1 Introduction

The American shad (Alosa sapidissima) is an economically and ecologically valuable anadromous herring native to the Atlantic coast of North America (Walburg and Nichols, 1967). In the last decades, American shad was introduced into China as an alternative species due to the almost extinction of Chinese shad (Tenualosa reevesii)and it is considered as an emerging aquaculture species in China. Most studies on early development of American shad were focused on the biological characteristics and rearing methods (Gao et al., 2015; Hong et al., 2011; Mi et al., 2014; Xu et al., 2012; Zhang et al., 2010). However,its relatively high mortality during intensive larval rearing has hampered the leap to industrial farming. Several studies have shown that there is a certain relationship between embryo and larval quality and biochemical composition (Farhoudi et al., 2011; Kestemont et al.,1999; Rainuzzo et al., 1997; Salze et al., 2005). An estimation of the utilization of endogenous nutrients from the yolk that occurs during embryonic and larval development can provide important information to the nutritional requirements of fish larvae (Abi-Ayad et al., 2000; Fraser et al., 1986; Mourente and Vázquez, 1996; Ostrowski and Divakaran, 1991; Sargent, 1995).

During pre-feeding developmental stages of fish, yolk is known to be ideally suited to meet the embryo’s requirements. In principle, fish eggs contain all the nutrients that the larvae utilize during the lecithotrophic phase,prior to exogenous feeding, to support both homeostasis and development (Mourente and Vázquez, 1996). Several studies have shown that the biochemical composition of fish eggs also varies between species and may change during the different stages of development according to ambient conditions, physiological events and energy demands (Cejas et al., 2004; Cruzado et al., 2013; Gimenez et al., 2008; Harlioğlu and Gölbaşi, 2013; Mourente and Vázquez, 1996; Sargent, 1995). It was observed that both lipids and protein can be catabolized as substrates for energy metabolism and structural components in membrane biogenesis (Finn et al., 1991, 1995; Heming and Buddington, 1988), although there was a clear preference in the utilization of lipid, protein and free amino acids(Cruzado et al., 2013; Gimenez et al., 2008; Rønnestad et al., 1998). Moreover, the amino acids play an important role in the formation of some structures during embryogenesis and early larval development (Finn et al.,1996; Rønnestad et al., 1999; Zhu et al., 2003). The fatty acids, especially polyunsaturated fatty acids (PUFA), are generally involved in the functional integrity and fluidity of cell membranes (Lauritzen et al., 2001), and the level of PUFA in eggs had a marked influence on the further larval survival (Eldridge et al., 1983; Fraser et al., 1988;Furuita et al., 2006). Larval nutrition was considered as one of the most important challenges in aquaculture, since a major difficulty in rearing fish larvae is the limited knowledge of their nutritional requirements. Moreover, the probable nutritional requirements of exogenous feeding larvae can be estimated by studying variations in the biochemical composition of eggs and larvae (Fraser et al.,1988; Desvilettes et al., 1997).

Within this context, we were interested in determining the changes in amino acids and fatty acids compositions of American shad during its embryogenesis and early larval development. The nutritional information can be used to gauge physiological condition and identify possible dietary deficiencies.

本文根据近两年湖南省不动产统一基础数据数字线划图成果进行过程质量检查及验收情况，对发现的较为突出的典型性问题以及普遍性问题进行总结和分析，找出生产过程中的关键节点及薄弱环节，旨在从生产环节尽量杜绝这些问题的出现，确保我省不动产数字线划图数据完整、真实、可靠。

2 Materials and Methods

2.1 Eggs and Yolk-Sac Larvae

Nutritional fortification is carried out in Zhongyang group, Jiangsu Province, from December to May next year in fresh water under natural photoperiod with an increasing temperature from 14 to 20℃. The dissolved oxygen was maintained at 6.8 ± 0.8 mg L−1. The feed for parent fish that includes 55% crude protein and 10%crude lipid contains 30% artificial feed, 35% trash fish,20% clam, 15% nereis, 0.5% Vitamin C (Vc), 0.5% Vitamin E (Ve), 0.5% fish oil and 0.1% soybean lecithin. The artificial feed that includes 50% crude protein and 8%crude lipid was consisted of 18% white fish meal, 30%red fish meal, 20% patent flour, 17% soybean meal, 3%beer yeast, 1% GuYuan flour, 1% squid powder, 0.1%Met, 0.3% Lys, 0.3% betaine, 2% vitamin mixture, 1.5%mineral mixture, 0.3% vitamin C phosphate, 0.2% bile acid, 0.6% calcium dihydrogen phosphate, 0.5% soybean lecithin, 0.2 fish oil, 0.2% soybean oil and 0.2% immune polysaccharide. One hundred female fish and sufficient male fish were selected and maintained in a circular spawning pond under natural photoperiod and room temperature. After artificial fertilization, fertilized eggs from 30 pairs of parents were collected and equally assigned into three 400 L hatch cylinder with a flowing-through fresh water system, and the volume of fertilized eggs per pair of parents was 200 g. Larvae were raised with the protocol described by Gao et al. (2016).

