Fish Sci (2011) 77:707–711 DOI 10.1007/s12562-011-0376-1 ORIGINAL ARTICLE Fisheries Isolation and characterization of 16 polymorphic microsatellite markers from Nibea albiflora Chunyan Ma • Hongyu Ma • Lingbo Ma Keji Jiang • Haiyu Cui • Qunqun Ma • Received: January 2011 / Accepted: 10 May 2011 / Published online: 11 June 2011 Ó The Japanese Society of Fisheries Science 2011 Abstract Nibea albiflora is a commercially important fish species in China Herein we report 16 novel polymorphic microsatellite markers in Nibea albiflora by using the 50 anchored polymerase chain reaction (PCR) technique The characteristics of these loci were estimated by using a sample of 30 individuals A total of 79 alleles were detected with an average of 4.9 alleles per locus The number of alleles per locus ranged from three to nine The polymorphism information content (PIC) values for the 16 microsatellite loci ranged from 0.3131 to 0.7910 The observed and expected heterozygosity per locus ranged from 0.2333 to 1.000 and from 0.3452 to 0.8421, with an average of 0.7248 and 0.6592, respectively Four loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction (P \ 0.0031), and no significant linkage disequilibrium between pairs of loci was found This study provides useful information for studies on genetic diversity and structure, construction of genetic linkage maps of N albiflora, and effective management of this fish resource Keywords Nibea albiflora Á Microsatellite markers Á Polymorphism Á 50 Anchored PCR C Ma Á H Ma Á L Ma (&) Á K Jiang Á H Cui Á Q Ma Key Lab of Marine and Estuarine Fisheries Resources and Ecology, Ministry of Agriculture, East China Sea Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Jungong Road 300, Shanghai 200090, China e-mail: malingbo@vip.sina.com H Cui Á Q Ma College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China Introduction Nibea albiflora is mainly distributed in northwest Pacific Ocean, including southern Japan and East China Sea [1] Because of its good taste and valuable nutrient content, N albiflora is commercially fished in China However, the wild resource of N albiflora has decreased sharply under the pressure of overfishing and pollution Genetic diversity in a given species is closely related to evolutionary potentiality, and genetic variation is a basic prerequisite for living organisms to cope with uncertainty in the environment [2] Therefore, it is of significant importance to analyze the genetic structure, divergence, and genetic diversity in N albiflora to reveal its genetic background To date, research on genetic diversity and population structure in N albiflora has been carried out by using amplified fragment length polymorphism (AFLP), mitochondrial DNA, and isozyme analyses [3–5] Microsatellites are short tandem-repeat DNAs with length of 1–6 bp, widely existing along the eukaryotic genome [6, 7] Microsatellite markers are popular genetic markers for use in molecular phylogeography and population genetics studies because of their high polymorphism, ease of genotyping, and codominant inheritance [8] Isolation of microsatellite markers has been carried out in many fish species [9, 10] Furthermore, more and more polymorphic markers have been applied in fishery science [11–13] To date, few microsatellite markers have been reported for this important fish species [14] Lack of sufficient microsatellite markers has hindered the evaluation of population genetic structure in N albiflora The 50 anchored PCR technique is considered a rapid and economical protocol for isolation of microsatellite markers [15–17] It permits genomic amplification with only one specific primer and produces libraries with highly enriched 123 708 single-locus microsatellites [15] In the present study, we isolated 16 novel polymorphic microsatellite markers in N albiflora using the 50 anchored PCR technique Materials and methods Sample collection and DNA extraction A total of 30 individuals of N albiflora were collected from Zhejiang Province, China Genomic DNA was extracted from muscle tissue using traditional phenol– chloroform extraction protocols as described by Ma et al [18] DNA was adjusted to 100 ng/ll concentration and stored at -20°C until use 50 Anchored primer design and PCR 50 Anchored primers were designed as follows: the seven nucleotides at 50 in the primers form the ‘‘anchor,’’ and the repeat parts of the primers can anneal to microsatellite loci in genomic DNA The sequences of four degenerate primers were KKDBDBD(AC)6, KKHBHBH(AG)6, KKVRVRV (CT)6, and KKRVRVR(GT)6, where K = G/T, D = G/A/ T, B = G/T/C, H = A/C/T, V = A/C/G, and R = A/G The primers were synthesized by Sangon Company (Shanghai) PCR amplification was performed in total reaction volume of 25 ll containing 2.0 mM MgCl2, 0.2 mM dNTP mix, 0.2 lM each primer, U Taq DNA polymerase (TaKaRa), 19 PCR buffer, approximately 100 ng template DNA, and deionized water The cycling parameters were as follows: initial denaturation of at 94°C, followed by 35 cycles of 45 s at 94°C, 45 s at annealing temperature, and 45 s at 72°C, and then final extra extension at 72°C for Amplification products were separated on 1.5% agarose gels (TaKaRa) and visualized by ultraviolet (UV) light Fish Sci (2011) 77:707–711 The PCR products were separated on 1.5% agarose gels (TaKaRa) After being identified, the positive clones were randomly selected for sequencing using an ABI3730XL sequencer (Applied Biosystems) Microsatellite sequences were searched using SSRHUNTER 1.3 software [19] Microsatellite primers were designed using Primer Premier 5.0 software (http://www premierbiosoft.com/primerdesign/) The major parameters for primer design were set as follows: primer length 19–25 nucleotides, size of PCR product 100–350 bp, and annealing temperature 50–65°C PCR amplification and polymorphism assessment The polymorphisms of microsatellite primers were tested in 30 individuals of N albiflora PCR amplification was performed in 25 ll volume containing 19 PCR buffer, 0.4 lM each primer, 0.2 mM dNTP mix, U Taq polymerase (TaKaRa), and 50 ng template DNA After denaturation for at 94°C, amplification proceeded for 35 cycles [94°C for 30 s, annealing temperature for each pair of primers (Table 1) for 40 s, 72°C for 45 s] and a final step at 72°C for The PCR products were separated on a 6% denaturing polyacrylamide gel and visualized by silver staining The ranges of allele size were determined by referring to the pBR322/MspI marker (TianGen Biotech Co., Ltd.) Data analysis Genetic diversity indexes including observed number of alleles (Na), observed (HO) and expected heterozygosity (HE), and polymorphism information content (PIC) Chi-square tests for Hardy–Weinberg equilibrium (HWE) were calculated using POPGENE version 1.31 software (http://www.ualberta.ca/*fyeh/) [20] Significance values for all multiple tests were corrected by the sequential Bonferroni procedure [21] The null allele frequency was estimated by MICRO-CHECKER version 2.2.3 software [22] Isolation of microsatellite markers After being reclaimed, DNA fragments ranging from 200 bp to 750 bp were ligated with pMD19-T vector (TaKaRa) and then transformed into Escherichia coli DH5a cells (TianGen Biotech Co., Ltd.) The positive clones were identified by PCR with vector-specific primers PCR amplification was performed in 25 ll reaction volume containing 2.0 mM MgCl2, 0.2 mM dNTP mix, 0.2 lM each primer, U Taq DNA polymerase (TaKaRa), 19 PCR buffer, and ll bacteria cultured overnight The cycling parameters were initial denaturation of at 94°C, followed by 35 cycles of 45 s at 94°C, 45 s at 55°C, and 45 s at 72°C, and final extra extension at 72°C for 123 Results In this study, microsatellites were isolated using four anchored primers After recovery, the PCR products (size ranging from 200 to 750 bp) were ligated into pMD19-T vector and transferred into DH5a competent cells A total of 105 recombinant clones were tested, and 87 positive clones were randomly selected to be sequenced using an ABI Prism 3730 automated DNA sequencer Of the 87 sequences, 63 contained microsatellite repeats Only 24 primer pairs could be designed using Primer Premier 5.0 software, as the remaining ones were too close to the (TG)6…(GT)7 (GAA)5 (CT)4 (AC)13TT(AC)6 (AC)6 (CA)17 (AC)4…(TG)4…(TG)7 (TG)10 (CA)4 (GA)13 (GA)6 (AC)7…(AC)9 (GA)13 (GT)14 (AC)18 (TC)5…(CTC)5…(CT)4…(TC)5…(AT)7…(CT)5 – Niba1 (HQ738540) Niba2 (HQ738541) Niba3 (HQ738542) Niba4 (HQ738543) Niba5 (HQ738544) Niba6 (HQ738545) Niba7 (HQ738546) Niba8 (HQ738547) Niba9 (HQ738548) Niba10 (HQ738549) Niba11 (HQ738550) Niba12 (HQ73851) Niba13 (HQ738552) Niba14 (HQ738553) Niba15 (HQ738554) Niba16 (HQ738555) Average F: 50 ACCGCTACTGTCTGGAATCAAAC 30 – R: 50 GGCAGCCATCATCAATCAGAGT 30 F: 50 AAACAGCTAAAGAGGCCAAAAACAC 30 R: 50 GTTTCCATACACACATTCACGGTCC 30 F: 50 TCTTTTTCTCCCCTTCATTGTCACT 30 R: 50 CACAAAAGCAGAACTCTCACCAT 30 F: 50 CACAACACTTAGCCAAGCACTCA 30 R: 50 GTGAAAAAGACGCAGGAGAGATT 30 F: 50 GAGATGTCAGATGCCTTGCCAGT 30 R: 50 ACTCACTGGGACCACTGATAAGA 30 F: 50 GGGAGATTACAGTGACTATTGCC 30 R: 50 TGTTCCATGACGTGTTGCC 30 F: 50 CTTCTCTGGAAAGACAAACAAAT 30 R: 50 AGAGGGTGAAAAAGACGCAGGAG 30 F: 50 GGAGATGTCAGATGCCTTGCCAG 30 R: 50 GAATACAACTGGAAGACAATAAACT 30 F: 50 CAGCAGACACTCACAATACACG 30 R: 50 GCCTGAGACTGGCAGAGAGAA 30 F: 50 ATAGGAGACGGCGGGAGAGGG 30 R: 50 ACCTCTCCCTCTCTTCCATCTTT 30 F: 50 TTTTGCTACATCAACCTGTCTAT 30 