Effects of feeding frequency and density on growth, energy budget and physiological performance of sea cucumber Apostichopus japonicus (Selenka)

Aquaculture 466 (2017) 26–32

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Effects of feeding frequency and density on growth, energy budget and physiological performance of sea cucumber Apostichopus japonicus (Selenka) Bin Xia a,b, Yichao Ren b, Jiying Wang a,⁎, Yongzhi Sun a, Zhendong Zhang c a b c

Key Laboratory of Marine Ecological Restoration, Shandong Marine Resource and Environment Research Institute, Yantai, Shandong 264006, China Marine Science and Engineering College, Qingdao Agricultural University, Qingdao, Shandong 266109, China National Fishery Technique Extension Center, Beijing 100125, China

a r t i c l e

i n f o

Article history: Received 14 June 2016 Received in revised form 14 September 2016 Accepted 23 September 2016 Available online 26 September 2016 Keywords: Apostichopus japonicus Feeding strategy Growth performance Endocrine response Energy budget

a b s t r a c t Feeding frequency and density are two of the most important factors that directly affect the growth and physiology of sea cucumber Apostichopus japonicus. In the present study, a 60-day experiment was conducted to evaluate the effects of feeding frequency (1 time, 2 times and 3 times per day) and stocking density (D10, D20, D40 and D60) on growth performance, feed utilization, endocrine response and energy budget of sea cucumber. The results revealed that there was no significant interaction between feeding frequency and stocking density on final weight and specific growth rate (SGR). The maximum SGR occurred at treatment of stocking density D10 with feeding thrice per day. Stepwise multiple regression analysis showed that SGRW and SGRE increased with increasing feeding frequency, and decreased with the increase of stocking density. Density had significant influence on coefficient of variation (CV) for the sea cucumber body weight. For D10 and D20, no significant difference in CV was found between different feeding frequencies. Feed intake, energy intake and feces production rate were significantly affected by feeding frequency, stocking density and their interaction. Density also had significant influence on food conversion efficiency and protein efficiency ratio. Apparent digestibility coefficients of crude protein and crude lipid exhibited remarkable descending trends as stocking density increased. The energy for growth decreased and energy required for metabolism increased with the increase of stocking density, suggesting that crowding stress modified their energy allocation by inhibiting the accumulation of growth energy and accelerating energy consumption of sea cucumber. Cortisol level in coelomic fluid of sea cucumber significantly increased with the increase of stocking density, which could generate energy to satisfy the increasing demand of stress-related energy. Lactate level significantly elevated, and glucose level in coelomic fluid and glycogen in muscle showed remarkable descending trends from D10 to D60, implying long-term energy consumption under high stocking density. Feeding frequency had significant influence on glucose, glycogen and cortisol levels. To some extent, increasing feeding frequency could effectively reduce coefficient of variation of sea cucumber, especially under high stocking density. Statement of relevance: • In the present study, a 60-day experiment was conducted to evaluate the effects of feeding frequency (1 time, 2 times and 3 times per day) and stocking density (D10, D20, D40 and D60) on growth performance, feed utilization, endocrine response and energy budget of sea cucumber. • Increasing feeding frequency could effectively reduce coefficient of variation of sea cucumber. • The present study provided valuable information for the intensive culture of A. japonicus.

© 2016 Elsevier B.V. All rights reserved.

1. Introduction

⁎ Corresponding author. E-mail address: [email protected] (J. Wang).

http://dx.doi.org/10.1016/j.aquaculture.2016.09.039 0044-8486/© 2016 Elsevier B.V. All rights reserved.

Sea cucumber Apostichopus japonicus (Selenka) (Echinodermata: Holothuroidea) has been used as a traditional remedy for wound healing, and extensively believed to be aphrodisiac and curative effects