2.2 Sample Collection

低钠血症是指血钠浓度<135 mmol/L，并引发一系列临床症状的疾病[1]。正常情况下，机体内钠离子的摄入与排出处于相对平衡的状态。随着年龄增加，调节水钠平衡机制的衰退，以及感染、心脑血管疾病、缺氧等多种因素导致老年患者经常出现低钠血症。低钠血症常缺乏特异性临床表现，易被误诊漏诊，预后不良。近年来对低钠血症机制等研究逐渐完善。现主要从病因、诊断等方面对低钠血症的诊治进展予以综述。

2.3 Biochemical Analysis

The change of amino acids content was similar to total protein, which exhibited a decreasing trend during embryogenesis and larval development (Table 2). A great reduction was detected in almost all amino acids after hatching except for glycine (P ＜ 0.05). During the yolksac phase (DAH0-DAH3), significant decrease was found in the content of cysteine, proline, tyrosine, valine, isoleucine, leucine and phenylalanine (P ＜ 0.05). The highest loss of amino acids content was found in proline (66.34%).Glycine was the only amino acid that maintained a constant content during the experimental period. Moreover,the content of essential amino acids (EAAs) is relative higher than that of non-essential amino acids (NEAAs).Both reduced significantly after hatching, exhibiting a similar trend during embryogenesis and larval development (Fig.1).

2.4 Statistical Analysis

由于信息不对称，建筑市场中的投机行为屡见不鲜。业主方面，经常出现业主让承包商垫资施工以降低资金成本和投资风险等现象。道高一尺魔高一丈，承包商也想出了一些诸如低价中标，高价索赔的妙招。无疑会给业主造成成本提高的后果。监理与承包商之间徇私舞弊、利益交易等也对工程质量造成严重的影响。这种恶性循环会造成工程质量越来越低，建筑行业高质量的开发商越来越少，使业主处于不利地位。这种情况长此以往发展下去也必然影响建筑市场的良性发展。

3 Results

Throughout the embryogenesis and early larval development, fish larvae obtain nutrients from the endogenous reserves of the yolk. As the major nutrients, lipid and protein are used both as substrates for energy metabolism and as structural components in membrane biogenesis(Finn et al., 1991, 1995; Li et al., 2015; Liu et al., 2013).Lipid and protein metabolism during the early life of fish may differ greatly among species. In the present study,the content of total protein and total lipid constantly decrease during the period studied with the decreasing content of dry weight. The dry weight decline is linked with the utilization of endogenous stores by the embryo and larvae. Based on the insignificant variations of total lipid content, it was likely that lipids were not used as the major energy source by embryos or larvae of American shad.In addition, significant decrease was found in the content of total protein and total amino acids, and the content of protein is far higher than that of lipid. As protein and lipid were major energy sources during ontogeny, and the lipid content of each individual remained stable while the content of protein and amino acids decreased significantly in this process, it was demonstrated that protein and amino acids may be the main source for energy production. A similar effect has been previously reported in a number of fish species such as Atlantic halibut (Hippoglossus hip-poglossus) (Zhu et al., 2003), brill (Scophthalmus rhombus L.) (Cruzado et al., 2013), barfin flounder (Verasper moseri) (Ohkubo and Matsubara, 2002) and gilthead seabream (Sparus aurata) (Rønnestad et al., 1994). On the other hand, a great decrease in total lipid content during embryogenesis and early larval development was also observed in some species such as pike (Esox lucius L)(Desvilettes et al., 1997), white seabream (Diplodus sargus) (Cejas et al., 2004), reflecting the utilization of lipid as the energy substrate. These data indicate that biochemical composition of yolk reserves in fish is species specific, and the precise sequence of consumption varies both qualitatively and quantitatively.

All statistical analyses were performed using SPSS18.0 for windows. Results are expressed as mean with standard error. The normality and variance homogeneity of the data were checked using Kolmogorov-Smirnov test and Levene’s test, respectively, prior to variance analysis.Percentage data were arcsin transformed to satisfy the parametric assumptions. One-way analysis of variance(ANOVA) was employed to distinguish statistical differences between different treatment and control groups.Tukey HSD tests were then used to find significant groupings within the data set. In all analyses, the significance level was set at P = 0.05.

The samples were freeze dried and weighed with electronic balance. The protein content was measured with Kjeldahl method (AOAC 2006) with a conversion factor of 6.25. The amino acid content was analyzed by HPLC-fluorescence following the method of Davies and Thomas(1973) and Pfeifer et al. (1983). Total lipid from original tissue was extracted with chloroform/methanol (2:1 v/v)containing 0.01% butylated hydroxytoluene (BHT) as antioxidant. The organic solvent was evaporated under a stream of nitrogen and the lipid content was determined gravimetrically. The fatty acid methyl esters were separated using a gas liquid chromatography, and peaks of individual fatty acids were identified by reference to authentic standards (Supelco; Sigma-Aldrich, St. Louis, MO,USA) and to well-characterized fish oil (Mehaden oil by Supelco; Sigma-Aldrich).

早春大棚茄子一般在3月中旬定植，定植时要求温室大棚内温度不低于10℃，土壤10 cm深度地温保持在13℃以上。在移栽之前苗床灌溉透水，在晴朗的早晨或者傍晚进行移栽，每亩定植2000-2500株。早春3月，由于外界温度和土壤层温度不稳定，因此应该选择大苗定植，确保幼苗成活以后，由营养生长向着生殖生长快速转变。

Table 1 The change of dry weight, total protein and total lipid contents in developing eggs and yolk-sac larvae of Alosa sapidissima during different development stages

Notes: Dry weight (mg egg−1 or mg larvae−1), total protein (μg egg−1 or μg larvae−1) and total lipid contents (μg egg−1 or μg larvae−1). Development stages: A, fertilized egg (2 h after fertilization); B, gastrul stage (12 h after fertilization); C, organ differentiation stage (30 h after fertilization); D, newly hatched larvae (DAH0); E, 1 day after hatching (1 DAH); F, 3 days after hatching (3 DAH, nearly absorbed yolk-sac). Data were expressed as mean ± SEM (n = 3). Different superscripts in the same row indicate statistical significances (P ＜ 0.05).