R:50 TCCTTTATCCAGAAGCAGAAACTCG 30 F: 50 CGACACCTCACACTTTCTTTTCTTA 30 R: 50 ATGTGCTGGTATGCGTGTCTGTC 30 F: 50 GGAGGAGAGGAAAGGAGGGAATA 30 R: 50 GTGAGGTCATTAGGCTGCCCATTAC 30 F: 50 TTCACCCATGATGCCCAGCTTTTAG 30 R: 50 TATCTGTATTTTTCTGCTCCTCG 30 F: 50 CTTATTTGCGTCACAGCATCTTG 30 R: 50 CCCTCTGTTCATTAGTATGCTGC 30 – 54 54 54 54 54 52 58 52 58 52 54 56 56 54 54 54 F: 50 GTAGAGAAGACAGCTACCTCCTGAC 30 R: 50 AACTACGGGATTTCTGATAGATTTT 30 Annealing temp (°C) Primer sequences (50 –30 ) * Significant deviation from Hardy–Weinberg equilibrium (HWE) after Bonferroni correction (P \ 0.0031) Repeat motif Locus (GenBank) – 306–318 237–249 131–147 101–123 213–229 141–153 107–119 125–137 223–235 245–257 261–279 183–199 123–135 127–139 224–236 124–140 Size range (bp) 79 6 4 4 4 Na 0.7248 0.4643 0.7407 0.5357 0.9655* 0.2333 0.6552 1.0000* 0.9310* 0.5714 0.5000 0.8148 0.6923 0.8929 1.0000* 0.6000 1.0000 HO 0.6592 0.6519 0.8092 0.7227 0.6848 0.3452 0.6782 0.5625 0.5402 0.5818 0.6835 0.8421 0.7172 0.6786 0.5625 0.6522 0.8348 HE Zhejiang population (N = 30) 0.5891 0.5738 0.7645 0.6688 0.6156 0.3131 0.6015 0.4511 0.4227 0.5410 0.6066 0.8060 0.6521 0.6033 0.4511 0.5626 0.7910 PIC Table Locus name, repeat sequence, primer sequences, annealing temperature (Ta), number of observed alleles (Na), allele size range, observed (HO) and expected heterozygosity (HE), polymorphism information content (PIC), and GenBank accession number for 16 polymorphic microsatellite loci in Nibea albiflora Fish Sci (2011) 77:707–711 709 123 710 flanking region of the sequences The polymorphism of these primers was assessed using 30 individuals of N albiflora Although the PCR conditions were optimized, eight pairs of primers had either amplified single PCR products of the expected size or smears In total, we isolated 16 novel polymorphic microsatellite markers in N albiflora A total of 79 alleles were identified in 30 individuals Allele size was between 101 and 318 bp The number of alleles per locus ranged from three to nine, with an average of 4.9 The PIC values for the 16 microsatellite loci ranged from 0.3131 to 0.7910 The observed and expected heterozygosity per locus ranged from 0.2333 to 1.000 and from 0.3452 to 0.8421, with an average of 0.7248 and 0.6592, respectively Significant deviation from Hardy–Weinberg equilibrium at four microsatellite loci (Niba3, Niba9, Niba10, and Niba13) was detected after Bonferroni correction (P \ 0.0031), and the MICRO-CHECKER analysis showed no evidence for scoring error or technical or statistical artifacts No significant genotypic linkage disequilibrium (LD) was found between all pairs of these 16 loci after Bonferroni correction (P [ 0.0031) None of these 16 sequences were similar to any of the sequences in GenBank by a homology search using the BLASTn program Discussion As one of most valuable fish species in China, N albiflora is of economical importance in fishing, but it has been overexploited and its natural resource has severely declined; the fisheries resource of N albiflora is at a rather critical level In recent years, artificial breeding and cage culture were carried out in Fujian and Zhejiang Provinces, China [5] For the purpose of developing rational strategies to protect the genetic resources and utilize valuable resources sustainable, it is of significant importance to analyze the genetic diversity in N albiflora to reveal its genetic background Some reports are available on population structure and genetic background of N albiflora using different genetic marker technique [3–5] Microsatellite markers have many advantages for understanding population genetics To date, only 13 polymorphic microsatellite markers have been reported in N albiflora The 50 anchored PCR technique offers a number of advantages such as more polymorphism than those from nonanchored primers and reduction of the cost of microsatellite discovery [15] In the present study, the 16 novel polymorphic microsatellite markers isolated in N albiflora by the 50 anchored PCR technique are different from those markers developed by fast isolation by AFLP of sequences containing repeats (FIASCO) method [14] Evaluating the 123 Fish Sci (2011) 77:707–711 variability in a sample of 30 individuals, all loci showed considerable variation in the Zhejiang population Gene heterozygosity is thought to be a good criterion to assess the genetic diversity of organisms The average observed heterozygosity of N albiflora was 0.7248 in Zhejiang population Compared with the heterozygosity of other species such as Epinephelus awoara (HO = 0.598) and Verasper moseri (HO = 0.60) [23, 24], the heterozygosity in our study was higher However, given the damaged resource and population structure, we should protect the genetic diversity of this species by decreasing environmental pollution and controlling fishing effort Significant deviation from Hardy–Weinberg equilibrium at four microsatellite loci was detected after Bonferroni correction (P \ 0.0031), which may be due to the small sample size or the presence of null alleles [22] Polymorphism information content (PIC) is considered as a measure of the usefulness of a molecular marker [25] According to the grades, twelve loci were shown to be highly informative (PIC [ 0.5), only four locus were shown to be intermediate informative (0.25 \ PIC \ 0.5), and no loci were shown to be low informative (PIC \ 0.25) Study on population structure is very important for successful and sustainable management of fish resources Determination of population genetic structure provides essential information to underpin resource recovery and aid in delineating and monitoring populations for fishery management [4] Molecular genetic techniques can offer more direct evidence to identify and delineate fish stock structure than phenotypic or behavioral characteristics can show [26] As a popular genetic marker, microsatellites have been used successfully to understand the structure of fish species [24, 27] These 16 loci will provide useful information for studies on genetic diversity and structure, construction of genetic linkage maps of N albiflora, and effective management of this fish resource Acknowledgments This study was supported by National NonProfit Institutes (East China Sea Fisheries Research Institute) (2008M04) References Leung AW (1994) The fish fauna of lobster Bay, Cape D’Aguilar, Hong Kong Master’s thesis, Hong Kong University Conrad M (1983) Adaptability: the significance of variability from molecular to ecosystem Plenum, New York Han ZQ, Gao TX, Wang ZY, Zhuang ZM, Su TF (2006) Analysis of genetic diversity of Nibea albiflora by AFLP markers J Fish China 5:640–646 Han ZQ, Gao TX, Yanagimoto T, Sakurai Y (2008) Genetic population structure of Nibea albiflora in Yellow Sea and East China Sea Fish Sci 74:544–552 Fish Sci (2011) 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fishery statistics system was improved through a cooperative project conducted by the Department of Fisheries and the Mekong River Commission between 1994 and 2000, especially in the seven provinces adjacent to Tonle Sap Lake However, the fisheries statistics were not effectively utilized for sustainable stock management After the cooperative project, fish catch data sorted by species or species group were collected at the province level in the seven provinces Another recent project also revealed the numbers of fishing gears that operated in the seven provinces The CPUEs of ten species in Kampong Thom Province—including Channa micropeltes and Cirrhinus spp.—could be calculated from 1994 to 2007, because these are caught solely using bamboo fence systems or barrages CPUE analysis clarified that stocks of high-price fishes such as Ch micropeltes, Hampala spp., and K Enomoto Á H Kurokura (&) Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan e-mail: akrkrh@mail.ecc.u-tokyo.ac.jp S Ishikawa School of Marine Science and Technology, Tokai University, 3-20-1 Orido, Shimizu-ku, Shizuoka, Shizuoka 424-8610, Japan M Hori Graduate School of Kuroshio Science, Kochi University, 2-5-1 Akebono, Kochi 780-8520, Japan H Sitha Á S L Song Á N Thuok Fisheries Adminisitration, Ministry of Agriculture Forestry and Fisheries, 186 Preah Norodom Blvd, 582 Phnom Penh, Kingdom of Cambodia Pangasius spp have deteriorated while those of relatively low-price fishes such as Cirrhinus spp., Cirrhinus microlepis, Cyclocheilichthys enoplos, and Channa striata have increased in recent decades Keywords Cambodian inland fisheries Á CPUE Á Stock assessment Á Tonle Sap Lake Introduction Inland fishes are vital as both food and income resources for Cambodians More than 81.5% of per-capita animal protein intake in Cambodia is supplied from fishery products [1], and approximately 85% of total fish catch comes from inland fisheries [2] In addition, a recent study revealed that small-scale fishing in Cambodia is quite an important income source for rural people [3] Almost all people have some fishery-related activities, especially around Tonle Sap Lake [4] Recently, several reports speculated about the possibility of exhaustion of fishery resources in the lake, and a relationship between stock of fishery resource and hydrological fluctuations in the Mekong water system was insinuated [5] However, those reports and discussions were not based on fluctuations of fishery resources but depended only on the trend of