B. Xia et al. / Aquaculture 466 (2017) 26–32

(Wu et al., 2015a). The natural distribution of A. japonicus covers the subtidal zone from 35°N to 44°N along the coast of Russia, China, Japan and Korea (Sloan, 1984; Yuan et al., 2009). With the overfishing of natural resources and increasing market demand, the farming scale of sea cucumber has been rapidly expanded in the last decades (Sun et al., 2012b). The total production of A. japonicus has reached 201,000 t in 2014 with an increase of 96.7% compared to that in 2009 (MOAC, 2010 and 2015). As an obligate deposit-feeding species (Liao, 1980), sea cucumber might take up organic matter in sediment as food sources, e.g. bacteria, prozotoa, benthic microalgae, detritus of macroalgae and sea grass (MacTavish et al., 2012; Slater and Jeffs, 2010). Previous studies regarding various aspects of A. japonicus, include its genetics (Du et al., 2012; Li et al., 2009), energetic (Bai et al., 2015; Yuan et al., 2009), nutrition (Xia et al., 2015a; Xia et al., 2013) and larval development (Li et al., 2010; Sun et al., 2012a), etc. However, few studies have been conducted on feeding strategy of sea cucumber (An et al. 2007), and it is crucial to set feeding ration and frequency with respect to feeding habit under laboratory condition and in aquaculture practice. An organism's morphology, behaviour, feeding and physiological characteristics tend to be adapted for maximum net energetic benefit, which is interpreted as the optimal foraging theory (Werner and Mittelbach, 1981; Wu et al., 2015b). Previous studies showed that increasing feeding frequency could improve growth, feed utilization and physiological performance of aquatic animals, e.g. fish (Dwyer et al., 2002; Wang et al., 2007) and invertebrates (Cárcamo, 2015; Cárvalho et al., 2006). Density is another important factor affecting the growth and endocrine response of aquatic animals (Façanha et al., 2016; Pei et al., 2012). High density can stimulate food competition, social hierarchy, depensatory growth, size heterogeneity and cannibalism (Ribeiro et al., 2015; Raimondo et al., 2013). In the present study, we hypothesised that increasing feeding frequency could effectively improve growth performance, reduced variation in individual growth and optimize energy allocation of sea cucumber, especially under high stocking density. The objective of this study was to investigate the effects of feeding frequency and stocking density on growth, energy budget and physiological performance of sea cucumber, providing valuable information for the intensive culture of A. japonicus. 2. Materials and methods 2.1. Experimental design Juvenile sea cucumbers (age 2) of similar wet weight of 22.14 ± 2.01 g were collected from a local farm in Dongying City, Shandong Province, and transported to the laboratory immediately. All animals were acclimated in a large tank for 3 weeks at 20 ± 0.5 °C, salinity was 30–32 PSU, dissolved oxygen was above 6.5 ml l−1 and a 14 h light: 10 h dark photoperiod, which were same with the experimental conditions. In this study, 05:00–19:00 was defined as “daytime” and 19:00–05:00 was defined as “nighttime”. During the period of the acclimation, the stocking density was approximately 500 g m−2, and the sea cucumbers were fed once (at 16:00) per day. After acclimation, the sea cucumbers were randomly divided into 3 groups, which were fed once (at 16:00), twice (at 08:00 and 16:00) and thrice (at 08:00, 16:00 and 24:00) per day, respectively. The feeding time was set by daily activity rhythm (Dong et al., 2011) and feeding habit (Sun et al., 2015) of sea cucumber. Each group contained four treatments of stocking densities, i.e., 10, 20, 40 and 60 individuals per cylinder aquarium (~400 l capacity, 100 cm height × 70 cm diameter), represented as D10, D20, D40 and D60, respectively. The corresponding densities were also expressed as 553.5, 1120.2, 2232.6 and 3335.7 g m−2. Each treatment had three replicates. The experiment lasted for 60 days. During the acclimation and experimental period, the sea cucumbers were fed with dry pellets (22.90% protein, 2.31% lipid, 38.21% ash and 12.06 kJ g−1 energy) and up to 5% of their total biomass per