Parameter Development stages A B C D E F Dry weight 0.41 ± 0.03a 0.39 ± 0.02a 0.38 ± 0.02a 0.28 ± 0.01b 0.28 ± 0.03b 0.26 ± 0.02b Total protein 333.11 ± 12.56a 314.93 ± 13.77a 305.10 ± 11.79b 214.36 ± 10.88b 209.66 ± 11.18bc 188.75 ± 9.64c Total lipid 28.38 ± 2.72 27.08 ± 2.58 26.24 ± 2.67 25.49 ± 1.92 25.33 ± 2.45 24.14 ± 2.13

Table 2 Amino acid content in developing eggs and yolk-sac larvae of Alosa sapidissima

Notes: Amino acid contents (μg egg−1 or μg larvae−1); Development stages: A, fertilized egg (2 h after fertilization); B, gastrul stage (12 h after fertilization); C, organ differentiation stage (30 h after fertilization); D, newly hatched larvae (DAH0); E, 1 day after hatching (1 DAH); F, 3 days after hatching (3 DAH, nearly absorbed yolk-sac). Results represent means ± SEM of three replicates. Treatment values in the same row followed by different superscript letters are significantly different (P ＜ 0.05). Asp, aspartic acid; Ser, serine; Glu, glutamic acid; Gly, glycine; Ala, alanine; Cys, cysteine; Pro, proline; Tyr, tyrosine; Val, valine; Met, methionine; Ile, isoleucine; Leu,leucine; Thr, threonine; Phe, phenylalanine; Lys, lysine; His, histidine; Arg, arginine; EAA, essential amino acid; TAA, total amino acid.

Amino acid Development stages A B C D E F Asp 22.38 ± 2.13a 22.0 ± 11.85a 20.66 ± 2.05a 14.79 ± 1.13b 14.73 ± 1.21b 13.50 ± 1.09b Ser 15.00 ± 1.03a 14.50 ± 1.24a 13.18 ± 1.19a 8.75 ± 0.64b 8.25 ± 0.56b 7.70 ± 0.62b Glu 37.31 ± 2.36a 37.05 ± 1.87a 35.01 ± 2.62a 23.27 ± 1.84b 23.57 ± 2.12b 21.49 ± 1.65b Gly 9.45 ± 0.67 9.44 ± 0.74 8.85 ± 0.43 8.44 ± 0.39 8.59 ± 0.48 8.35 ± 0.53 Ala 25.79 ± 1.89a 25.64 ± 2.25a 24.38 ± 1.83a 14.47 ± 1.29b 12.55 ± 1.08b 11.73 ± 1.05b Cys 1.54 ± 0.16a 1.40 ± 0.08b 1.33 ± 0.11a 0.80 ± 0.03c 0.65 ± 0.02d 0.65 ± 0.03d Tyr 11.64 ± 0.79a 11.48 ± 1.06a 10.86 ± 0.95a 8.80 ± 0.64b 8.39 ± 0.56b 7.05 ± 0.62c Pro 18.73 ± 1.53a 15.86 ± 1.15b 17.16 ± 1.62ab 10.66 ± 0.86c 8.76 ± 0.54d 6.31 ± 0.49e NEAA 141.84 ± 10.52a 137.39 ± 10.38a 131.42 ± 10.67a 89.97 ± 6.93b 85.49 ± 6.44bc 76.78 ± 5.88c Thr 17.56 ± 1.18a 17.13 ± 1.23a 15.95 ± 1.12a 10.20 ± 1.06b 9.80 ± 0.67b 8.69 ± 0.63b Val 18.86 ± 1.56a 19.15 ± 1.63a 18.42 ± 1.44a 11.17 ± 0.88b 10.88 ± 1.02bc 9.53 ± 0.59c Met 8.07 ± 0.68a 7.97 ± 0.56a 7.52 ± 0.58a 6.15 ± 0.34b 5.87 ± 0.25b 5.43 ± 0.38b Ile 19.18 ± 1.76a 19.65 ± 1.54a 18.99 ± 1.55a 11.42 ± 1.09b 10.94 ± 0.87bc 9.22 ± 0.58c Leu 29.60 ± 2.61a 29.59 ± 1.89a 28.06 ± 2.67a 18.49 ± 1.41b 17.48 ± 1.33b 14.80 ± 1.21c Phe 11.76 ± 0.98a 12.11 ± 1.06a 11.51 ± 1.01a 9.80 ± 0.75ab 9.58 ± 0.68bc 8.41 ± 0.52c Lys 20.27 ± 1.78a 20.11 ± 1.91a 19.10 ± 1.55a 14.42 ± 1.34b 14.37 ± 1.35b 13.16 ± 1.12b His 7.26 ± 0.37a 7.27 ± 0.53a 6.95 ± 0.46a 6.07 ± 0.27b 6.06 ± 0.33b 5.72 ± 0.24b Arg 14.31 ± 1.14a 14.35 ± 1.05a 13.52 ± 1.26a 11.03 ± 1.05b 10.85 ± 0.96b 10.91 ± 0.81b EAA 146.87 ± 10.07a 147.33 ± 11.33a 140.00 ± 11.46a 98.75 ± 8.21b 95.83 ± 7.49b 85.86 ± 6.32b TAA 288.70 ± 20.32a 284.72 ± 19.91a 271.42 ± 20.67a 188.72 ± 13.78b 181.32 ± 12.81bc 162.64 ± 10.14c EAA/NEAA 1.04 ± 0.03 1.07 ± 0.05 1.07 ± 0.03 1.10 ± 0.04 1.12 ± 0.08 1.12 ± 0.06