total fish catch or correlation analysis between total fish catch by several fishing methods and hydrological records [6] The Cambodian fisheries authorities recognized the importance of stock assessment, and in order to obtain an accurate status of the fish stock, the government, the Food and Agriculture Organization (FAO), and the Mekong River Commission (MRC) conducted a fisheries statistics improvement program in the seven provinces adjacent to Tonle Sap Lake from 1994 to 2000 Although the FAO 123 714 statistics database, named ‘‘FAO Fish STAT,’’ reflected the improvements after the project, all fish catch data were summed up as a total in the database, and records of fishing effort were not available Catch data sorted by species or species groups might have been collected under the project at the local level [7–9], but these detailed data were not utilized Even as the statistics improvement project was underway, the government and the MRC implemented another project to collect information on Cambodia’s fishing gear [10] This project revealed the existence of a strong link between the type of fishing gear and the target species Therefore, stock assessment using the catch per unit effort (CPUE) method is possible for the species for which records of both catch volume and fishing effort are available from several local fisheries management organizations The survey reported in this paper comprises two steps: data mining of fishing records in provinces around Tonle Sap Lake, and CPUE analysis and evaluation of the present stock of each species Materials and methods Data mining Domestic fisheries statistics data were officially collected by the Department of Fisheries of the Government of Cambodia [DOF, currently called the Fisheries Administration (FiA)] and compiled as Fisheries Statistics Year Books We collected as many yearbooks as possible from the headquarters of the DOF in Phnom Penh in February 2005 In addition, we gathered information from the current fisheries statistics data collection system through interviews with government officers in charge at the DOF We conducted field surveys in the five provinces directly connecting with Tonle Sap Lake, namely Siem Reap, Kampong Thom, Kampong Chhnang, Pursat, and Battanbang (Fig 1) from February to April 2005 In the collaborative project by the DOF and MRC [7–9], the provincial government had collected monthly catch and effort data on large-scale fishing, i.e., commercially operated fisheries, in demarcated areas called fishing ‘‘lots.’’ We tried to gather the actual primary records of fish catch and effort data for each fishing lot, which had been collected by the provincial governments Simultaneously, we also asked the provincial governments about the current data collection systems conducted by them, including the person who reported the catch amount of each species, the person who described the record, and how the accuracy of the reports was confirmed Simultaneously, the relationship between the local classification of fish and scientific classification was confirmed by direct interview using picture books and fish samples Then, taxonomic group classifications written in Khmer 123 Fish Sci (2011) 77:713–722 Fig Five provinces around Tonle Sap Lake and location of fishing lots B Battanbang, C Kampong Chhnang, P Pursat, S Siem Reap, T Kampong Thom were translated to species or species groups according to the contemporary system on the basis of published field guides and biological reviews [11–13] The species composition of each fishing gear’s yield was examined on the basis of outcomes of the MRC project compiled in the Information of Major Fishing Gears in Cambodia [14] As the large-scale fishing season extends from the previous October to May of the current year, all annual data were calculated by summing up the monthly data for the same period CPUE analysis and evaluation of present stock of each species Before CPUE analyses, the correlation between percentages of catch volume in a lot to the total catch volume in the province in a year and number of fishing gears, size of the gears, and duration of season of the fishing method in the year in the lot were analyzed, and the catch effort data with the highest correlation to the contribution of each species were selected as parameters of fishing effort We selected Kampong Thom Province as the target area for CPUE analysis because of the existence of reliable raw data CPUEs of Barbodes gonionotus, Channa micropeltes, Channa striata, Cirrhinus microlepis, Cirrhinus spp., Cyclocheilichthys enoplos, Hampala spp., Osteochilus melanopleurus, and Pangasius spp., in lots no 3, 4, and 5, were calculated by dividing the catch volume of each fishing year (ton) by the total length of the bamboo fence systems (km) in the lot as an index of fishing effort The bamboo fence system is a huge barrier made from bamboo along the fringe of flooding forest For migratory fish Fish Sci (2011) 77:713–722 715 species such as Cirrhinus spp and Trichogaster microlepis, additional CPUEs were performed in lots 1, 2, and using the maximum width of the water current (m) where the barrages (set nets) were set in the river A barrage is a setting net with a bag net and wing net, being set across the flow of the river or canal between both shores The target species for each fishing gear were selected on the basis of catch amount The species caught in analyzable amounts by each gear were selected for CPUE analysis Among them, Cirrhinus spp is a target species for both bamboo fence and barrage This is because analyzable amounts of Cirrhinus spp are also caught by bamboo fence due to their huge biomass, although they are migratory species mainly caught by barrage Statistical analysis The distribution pattern of the species among the lots in Kampong Thom Province were categorized by cluster analysis In the analysis, the Bray–Curtis similarity coefficient was used as the similarity index, and the neighborjoining method was used for clustering of species The fish groups were divided at 85% similarity level In the correlation analysis for clarification of CPUE trends, fishes were divided at 0.05 risk level without Bonferroni correction Results Data mining Although the raw data were not compiled in provincial offices, species-wise annual fish catch data from each fishing lot in five provinces around Tonle Sap Lake were reported in the annual year books from 1995 to 1997, and Table Local and scientific names of fish groups the records of total catch of each species in all fishing lots in 1998 existed in all provinces Among them, only Kampong Thom and Battanbang Provinces maintained primary fish catch records We could obtain further fish catch data for 2000–2007 in Kampong Thom Province from records kept in the provincial office The data recorded catch volumes of each fish species in each fishing lot Subsequently, we could collect catch data for each species in each of five provinces from 1995 to 1998 Thus, we had catch data for each species in different fishing lots in Kampong Thom Province from 1995 to 2007 except 1999 The precise numbers of fishing gears used for largescale fishing in the fishing lots were also described in the domestic fisheries statistics for more than 10 years No consistent increase or decrease in catch volumes of any species and fixed ratio among catch volume of species throughout the recorded period were noticed in any fishing record of the five provinces We selected ten species for which multiyear catch records were obtainable in the five provinces The species names were written in Khmer, and we estimated the scientific name of each species based on interview surveys referring to several previous field guides [11, 12] Their scientific names and local names in Khmer are shown in Table Among them, the fish called trey riel was mainly Cirrhinus siamensis, and other species in the genera Cirrhinus and Henicorhynchus were included in trey riel However, among Cirrhinus, Ci microlepis was classified as other species and called trey pruol by local people Therefore, we classified trey riel as Cirrhinus spp and trey pruol as Ci microlepis Hampala spp (trey khmann in Khmer) mainly consisted of H dispar, although small amounts of H macrolepidota were included Pangasius spp (trey pra in Khmer) included P hypophthalmus, P djambal, and other species in Pangasius Trey raws included several species in the genus Channa, such as Khmer name Latin name Trey chhipin Barbodes gonionotus Trey chhdaur (diep: juvenile) Channa micropeltes Trey raws (ptuok: juvenile) Channa striata Trey pruol (kralang: juvenile) Cirrhinus microlepis Trey riel Cirrhinus spp (Ci siamensis, other species in Cirrhius and Henicorhynchus except Ci microlepis) Trey chhukok Cyclocheilichthys enoplos Trey khmann Hampala spp H dispar, H macrolepidota) Trey krum Osteochilus melanopleurus Trey pra Pangasius spp Trey kamphleanh Trichogaster microlepis (P hypophthalms, P djambal, others) 123 716 Fish Sci (2011) 77:713–722 Fig Location of lots in Kampong Thom Province The numbers in the figure indicate lots Fig Interannual fluctuation of catch amount in Battanbang (solid squares), Kampong Chhmang (open triangles), Pursat (solid triangles), Siem Reap (solid circle), and Kampong Thom Provinces (open circles) The fluctuation is expressed as the sum of the catch amount of 10 species and the catch