27

day. According to our previous studies (Liu et al., 2009; Xia et al., 2015b), this ration size could ensure the satiation of this species and minimize the feed waste. Each meal amount was equal for sea cucumber those were fed more than once each day. Feeding ration was adjusted once every ten days based on its growth performance. Uneaten feed residues and feces were collected by siphoning before next feeding, and dried at 60 °C to a constant weight for further analysis (Yuan et al., 2006). The weight of uneaten feed was adjusted by the leaching ration of diets in water (Shi et al., 2015). All diets contained 500 mg kg−1 yttrium oxide as an inert marker for determining digestibility. 2.2. Sample collection and determination At the end of the experiment, the sea cucumbers were starved for 24 h prior to sampling. Ten sea cucumbers were collected randomly from each aquarium, weighted and counted for calculation of growth performance. After weighting, the sea cucumbers were dried at 60 °C individually for at least 72 h to a constant weight to determine feed utilization. The dried samples were ground to fine and homogeneous powder, which was tightly sealed in a glass Petri dish and stored at − 80 °C for further analysis. Proximate composition of the diets was analyzed according to the standard methods of AOAC (1995). Crude protein (N × 6.25) was determined by the Kjeldahl method after acid digestion. Crude lipid was determined by the ether-extraction method. Moisture was determined by oven drying at 105 °C for 24 h. Ash was determined by using a muffle furnace at 550 °C for 6 h. Apparent digestibility coefficients (ADC) were measured following the method by Xia et al. (2015a). Yttrium concentrations in the diets and feces were determined with an inductively coupled plasma atomic emission spectrophotometer (ICP-OES, VIATAMPX) after perchloric acid digestion. The energy content of the diets, feces and animal samples were measured by a bomb calorimeter (Parr 6100, Parr Instrument Company). Coelomic fluid was sampled by puncturing the abdomen with a 1 ml disposable syringe and then separated the supernatant by centrifugation (200 rpm) for 10 min at 4 °C and stored at −80 °C for further analysis. Glucose in coelomic fluid was determined using Glucose Diagnostic Kits (Rsbio, China). Cortisol level in coelomic fluid was determined using Lodine [125I]-Cor RIA Kits (Jiuding Diagnostic, China) by radioimmunoassay and lactate was determined enzymatically using Sigma Diagnostic Kits (Sigma, USA). Glycogen in muscle was determined according to Chen (2013) with assay kits (Nanjing Jiancheng Biotech Company, China). 2.3. Data calculation Weight gain, specific growth rate in terms of weight (SGRW) and energy (SGRE), coefficient of variation for body weight (CV), feed intake (FI), feces production rate (FPR), food conversion efficiency (FCE) and protein efficiency ratio (PER) were calculated as follow: Weight gain ð%Þ ¼ ðW f −W i Þ=W f  100   −1 ¼ ln ðW f =W i Þ=t  100 SGRW % d   −1 ¼ ln ðE f =Ei Þ=t  100 SGRE % d CV ð%Þ ¼ SD=W m  100   −1 ¼ I=½ðW f þ W i Þ=2  t  FI g g−1 d   −1 FPR g g−1 d ¼ F=½ðW f þ W i Þ=2  t  FCE ð%Þ ¼ ðW f −W i Þ=I  100

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B. Xia et al. / Aquaculture 466 (2017) 26–32

Table 1 Survival and growth performance of A. japonicus at different treatments.1 Feeding frequency

Density

Survival (%)

Final weight (g)

1 time

D10 D20 D40 D60 D10 D20 D40 D60 D10 D20 D40 D60

0.83 0.88 0.90 0.90 0.87 0.90 0.89 0.91 0.87 0.88 0.90 0.92

46.44 40.67 35.13 28.12 47.88 48.19 36.31 29.07 50.06 48.36 39.39 29.54

2 times

3 times

± ± ± ± ± ± ± ± ± ± ± ±

0.06 0.03 0.03 0.04 0.12 0.05 0.04 0.06 0.06 0.03 0.03 0.04

Weight gain (%)

± 8.06a ± 8.35ab ± 10.56ab ± 7.55b ± 10.12a ± 9.87a ± 7.27ab ± 6.78b ± 9.88a ± 8.33a ± 7.19ab ± 6.91b

51.23 43.20 37.61 26.17 54.20 51.34 35.68 26.16 55.92 53.32 39.05 26.20

± 12.12a ± 10.08ab ± 19.22ab ± 9.29b ± 11.09 ± 13.66 ± 17.21 ± 9.20 ± 16.83a ± 15.47a ± 10.27ab ± 8.79b

Two-way ANOVA2 Feeding frequency Density Interaction

ns ns ns

ns *** ns

ns *** ns

1 Data are mean ± SD. Different superscripted lowercase letters within the same column mean significant differences between stocking densities at the same feeding frequency (p b 0.05). 2 ⁎ p b 0.05, ⁎⁎ p b 0.01, ⁎⁎⁎ p b 0.001, ns: non-significant.