Table 3 Fatty acids composition in developing eggs and yolk-sac larvae of Alosa sapidissima (% total fatty acids)

Notes: Development stages: A, fertilized egg (2 h after fertilization); B, gastrul stage (12 h after fertilization); C, organ differentiation stage (30 h after fertilization); D, newly hatched larvae (DAH0); E, 1 days after hatching (1 DAH); F, 3 days after hatching (3 DAH,nearly absorbed yolk-sac). Results represent means ± SEM of three replicates. Treatment values in the same row followed by different superscript letters are significantly different (P ＜ 0.05). SFA, Saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; HUFA, highly unsaturated fatty acids; EPA, eicosapentaenoic acid (20:5n-3); DHA, docosahexaenoic acid (22:6n-3);ARA, arachidonic acid (20:4n-6).

Fatty acid A B C D E F Development stages C14:0 0.93 ± 0.08 0.96 ± 0.06 0.94 ± 0.04 1.09 ± 0.10 1.01 ± 0.09 1.01 ± 0.08 C15:0 0.22 ± 0.03 0.22 ± 0.02 0.22 ± 0.02 0.23 ± 0.01 0.22 ± 0.03 0.24 ± 0.02 C16:0 24.41 ± 1.08 24.40 ± 1.95 24.28 ± 1.28 24.27 ± 1.17 24.10 ± 1.13 21.61 ± 1.86 C16:1n-7 2.46 ± 0.16 2.47 ± 0.27 2.47 ± 0.24 2.69 ± 0.19 2.55 ± 0.25 2.48 ± 0.18 C17:0 0.48 ± 0.04 0.48 ± 0.04 0.48 ± 0.02 0.44 ± 0.03 0.43 ± 0.03 0.47 ± 0.02 C18:0 8.87 ± 0.37 8.97 ± 0.43 8.91 ± 0.39 8.27 ± 0.27 8.68 ± 0.36 8.44 ± 0.54 C18:1n-9 19.47 ± 1.71 19.44 ± 1.36 19.53 ± 1.82 19.73 ± 1.62 19.51 ± 1.60 20.64 ± 1.77 C18:2n-6 7.68 ± 0.56a 7.62 ± 0.32a 7.60 ± 0.28a 7.76 ± 0.34a 7.47 ± 0.28a 6.70 ± 0.25b C18:3n-3 0.39 ± 0.04a 0.39 ± 0.03a 0.39 ± 0.03a 0.43 ± 0.02a 0.37 ± 0.0 a 0.58 ± 0.07b C18:3n-6 2.43 ± 0.12a 2.46 ± 0.16a 2.51 ± 0.17a 2.30 ± 0.21ab 2.14 ± 0.15bc 1.87 ± 0.18c C20:0 0.11 ± 0.03 0.09 ± 0.05 0.12 ± 0.04 0.19 ± 0.05 0.19 ± 0.04 0.19 ± 0.05 C20:1n-9 0.65 ± 0.03a 0.65 ± 0.05a 0.66 ± 0.02a 0.59 ± 0.05ab 0.59 ± 0.08ab 0.52 ± 0.04b C20:4n-6 1.08 ± 0.09a 1.07 ± 0.10a 1.07 ± 0.11a 1.15 ± 0.10a 1.23 ± 0.13a 1.69 ± 0.15b C20:5n-3 3.51 ± 0.23 3.53 ± 0.31 3.50 ± 0.24 3.42 ± 0.22 3.31 ± 0.28 3.58 ± 0.25 C22:5n-3 1.66 ± 0.13 1.66 ± 0.17 1.66 ± 0.14 1.57 ± 0.17 1.58 ± 0.11 1.55 ± 0.16 C22:6n-3 20.98 ± 1.51a 20.96 ± 1.37a 21.00 ± 1.44a 21.82 ± 1.25a 22.43 ± 1.26ab 24.25 ± 1.15b SFA 35.02 ± 1.19a 35.12 ± 1.23a 34.94 ± 1.44a 34.49 ± 1.38a 34.64 ± 1.49a 31.97 ± 1.07b Mono 22.59 ± 1.86 22.55 ± 2.49 22.66 ± 1.67 23.00 ± 1.96 22.64 ± 1.72 23.64 ± 1.83 PUFA 37.73 ± 2.26 37.68 ± 1.83 37.74 ± 2.35 38.43 ± 2.31 38.53 ± 2.13 40.21 ± 2.29 n-3 26.54 ± 1.34a 26.53 ± 1.41a 26.56 ± 1.67a 27.23 ± 1.53ab 27.69 ± 1.71ab 29.96 ± 1.45b n-6 11.18 ± 1.01 11.15 ± 1.03 11.18 ± 0.90 11.20 ± 0.80 10.84 ± 0.80 10.26 ± 0.60 n-3HUFA 26.15 ± 1.72 26.14 ± 2.11 26.17 ± 1.83 26.80 ± 2.23 27.32 ± 2.16 28.88 ± 2.37 n-3/n-6 2.37 ± 0.13a 2.38 ± 0.15a 2.38 ± 0.16a 2.43 ± 0.22a 2.55 ± 0.19a 2.92 ± 0.14b EPA / DHA 0.17 ± 0.01 0.17 ± 0.01 0.17 ± 0.01 0.16 ± 0.01 0.15 ± 0.01 0.15 ± 0.01 EPA/ARA 3.26 ± 0.23a 3.28 ± 0.19a 3.28 ± 0.24a 2.99 ± 0.26ab 2.69 ± 0.22b 2.12 ± 0.17c EPA+DHA 24.49 ± 1.48 24.49 ± 1.56 24.51 ± 1.72 25.24 ± 1.65 25.74 ± 1.53 27.33 ± 1.42

