's post-hoc test with statistical level of significance Î±=0.05 were used.
The microarray data were first normalized (Huber et al., 2002) and corrected (Cleveland and Devlin, 1988). The differentially expressed genes were extracted using Q-values (Storey, 2002) and the control false discovery rate at the level of 10 % (Benjamini and Hochberg, 1995, 2000). The up and down regulated genes were associated with gene ontology terms to see which metabolic pathways were influenced by the sesamin treatment.
Summary of results
The lipid content and composition varied considerably among the samples from different parts of carp fillet. The lowest fat content was found in dorsal white muscle (0.95Â±0.14 %), medium in red muscle (16.7Â±5.0 %), whereas the highest was found in abdominal wall (30.2Â±7.8 %). Lipid classes composition of these different fillet parts showed that samples with higher fat content had increased proportion of triacylglycerols. Consequently, abdominal wall with the highest fat content was dominated by triacylglycerols whereas the white muscle had the highest contribution of phospholipids. The total lipid fatty acid composition differed greatly depending on the lipid content and ratio between phospholipids and triacylglycerols. The fatty acid composition of the leanest part, the white muscle contained a large proportion of n-3 highly unsaturated fatty acids and a ratio n-3/n-6 = 1.1, having a high proportion of phospholipids. The abdominal wall was rich in monounsaturated FA and had a lower ratio n-3/n-6 = 0.5.
The lipid content in white muscle of the cross M2 x M2 (1.40 %) was slightly lower than the three other crosses (M2 x L15 =1.80, M2 x M72 = 1.65, M2 x Dor70 = 1.85), although not significantly different. The total lipid fatty acid composition of the different crosses varied. In the M2 x M2 group, the proportion of n-3 PUFA was higher than in the other groups. However, there were no substantial differences when the fatty acid profiles of phospholipids and triacylglycerols were compared separately among the crosses. There was an inverse correlation between the lipid content and n-3 PUFA. So the differences seen in the total lipid fatty acid composition are most likely to be caused by the differences in lipid content and thus by a different proportion of phospholipids and triacylglycerols.
The zooplankton sampled from the pond had a very favorable composition with high proportion of n-3 PUFA, especially EPA and DHA and a high ratio n-3/n-6 (2.4-7.5).
No significant differences were found in fish survival, specific growth rate, food conversion ratio and lipid content in white muscle. Experimental diet with sesamin increased the percentage of 18:3 n-3 and decreased 18:4 n-3, 22:5 n-3, 22:6 n-3 as well as the desaturation index reflecting the ratio of n-3 highly unsaturated fatty acids/18:3 n-3 in the triacylglycerol fraction. Sesamin increased total cytochrome P450 content in hepatopancreatic microsomes as well as 7-ethoxyresorufin O-deethylase activity. However, a detailed microarray analysis using 26K gene probes failed to establish any significant pattern of transcriptional response, including lipid biosynthetic genes. Together these results indicate that under conditions of our study sesamin was ineffective in the common carp as a means of achieving the changes in tissue lipid composition as were seen in salmonids.
The FA composition is different in PL and TAG fraction. PL have a higher level of n-3 HUFA in comparison with TAG. FA composition in total lipid of different parts of the fillet depends strongly on the lipid content, because it influences ratio of the two main fractions - PL and TAG.
No significant differences were found between in fish weight, specific growth rate, food conversion ratio and lipid content. Addition of sesamin increased the percentage of 18:3 n-3 and decreased 18:4 n-3, 22:5 n-3, 22:6 n-3 as well as the desaturation index reflecting the ratio of n-3 highly unsaturated fatty acids/18:3 n-3 in the triacylglycerol fraction. Sesamin increased total cytochrome P450 content in hepatopancreatic microsomes as well as EROD activity. However, a detailed microarray analysis using 26K gene probes failed to establish any significant pattern of transcriptional response, including lipid biosynthetic genes. Together these results indicate that sesamin is ineffective in the common carp as a means of achieving a different tissue lipid composition.
This thesis evaluated several factors influencing lipid content and composition in common carp muscle. Some specific areas of future interest are:
Influence of supplemental diets based on rapeseed and linseed on the lipid composition of common carp cultured in ponds.
The effect of starvation period on carp lipid metabolism and composition.
Differences in lipid metabolism between two common carp subspecies, C. c. carpio and C. c. haematopterus.
The use of finishing feeding strategy in common carp
Comparison of carp juveniles and adults response to sesamin supplementation.
Comparison of carp and perch lipid metabolism as a response on plant oil and sesamin.
Screening for other bioactive substance which could be modulators of lipid metabolism.