Effects of Dietary Aqueous Extract from Eucommia ulmoides Oliver on Growth, Muscle Composition, Amino Acid Composition and Fatty Acid Composition of Rainbow Trout (Oncorhynchus mykiss)

Li Yao1,2,3, AnQing Zhang1,2,3, HaiYan Zhang2, Jian Shao1,2,3, Ming Wen2,4, ChangAn Wang5, HaiBo Jiang1,2,3,* and Ming Li6,* 1Breeding and Reproduction in The Plateau Mountainous Region, Ministry of Education (Guizhou University), Guiyang 550025, China 2College of Animal Science, Guizhou University, Guiyang 550025, China 3Special Fisheries Research Institute, Guizhou University, Guiyang 550025, China 4Key Laboratory for Animal Diseases and Veterinary Public Health of Guizhou Province, Guiyang 550025, China 5Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China 6School of Marine Sciences, Ningbo University, Ningbo 315211, China Article Information Received 19 December 2019 Revised 12 Februaary 2020 Accepted 04 March 2020 Available online 26 June 2020


INTRODUCTION
I n developed and developing countries, fish constitutes an important food source due to its rich nutrient content. Consumers demand healthy, high-quality, natural, and fresh fish products. Rainbow trout (Oncorhynchus mykiss), as an important worldwide cold water fish species (Rezaei and Hosseini, 2008), is one of the main sources of protein, minerals, vitamins, and n-3 long-chain polyunsaturated fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Abedi and Sahari, 2014; O n l i n e

F i r s t A r t i c l e
vitamin E (Kamireddy et al., 2011), zinc (Wu et al., 2015), selenium yeast (Wang et al., 2018), astaxanthin and canthaxanthin (Cui et al., 2009). Eucommia ulmoides Oliver (EUO) ("Du Zhong" in Chinese, "Tu Chung" in Korean and "Tuchong" in Japanese), the sole species in the genus Eucommia and family Eucommiaceae, is a deciduous tree indigenous in China and widely cultured in eastern Asian countries (Si et al., 2013). In China, it is distributed mainly in Guizhou, Hubei, Shanxi, Gansu, and Henan Provinces with a total area about 67 000 hectares (Jiao et al., 2015). It is highly valued and commonly used in traditional medicines to treat various diseases, especially to relieve hypertension, protect the nervous system, treat diabetics, regulate lipid metabolism, inhibits oxidative stress (Okada et al., 1994;Geun et al., 2004;Kwon et al., 2014). The inclusion of EUO in diet improved the flesh quality of pig (Wang et al., 2007), chickens (Wang et al., 2012), grass carp, Ctenopharyngodon idella Sun et al., 2017a, b), and eel, Anguilla japonica (Tanimoto et al., 1993a, b).
In China, rainbow trout aquaculture production was from 8,818 to 38,606 tones in recent ten years (MOAC, 2019). But the flesh quality of rainbow trout has been declining in recent years, showing a loose texture due to the high stocking density and the rapid growth in China. Until now, there has been no scientific report concerning the effect of EUO as a supplement in rainbow trout diet to improve flesh quality. Water is a main solvent of decocting medicinal herbs in traditional Chinese medicine. The objective of the present study was to investigate the effects of dietary aqueous extract from EUO on the growth performance and flesh quality of rainbow trout, to supply some nutritional strategies for improving the flesh quality of cultured fish.

Experimental diets and design
During the study, the fish were fed a commercial rainbow trout feed produced by Shengsuo Fishery Feed Research Centre (Shandong, China) as the basal diet. The proximate composition was as follows: crude protein 420 g kg -1 , lipid 180 g kg -1 , crude fibre 60 g kg -1 , and crude ash 80 g kg -1 . The diets were prepared to contain 0 g kg -1 (control, EUO-0), 5.00 g kg -1 (EUO-5), 10.00 g kg -1 (EUO-10), 20.00 g kg -1 (EUO-20) and 40.00 g kg -1 (EUO-40) Eucommia ulmoides Oliver barks extract (EUO). The main nutritional component and active substance content of Eucommia ulmoides Oliver bark aqueous extract is in Table I. Commercial rainbow trout feed was ground through 40-mesh size and then was weighed. EUO was purchased from a local hospital, which was cut and dried at room temperature for 3 days. After dried, the EUO (0 g, 5.0 g, 10.0 g, 20.0 g or 40.0 g, respectively) was lightly boiled in 1000ml of distilled water for 4 h, concentrated to 100ml, and then aqueous extract from EUO (10%, v/w) and water (30%, v/w) were added to feed form a soft dough, and then were mixed to facilitate pelleting by a granulator (330; Ou-siqi Mechanical and Electrical Equipment Co., Ltd, Zhejiang, China). The pellets (1.0 mm diameter) were extruded and air-dried and stored at -20 o C until use.