amount of Cirrhinus spp., Channa micropeltes, Channa striata, and Trichogaster microlepis Ch marulia and Ch striata It was mentioned in the field guide [12] that the most common snakehead in Cambodia was Ch striata, and a review [11] reported that Ch striata was distributed mainly in the lake We also confirmed the scientific name of trey raws in Tonle Sap Lake as Ch striata through interviews with DOF staff For these reasons, we assumed that trey raw in the statistics records of these provinces mainly included Ch striata Figure presents the interannual fluctuation in catch from 1995 to 2007 in five provinces The figure shows the sum of catch amount of the above-mentioned ten species, and the catch amount of Cirrhinus spp., Channa micropeltes, Chana striata, and Trichogaster microlepis The total catch weight in Kampong Chhnang Province was several times higher than that in other provinces The catch volumes of Cirrhinus spp were prominently higher than those of other species, especially in Kampong Chhnang and Kampong Thom Provinces The catch volume of Ch micropeltes was high before 1998 and decreasing after 1999 Chana striata 123 and Trichogaster microlepis were distributed mainly in Battanbang Province In Kampong Thom Province, there are seven fishing lots The geographical features of the lots are different from each other (Fig 3) The lots could be roughly divided into two types according to their geographical features Lots no 1, 2, and are located in large rivers or near the river mouth We categorized this type as river-type lots On the contrary, lots no 3, 4, and are located not in rivers but in flooding areas in the high-water season We categorized this type as lake-type lots Lot no is of intermediate type, located in both river and flooding areas Two types of fishing gear were used in these fishing lots, namely bamboo fence and barrage Bamboo fence is a long barrier made with bamboo along the flooding area, and fish are caught inside the barrier when water depth decreases Barrage is a set net used in the river with a bag net and wings across the flow Bamboo fences were used in fishing lots 3, 4, 5, and 6, and barrages were used in lots 1, 2, 6, and Therefore, fish in lots 3, 4, and were caught solely by bamboo fences, and in lots 1, 2, and they were solely caught by barrages The annual catch of the seven fishing lots had been approximately stable for 10 years from 1995, fluctuating from 2437 to 4434 tons; significantly increasing or decreasing trends were not observed However, the main fish species caught showed significant variation In particular, the catch of Cirrhinus spp (before 1997 this genus was categorized as Henicorhynchus [15]) had increased during the period, with several fluctuations, whereas that of Ch micropeltes constantly diminished, except in 1999 The catch volumes of the seven fishing lots showed different fluctuation patterns Fishing lot no had the highest catch for the 10 years except 1995 The catches of lots and Fish Sci (2011) 77:875–882 DOI 10.1007/s12562-011-0387-y ORIGINAL ARTICLE Chemistry and Biochemistry Occurrence of all-cis-5,8,11,14,17,20,23-hexacosaheptaenoic acid (26:7n-3) in roughscale sole Clidoderma asperrimum flesh lipids Yuuki Fukuda • Yasuhiro Ando Received: 10 May 2011 / Accepted: 28 June 2011 / Published online: 13 July 2011 Ó The Japanese Society of Fisheries Science 2011 Abstract Fatty acid analysis of roughscale sole Clidoderma asperrimum flesh lipids was carried out by gas chromatography An unidentified peak appeared in the chromatogram in the elution region of CC24 fatty acids After enrichment by solvent partitioning, reversed-phase thin-layer chromatography (TLC), and argentation TLC, the peak component was subjected to structural analyses The partially hydrogenated products after reaction with hydrazine hydrate gave seven isomers of cis-hexacosenoic acid (26:1) Gas chromatography-mass spectrometry (GC–MS) analyses of their dimethyl disulfide adducts identified the monounsaturates as 5-, 8-, 11-, 14-, 17-, 20-, and 23-26:1 The peak component was assigned to allcis-5,8,11,14,17,20,23-hexacosaheptaenoic acid (26:7n-3) GC–MS analyses of the 4,4-dimethyloxazoline derivative and methyl ester confirmed this structure This fatty acid is a rare, very long-chain polyunsaturated fatty acid (VLCPUFA) The concentrations of the acid found in roughscale sole were 0.69 ± 0.34% (N = 5) of total fatty acids in flesh lipids Roughscale sole appears to be characterized by the occurrence of 26:7n-3, which is lacking in popular sources of methylene-interrupted VLCPUFA, such as vertebrate retina, spermatozoa, and herring Keywords Hexacosaheptaenoic acid Á Very long-chain polyunsaturated fatty acid Á Fatty acid Á Roughscale sole Á Clidoderma asperrimum Á GC Á GC–MS Y Fukuda Á Y Ando (&) Division of Marine Life Sciences, Faculty of Fisheries Science, Hokkaido University, 3-1-1 Minato-cho, Hakodate, Hokkaido 041-8611, Japan e-mail: ando@fish.hokudai.ac.jp Introduction Very long-chain polyunsaturated fatty acids (VLCPUFA) occur in many species of animals, plants, and lower organisms [1–4] Mammalian retinas [5, 6], brains [7–9], testes [10], and spermatozoa [11, 12] include methyleneinterrupted VLCPUFA of the n-3 and n-6 series up to C34–C40 Aquatic organisms also contain VLCPUFA [13] Baltic herring include those up to C28 [14–16], some of which are represented in the fatty acids of the predatory ringed seal [17] Bottom-living flathead flounder is rich in 24:6n-3 [18, 19], which presumably originated from their diet of brittle star [20–22] Additionally, dinoflagellates and some species of microalgae include 28:8n-3 and 28:7n6 [4], and even- and odd-numbered VLCPUFA up to C36 were observed in a species of dinoflagellate [23, 24] All-cis-5,8,11,14,17,20,23-hexacosaheptaenoic acid (26:7n-3) is also a VLCPUFA However, this fatty acid is a very rare one among the VLCPUFA of n-3 series To the best of our knowledge, 26:7n-3 has been found in only species of microalgae (Cryptophyceae, Prymnesiophyceae, and Dinophyceae), at less than 0.3% of total fatty acid content [23–25] This fatty acid also cannot be prepared from popular sources of n-3 and n-6 VLCPUFA standards such as retina, sperm, and herring [1] A detailed assignment of the structure has also not been reported In the present study, the fatty acids of roughscale sole Clidoderma asperrimum, i.e., samegarei in Japanese, which is one of the edible fish in Japan, were investigated for VLCPUFA Fatty acid analysis of the sole revealed the occurrence of 0.3–1.3% of 26:7 in their flesh lipids This paper reports the structural assignment of this fatty acid as 26:7n-3 along with the enrichment process and the fatty acid composition of the roughscale sole 123 876 Fish Sci (2011) 77:875–882 Materials and methods Derivatization for structural analysis Materials Partial hydrogenation of the polyunsaturated fatty acids was carried out using hydrazine hydrate [30, 31] A mixture of mg free fatty acids and 10% (v/v) hydrazine hydrate in methanol (5 ml) was stirred at 50°C for h with aeration The products, which were extracted with diethyl ether, were converted to methyl esters using 7% BF3methanol The monounsaturated fatty acids were isolated from the products by Ag-TLC on 5% silver nitrateimpregnated silica gel 60G with hexane/acetone (95:5, v/v), and then fractionated according to the olefinic bond position by Ag-TLC on 15% silver nitrate-impregnated silica gel 60G with hexane/toluene (50:50, v/v) [32, 33] Dimethyl disulfide (DMDS) adducts of monounsaturated fatty acids were prepared following the procedure of Shibahara et al [34, 35] The methyl esters were reacted with DMDS (1 ml) in the presence of catalytic I2 (13 mg) for h at 35°C before adding 30% aqueous NaHSO3 The resulting adducts that were extracted by hexane/diethyl ether (50:50, v/v) were purified by TLC on a silica gel G plate with hexane/diethyl ether/acetic acid (80:20:1, v/v/v) for development 4,4-Dimethyloxazoline (DMOX) derivatives of fatty acids were prepared by the procedure reported by Christie [36] Acid chlorides formed from the free fatty acids by a reaction with oxalyl chloride were reacted with 10 mg/ml solution of 2-amino-2-methyl-1-propanol in dichloromethane (0.5 ml) for h at room temperature After the solvent was evaporated, trifluoroacetic anhydride was added to the residue, and the mixture was left at 40°C for h The DMOX derivatives were purified by TLC on a silica gel G plate with hexane/diethyl ether/acetic acid (50:50:1, v/v/v) for development Five individuals of roughscale sole caught in the Pacific waters off Hidaka, Hokkaido, Japan, and in the Sea of Okhotsk off Shari, Hokkaido, Japan, were purchased in stores in May 2009 Two of the Pacific samples were obtained as whole fish (females, body lengths 39.8 and 41.2 cm; body weights 2.4 and 2.8 kg) and another in frozen form without skin and viscera (body length 34.8 cm) The two Okhotsk samples were obtained in frozen form without head, skin, or viscera (lengths of trunk and tail 24.0 and 25.8 cm) The flesh was removed, minced, and stored at -30°C before lipid extraction Fatty acid methyl esters Total lipids were extracted from 150 g flesh by the method reported by Bligh and Dyer [26] Fatty acid methyl esters were prepared from the lipids by transesterification with 7% BF3-methanol for h at 100°C under nitrogen atmosphere Methyl