Fig.1 Changes in amino acids in developing American shad eggs and larvae. Development stages: A, fertilized egg (2 h after fertilization); B, gastrul stage (12 h after fertilization);C, organ differentiation stage (30 h after fertilization); D,newly hatched larvae (DAH0); E, 1 d after hatching (1 DAH); F, 3 d after hatching (3 DAH, nearly absorbed yolk-sac). Different superscripts in the same development stage indicate statistical significances (P ＜ 0.05).

4 Discussion

Table 1 gives mean values of dry weight, total protein and total lipid at each developmental stage. The content of dry weight remained relatively constant during embryonic development but declined significantly after hatching (P ＜ 0.05). The content of total protein exhibited a decreasing trend during embryogenesis and larval development, and a significant reduction was detected after hatching (P ＜ 0.05). However, the total lipid content was less than protein and remained stable during embryogenesis and larval development.

The change of amino acids content was similar to total protein, which exhibited a decreasing trend during embryogenesis and larval development. Additionally, a great reduction was detected in almost all amino acids after hatching. It was also demonstrated that amino acid may be utilized as the major energy sources during this phase.During the yolk-sac phase, significant decrease was found in the content of NEAA (cysteine, proline and tyrosine)and EAA (valine, isoleucine, leucine and phenylalanine).Therefore, a selective use of amino acid was noted during this phase. Similar result was also observed in other species with the way of selective utilizing amino acid, but the amino acids selected were not coincident among different species (Fyhn and Govoni, 1995; Rønnestad et al., 1994).

The fatty acids composition in developing eggs and yolk-sac larvae of Alosa sapidissima (% total fatty acids)has been presented in Table 3. The highest content is PUFA, followed by saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA). During the period of embryogenesis, all fatty acids remained stable. After hatching, significant decrease was found in the content of C18:2n-6, C18:3n-6, SFA and ratio of EPA (eicosapentaenoic acid)/ARA (arachidonic acid) (P ＜ 0.05), while significant increase was found in the content of C18:3n-3,C20:4n-6, C22:6n-3 and ratio of n-3/n-6 (P ＜ 0.05). Furthermore, increasing level of n-3 PUFA was observed at stage of nearly absorbed yolk-sac compared to fertilized egg stage (P ＜ 0.05).

Moreover, although the content of EAA is relatively higher than NEAA, both reduced significantly after hatching with a similar trend during embryogenesis and larval development. It was demonstrated that EAA was not preferentially preserved. Nevertheless, opposite changes occurred in the Atlantic halibut, where the NEAA with the selective utilization of serine and alanine was utilized more than EAA (Zhu et al., 2003). The retained EAA was presumed to be used for protein synthesis, since the amino acid can be utilized both as energy substrates, as well as the precursors for synthesis of lipid, glucose, protein and some other quantitatively unimportant N-rich substances (Zhu et al., 2003). In the present study, glycine was the only amino acid that maintained a constant content during the experimental period. It is likely that this amino acid may play an important role as a structure component during embryogenesis and larval development.This was similar with previous results that observed in brill, where glycine remained steady over the embryonic and yolk-sac larval periods (Cruzado et al., 2013). On the other hand, substantial reduction in content in proline may be due to the rupture of the zona pellucida proteins during embryonic development. This amino acid as an important structure component of zona pellucida proteins has also been noted in other species (Cruzado et al., 2013;Finn et al., 1996).

Fatty acid composition may be important parameters in evaluating egg quality as they are important for fish larval performance (Salze et al., 2005; Xu et al., 2016). The preferential utilization of fatty acids during embryogenesis and early larval development has been observed in some species (Gimenez et al., 2008; Cejas et al., 2004;Desvilettes et al., 1997). In our work, no significant fluctuation of fatty acids was observed throughout the period before hatching, indicating the fatty acids were not important energy substrate. However, significant decrease was found in the content of C18:2n-6, C18:3n-6, and ratio of EPA/ARA, while significant increase was found in the content of C18:3n-3, C20:4n-6, C22:6n-3, and ratio of n-3/n-6 during early larval development. This selective use of fatty acids may be related to the energy utilization and function during early larval development.

Samples from six developmental stages were collected for analysis of moisture, total protein and amino acid contents, total lipids and fatty acids. The samples included the fertilized eggs 2 h, 12 h, 30 h after fertilization,newly hatched larvae (DAH0), 1 day after hatching (1 DAH) and 3 days after hatching (3 DAH, nearly absorbed yolk-sac). Totally 2000 eggs or larvae were collected from each hatch cylinder per time. The samples collected were stored at −80℃ for further analysis. All samples were prepared in triplicate.

Moreover, the PUFA dominated in all samples, and exhibited an increasing trend throughout the period studied in this work, especially significant increase in the level of n-3 PUFA. PUFA are generally involved in the functional integrity and fluidity of cell membranes, and are conserved by the developing larvae at the expense of other less functionally essential fatty acids (Lauritzen et al.,2001). The deficiency of PUFA may lead to malpigmentation, visual impairment, and behavioral abnormalities in larval stages (Penney et al., 2006; Yanes-Roca et al., 2009).Moodie et al. (1989) also concluded that the level of PUFA in eggs had a marked influence on the size and survival of walleye (Stizostedion vitreum) larvae.