Fish and sampling
The feeding activities were performed for 70 d at a private rainbow trout farm (Dashan rainbow trout farm, Dafang, Guizhou, China). A total of 450 rainbow trout with an initial body weight of 145.56 ± 4.12 g were randomly allocated in 15 cages (1 m × 1 m × 1.2 m) with a water depth of 0.8 m and 30 fish per cage. The cages were located in three outdoor cement pools (18 m × 3.8 m × 1.2 m) with each pool containing five cages from five treatments. During the feeding period, the fish were fed manually to apparent satiation four times per day (08: 00, 12:00, 15:00, 18: 00) with a daily feeding rate of 2.0% -4.0% of body weight. Fish in each cage were weighed every 2 weeks for feed adjustments throughout the experimental period. Dissolved oxygen, temperature, pH, ammonia nitrogen, and velocity of water were > 7.5 mg L -1 , 12.8 -14.1 o C, 7.6 -8.4, < 0.05 mg L -1 , and 0.04 m s -1 .

O n l i n e F i r s t A r t i c l e
All fish in each cage were weighed and counted at the beginning and at the end of the experiment. Dead fish were removed and recorded daily. At the end, twelve fish from each cage were randomly collected and were anesthetized with 200 mg L -1 MS-222. Six fish were used for the analysis of muscle composition, and six fish were used to obtain the weights of whole body, viscera and liver. All samples were stored at -80 o C before analysis.

Muscle and diets proximate composition
Chemical compositions of diets and muscle including lipid, protein, moisture and ash were analyzed according to standard methods (AOAC, 2005). All analyses were performed in five replicates.

Muscle amino acids and fatty acids
The amino acid compositions of ingredients were determined in triplicate with an automatic amino acid analyzer (LKB Biochrom Ltd., Cambridge, UK). Total lipid of muscle samples were extracted using chloroform: methanol (2:1, v/v) according to the method of Folch et al. (1957). The saponifiable lipids were converted to methyl esters by using the standard boron tri-fluoridemethanol method (Morrison and Smith, 1964). Fatty acids methyl esters (FAME) were analysed on an Agilent 6890 gas chromatograph (Agilent Technologies, Santa Clara, CA, USA), equipped with a flame ionization detector (FID) and a SP-2560 fused silica capillary column (100 m long, 0.25 mm i.d. and 0.20 μm thick). Injector and detector temperatures were 270 and 280 o C, respectively. Column temperature was held at 120 o C for 5 min then programmed to increase at 3 o C min -1 up to 240 o C, where it was maintained for 20 min. Carrier gas was helium (2 mL min -1 ), and the split ratio was 30:1. Identification of fatty acids was carried out by comparing the sample FAME peak relative retention times with Sigma-Aldrich (St. Louis, MO, USA) standards. The individual FAME was identified by comparing the retention times of the authentic standard mixtures. The amount of each fatty acid was calculated using the peak area normalization method as a relative per cent value to the total fatty acids.

Statistical analysis
Data were expressed as means ± standard deviation (SD). All statistical evaluations were analyzed using oneway analysis of variance (ANOVA) by the software SPSS 11.0 (SPSS Inc., Chicago, IL, USA) for Windows. Prior to the statistical tests, data were examined for normality and homogeneity of variances. Differences between the means were tested by Duncan' s multiple-range tests. The level of significance was set at p<0.05.

Growth performance
Results of growth performance are presented in Table II. FBW, WG, SGR, HSI, VSI, CF and SR show no significant difference among all treatment (p > 0.05).

Muscle composition
Muscle composition (g kg -1 , wet basis) of the rainbow trout fed with different aqueous extract from EUO levels is presented in Table III. The fish fed EUO-40 diet showed significantly higher muscle crude lipid content than the fish fed other diets (p < 0.05), and the fish fed EUO-5, EUO-10, or EUO-20 diet showed significantly higher muscle crude lipid content than the EUO-0 group (p < 0.05). For moisture, crude protein and crude ash, there were no significant differences among all treatments (p > 0.05).