esters were purified by thin-layer chromatography (TLC) on silica gel G plates (10 10 cm, 0.25 mm thickness; Analteck, Newark, USA) with hexane/diethyl ether (85:15, v/v) for development For quantity preparation, the total lipids were saponified by reflux with M KOH in ethanol for h; the unsaponifiable portion was extracted with diethyl ether Following acidification of the aqueous phase using dilute HCl, the fatty acids were recovered by ether extraction The fatty acids were converted to methyl esters by refluxing them with 7% BF3-methanol at 70°C for 14 Instrumental analysis Fractionation of fatty acids The fatty acid methyl esters were divided into two fractions obtained through a solvent partition method using a solvent system of 2,2,4-trimethylpentane and ethanol/water (1:1, v/v) containing 0.25 g/ml silver nitrate [27, 28] The methyl esters were fractionated according to their partition number by reversed-phase TLC (RP-TLC) on Partisil KC18F plates (20 20 cm, 0.2 mm thickness; Whatman, Maidstone, England) with acetonitrile/water (95:5, v/v) for double developments [29] The methyl esters were fractionated according to the degree of unsaturation by argentation TLC (Ag-TLC) on 10% silver nitrate-impregnated layers of silica gel 60G (20 20 cm, 0.5 mm thickness; Merck, Darmstadt, Germany) with hexane/acetone (70:30, v/v) for double developments [29] 123 The fatty acid methyl esters were analyzed by GC using a Shimadzu GC-18A gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a flame ionization detector and a Restek FAMEWAX column (30 m 0.32 mm i.d., 0.25 lm film thickness; Restek, Bellefonte, USA) The column temperature was programmed either to increase from 170°C to 240°C at rate of 4°C/min, or to remain isothermal at 240°C The injector and detector temperatures were 240°C, and the carrier gas was helium (85 kPa) Peak area percentages were obtained using a Shimadzu C-R6A integrator The monounsaturated fatty acids isolated from the hydrazine hydrogenation products were analyzed using a Shimadzu GC-17A gas chromatograph equipped with a flame ionization detector and a SLB-IL100 column (60 m 0.32 mm i.d., 0.26 lm film thickness; Supelco, Fish Sci (2011) 77:875–882 877 Bellefonte, USA) [37] The column temperature was isothermal at 200°C, the injector and detector temperatures were 240°C, and the carrier gas was helium (117.5 kPa) The peaks were monitored using a Shimadzu C-R3A integrator GC–MS analysis was carried out using an HP 6890 series gas chromatograph (Hewlett-Packard, Palo Alto, USA) linked to a JEOL JMS-700TZ mass spectrometer (JEOL, Tokyo, Japan) The latter was used in electron impact mode at 70 eV with source temperatures of 240°C for methyl esters, 270°C for DMOX derivatives, and 280°C for DMDS adducts The GC was fitted with split/splitless injection For the analyses of the methyl esters and DMOX derivatives, a DB-23 column (30 m 0.25 mm i.d., 0.25 lm film thickness; Agilent, Santa Clara, USA) was used The column temperatures were programmed from 40°C to 230°C and from 40°C to 270°C at 20°C/min for the methyl esters and DMOX, respectively For the analysis of the DMDS adducts, a Zebron ZB-1 ms column (30 0.25 mm i.d., 0.25 lm film thickness; Phenomenex, Torrance, USA) was used The column temperature was held at 40°C for then raised to 175°C at 40°C/min and then to either 265°C at 5°C/min or to 280°C at 20°C/ Helium was used as the carrier gas Fourier-transform infrared spectra were measured in CCl4 solutions using a JASCO FT-IR 5300 spectrometer (JASCO, Tokyo, Japan) Fatty acid composition determined by GC of methyl esters was presented as mean ± standard deviation of the five individuals of roughscale sole 14:0 16:0 16:1 18:1 18:1 n-7 n-9 n-7 20:1 n-9 22:1 n-11 The GC analysis of the fatty acid methyl esters derived from roughscale sole flesh lipids showed two remarkable peaks, A and B, after elution of 24:1n-9 (Fig 1) Their equivalent chain lengths (ECLs) on FAMEWAX at 240°C were 26.14 and 28.30, for peak A and B, respectively The ECL of peak A was in fair agreement with that of 24:6n-3, which was previously found in flathead flounder [18, 19] and brittle star [20, 21] The component of peak B was confirmed to be hexacosaheptaenoic acid (26:7) since the mass number of the molecular ion peak obtained under high-resolution conditions agreed with that calculated for a 26:7 methyl ester; the m/z found was 396.30280, and the value calculated was 396.30283 for C27H40O2 For structural analysis, enrichment of 26:7 was conducted by the solvent partition procedure, RP-TLC, and 10% Ag-TLC, in that order (Table 1) The concentration of 26:7, which started at 1.3% of total fatty acids, was boosted to 10.4% by the solvent partition, 27.7% by RP-TLC, and finally 83.0% by Ag-TLC A coexistent minor component of the final fraction was 24:6n-3 (17.0%) Structure of 26:7 The infrared spectrum of the 26:7 concentrate showed absorptions at 1650 cm-1 and 710 cm-1, but not that near 12 16 Peak A 24:6 n-3 22:6 n-3 22:5 n-3 20:4 n-6 GC and enrichment of 26:7 20:5 n-3 20:1 n-11 18:0 Results Peak B 26:7 n-3 24:1 n-9 24 20 32 28 Fig Gas chromatogram of fatty acid methyl esters formed from roughscale sole flesh lipids (Restek FAMEWAX, 170 to 240°C at 4°C/min) Table Enrichment of 26:7 from the fatty acids of roughscale sole flesh lipids ND not detected a Fatty acid methyl esters prepared from a Pacific sample (41.2 cm, 2.8 kg) Fatty acid Intacta Solvent partition RP-TLC Ag-TLC Concentration (wt%) 20:5n-3 7.7 48.4 0.4 ND 21:5n-3 0.2 1.1 0.5 ND 22:5n-3 0.7 2.0 6.8 ND 22:6n-3 2.0 15.7 ND ND 24:6n-3 3.1 16.4 62.1 17.0 26:7n-3 1.3 10.4 27.7 83.0 Others 85.0 6.0 2.5 0.0 123 878 970 cm-1 which is characteristic of a trans-olefinic bond Hydrazine hydrogenation yielded n-26:0 as a saturated fatty acid These results indicate that the fatty acid is normal-chain 26:7 with all-cis geometry The monounsaturated fatty acids which were produced by the partial hydrogenation of 26:7 separated into four fractions on the 15% Ag-TLC plate The GC analyses of these fractions showed a total of seven peaks corresponding to 26:1 isomers with ECLs in the range of 26.41–27.23 on SLB-IL100 at 200°C The mass spectra of the DMDS adducts of the 26:1 isomers gave a molecular ion at m/z 502 corresponding to the DMDS adduct of 26:1 methyl ester and a series of key fragment ions showing the olefinic bond position in 26:1 (Fig 2) In Fig 2a, the fragment ions at m/ z 161 and 341 indicate cleavage between the methylthiosubstituted carbons of C5 and C6 The fragment ion at m/ z 129 was due to the loss of methanol (m/z 32) from the ion at m/z 161 A set of the fragment ions indicated the structure of 5-26:1 In the same manner, the other isomers were identified as 8-, 11-, 14-, 17-, 20- and 23-26:1, as shown in Fig 2b–g Hydrazine reduces olefinic bonds without positional and geometrical isomerization of the remaining olefinic bonds [30] The structure of 26:7 was assigned as all-cis-5,8,11,14,17,21,23-hexacosaheptaenoic acid (26:7n-3) The mass spectrum of the DMOX derivative of 26:7 showed irregular intervals of m/z 12 between the maxima in the fragment ion peaks for each carbon atom as follows: C7 (m/z 180, intensity 19.0%)–C8 (m/z 192, 9.7%); C10 (m/z 220, 23.2%)–C11 (m/z 232, 15.0%); C13 (m/z 260, 16.5%)–C14 (m/z 272, 8.6%); C16 (m/z 300, 13.7%)–C17 (m/z 312, 7.3%); and C19 (m/z 340, 15.6%)–C20 (m/z 352, 9.8%) (Fig 3a) These fragments indicate the occurrence of olefinic bonds at the D8, 11, 14, 17, and 20 positions in 26:7 [29] An olefinic bond at the D5 position was shown by the fragment ion due to cleavage at this position (m/ z 152, 17.6%) accompanied by an intense odd-numbered peak at m/z 153 (32.3%) [38] GC–MS analysis of the 26:7 methyl ester revealed fragment ions characteristic of n-3 and D5 series polyunsaturated fatty acids at m/z 108 (33.4%) and 180 (14.1%), respectively [29], confirming the structure of 26:7n-3 (Fig 3b) Fatty acids of the roughscale sole flesh lipids The fatty acid composition of the roughscale sole flesh lipids is presented in Table The major fatty acids ([5% of total lipids) were 14:0, 16:0, 16:1n-7, 18:1n-9?18:1n11, 20:1n-11?20:1n-13, and 20:5n-3 The proportions of these fatty acids were not very different from those previously reported for deep-sea flounders [39], in which the monounsaturated fatty acids were rich in the liver and flesh neutral lipids The major highly unsaturated fatty acids of 123 Fish Sci (2011) 77:875–882 100 341 a 129 161 129 50 43 57 5-26:1 502 100 200 150 250 300 400 350 450 203 171 299 203 8-26:1 8395 123 100 502 150 200 250 245 c 300 400 350 450 213 257 245 11-26:1 74 97 50 100 502 150 200 215 d 250 50 100 300 255 287 150 200 250 173 300 450 500 m/z 329 83 173 100 329 150 200 250 300 131 339 74 17-26:1 400 350 450 83 500 m/z 131 CH3O-CO(CH2)18CH(SCH3)-CH(SCH3)(CH2)4CH3 371 100 502 371 339 41 150 200 250 300 350 20-26:1 400 450 502 500 m/z 413 g 381 381 50 55 41 400 297 50 100 215 CH3O-CO(CH2)15CH(SCH3)-CH(SCH3)(CH2)7CH3 50 50 m/z 14-26:1 350 297 55 55 500 502 e f 450 CH3O-CO(CH2)12CH(SCH3)-CH(SCH3)(CH2)10CH3 74 100 400 350 287 255 74 55 50 43 83 95 43 m/z CH3O-CO(CH2)9CH(SCH3)-CH(SCH3)(CH2)13CH3 43 50 55 50 500 257 213 100 m/z CH3O-CO(CH2)6CH(SCH3)-CH(SCH3)(CH2)16CH3 171 50 100 500 299 b 50 43 57 100 161 87 74 50 100 341 CH3O-CO(CH2)3CH(SCH3)-CH(SCH3)(CH2)19CH3 89 89 74 CH3O-CO(CH2)21CH(SCH3)-CH(SCH3)CH2CH3 413 23-26:1 502 