In consideration of the decrease of C18:2n-6 and C18:3n-6 contents, it is likely that these two fatty acids are used as energy sources during early development.ARA is an important constituent of phospholipids, and may be involved in determining general egg quality (Gallagher et al., 1998; Salze et al., 2005). The content of ARA increased in the present study, possibly due to the importance of this fatty acid as the major eicosanoid precursor in fish cells. It has been observed that eicosanoids intervene in numerous physiological processes, and probably it is involved in embryonic development of the immune system, hatching and early larval performance(Mustafa and Srivastava, 1989; Sorbera et al., 1998). In addition, the decrease in the ratio of EPA/ARA may indicate that ARA was relatively less utilized than EPA,which agreed with several reports for other species (Cejas et al., 2004; Cruzado et al., 2013). Docosahexaenoic acid(DHA) may be related to the important role of this lipid as structural component in cell membranes, especially in the processes of synaptogenesis and retinogenesis during early larval development of fish (Lauritzen et al., 2001;Sargent et al., 1993; Tocher, 2003). A high content has been related to specific requirements for DHA during the early larval development of neural tissues including brain and retina, as these tissues are highly enriched in DHA(Morais et al., 2004; Silversand et al., 1996; Villalta et al.,2005). The DHA conservation as observed here in larvae of American shad has also been observed in other fishes(Mourente and Vázquez, 1996; Xu et al., 2013; Jin et al.,2014). On the other hand, DHA can be also utilized as energy substrate in the growth of larvae to fulfill energy requirement (Cejas et al., 2004; Rønnestad et al., 1994).

In conclusion, the combined data suggest American shad utilizes protein as preferential energy substrates during embryonic and early larval development, while some specific amino acids were also consumed. Signifi-cant decrease was found in the content of NEAA (cysteine, proline and tyrosine) and EAA (valine, isoleucine,leucine and phenylalanine) during the yolk-sac phase.Substantial reduction was found in the content of proline.Glycine was the only amino acid that maintained a constant content during the experimental period. The content of lipids and fatty acids usually remained stable, while the content of PUFA, especially ARA and DHA, increased,indicating their important role as structure component during embryogenesis and larval development. The results in this study can lead to a better understanding of nutritional requirement of American Shad, and can help to design feed formulations for the fish larvae.

于手术前、手术后1、3、7天早晨采集静脉血并进行离心处理,离心速度为3000r/min,持续10分钟。检测患者的血浆凝血酶时间(TT)、血小板(PLT)水平、凝血酶原时间(PT)、活化部分凝血酶时间(APTT)、D-二聚体(D-D)及血浆纤维蛋白原(FIB)等。

Acknowledgements

This work was supported by the Jiangsu Province Technology Infrastructure Construction Project (No. BM2013 12), the Qingdao postdoctoral application research project(No. Q51201611) and the State Level Commonweal Project of Research Institutes (No. 20603022015005). The authors thank the Jiangsu Zhongyang Group for providing the eggs and larvae for the present study, and thank Yellow Sea Fisheries Research Institute for the excellent technical assistance.

References

Abi-Ayad, S., Kestemont, P., and Mélard, C., 2000. Dynamics of total lipids and fatty acids during embryogenesis and larval development of Eurasian perch (Perca fluviatilis). Fish Physiology and Biochemistry, 23: 233-243.

AOAC (Association Official of Analytical Chemist), 2006. Official Methods of Analysis of AOAC International. 18th edition. AOAC International, Gaithersburg, MD, 66-88.

Cejas, J. R., Almansa, E., Jérez, S., Bolaños, A., Felipe, B., and Lorenzo, A., 2004. Changes in lipid class and fatty acid composition during development in white seabream (Diplodus sargus) eggs and larvae. Comparative Biochemistry and PhysiologyPart B: Biochemistry and Molecular Biology, 139:209-216.

Cruzado, I. H., Rodríguez, E., Herrera, M., Lorenzo, A., and Almansa, E., 2013. Changes in lipid classes, fatty acids, protein and amino acids during egg development and yolk-sac larvae stage in brill (Scophthalmus rhombus L.). Aquaculture Research, 44: 1568-1577.

Davies, M. G., and Thomas, A. J., 1973. An investigation of hydrolytic techniques for the amino acid analysis of foodstuffs. Journal of the Science of Food and Agriculture, 24:1525-1540.

Desvilettes, C., Bourdier, G., and Breton, J. C., 1997. Changes in lipid class and fatty acid composition during development in pike (Esox lucius L) eggs and larvae. Fish Physiology and Biochemistry, 16: 381-393.

Eldridge, M. B., Joseph, J. D., Taberski, K. M., and Seaborn, G.T., 1983. Lipid and fatty acid composition of the endogenous energy sources of striped bass (Morone saxatilis) eggs. Lipids,18: 510-513.

Farhoudi, A., Kenari, A. A., Nazari, R. M., and Makhdoomi, C.H., 2011. Study of body composition, lipid and fatty acid profile during larval development in caspian sea carp (Cyprinuscarpio). Journal of Fisheries and Aquatic Science, 6: 417-428.

Finn, R. N., Fyhn, H. J., and Evjen, M. S., 1991. Respiration and nitrogen metabolism of Atlantic halibut eggs (Hippoglossus hippoglossus). Marine Biology, 108: 11-19.