ADC of nutrients ð%Þ ¼ ½1−ðdietary Y2 O3 =faecalY2 O3 Þ  ðfaecalnutrients=dietary nutrientsÞ  100

2.0

D10 D20 D40 D60

1.6 a

a -1

where Wi and Wf are the initial and final dry weights of the sea cucumbers (g), t is the duration of the experiment (d), Ei and Ef are the initial and final energy contents of the sea cucumbers (kJ g−1 d−1), SD is the standard deviation in dry weight of the sea cucumbers, Wm is the mean weight (g), I is the dry weight of food consumed, F is the dry weight of feces, P is the protein intake. Apparent digestibility coefficients (ADC) for dry matter (ADMD), crude protein (ACPD) and crude lipid (ACLD) were calculated by the following equation:

to determine significant differences among means. Prior to analysis, raw data were diagnosed for normality of distribution and homogeneity

SGRW (% d )

PER ¼ ðW f −W i Þ=P

a Ba Ba

1.2 Ab

b

b b

0.8

c c c

Energy budget was constructed according to the equation as follows:

0.4

C ¼Gþ F þUþR 0

where C is energy consumed, G is energy for growth, F represents energy of feces produced, U is energy lost in ammonia excretion and R stands for energy lost in respiration. The estimation of U was based on the nitrogen budget equation:

1 time

2.0

D10 D20 D40 D60

Ba

1.6

ABa Aa

SGRE (% d-1)

R ¼ C−G−F−U

3 times

Feeding frequency

U ¼ ðC N −GN −F N Þ  24; 830 (Liu et al., 2009), where CN is the nitrogen consumed from the diet, GN is the nitrogen deposited in animal body, FN stands for the nitrogen lost in feces and 24,830 is the energy content in excreted ammonia (J g−1). The value of R was calculated by the energy budget equation:

2 times

1.2

Bb

Bb

b

b

b Ab

0.8

c

c

2.4. Statistical analysis

c

0.4

All statistical analysis were performed with the software SPSS for windows release 16.0 (SPSS Inc., 2008). Two-way analysis of variance (ANOVA) was used to test for the effect of the interaction between feeding frequency and density on growth, feed utilization, digestibility, endocrine response and energy budget of sea cucumber. The co-effects of feeding frequency and density on SGR were tested using stepwise multiple regression analysis. The probability level of 0.05 was used for rejection of the null hypothesis. Tukey multiple range tests were used

0 1 time

2 times

3 times

Feeding frequency Fig. 1. Specific growth rate (SGR) of A. japonicus in terms of weight (SGRW) and energy (SGRE) at different treatments.

B. Xia et al. / Aquaculture 466 (2017) 26–32

D10 D20 D40 D60

40 Bb Bab

CV (%)

30

Ba

Cb

Ba Ba Ca

Bab

20

Ca

Ba A

A

A

10

A

0 initial

1 time

increase of stocking density. The relationship of SGR, feeding frequency (FQ) and density (D) can be expressed as the following regression equation:
SGRW ¼ 1:306 þ 0:099FQ−0:017D r2 ¼ 0:965

Cb

Ba

29

2 times

3 times

Feeding frequency Fig. 2. Coefficient of variation (CV) in weight of A. japonicus at different treatments. Data are mean ± SD. Different superscripted capital letters mean significant differences between feeding frequencies at the same stocking density (p b 0.05), different lowercase letters mean significant differences between stocking densities at the same feeding frequency (p b 0.05).

of variance with Kolmogorov-Smirnov test and Levene's test, respectively (Zar, 1999).


SGRE ¼ 1:244 þ 0:093FQ−0:015D r2 ¼ 0:859 Coefficients of variation (CVs) for the body weight of sea cucumber at the end of experiment were much higher than initial CVs (p b 0.05) (Fig. 2). There was significant interaction between feeding frequency and stocking density on CV of A. japonicus (p b 0.05). Density had significant influence on CVs (p b 0.05). For D40 and D60, the CVs at treatments of feeding twice and thrice were significantly higher than that of sea cucumber feeding once per day (p b 0.05), while no significant difference was found between feeding frequencies in D10 and D20 (p N 0.05). Feed intake (FI) and feces production rate (FPR) were significantly affected by feeding frequency, stocking density and their interaction (p b 0.05) (Table 2). FI and FPR both decreased with increasing density at the same feeding frequency (p b 0.05), and increased with increasing feeding frequency at the same density except D10 (p b 0.05). Density had significant influence on food conversion efficiency (FCE) and protein efficiency ratio (PER) (p b 0.05), however, no significant interaction between feeding frequency and stocking density was found on FCE and PER (p N 0.05).