50 100 150 200 250 300 350 400 450 500 m/z Fig Mass spectra of dimethyl disulfide (DMDS) adducts of 26:1 isomers formed by hydrazine hydrogenation of the roughscale sole 26:7 acid the roughscale sole were 20:5n-3, 22:5n-3, 22:6n-3, 24:6n3, and 26:7n-3 The content of 26:7n-3 was 0.69 ± 0.34% of total lipids, ranging from 0.33% to 1.26% among the five individuals The lipid content of the flesh was 30.5 ± 4.6% Fish Sci (2011) 77:875–882 Fig Mass spectra of 4,4dimethyloxazoline (DMOX) derivative (a) and methyl ester (b) of the roughscale sole 26:7 acid 879 a 100 113 232 192 N 272 352 312 O 180 126 220 260 300 340 50 435 98 41 55 67 152 b 79 206 220 166 286 246 366 180 260 232 300 326 340 352 272 192 312 380 150 100 50 100 153 79 200 250 300 350 420 406 400 450 m/z 91 O 67 CH3 O 105 119 50 108 41 131 55 145 159 180 199 50 Table Fatty acid composition of the roughscale sole flesh lipids (wt%) Fatty acid 300 Fatty acid 400 m/z Composition 0.06 ± 0.02 18:4n-3 0.37 ± 0.06 18:4n-1 0.10 ± 0.02 14:1n-5 0.28 ± 0.03 20:0 0.14 ± 0.03 6.50 ± 1.38 Iso-15:0 0.24 ± 0.08 20:1n-11?n-13 Anteiso-15:0 0.10 ± 0.04 20:1n-9 2.63 ± 0.34 15:0 0.31 ± 0.01 20:1n-7 0.76 ± 0.16 0.12 ± 0.03 20:2n-6 0.18 ± 0.02 12.05 ± 1.44 20:3n-6 0.02 ± 0.02 16:1n-9 0.33 ± 0.04 20:4n-6 0.83 ± 0.13 16:1n-7 9.30 ± 0.65 20:3n-3 0.10 ± 0.02 16:1n-5 0.20 ± 0.03 20:4n-3 0.23 ± 0.03 Iso-17:0 0.52 ± 0.19 20:5n-3 8.31 ± 0.46 Anteiso-17:0 0.11 ± 0.03 22:0 0.07 ± 0.01 16:2n-4 0.15 ± 0.04 22:1n-11?n-13 3.07 ± 0.59 17:0 16:3n-4 0.18 ± 0.02 0.08 ± 0.03 22:1n-9 22:1n-7 0.93 ± 0.14 0.22 ± 0.05 0.05 ± 0.03 17:1n-7 0.32 ± 0.02 22:2n-6 16:4n-1 0.19 ± 0.09 21:5n-3 0.24 ± 0.05 18:0 2.12 ± 0.13 22:5n-6 0.13 ± 0.05 18:1n-13 Determined by gravimetry (% on wet-weight base) 250 5.97 ± 0.64 18:1n-9?n-11 b Compositiona 200 12:0 Iso-16:0 Mean ± standard deviation (SD) of the five individuals caught in the Pacific water and the Sea of Okhotsk around Hokkaido, Japan 150 14:0 16:0 a 100 396 350 0.69 ± 0.15 22:5n-3 0.80 ± 0.11 23.54 ± 1.48 22:6n-3 2.88 ± 0.74 18:1n-7 4.34 ± 0.25 24:1n-9 0.75 ± 0.11 18:1n-5 0.60 ± 0.07 24:6n-3 3.75 ± 1.02 18:2n-6 0.33 ± 0.05 26:7n-3 0.69 ± 0.34 18:2n-4 0.13 ± 0.04 Others 3.18 ± 0.34 18:3n-6 0.02 ± 0.02 Lipid content (%)b 30.5 ± 4.5 18:3n-3 0.15 ± 0.03 123 880 Fish Sci (2011) 77:875–882 on wet-weight base, much higher than the previous datum (5.6%) observed for smaller-sized roughscale sole (mean body length 22.3 cm; mean body weight 199.8 g) [39] time, the occurrence of 26:7n-3 in brittle star is unknown Although the concentrations of 26:7n-3 in the flesh lipids (0.3–1.3%) were not very high, there was no sample where 26:7n-3 was not found Discussion Acknowledgments The authors wish to thank Ms Seiko Oka, Instrumental Analysis Division, Equipment Management Center, Creative Research Institution, Hokkaido University, for the GC–MS analyses In the present study, 26:7n-3 and 24:6n-3 were found in the roughscale sole flesh lipids as their VLCPUFA Methyleneinterrupted VLCPUFA of n-3 series are typical of vertebrate retinas (up to 36:6n-3) [5, 6], spermatozoa (up to 34:6n-3 in chain length and 32:7n-3 in unsaturation) [11, 12], and Baltic herring (generally up to 28:7n-3) [14–16] These tissues or fish have been recognized as convenient sources of VLCPUFA standards for the n-3 and n-6 series [1] One of the shorter-chain VLCPUFA, 24:6n-3, was rich in flathead flounder [18, 19] and brittle stars [20–22], and was also found in sea lilies [20], coelenterates [40, 41], gorgonians [42], jellyfish [43], and gastropods [44] Freshwater crustacea of the order Bathynellacea were reported to contain more than 50 VLCPUFA up to 40:8n-3 [45, 46] Marine dinoflagellates contain 28:8n-3 together with 28:7n-6 [23–25, 47–53] Fatty acids of the dinoflagellate Amphidinium carterae included even- and oddnumbered-chain VLCPUFA up to 36:8n-3 [23, 24] As a tentatively identified component, 26:7n-3 were found in Dinophyceae (Heterocapsa niei, 0.2% of total fatty acids; Amphidinium carterae, trace amounts), Prymnesiophyceae (Pavlova pinguis, up to 0.3%), and Cryptophyceae (Proteomonas sulcata and Phodomonas salina, each in trace amounts) [25] In the fatty acids of the dinoflagellate Amphidinium carteae, 26:7n-3 was found at concentration of 3.2% in the heptaenoic ? octaenoic acid concentrate [23], which corresponds to 0.064% of total fatty acids The same acid was also found at 1.40% in a concentrate of VLCPUFA with more than olefinic bonds [24] To the best of our knowledge, there has been no other report describing the occurrence of 26:7n-3 in nature While the analogous 26:6n-3 was observed in vertebrate retina [5, 6], murine testis [10], and ram spermatozoa [11], the fatty acids of these tissues were not reported to include 26:7n-3 Baltic herring [15], seal [17], and crustacea (Bathynellacea) [45, 46] contain both analogous 26:6n-3 and homologous 28:7n-3, but not 26:7n-3 Therefore, the roughscale sole flesh lipids are characterized by the occurrence of the rare VLCPUFA, 26:7n-3 This fatty acid seems to be formed from coexistent 24:6n-3 via two-carbon chain elongation followed by D5-desaturation On GC 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(2003) On the distribution and biology of roughscale sole Clidoderma asperrimum (Temminck et Schlegel, 1846) in the Pacific waters off the northern Kuril Islands and southeastern Kamchatka Bull Sea Fish Inst 159:67–80 123 Fish Sci (2011) 77:875–882 55 Fujita T (1996) Bathymtric distribution of Ohiuroids (Echinodermata) off Sendai Bay, northern Japan, with notes on the diet of the roughscale sole Clidoderma asperrimum (Pisces, Pleuronectidae) Mem Natn Sci Mus Tokyo 29:209–222 Fish Sci (2011) 77:883–889 DOI 10.1007/s12562-011-0386-z ORIGINAL ARTICLE Food Science and Technology Beneficial effects on meat quality of yellowtail Seriola quinqueradiata induced by diets containing red pepper Itaru Shioya • Koji Inoue • Akihisa Abe Akira Takeshita • Takahiro Yamaguchi • Received: 21 March 2011 / Accepted: 17 June 2011 / Published online: 16 July 2011 Ó The Japanese Society of Fisheries Science 2011 Abstract The meat quality of farmed yellowtail Seriola quinqueradiata fed on extruded pellets (EP) containing 0.5% (v/v) red pepper (experimental group) was compared with yellowtail of the same age fed on EP (control group) In 1-year-old yellowtail, the crude lipid content of the dorsal muscle of the experimental group tended to be lower than that of the control group In contrast, there was no difference in the lipid content of the dorsal muscle between the control group and the experimental group in 2-year-old yellowtail The muscle texture of the experimental group was significantly firmer than that of the control group, with the effect of red pepper unrelated to fish age and lipid content Color change of red muscle of the experimental group was significantly lower than that of the control group, and the content of thiobarbituric acid-reactive substances in the red muscle was significantly lower in the I Shioya (&) Central Research Laboratory, Nippon Suisan Kaisha, Ltd., 1-32-3 Nanakuni, Hachioji, Tokyo 192-0991, Japan e-mail: i-shio@nissui.co.jp K Inoue Atmosphere and Ocean Research Institute, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8564, Japan A Abe Bio-Production Research Center, Nippon Suisan Kaisha, Ltd., 1-32-3 Nanakuni, Hachioji, Tokyo 192-0991, Japan A Takeshita Kurose Suisan Co., Ltd., 2-15-7 Nishihama, Kushima, Miyazaki 888-0012, Japan T Yamaguchi Graduate School of Agriculture, Tohoku University, 1-1 Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai, Miyagi 981-8555, Japan experimental group than in the control group These results are the first to demonstrate that the inclusion of red pepper in the diet is able to reduce the loss of muscle texture firmness and to slow down color change in red muscle of yellowtail Keywords Yellowtail Á Red pepper Á Ordinary muscle Á Red muscle Á Crude lipid Á Texture Á Color difference Introduction The domestic production of yellowtail, Seriola quinqueradiata, an economically important fish species marketed on a large scale in Japan, was about 233,277 t in 2009 based on the 2010 annual report of the Fisheries Agency (http://www.maff.go.jp/) Aquaculture production accounted for 154,943 t and the wild fish catch for 78,334 t, demonstrating that the production of yellowtail by aquaculture is higher Meat texture is important in many kinds of fish, including yellowtail, when the meat is eaten raw, such as ‘‘sushi’’ and ‘‘sashimi’’ Ando et al [1] reported that the texture of sashimi shows a loss of firmness that is contrary to the progress of rigor mortis after death of the fish Therefore, it is important to control the texture—that is, the rate of decrease in the firmness of the skeletal muscles—to maintain the commercial value of slices of sashimi The accumulation of extra fat in the muscle may be associated with a loss of muscular texture since muscle neutral lipid content is negatively correlated with the breaking strength [2] In addition, the red muscle of yellowtail undergoes a color change after death of the fish [3, 4] which remarkably reduces the economic value of the yellowtail Therefore, it can be said that the commercial value of yellowtail 123 884 increases when the muscular texture is firm and the color change of the red muscle is slow The addition of a variety of plant materials into fish diets