Finn, R. N., Fyhn, H. J., Henderson, R. J., and Evjen, M. S.,1996. The sequence of catabolic substrate oxidation and enthalpy balance of developing embryos and yolk-sac larvae of turbot (Scophthalmus maximus L.). Comparative Biochemistry and PhysiologyPart A: Physiology, 115: 133-151.

Finn, R. N., Rønnestad, I., and Fyhn, H. J., 1995. Respiration,nitrogen and energy metabolism of developing yolk-sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.). Comparative Biochemistry and PhysiologyPart A: Physiology,111: 647-671.

Fraser, A. J., Gamble, J. C., and Sargent, J. R., 1988. Changes in lipid content, lipid class composition and fatty acid composition of developing eggs and unfed larvae of cod (Gadus morhua). Marine Biology, 99: 307-313.

Fraser, A. J., Sargent, J., Gamble, J., and MacLachlan, P., 1986.Lipid class and fatty acid composition as indicators of the nutritional condition of larval Atlantic herring. American Fisheries Society Symposium Series, 2: 144-150.

Furuita, H., Unuma, T., Nomura, K., Tanaka, H., Okuzawa, K.,Sugita, T., and Yamamoto, T., 2006. Lipid and fatty acid composition of eggs producing larvae with high survival rate in the Japanese eel. Journal of Fish Biology, 69: 1178-1189.Fyhn, H., and Govoni, J., 1995. Endogenous nutrient mobilization during egg and larval development in two marine fishes-Atlantic menhaden and spot. ICES Marine Science Symposia,201: 64-69.

Gallagher, M. L., Paramore, L., Alves, D., and Rulifson, R. A.,1998. Comparison of phospholipid and fatty acid composition of wild and cultured striped bass eggs. Journal of Fish Biology, 52: 1218-1228.

Gao, X. Q., Hong, L., Liu, Z.F., Guo, Z. L., Wang, Y. H., and Lei, J. L., 2015. The study of allometric growth pattern of American shad larvae and juvenile. Acta Hydrobiologica Sinica, 39: 638-644

Gao, X. Q., Hong, L., Liu, Z. F., Guo, Z. L., Wang, Y. H., and Lei, J. L., 2016. An integrative study of larval organogenesis of American shad Alosa sapidissima in histological aspects.Chinese Journal of Oceanology and Limnology, 34: 136-152.

Gimenez, G., Estévez, A., Henderson, R. J., and Bell, J. G.,2008. Changes in lipid content, fatty acid composition and lipid class composition of eggs and developing larvae (0-40 days old) of cultured common dentex (Dentex dentex Linnaeus 1758). Aquaculture Nutrition, 14: 300-308.

Harlioğlu, A. G., and Gölbaşi, S., 2013. Changes in fatty acid composition, cholesterol and fat-soluble vitamins during development of eggs and larvae in shabbout (Barbus grypus,Heckel 1843). Journal of Applied Ichthyology, 29: 1357-1360.

Heming, T. A., and Buddington, R. K., 1988. 6 yolk absorption in embryonic and larval fishes. Fish Physiology, 11: 407-446.

Hong, X. Y., Zhu, X. P., Chen, K. C., Pan, D. B., and Li, K. B.,2011. Study on the development of the embryo and larvae of American shad, Alosa sapidissima. Acta Hydrobiologica Sinica, 35: 153-162.

Jin, Y. H., Xie, Z. G., Lou, B., and Shi, H. L., 2014. The vary of amino acid, free amino acid and fatty acid of Nibea albiflora larvae during ontogeny. Journal of Zhejiang Ocean University (Natural Science), 34: 53-58 (in Chinese with English abstract).

Kestemont, P., Cooremans, J., Abi-Ayad, A., and Mélard, C.,1999. Cathepsin L in eggs and larvae of perch Perca fluviatilis: Variations with developmental stage and spawning period. Fish Physiology and Biochemistry, 21: 59-64.

Lauritzen, L. A., Hansen, H. S., Jørgensen, M. H., and Michaelsen, K. F., 2001. The essentiality of long chain n-3 fatty acids in relation to development and function of the brain and retina. Progress in Lipid Research, 40: 1-94.

Li, M., Mai, K., Ai, Q. H., He, G., Xu, W., Zhang, W. B., Zhang,Y. J., Zhou, H. H., and Liufu, Z. G., 2015. Effect of dietary lipid on the growth, fatty acid composition and Δ5 Fads expression of abalone (Haliotis discus hannai Ino) hepatopancreas. Journal of Ocean University of China, 14: 317-324.

Liu, W., Li, Q., and Kong, L., 2013. Reproductive cycle and seasonal variations in lipid content and fatty acid composition in gonad of the cockle Fulvia mutica in relation to temperature and food. Journal of Ocean University of China, 12: 427-433.

Mi, G. Q., Lian, Q. P., Wang, Y. P., Hu, T. J., and Li, Q., 2014.A study on embryonic development of Alosa sapidissima.Journal of Jiangxi Agricultural University, 36:1343-1348.

Moodie, G., Loadman, N. L., Wiegand, M. D., and Mathias, J.A., 1989. Influence of egg characteristics on survival, growth and feeding in larval walleye (Stizostedion vitreum). Canadian Journal of Fisheries and Aquatic Sciences, 46: 516-521.

Morais, S., Narciso, L., Dores, E., and Pousao-Ferreira, P., 2004.Lipid enrichment for Senegalese sole (Solea senegalensis)larvae: Effect on larval growth, survival and fatty acid profile.Aquaculture International, 12: 281-298.

Mourente, G., and Vázquez, R., 1996. Changes in the content of total lipid, lipid classes and their fatty acids of developing eggs and unfed larvae of the Senegal sole, Solea senegalensis Kaup. Fish Physiology and Biochemistry, 15: 221-235.