3. Results 3.2. Apparent digestibility coefficients 3.1. Growth performance and feed utilization During the period of 60 d experiment, no significant difference in survival of sea cucumber was observed between feeding frequencies and between stocking densities (p N 0.05) (Table 1). Individuals had increased their average wet weight by between 22.20% and 55.92%. The results of two-way ANOVA analysis showed no significant interaction between feeding frequency and stocking density on final weight and weight gain (p N 0.05). However, final weight and weight gain of A. japonicus were both significantly affected by density (p b 0.05), which decreased with the increase of stocking density. The maximum SGR (SGRW and SGRE) occurred at treatment (3 times, D10) (Fig. 1). There was no significant interaction between feeding frequency and stocking density on SGR of sea cucumber (p N 0.05). Stepwise multiple regression analysis showed that SGRW and SGRE increased with increasing feeding frequency, and decreased with the

Apparent digestibility coefficients (ADCs) for dry matter (ADMD), crude protein (ACPD), crude lipid (ACLD) of A. japonicus at different treatments were present in Table 3. There was no significant interaction between feeding frequency and stocking density on ADMD, ACPD and ACLD (p N 0.05). ACPD was significantly affected by feeding frequency and density (p b 0.05), and decreased with the increase of stocking density. ACLD at 2 times treatment was significantly affected by density (p b 0.05), and showed a remarkable descending trend from D10 to D60. 3.3. Glucose, lactate, cortisol and glycogen levels Glucose, lactate and cortisol levels in coelomic fluid and glycogen level in muscle of A. japonicus at different treatments were present in Table 4. The results showed significant interaction between feeding frequency and stocking density on glucose and glycogen (p b 0.05).

Table 2 Feed utilization of A. japonicus at different treatments.1 Feeding frequency

Density

FI (g g−1 d−1)

1 time

D10 D20 D40 D60 D10 D20 D40 D60 D10 D20 D40 D60

0.35 0.29 0.25 0.21 0.34 0.28 0.28 0.24 0.34 0.33 0.30 0.28

2 times

3 times

± ± ± ± ± ± ± ± ± ± ± ±

0.02a 0.02Ab 0.03Ac 0.01Ad 0.03a 0.02Ab 0.02ABb 0.01Bc 0.01a 0.01Ba 0.01Bb 0.02Cc

FPR (g g−1 d−1) 0.25 0.21 0.19 0.17 0.25 0.20 0.20 0.19 0.24 0.23 0.24 0.21

± ± ± ± ± ± ± ± ± ± ± ±

0.01a 0.02ABb 0.02Ac 0.01Ac 0.02a 0.01Ab 0.01Ab 0.01Bb 0.01a 0.01Ba 0.01Ba 0.01Cb

FCE (%) 3.68 3.44 2.70 3.03 3.85 3.68 3.44 2.95 3.93 3.76 2.46 2.78

± ± ± ± ± ± ± ± ± ± ± ±

PER 0.43a 0.25ab 0.25c 0.38abc 0.62 0.43 0.25 0.49 0.49a 0.38a 0.43b 0.38b

0.15 0.14 0.11 0.12 0.16 0.15 0.14 0.12 0.16 0.15 0.10 0.11

± ± ± ± ± ± ± ± ± ± ± ±

0.02a 0.01ab 0.01Ac 0.02bc 0.03 0.02 0.01B 0.02 0.02a 0.02a 0.02Ab 0.02b

Two-way ANOVA2 Feeding frequency Density Interaction

*** *** **

*** *** *

ns *** ns

ns *** ns

1 Data are mean ± SD. Different superscripted capital letters within the same column mean significant differences between feeding frequencies at the same stocking density (p b 0.05), different lowercase letters mean significant differences between stocking densities at the same feeding frequency (p b 0.05). 2 ⁎ p b 0.05, ⁎⁎ p b 0.01, ⁎⁎⁎ p b 0.001, ns: non-significant.