Muscle amino acid and fatty acid composition
The effects of the dietary aqueous extract from EUO on the amino acid profile in the muscle of rainbow trout are shown in Table IV. These results indicated that most of the amino acid concentrations in muscle were not significantly affected by dietary EUO levels compared with control group (EUO-0) (p >0.05), except for Val, Phe, Gly, Ala and DAA (p <0.05). Fish fed EUO-5 diet had lower Val  Values in the same row with different superscripts alphabets indicate significant differences (P < 0.05) (n = 3). EAA, essential amino acids; NEAA, nonessential amino acids; TEAA, total essential amino acids; TNEAA, total non-essential amino acids; DAA (Asp, Gly, Glu, Ala), delicious amino acids; TAA, total amino acids.

O n l i n e F i r s t A r t i c l e
L. Yao et al.

DISCUSSION
The current study showed no improvement in growth performance by dietary aqueous extract of EUO. Similarly, Sun et al. (2018) found that dietary 20.00 g kg -1 aqueous extract (water-soluble) and residue (water-insoluble) of EUO could not cause positive effect of the growth in Ctenopharyngodon idella (initial body weight, 47.1 ± 0.8 g). However, some studies showed the positive effects of dietary EUO on the growth performance of culture animals. Another study by Sun et al. (2017b) indicated that WG of C. idella (initial body weight, 95.2 ± 0.6 g) was significantly increased by 20.00 g kg -1 aqueous extract (water-soluble) and residue (water-insoluble) of EUO. The inconsistent results of C. idella were probably due to the differences in body size, producing area, processing method, storing time and active compound content in EUO. And dietary 20.00 and 30.00 g kg -1 aqueous extract (water-soluble) and residue (water-insoluble) of EUO significantly increased the WG of Litopenaeus vannamei (Liu, 2013). In the study by Wang et al. (2007), 1.5 g kg -1 dietary EUO promoted the daily weight gain in pig. Meng et al. (2007) found that 1.5 g kg -1 EUO powder significantly increased WG in C. idella (initial body weight, 37.0 ± 3.0 g). The supplementation of 40.0 g kg -1 EUO leaf powder significantly increased WG in C. idella (initial body weight, 425.8 ± 37.6 g) , and 1.5 g kg -1 EUO leaf extract significantly increased SGR in Carassius autatus gibelio (Shi et al., 2008). The inconsistent reports of different species were probably ascribed to the species, body size, diet composition, environment, feeding period, active compounds contents of EUO from different parts and locations, etc. Zhou et al. (2009) reported that there are significant differences in the components and their contents of the relevant essential oil and flavones from EUO barks and leaves. Liu et al. (2015) found that there are some regional differences of contents of five main effective components (aucubin, geniposidic acid, chlorogenic acid, catechin and rutin) in EUO leaf. There were no significant differences in HSI, VSI, and CF among all groups in this study, which were similar to the report of crucian carp (Shi et al., 2008) and grass carp (Sun et al., 2017a(Sun et al., , b, 2018. In the present study, no effect of dietary EUO was observed in muscle moisture, crude protein, and crude ash contents of rainbow trout, as reported for C. autatus gibelio (Meng et al., 2007), L. vannamei (Liu, 2013), C. idella (Sun et al., 2017a, b). The lipid storage is one of the key factors in determining skeletal muscle quality. In previous studies, Sun et al. (2018) found that dietary EUO could decreased muscle lipid content of C. idella. However, the other studies by Sun et al. (2017a, b) indicated that there was no significant difference in muscle lipid content of C. idella fed EUO-supplemented diet and control diet, which was similar to the studies of L. vannamei (Liu, 2013), C. autatus gibelio (Shi et al., 2008), Angulla japonica (Tanimoto et al., 1993a). At present, the result showed that dietary EUO significantly increased the lipid content in muscle. The result was in line with previous studies in some domestic animals such as pig (Wang et al., 2007), sheep (Yang et al., 2017). It is probably due to the regulation of lipid metabolism and distribution by EUO, which could lower serum lipid levels and increase the muscle crude lipid contents (Yang et al., 2017).
Amino acids are known as anabolic factors, which induce protein gain by stimulating protein synthesis while inhibiting proteolysis Métayer et al., 2008). Sun et al. (2017a) found that the supplementation of EUO into diet showed significantly improved the threonine (Thr), valine (Val), isoleucine (Ile), leucine (Leu), lysine (Lys), arginine (Arg), proline (Pro), total amino acids (TAA) level in muscle of C. idella. The study of Sun et al. (2018) reported that dietary EUO significantly promoted the muscle Arg, aspartic acid (Asp), tyrosine (Tyr), Pro, total nonessential amino acids (TNEAA), delicious amino acids (DAA), TAA levels in C. idella. In the study by Wang et al. (2012), dietary EUO promoted the generation of serine (Ser), glutamic acid (Glu) and lys in O n l i n e