has been examined with the aim improving the meat quality of farmed fish The results of such studies show that addition of kelp Laminaria digitata meal to the diet improves the meat quality of yellowtail [5] and that Ulva meal supplement is associated with the activation of lipid metabolism in black sea bream Acanthopagrus schlegeli [6] and red sea bream Pagrus major [7] Spirulina meal was found to decrease lipid accumulation in red sea bream muscle [8], tochu leaf powder to improve the texture of eel Anguilla japonica [9], and red clover extract to increase the content of protein and lipid in muscle of African catfish Clarias gariepinus [10] Red pepper Capsicum annuum is used world-wide as a food material The antioxidative effects and lipid metabolic promotion action of red pepper have been reported in humans and rats [11–15], while in fish there are reports its effect on body coloration [16, 17] However, there is as yet insufficient data on the effect of red pepper on fish meat quality The aim of the study reported here was to assess the improved meat quality when red pepper was added to the diet of farmed yellowtail Materials and methods Diets The proximate composition of commercial extruded pellets (EP; diameter 14 mm) for yellowtail (Nippon Suisan Kaisha, Tokyo, Japan) was 6.0% moisture, 45.8% crude protein, 23.7% crude lipid, and 24.5% ash The control group was fed on a commercial EP only, and the experimental group was fed on EP diets supplemented with red pepper 0.5% (v/v) Nakagawa et al [5] suggested that diets containing 0.5% (v/v) kelp Laminaria digitata meal were able to improve the meat quality of yellowtail Thus, in this study, the experimental diets contained the same amount of red pepper (0.5%, v/v) The content of capsaicin in the experimental diets was 0.68 lg/g, as measured by high-performance liquid chromatography (HPLC) [18] Fish and rearing condition One-year-old yellowtail S quinqueradiata with a mean body weight of 1.6 kg were placed into one of two sea cages (8 9 m; about 435 fish per cage) at the Oita Marine Biological Technology Center (Nippon Suisan Kaisha) in Oita Prefecture, Japan Fish were reared for months (July–September) and fed on the commercial EP 123 Fish Sci (2011) 77:883–889 One sea cage was used for the control group and the other for the experimental group; the mean body weight of 30 randomly selected fish from each cage was 2.2 kg The experimental group was fed on EP diets containing red pepper for 30 days Two-year-old yellowtail were fed on red pepper-supplemented EP (diameter 16 mm; same composition as pellets for 1-year-old fish) for months (August–October) in a fish farm in Ehime Prefecture and transported to the stock facilities (Nippon Suisan Kaisha) at Misaki fishing port in Kanagawa Prefecture by ship The final mean body weight of the experimental and control group fish was 3.7 kg The fish were slaughtered instantly by cutting the spinal bulb to avoid handling stress, and various measurements were performed, as described below Biochemical analyses Five fish were randomly selected from each group of 1- and 2-year-old yellowtail and individually analyzed The muscle was dissected immediately after slaughter and divided into left and right lateral muscles The left lateral muscle was cut out from the seventh to tenth vertebra and divided into dorsal and ventral portions The red muscle and subcutaneous adipose tissue were then separated from the ordinary muscle Lipids were extracted from the dorsal or ventral portion with chloroform:methanol (2:1, v/v) according to Bligh and Dyer [19] and was estimated gravimetrically The composition of the fatty acid in total lipid extracted from the dorsal portion of the ordinary muscle was analyzed according to Inoue et al [20] Crude protein was measured using the Kjeldahl method ATP and related compounds ATP, ADP, AMP, IMP, inosine (HxR), and hypoxanthine (Hx) were analyzed using modifications of the methods of Iwamoto et al [21] in five fish stored at 0°C for 24 h after slaughter Briefly, about g of the tissue was dissected from the musculus latero-dorsalis and dispersed into ml of ice chilled 10% perchloric acid (PCA) The mixture was added to ml ice-chilled 5% PCA and centrifuged at 12109g for The supernatant was adjusted to pH 6.4 using 10 N KOH and N KOH, and the precipitation was removed by centrifugation ATP and its related compounds were determined in the supernatant using a SHIMADZU LC-10AD HPLC equipped with a SHIMADZU SPD-10A UV–Vis detector and a SHIMADZU C-R7A plus data processor (Shimadzu, Kyoto, Japan) The amounts of ATP and its related compounds were presented as the proportion of each component relative the total of the six components Fish Sci (2011) 77:883–889 885 Texture Results The dorsal muscles were dissected from five fish randomly selected from each group and sliced into 10-mm-thick sashimi at 24 h after slaughter Texture was measured using a Yamaden Rheoner RE-3305 with a wedge-shaped plunger and a kg load cell (Yamaden, Tokyo, Japan) according to Inoue et al [20] The texture was analyzed using the default conditions and detected as the ‘‘hardness load’’ when the plunger was inserted into the 80% thickness of the sample cut in the orientation of muscle fibers Five specimens were analyzed for each individual and the data were described as firmness (N) Proximate composition of muscles Observation of adipose tissue Lipid staining of cross slices of fish was performed using modifications of the methods of Yamaguchi et al [22] Briefly, the right lateral muscles between the seventh and eighth vertebrae were used for the histological observations of adipose tissue The tissues were fixed in 20% neutral buffered formalin (Mildform 20 N; Wako Pure Chemical Co, Osaka, Japan) at room temperature Cross slices, 15 mm thick, were prepared and washed in running water The slices were stained with oil red O (Nakalai Tesque Co, Kyoto, Japan), rinsed in 60% isopropanol, and the stained adipose tissue observed under the light microscope Color of the red muscle and thiobarbituric acid-reactive substances Fish fillets from five fish were sliced into thicknesses of 10 mm at h after slaughter The slices were preserved in a box in which the temperature was kept at 4°C The color of the red muscle was measured at and 32 h after slaughter using a colorimeter (CR-200; Minolta, Tokyo, Japan) with CIELAB values (CIE) The color difference (DE*ab) of each group was calculated with L*, a*, and b* referring to the initial color values (7 h after slaughter), as shown in the following formula DEà ab h i1=2 ¼ ðLà 7hÀLà 32hÞ2 þðaà 7hÀaà 32hÞ2 þðbà 7hÀbà 32hÞ2 The thiobarbituric acid-reactive substances (TBARS) of red muscle were measured according to Sohn et al [4] at 24 h after slaughter The initial body weight was 2.2 kg and the final body weight was 2.6 kg in both groups The growth of each group was equal throughout the experimental period of 30 days The experimental group actively fed on red pepper EP at the start of the study, and a decrease in appetite due to the red pepper supplement was not observed The average initial crude lipid content in the dorsal and ventral muscle was 9.5 and 15.3%, respectively; after 30 days, these values were 14.5 and 18.7%, respectively, in the control fish (Table 1) In the control group, lipid content increased by 5.0 and 3.4% in the dorsal and ventral muscle, respectively, during the 30-day experiment In comparison, in the experimental group, crude lipid level was 10.3 and 18.9% in the dorsal and ventral muscle, respectively (Table 1); this represents a 3.6% increase in the ventral muscle relative to the initial level, but there was hardly any increase in the dorsal muscle (0.8%) Adipose tissue developed in the subcutaneous, the musculus carinatus dorsalis, the musculus carinatus ventralis, and the red muscle surroundings in the initial samples (Fig 1a), but the development of adipose tissue was not remarkable in the musculus latero-dorsalis and the musculus latero-ventralis (Fig 1a) Lipid accumulated in the adipose tissue of the control group compared with the initial samples over the entire muscle after 30 days (Fig 1b) The experimental group differed from the control group in terms of lipid accumulation: adipose tissue in the surroundings of the horizontal septum did not develop, and this was particularly remarkable in the m latero-dorsalis (Fig 1c) In 2-year-old yellowtail, the average crude lipid content of the dorsal muscle of the control and experimental group was 19.9 ± 1.8 and 21.1 ± 2.6%, respectively (Table 1); the average crude lipid of the ventral muscle of the control and experimental group was 26.7 ± 2.8 and 29.2 ± 2.4%, respectively (Table 1) There were no significant differences between groups Moreover, adipose tissue developed throughout the entire muscle in both groups Fatty acid composition Table shows the fatty acid composition (%) of the muscle Because the lipid source in each diet was the same, the fatty acid composition of the muscle was also almost the same in both groups ATP and related compounds Statistical analysis Statistical analysis was performed using Student’s t test Data are given as means ± standard deviation (SD) In the 1-year-old yellowtail, the proportion of ATP in all fish became \1% at 24 h after slaughter; however, compared to the intial value, this decrease was not significant 