30

B. Xia et al. / Aquaculture 466 (2017) 26–32

Table 3 Apparent digestibility coefficients (ADCs) for dry matter (ADMD), crude protein (ACPD) and crude lipid (ACLD) of A. japonicus at different treatments.1 Feeding frequency

Density

ADMD (%)

ACPD (%)

1 time

D10 D20 D40 D60 D10 D20 D40 D60 D10 D20 D40 D60

57.88 58.42 56.91 57.02 58.28 60.01 57.43 57.14 59.88 56.90 57.83 60.46

68.28 67.36 64.23 63.40 66.44 64.73 64.78 63.27 64.72 64.59 63.32 61.40

2 times

3 times

± 2.31 ± 1.70 ± 1.33 ± 1.80 ± 3.05 ± 1.22 ± 1.69 ± 1.29 ± 2.47 ± 1.21 ± 2.22 ± 3.28

ACLD (%)

± 1.89a ± 1.39Aa ± 2.00b ± 1.20b ± 2.46 ± 0.65B ± 2.56 ± 2.24 ± 1.35a ± 1.37Ba ± 0.60ab ± 0.99b

88.51 88.99 86.73 88.70 90.57 88.39 86.91 86.38 88.81 89.62 86.33 88.59

± 2.59 ± 1.96 ± 1.37 ± 2.07 ± 1.40a ± 1.23ab ± 1.94b ± 1.47b ± 3.15 ± 2.06 ± 1.32 ± 1.10

Two-way ANOVA2 Feeding frequency Density Interaction

ns ns ns

* *** ns

ns * ns

1 Data are mean ± SD. Different superscripted capital letters within the same column mean significant differences between feeding frequencies at the same stocking density (p b 0.05), different lowercase letters mean significant differences between stocking densities at the same feeding frequency (p b 0.05). 2 ⁎ p b 0.05, ⁎⁎ p b 0.01, ⁎⁎⁎ p b 0.001, ns: non-significant.

Significant differences in glucose, lactate, cortisol and glycogen levels were observed between densities (p b 0.05). Lactate and cortisol levels increased with the increase of stocking density at the same feeding frequency, while glucose and glycogen decreased with increasing density. Feeding frequency had significant influence on glucose, cortisol and glycogen levels except lactate level (p b 0.05). The lowest values of glucose and glycogen levels and highest value of cortisol level occurred at treatment of feeding 1 time and D60. 3.4. Energy budget Energy parameters and budget of A. japonicus in different treatments were present in Table 5. There was significant interaction between feeding frequency and stocking density on energy intake (C) (p b 0.05). C decreased with the increase of stocking density at the same frequency (p b 0.05), and increased with increasing feeding frequency at the same density except D10 (p b 0.05). The results showed no significant interaction between feeding frequency and stocking density on energy for growth (G), energy lost in feces (F), excretion (U) and respiration (R) (p N 0.05). However, G, F, U and R were significantly affected by density (p b 0.05). U and R showed obvious ascending trends with the increase of stocking density, while G decreased with increasing density

at the same feeding frequency. Only in D20, significant differences in G were observed between feeding frequencies (p b 0.05). 4. Discussion In the present study, two-way ANOVA analysis showed no significant interaction between feeding frequency and stocking density on final weight, weight gain and SGR of A. japonicus. However, density had significant influence on final weight and SGR. Previous studies showed that stocking density might affect the growth performance of aquatic animals from three aspects, water quality, food competition and crowding stress (Kebus et al., 1992; Li et al., 2006). Stocking density has been shown to affect the physiological function of individual organism (Ellis et al., 2002), and crowding stress significantly reduced the specific growth rate (Pei et al., 2012). Our results of stepwise multiple regression analysis also demonstrated that SGRW and SGRE of the sea cucumbers decreased with the increase of stocking density. Similar trends were observed in feed intake and energy intake, despite sufficient food supplied to sea cucumber. Furthermore, it appeared that food conversion efficiency and protein efficiency ratio decreased with increasing density. Although apparent digestibility coefficient of dry matter was not significantly affected by density, apparent digestibility

Table 4 Glucose, lactate, cortisol and glycogen levels of A. japonicus at different treatments.1 Feeding frequency

Density

Glucose (mmol l−1)

1 time

D10 D20 D40 D60 D10 D20 D40 D60 D10 D20 D40 D60

0.68 0.56 0.44 0.35 0.64 0.56 0.50 0.37 0.74 0.50 0.53 0.57

2 times

3 times

± ± ± ± ± ± ± ± ± ± ± ±

0.05ABa 0.05ab 0.04Abc 0.10Ac 0.04Aa 0.06ab 0.02ABb 0.06Ac 0.04Ba 0.06b 0.04Bb 0.03Bb

