Proximate Composition and Fatty Acid Profile in Different Tissues of Wild Adult Female Coilia mystus in Yangtze Estuary

Osbeck’s grenadier anchovy (Coilia mystus Linnaeus 1758) is a well-known estuarine migratory species and distributes most widely along the Chinese coasts (Chen and Cheng, 2012; Cui et al., 2015; Jiang et al., 2019). It inhabits China’s Pacific coastal waters from Hainan in the south to Port Arthur in the north and perhaps also north to Korea (He et al., 2008). C. mystus is a short-distance anadromous fish. In spring, adult fish migrate to estuarine brackish water to spawn and are subjected to an extensive fishery. After spawning, most of the adult population and the larvae grow in Yangtze Estuary, mainly during August and September (He et al., 2008; Cheng et al., 2008). In October and November, when the water becomes cold, the adult population and the juveniles start to move offshore and pass the winter in deep waters from December to April (He et al., 2008). C. mystus is commercially the most important species in Yangtze Estuary, which provides approximately 48.6% of the total fish and shrimp catch in the estuarine area (He et al., 2008; Cui et al., 2015). Despite the commercial importance of C. mystus, studies on its nutritional composition in different tissues are few. Information to date includes resource assessment and population structure (Liu et al., 2013; Yang et al., 2019 a), reproductive biology (Wang et al., 2016), early resources (Zhou et al., 2011), feeding and migration habits (Yang et al., 2006; Liu et al., 2012; Yang et al., 2019 b). In the existing studies, there were only preliminary studies about the muscle and ovary (Liu et al., 2009 b) and the whole (Song et al., 2018 a) nutritional composition of C. mystus, but no studies on fat and fatty acid profile in different tissues of the broodstock for this species.

Previous studies showed that broodstock nutrition is of key importance for reproductive performance of broodstock and larval quality (Izquierdo et al., 2001). There have been many studies about the effects of proximate and fatty acid composition on broodstock and hatched larvae, among which proximate mainly including moisture, fat, protein and ashes and essential fatty acids mainly referring to n-3 highly unsaturated fatty acids (HUFA) of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Song et al., 2014; Song et al., 2019). Such information on broodstock, particularly the fat and EPA and DHA, would provide a better understanding of the reproductive performance (Furuita et al., 2002; Li et al., 2005).
 
To date, little information has been gathered concerning the nutrient composition of the wild adult female C. mystus. In order to understand the nutritional status of the wild broodstock and to analyze their reproductive performance, the proximate and fatty acid profile in the different tissues of this species were studied. The paper would provide a reference for the C. mystus broodstock culture, resource development and utilization in Yangtze Estuary.

Study animals
 
The adult wild female C. mystus were sampled from Yangtze Estuary in July 2019. The C. mystus were 14.16 ± 0.83 cm in body length and 12.01± 4.21 g in body weight. The ovaries before spawning were developed to stage IV, with light yellow color. In this study, the samples got from 3 tissues (muscle, liver, ovary) and the proximate composition and fatty acid profile in these 3 tissues were analyzed in East China Sea Fisheries Research Institute by the biochemical analysis methods, as follows and the reagents are from the Sinopharm Chemical Reagent Co., Ltd.
 
Proximate composition analyses
 
Moisture content was determined by drying sample in an oven at 105°C until a constant weight was obtained (AOAC, 1990). Total fat was determined by using the Soxhlet extraction method (AOAC, 1990).
 
Fatty acid analyses
 
Fatty acids were extracted and fatty acid methyl esters (FAMEs) were prepared according to the ISO 5509 method: first, Soxhlet extraction and then, saponification, followed by esterification and finally, extraction of FAMEs in hexane. FAMEs were subsequently analyzed by capillary gas chromatography (column: 30 m × 0.25 mm I.D., 0.5 µm film thickness; Supelco. Flame ionisation detected temperature at 210°C; carrier gas N2 at 1.0 ml/min; injector temperature at 210°C; oven temperature programmed from 180 to 250°C) using an Agilent 6890 capillary gas chromatograph (Song et al., 2019). Quantitative data were calculated using the peak area ratio (% total fatty acids) (Song et al., 2014).
 
Statistical analysis
 
Homogeneity of variance of data was tested with Levene’s test. One-way ANOVA was used to determine the differences among these 3 tissues. The significant means were compared by Tukey’s multiple range tests. Statistical analysis Data are presented as means ± SD and p<0.05 was regarded as the statistically significant level. All statistics were performed using SPSS package (version 22.0).