123 886 Fish Sci (2011) 77:883–889 Table Proximate composition of dorsal and ventral muscle in yellowtail (Seriola quinqueradiata) Composition One-year-old yellowtaila Initial Two-year-old yellowtailb Control Experimental Control Experimental Dorsal Ventral Dorsal Ventral Dorsal Ventral Dorsal Ventral Dorsal Ventral Moisture (%) 66.4 62.0 63.1 58.8 65.7 60.1 58.3 ± 2.0 53.5 ± 1.7 58.1 ± 2.3 52.5 ± 2.1 Crude protein (%) 22.7 21.2 22.0 21.3 22.6 20.8 22.2 ± 0.5 20.0 ± 0.7 21.4 ± 0.5 18.9 ± 0.4 9.5 15.3 14.5 18.7 10.3 18.9 19.9 ± 1.8 26.7 ± 2.8 21.1 ± 2.6 29.2 ± 2.4 Crude lipid (%) a In 1-year-old yellowtail, an equal amount of muscle from five fish was mixed and analyzed b Data are shown as the mean ± standard deviation (n = 5) Fig Accumulation of lipid in the muscle of yellowtail The cross slices of yellowtail muscles at the position between the 7th and 8th vertebra was stained with oil red O a Muscle at the beginning of the experiment Deposition of lipid is observed in the subcutaneous (arrow), the m carinatus dorsalis (asterisk), the m carinatus ventralis (dagger), and the red muscle (filled circle) b Muscle of the control group c Muscle of the experimental group Arrowheads in c show lipid is accumulated less in the experimental group than in the control group Deposition of lipid surrounding the horizontal septum (plus sign) did not develop Table Fatty acid composition of the muscle (Fig 2a) There was no difference in other ATP-related compounds in both groups In the 2-year-old yellowtail, the proportion of ATP in the ATP-related compounds 24 h after slaughter was 2.0 ± 1.4% in the control group and 1.1 ± 0.4% in the experimental group; this difference was not statistically significant (Fig 2b) There was no difference in other ATP-related compounds in both groups, and there was no age-related difference for ATP and related compounds Fatty acids C14:0 Control (%) 5.0 ± 0.3 Experimental (%) 5.0 ± 0.1 C15:0 0.4 ± 0.0 0.4 ± 0.0 C16:0 16.4 ± 0.7 16.6 ± 0.6 C16:1n-7 7.1 ± 0.4 7.2 ± 0.3 C18:0 4.2 ± 0.2 4.3 ± 0.2 22.1 ± 0.2 22.3 ± 0.3 C18:1n-9 C18:2n-6 4.6 ± 0.3 4.6 ± 0.1 C18:3n-6 0.0 ± 0.0 0.0 ± 0.0 C18:4n-6 1.2 ± 0.1 1.1 ± 0.1 C20:1n-11 6.1 ± 0.4 6.4 ± 0.3 C20:4n-6 C20:4n-3 4.9 ± 0.2 0.7 ± 0.0 5.1 ± 0.3 0.7 ± 0.0 C20:5n-3 7.3 ± 0.3 6.9 ± 0.3 C22:5n-3 3.0 ± 0.1 3.1 ± 0.0 C22:6n-3 12.6 ± 1.2 11.8 ± 0.5 4.5 ± 0.1 4.6 ± 0.1 Others Data were obtained individually from five fish and are shown as the mean ± SD 123 Texture In 1-year old yellowtail, the texture of the control and experimental group was 8.2 ± 1.2 and 10.9 ± 0.6 N, respectively (Fig 3a); in 2-year-old yellowtail the texture of the control and experimental group was 5.4 ± 2.2 and 10.6 ± 2.9 N, respectively (Fig 3b) Handling of the fish and the proportion of ATP level until measuring was equal (Fig 2a, b); the texture of the experimental group was significantly firmer than that of the control group (p \ 0.01) for both 1- and 2-year-old fish (Fig 3) Fish Sci (2011) 77:883–889 Color of the red muscle and TBARS The DE*ab was 1.1 ± 0.1 between the control group and the experimental group at h after slaughter; thus, there was no difference in color between the two groups The color of the red muscle changed with increasing time after slaughter, with the red color decreasing in intensity and the brightness and yellow coloration increasing In 1-year-old fish, the DE*ab in the control and experimental group at 32 h after slaughter (i.e., between and 32 h after slaughter) was 6.2 ± 0.3 and 4.0 ± 1.1, respectively (Fig 4a) The DE*ab of the experimental group was significantly lower than that of the control group (p \ 0.05) TBARS of red a 887 muscle of the experimental group was significantly lower than that of the control group (Table 3) Similarly, in 2-year-old yellowtail, the DE*ab of the control group was 4.9 ± 1.7 and that of the experimental group was 2.7 ± 0.7; this difference was significant (p \ 0.05, Fig 4b) Discussion In 1-year-old yellowtail, the crude lipid of the dorsal muscle and ventral muscle increased after 30 days from the start of the study in the control group, while the yellowtail b Fig ATP and related compounds in the dorsal muscle of yellowtail at 24 h after slaughter ATP-degraded compounds were analyzed as ADP, AMP, IMP, inosine (HxR), and hypoxanthine (Hx) Analytical results are given for 1-year-old yellowtail (a) and 2-year-old yellowtail (b) a b Fig Texture of dorsal muscle of yellowtail at 24 h after slaughter Closed bar Control group, open bar experimental group Significant difference (p \ 0.01) is indicated with double asterisks a One-year-old yellowtail, b 2-year-old yellowtail a b Fig Effect of color changes in the red muscle of yellowtail at 32 h after slaughter Significant difference between control and experimental group is indicated with an asterisk (p \ 0.05) a One-year-old yellowtail, b 2-year-old yellowtail 123 888 Fish Sci (2011) 77:883–889 Table Comparison of thiobarbituric acid-reactive substances (TBARS) of red muscle of yellowtail TBARS (nmol/g muscle) Control Experimental 4.95 ± 0.55 3.05 ± 0.87 Data are shown as the mean ± SD TBARS content different between control group and experimental group is signficantly different at p \ 0.01 grew from about 2.2 to about 2.6 kg While the experimental group showed no differences in growth and in crude lipid content in the ventral muscle compared with the control group, the crude lipid of the dorsal muscle of the former tended to be lower than that of the control group On the other hand, there was no difference in lipid content between the control and experimental group in 2-year-old yellowtail Therefore, it would appear that red pepper has the potential to suppress lipid deposition in the muscle of yellowtail with little muscle lipid However, it is believed that this effect is minimal, and it cannot be expected that lipid deposition in yellowtail with a high content of muscle lipid would be decreased The texture of fish muscle during chilled storage proceeds independently of rigor, with the texture decreasing for some time immediately after slaughter [1] When muscular ATP decreases to about lmol/g, fish show full rigor [23] The slaughter condition influences muscular ATP, and the decrease in ATP differs between unstressed and stressed fish [24–26] In two studies on 1- and 2-yearold yellowtail [27, 28], no differences were observed in the change of the ATP ratio and of ATP-related compounds between the two groups at 24 h after slaughter, indicating that the slaughter conditions of both groups were equal For that reason, the texture of the muscle clearly decreased until 24 h after slaughter, becoming stable thereafter [27, 28] Therefore, in our study, we measured the texture at 24 h after slaughter Because the texture of the experimental group was significantly higher in at both measurements, it was thought that the muscle became firmer due to the consumption of red pepper in the yellowtail We demonstrated that the effect of red pepper was not related to fish age and lipid content because there was no difference in lipid content in 2-year-old yellowtail Texture and collagen content are highly correlated in the muscle [29] In the broiler and the eel, it was reported that a diet containing tochu leaf power increases collagen content and improves muscle texture [9, 30] There is a also a possibility that feed containing red pepper exerts some influence on the extracellular matrix in yellowtail In young yellowtail, the replacement of dietary fish oil with olive oil was found to prevent the red muscle from discoloring because the muscular n-3 highly unsaturated fatty acid level decreased [31] However, in our study, the 123 color change of the red muscle of the experimental group fed on red pepper was slow even though the fatty acid composition of muscle was almost the same between the control group and the experimental group It is possible that red pepper has an antioxidative potential because the TBARS content of the experimental group was significantly lower than that of the control group Sohn et al [4] showed that the accumulation of lipid hydroperoxide is related to the color change of the red muscle of yellowtail Dairam et al [15] suggested that capsaicin can reduce Fe2?-induced lipid peroxidation in rat brain homogenate Furthermore, Sim and Sil [32] showed that red pepper pericarp extract is highly effective in scavenging free radicals and that red pepper seed extract is highly effective as a superoxide anion scavenger and in terms of superoxide dismutase activity In this study, the accumulation of the lipid hydroperoxide of the red muscle of yellowtail may have been suppressed by including red pepper in the diet These results are the first to demonstrate that dietary red pepper is able to reduce the loss of muscle texture firmness and to slow down color change in red muscle of yellowtail and that the effect of red pepper, although slight on the muscle of yellowtail, is due to reduced lipid deposition The inclusion of red pepper in the diet of farmed fish should, therefore, improve the quality of the yellowtail as food when served as raw fish (sashimi) However, the relation between the amount of red pepper content in EP and the effect of the improvement of meat quality is not yet clear Future studies need to focus on the appropriate red pepper 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