Lactate (mmol l−1) 0.11 0.14 0.17 0.17 0.13 0.13 0.18 0.18 0.12 0.13 0.15 0.17

± ± ± ± ± ± ± ± ± ± ± ±

0.02a 0.02a 0.01b 0.02b 0.01a 0.02a 0.02b 0.01b 0.02a 0.01ab 0.01bc 0.02c

Cortisol (mmol l−1) 4.38 4.70 5.61 6.09 4.23 4.25 5.09 5.97 4.28 4.29 5.11 5.44

± ± ± ± ± ± ± ± ± ± ± ±

0.18a 0.24Aa 0.24Ab 0.31Ac 0.13a 0.14Ba 0.21Bb 0.23Ac 0.20a 0.14Ba 0.14Bb 0.23Bb

Glycogen (mg g−1) 20.57 19.31 15.38 13.50 20.64 20.44 17.47 14.11 21.19 21.25 18.57 17.28

± 0.85a ± 0.54Aa ± 1.09Ab ± 0.28Ac ± 0.62a ± 0.38Ba ± 0.60Bb ± 0.32Ac ± 1.52a ± 0.25Ba ± 0.64Bb ± 0.27Bb

Two-way ANOVA2 Feeding frequency Density Interaction

** *** **

ns *** ns

*** *** ns

*** *** **

1 Data are mean ± SD. Different superscripted capital letters within the same column mean significant differences between feeding frequencies at the same stocking density (p b 0.05), different lowercase letters mean significant differences between stocking densities at the same feeding frequency (p b 0.05). 2 ⁎ p b 0.05, ⁎⁎ p b 0.01, ⁎⁎⁎ p b 0.001, ns: non-significant.

B. Xia et al. / Aquaculture 466 (2017) 26–32

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Table 5 Energy budgets of A. japonicus at different treatments.1 Feeding frequency

Density

C (kJ g−1 d−1)

1 time

D10 D20 D40 D60 D10 D20 D40 D60 D10 D20 D40 D60

5.41 4.49 4.04 3.28 5.25 4.28 4.34 3.72 5.15 5.05 4.84 4.32

2 times

3 times

± ± ± ± ± ± ± ± ± ± ± ±

0.23a 0.32Ab 0.42Ab 0.20Ac 0.39a 0.31Ab 0.23ABb 0.18Bc 0.18a 0.15Ba 0.23Ba 0.10Cb

G (% C−1) 6.25 4.42 2.92 1.90 6.38 5.07 2.84 1.51 6.31 5.34 2.43 1.63

± ± ± ± ± ± ± ± ± ± ± ±

0.51a 0.44Ab 0.69c 0.52c 0.32a 0.42ABb 0.52c 0.50d 0.46a 0.30Bb 0.23c 0.37d

F (% C−1)

U (% C−1)

66.16 67.04 67.10 65.84 68.58 69.07 68.00 62.64 66.94 68.32 67.86 64.65

2.39 2.79 3.85 3.73 2.63 3.13 3.96 4.21 2.82 2.92 3.42 4.19

± 2.15 ± 3.46 ± 1.77 ± 3.08 ± 3.99a ± 1.27a ± 1.60a ± 1.58b ± 2.40 ± 3.12 ± 4.41 ± 2.19

± ± ± ± ± ± ± ± ± ± ± ±

0.18a 0.21a 0.34b 0.31b 0.22a 0.36b 0.17c 0.28c 0.13a 0.10ab 0.53b 0.19c

R (% C−1) 25.21 25.75 26.13 28.53 22.41 22.73 25.20 31.65 23.93 23.42 26.29 29.53

± 2.53 ± 3.95 ± 1.24 ± 2.81 ± 4.16a ± 0.60a ± 2.17a ± 1.53b ± 1.97 ± 3.41 ± 5.00 ± 1.86

Two-way ANOVA2 Feeding frequency Density Interaction

*** *** **

ns *** ns

ns * ns

ns *** ns

ns *** ns

1 Data are mean ± SD. Different superscripted capital letters within the same column mean significant differences between feeding frequencies at the same stocking density (p b 0.05), different lowercase letters mean significant differences between stocking densities at the same feeding frequency (p b 0.05). 2 ⁎ p b 0.05, ⁎⁎ p b 0.01, ⁎⁎⁎ p b 0.001, ns: non-significant.