F i r s t A r t i c l e
Effects of Eucommia ulmoides Oliver on Rainbow Trout 7 the muscle of chickens. The present results indicated that phenylalanine (Phe), glycine (Gly), alanine (Ala), DAA in muscle were increased by the supplementation of high levels of EUO. The higher EAA content in muscle means better nutritional value for human. The fresh taste of meat is fundamentally influenced by the content of DAA.Thus it can be seen that dietary EUO can improve the contents of part EAA and DAA in muscle of animals. This may be due to the chlorogenic acid and geniposidic acid, which are the most important bioactive compounds in EU. Sun et al. (2017a) showed that the supplementation of 400-800 mg/kg chlorogenic acid significantly increased the contents of TEAA and TAA in muscle of grass carp. Zheng et al. (2014) found that dietary chlorogenic acid increased serum Gly level and liver glutathione level of rat. Sun et al. (2018) reported that TNEAA, TAA, DAA in muscle of grass carp were increased by the supplementation of high levels of geniposidic acid. However, we do not know the mechanism of the increased amino acids by EUO or active ingredients of EUO. Fatty acid composition is an important characteristic of lipid and oil. In this study, 22:1n-9 content in muscle examined are positively correlated to high level of EUO in diet. This is most likely because of EUO contains 22:1n-9 (Liu, 2013), which were accumulated in muscle of fish fed EUO diets. Our results showed that 18:3n-6 content in muscle of fish fed EUO diet was lower than that of fish fed control diet, especial for EUO-5 and EUO-40 groups. Similarly, 18:2n-6 content in muscle was decreased with the increasing of EUO level in diets. Contrary to 18:2n-6 and 18:3n-6, 20:4n-6 content in muscle of EUO-10 and EUO-40 groups examined were slightly above that of control group. It is possible that rainbow trout has capacity to convert 18:2n-6 to 20:4n-6 (from 18:2n-6 to 18:3n-6, and eventually to 20:4n-6) (Thanuthong et al., 2011;Dernekbasi and Karatas, 2020). At present, there has been no study on the effect of EUO on muscle fatty acid composition of aquatic animal. Results of this report showed that muscle 22:5n-3 and 22:6n-3 contents were significantly increased by the supplementation of EUO-40 in diet. This may be due to the chlorogenic acid in EUO. Kühn et al. (2017) found that there was a tendency to increase C22:6n-3 levels in the liver of Atlantic salmon fed chlorogenic acid diets. The chlorogenic acid is an ester of caffeic acid and quinic acid present in coffee and various other plants, such as EUO, fruits, vegetables, black teas, soybeans and wheat (Sun et al., 2017b). Chlorogenic acid inclusion seems to affect lipid metabolism. The antioxidant capacity of Chlorogenic acid may delay the oxidation of lipids. And it not only promotes β-oxidation of lipids but also induces n-3 LC-PUFA syntheses via transcriptional control of fatty acid elongases and desaturases through increased the expression of the perxisome proliferator-acivated receptor ɑ (Rakhshandehroo et al., 2007;Kühn et al., 2017). However, it did not showed dose-dependent between chlorogenic acid addition level and muscle n-3 LC-PUFA level in this study.
In conclusion, the present study demonstrates that the supplementation of EUO could improve flesh quality without negative effects on growth performance of juvenile rainbow trout, Oncorhynchus mykiss. The supplemental level of EUO was estimate to be a 40.00 g kg -1 diet.

O n l i n e F i r s t A r t i c l e O n l i n e F i r s t A r t i c l e
Effects of Eucommia ulmoides Oliver on Rainbow Trout