Proximate composition mainly refers to the content of moisture, fat, protein and ashes. Previous studies have shown that the moisture and protein content in muscle is higher, while the fat content in ovary is higher for broodstock (Shi et al., 2008; Dos Santos et al., 2016). Table 1 showed that the highest moisture content is in the muscle and the lowest in the ovary, while the highest fat content is in the ovary and the lowest in the muscle, which showed similar results with the previous studies (Song et al., 2014; Song et al., 2019). Fat are the chemical components that most greatly affect the composition of eggs and the broodstock fatty acid profile was associated with diets fatty acid composition (Izquierdo et al., 2001). Fatty acid nutritional level of broodstock would affect reproductive performance and quality of produce fertilized eggs and larvae (Mazorra et al., 2003). In this study, the content of fat in the muscle of C. mystus was lower than that of the C. ectenes and C. ectenes taihuensis, but that in liver and ovary were higher than that in the muscle of C. ectenes and C. ectenes taihuensis (Liu et al., 2009 a), which showed that the content of the total fat in different tissues of the Coilia is closely related to their species, tissues distribution, the environmental conditions and the nutrition intakes. The fat content was significant different in different tissues, which is related to their different physiological function. In the process of vitellogenesis to contribute to egg reserves, fat and fatty acids synthesized in the liver and transferred to the ovaries (Tocher, 2003; Al Nouri et al., 2016). The same rule was also found in Pampus cinereus, which may be related to the period of ovarian structure (Shi et al., 2008). Stage IV ovarian tissue oocytes had experienced the large growth stage, fat and other nutrients accumulated largely in the oocyte.



Table 2 shows that the most abundant SFA is 16: 0, with the same results reported for Scorpaena porcus muscle (Kaya and Kocatepe, 2014), the predominant MUFAs is C18:1n-9c and 16:1, with the same results reported for Oncorhynchus mykiss and Siganus guttatus muscle (Sarma et al., 2015; Song et al., 2018 b) and the most abundant PUFAs is DHA and EPA, with the same results reported for Cynoglossus gracilis (Song et al., 2019). Previous studies have shown that the n-3 HUFA of DHA and EPA are relatively higher in different tissues of marine fishes (Guil-Guerrero et al., 2011; Norambuena et al., 2012). Table 2 demonstrated that fatty acid profile of C. mystus in different tissues were similar to the marine fish, as DHA and EPA contents were significantly higher than C18: 2n6c and 18:3n3 (ALA) (Shi et al., 2008; Norambuena et al., 2012). EPA and DHA in n-3 HUFA of marine fish are regarded as essential fatty acid due to the inability of most marine fish being barely able to convert ALA to EPA and DHA (Zakeri et al., 2011; Castro et al., 2012). The n-3 HUFA content of broodfish gonads has an important impact on the reproductive performance (Li et al., 2005). After the broodstock was fed with the EPA and DHA fortified diet, the quality of their eggs and larvae can be effectively improved (Watanabe and Vassallo-Agius, 2003; Lund et al., 2007). In this study, the adult female C. mystus broodstock are rich in DHA and EPA in the ovary. As there are plentiful natural food organisms for C. mystus in Yangtze Estuary, i.e. copepods, bran shrimp, decapod and fishes (Liu et al., 2012), in which are full of DHA and EPA (e.g. EPA 12.11%, DHA 13.94% in copepods; EPA 9.41%, DHA 18.95% in bran shrimp). C. mystus in Yangtze Estuary can accumulate the PUFA nutrients from these prey by the food chain enrichment effect and then the selected fatty acids were transferred to the ovaries providing some material basis for their reproduction.

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Some studies found that the ratio of DHA/EPA was a very important nutritional indicator for broodstock, larvae and the juvenile baits (Bell and Sargent, 2003). There were differences of the ratio of DHA/EPA in different tissue among different fish species. The ratio of DHA/EPA in muscle, liver and ovary of Acipenser sinensis and Cynoglossus gracilis were 2.55/1, 3.15 / 1, 3.31/ 1 and 1.05/1, 1.11 / 1, 1.26 / 1, respectively (Song et al., 2014; Song et al., 2019). In this study, the DHA/EPA ratio in muscle, liver and ovary was 1.86/1, 1.56/1 and 1.85/1, respectively. The materials all were from the Yangtze Estuary, which ovarian developmental stages were substantially IV. The studies suggested that both the concentration and ratio of these two n-3 HUFA of DHA and EPA, were important in larval nutrition and the optimum ratio would be species and tissues specific (Bell and Sargent, 2003; Heinsbroek et al., 2013). The ratio of n3/n6 in muscle, liver and ovary tissues of C. mystus accounted for 7.78, 4.47 and 9.87, respectively. The ratio is close to that of marine fishes with that from 4.7 to 14.4 (Kleimenov, 1971; Guil-Guerrero et al., 2011), which are a crucial factor in the nutritional value of fish (Das et al., 2009).

The fat and fatty acids profile of the broodstock can affect their reproductive performance and the quality of their eggs and the hatched larvae. In this study, the fat and fatty acid profile in the ovary of the wild C. mystus broodstock was analyzed and the gonad development situation was known. The ovary contains the highest fat, PUFA, n-3PUFA, n-3HUFA, EPA+DHA content, which is suitable for its reproductive function. The information obtained here can be used to determine the nutritional status of the wild C. mystus broodstock in their reproductive stage in Yangtze Estuary and to further speculate the quality of their eggs and the hatched larvae.

This work was supported by special research fund for the national non-profit institutes (East China Sea Fisheries Research Institute) (NO. 2019T05), Shanghai Nature Fund Project (19ZR1470200) and the key project of promoting agriculture by science and technology in Shanghai (2017-02-08-00-07-F00075).