coefficients of crude protein and crude lipid exhibited remarkable descending trends as stocking density increased. Nga et al. (2005) found that crowding effects through physical and/or chemical interference was an important factor in reducing growth of Penaeus monodon postlarvae. It is generally believed that increasing feeding frequency has immediate benefits, including reduce nutrient leaching, improve feed utilization and increase growth of aquatic animals (Luo et al., 2015; Velasco et al., 1999; Wu et al., 2015b). Fang et al. (2014) reported that SGR, feed intake and food conversion efficiency of sea cucumber at treatments of feeding frequency (3 times and 4 times per day) were significantly higher than those of other treatments (1 time and 2 times per day), which however was conducted in a very low density of only 3 individuals each aquarium. In our study, feeding frequency had no influence on SGR, food conversion efficiency, protein efficiency ratio and apparent digestibility coefficients for dry matter at different stocking densities, while feed intake, energy intake and feces production rate all significantly increased with increasing feeding frequency in densities of D20, D40 and D60, suggesting that the effect of feeding frequency on improving growth performance and feed utilization of sea cucumber had been reduced under high stocking density. Energy budget provide a framework for the evaluation of various ways in which nutrients are utilized (Lawrence and Lane, 1982). In the present study, the average formulas of energy allocation for sea cucumber were: 100C = 3.9G + 66.8F + 3.3 U + 26.0R. The energy lost in feces and respiration was the main proportion of energy intake, as reported by other studies (Pei et al., 2012; Shi et al., 2013; Yuan et al., 2006). The energy for growth decreased and energy required for metabolism increased with the increase of stocking density, suggesting that crowding stress modified their energy allocation by inhibiting the accumulation of growth energy and accelerating energy consumption of sea cucumber. There was no significant difference between feeding frequencies in all the energy parameters, which might be the basis for the non-discrepancy in growth performance of sea cucumber. Previous studies showed that coefficient of variation in weight of A. japonicus was significantly affected by stocking density (Dong et al., 2010; Liang et al., 2010). The present study also found that individual growth variation of sea cucumber significantly increased with the increase of stocking density, and crowding stress significantly enlarged coefficients of variation. However, it was interesting that coefficients of variation decreased as feeding frequency increased, especially under high stocking densities (D40 and D60), which were significantly affected by interaction between stocking density and feeding frequency. It might be induced by differentiation in endocrine response of sea cucumber (Pei et al., 2012). Plasma cortisol in many aquatic animals

increased under crowding stress (Ruane et al., 2002; Xia et al., 2015a). Our results showed that cortisol level in coelomic fluid of sea cucumber significantly increased with the increase of stocking density, which could generate energy to satisfy the increasing demand of stress-related energy through the process of glyconeogenesis and fat degradation (Vijayan et al., 1990). Glucose and glycogen are essential energy substances for animal metabolism and lactate is an intermediate production of energy metabolism (Barnett and Pankhurst, 1998). Lactate level significantly elevated, and glucose level in coelomic fluid and glycogen in muscle of A. japonicus showed remarkable descending trends as stocking density increased, which implied long-term energy consumption under high stocking density, as described by results of energy allocation. However, feeding frequency had significant influence on glucose, glycogen and cortisol levels. Glucose and glycogen levels significantly increased and cortisol level significantly decreased as feeding frequency increased in D40 and D60, which might be used to explain the relatively lower coefficients of variation induced by feeding frequency. In conclusion, our results reconfirmed that stocking density could significantly affected growth performance, feed utilization, endocrine response and energy budget of sea cucumber. Growth performance, apparent digestibility coefficient and energy budget were not significantly affected by interaction between feeding frequency and stocking density. To some extent, increasing feeding frequency could effectively reduce coefficient of variation by differentiation in endocrine response of sea cucumber, especially under high stocking density. The present study provided valuable information for the intensive culture of A. japonicus. Acknowledgments The research was funded by the grants from the Promotive Research Fund for Young and Middle-aged Scientists of Shandong Province (BS2015HZ004), the Doctor Science Research Foundation of Qingdao City (15-9-1-89-jch), and the Modern Agriculture Industry System of Shandong Province of China: Industrial Innovation of Sea cucumber (SDAIT-22-06). References An, Z.H., Dong, Y.W., Dong, S.L., 2007. Temperature effects on growth-ration relationships of juvenile sea cucumber Apostichopus japonicus (Selenka). Aquaculture 272, 644–648. AOAC, 1995. Official methods of analysis of AOAC International. In: Official Analytical Chemists. 16th edn. AOAC International, Arlington, Virginia, p. 1141. Bai, Y.C., Zhang, L.B., Liu, S.L., Ru, X.S., Xing, L.L., Cao, X.B., Zhang, T., Yang, H.S., 2015. The effect of salinity on the growth, energy budget and physiology performance of

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