Mustafa, T., and Srivastava, K. C., 1989. Prostaglandins (eicosanoids) and their role in ectothermic organisms. Advances in Comparative and Environmental Physiology, 5: 157-207.

Ohkubo, N., and Matsubara, T., 2002. Sequential utilization of free amino acids, yolk proteins and lipids in developing eggs and yolk-sac larvae of barfin flounder Verasper moseri. Marine Biology, 140: 187-196.

Ostrowski, A. C., and Divakaran, S., 1991. Energy substrates for eggs and prefeeding larvae of the dolphin Coryphaena hippurus. Marine Biology, 109: 149-155.

Penney, R. W., Lush, P. L., Wade, J., Brown, J. A., Parrish, C.C., and Burton, M. P., 2006. Comparative utility of egg blastomere morphology and lipid biochemistry for prediction of hatching success in Atlantic cod, Gadus morhua L. Aquaculture Research, 37: 272-283.

Pfeifer, R., Karol, R., Korpi, J., Burgoyne, R., and McCourt, D.,1983. Practical application of HPLC to amino acid analysis.American Laboratory, 15: 78-84.

Rainuzzo, J. R., Reitan, K. I., and Olsen, Y., 1997. The significance of lipids at early stages of marine fish: A review. Aquaculture, 155: 103-115.

Rønnestad, I., Koven, W., Tandler, A., Harel, M., and Fyhn, H.J., 1998. Utilization of yolk fuels in developing eggs and larvae of European sea bass (Dicentrarchus labrax). Aquaculture, 162: 157-170.

Rønnestad, I., Koven, W. M., Tandler, A., Harel, M., and Fyhn,H. J., 1994. Energy metabolism during development of eggs and larvae of gilthead sea bream (Sparus aurata). Marine Biology, 120: 187-196.

Rønnestad, I., Thorsen, A., and Finn, R. N., 1999. Fish larval nutrition: A review of recent advances in the roles of amino acids. Aquaculture, 177: 201-216.

Salze, G., Tocher, D. R., Roy, W. J., and Robertson, D. A., 2005.Egg quality determinants in cod (Gadus morhua L.): Egg performance and lipids in eggs from farmed and wild broodstock. Aquaculture Research, 36: 1488-1499.

Sargent, J. R., Bell, M. V., and Tocher, D. R., 1993. Docosahexaenoic acid and the development of brain and retina in marine fish. In: Omega-3 Fatty Acids: Metabolism and Biological Effects. Birkhauser Verlag, Basel, 139-149.

Sargent, J. R., 1995. Origins and functions of egg lipids: Nutritional implications. In: Broodstock Management and Egg and Larval Quality. Bromage, N. R., and Roberts, R. J., eds.,Blackwell Science Ltd., Oxford, 353-372.

Silversand, C., Norberg, B., and Haux, C., 1996. Fatty-acid composition of ovulated eggs from wild and cultured turbot(Scophthalmus maximus ) in relation to yolk and oil globule lipids. Marine Biology, 125: 269-278.

Sorbera, L. A., Zanuy, S., and Carrillo, M., 1998. A role for polyunsaturated fatty acids and prostaglandins in oocyte maturation in the sea bass (Dicentrarchus labrax). Annals of the New York Academy of Sciences-Paper Edition, 839: 535-537.

Tocher, D. R., 2003. Metabolism and functions of lipids and fatty acids in teleost fish. Reviews in Fisheries Science, 11:107-184.

Villalta, M., Estévez, A., Bransden, M. P., and Bell, J. G., 2005.The effect of graded concentrations of dietary DHA on growth, survival and tissue fatty acid profile of Senegal sole(Solea senegalensis) larvae during the Artemia feeding period.Aquaculture, 249: 353-365.

Walburg, C. H., and Nichols, P. R., 1967. Biology and Management of the American Shad and Status of the Fisheries,Atlantic Coast of the United States, 1960. U.S. Department of the Interior, Bureau of Commercial Fisheries, Washington,105pp.

Xu, G. C., Zhang, C. X., Zheng, J. L., and Gu, R. B., 2012. Artificial propagation and embryonic development of American shad, Alosa sapidissima. Marine Science, 36: 89-96.

Xu, H. G., Dong, X. J., Zuo, R. T., Mai, K. S., and Ai, Q. H.,2016. Response of juvenile Japanese seabass (Lateolabrax japonicus) to different dietary fatty acid profiles: Growth performance, tissue lipid accumulation, liver histology and flesh texture. Aquaculture, 461: 40-47.

Xu, S. L., Wang, Y. J., Wang, D. L., and Yan, X. J., 2013. The study of fatty acid components in early developmental stage of Oplegnathus fasciatus. Oceanologia et Limnologia Sinica,44: 438-444.

Yanes-Roca, C., Rhody, N., Nystrom, M., and Main, K. L.,2009. Effects of fatty acid composition and spawning season patterns on egg quality and larval survival in common snook(Centropomus undecimalis). Aquaculture, 287: 335-340.

Zhang, C. X., Xu, G. C., Xu, P., Zheng, J. L., and Gu, R. B.,2010. Morphological development and growth of American shad (Alosa sapidissima) at larvae, fry and juvenile stages.Journal of Fishery Sciences of China, 17: 1227-1235.

Zhu, P., Parrish, C. C., and Brown, J. A., 2003. Lipid and amino acid metabolism during early development of Atlantic halibut(Hippoglossus hippoglossus). Aquaculture International, 11:43-52.