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Article

Optimal Dietary Carbohydrates to Lipids Ratio for Fast and Coordinated Test Growth of Juvenile Sea Urchin (Strongylocentrotus intermedius)

by
Panke Gong
†,
Haijing Liu
†,
Dan Gou
,
Weixiao Di
,
Jiahao Cao
,
Jun Ding
,
Yaqing Chang
and
Rantao Zuo
*
Key Laboratory of Mariculture and Stock Enhancement in North China’s Sea (Ministry of Agriculture and Rural Affairs), Dalian Ocean University, Dalian 116023, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Fishes 2025, 10(2), 57; https://doi.org/10.3390/fishes10020057
Submission received: 20 December 2024 / Revised: 21 January 2025 / Accepted: 28 January 2025 / Published: 30 January 2025
(This article belongs to the Special Issue Development of Low-Crop, Low-Fishmeal or Low-Fish Oil Feeds for Aquaculture)
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Abstract

Rapid and coordinated test growth is crucial for maintaining the normal body shape of Strongylocentrotus intermedius juveniles. In total, 270 S. intermedius (1.19 ± 0.01 g) were randomly assigned to 18 floating cages. Three cages of sea urchins were fed kelp (Saccharina japonica) (control diet) or one of five formulated feeds with different carbohydrate-to-lipid ratios (C/L) (1, 2, 4, 8, and 16) for 90 days. The results suggested that the weight gain rate of S. intermedius fed C/L4 was markedly greater than that fed C/L1 and C/L16 except for kelp, C/L2, and C/L8. The test diameter (TD) and test height (TH) gain rates of S. intermedius fed C/L4 were markedly greater than those fed other dry feeds except for C/L2. The TH/TD of S. intermedius fed kelp was markedly greater than that fed dry feeds, except for C/L1 and C/L2. Juvenile S. intermedius fed C/L2 showed higher test magnesium content, larger holes, and longer and thinner trabeculae than those fed other dry feeds. Overall, juvenile S. intermedius fed C/L4 had the highest body weight gain rate and test growth rate among the formulated feed groups. Juveniles fed C/L2 had the most coordinated test growth reflected by TD/TH, which is comparable to those fed kelp. Therefore, the optimal C/L for rapid and coordinated test growth of juvenile S. intermedius should be higher than 2 but lower than 4.
Keywords:
sea urchin; carbohydrate-to-lipid ratio; feeding; somatic growth; test growth; test characteristics
Key Contribution: This study was expected to precisely quantify the optimal C/L for achieving the fastest and most coordinated test growth of juvenile S. intermedius. These findings can provide a reference for formulating feeds suitable for the effective and efficient production of juvenile S. intermedius with healthy body shapes, reflected by coordinated test height/test diameter, especially under intensive farming patterns.

1. Introduction

The sea urchin (Strongylocentrotus intermedius), as a member of the family Strongylocentridae, is naturally distributed in northern Japan and Russia [1,2,3,4,5,6]. Sea urchin gonads are commonly accepted for their delicious taste and rich long-chain polyunsaturated fatty acids, which can prevent most cardiovascular, neurodegenerative, and proinflammatory disorders [7,8,9,10,11,12,13]. The worldwide overfishing of sea urchins, coupled with habitat destruction and global climate change, has led to the severe decline of wild resources [1,2,8,14,15,16]. During recent decades, aquaculture has proven to be effective in alleviating the fishing pressure on their wild populations. Different from most fish and crustaceans, S. intermedius need three years or even more time to become a market-sized aquatic product. From this background, producing large-sized S. intermedius juveniles (test diameter ≥ 20 mm) is extremely important for shortening the period and reducing the risk of farming of this species [2]. The shell of sea urchins includes test (the main part of the shell) and spines [17]. Rapid and coordinated growth of the test height and test diameter is important for producing high-quality juveniles. However, imbalanced growth between the test diameter and height of juvenile S. intermedius is usually observed when they are fed formulated feeds [18]. Thus, it is imperative to elucidate the relationship between nutrients and test growth of S. intermedius.
Proteins, as the most expensive energy substances, will generate nitrogen-containing waste, which has detrimental effects on the growth and health of aquatic animals. Carbohydrates and lipids possess the advantages of low costs and nitrogen-free excrement [19,20]. Carbohydrates are primarily utilized as the energy sources for the daily activities and gametogenesis of sea urchins, while lipids mainly provide essential fatty acids for the normal growth and development of sea urchins [21,22]. Sea urchins, as marine omnivorous invertebrates, can normally accept starch- and cellulose-rich foods such as maize, spinach, pumpkin, and kelp, and consume a certain amount of lipid-rich foods such as fish and shellfish [23,24]. To date, the requirements for carbohydrates and lipids have been reported in several sea urchin species. Juvenile sea urchin Lytechinus variegatus (initial body weight, IBW, of approximately 3.95 g) fed diets containing 18% carbohydrates showed a significantly higher weight gain rate (WGR) than those fed diets containing 12% carbohydrates [25]. Juvenile L. variegatus (IBW of approximately 5.60 g) fed diets with 19% carbohydrates had higher WGR and gonadosomatic index (GSI) values than those fed diets with 26% and 38% carbohydrates [22]. As for lipid requirements, it was found that formulated feeds with above 9% lipids negatively influenced the WGR of juvenile L. variegatus (IBW of approximately 0.09 g) [26]. Diets with 5–6% lipids are optimal for achieving the highest WGR of juvenile L. variegatus (IBW of approximately 0.07–0.10 g) [27]. Formulated feeds with 6% lipids resulted in the highest GSI of adult Paracentrotus lividus (IBW of approximately 35 g) [21].
It is understood that an inseparable bond exists between lipids and carbohydrates, and an imbalance can adversely impact the feed conversion and somatic growth of aquatic animals [19,28,29,30]. Since lipids and carbohydrates are crucial for bone growth and development, it is essential to ascertain a suitable carbohydrate-to-lipid ratio (C/L) to achieve fast and coordinated test growth of sea urchins. A previous study found that juvenile L. variegatus fed dry feeds with C/L2 exhibited markedly lower final test diameters than that of individuals fed dry feeds with C/L2.56–6.88 [26]. It was also shown that Chinese longsnout catfish (Leiocassis longirostris) fed diets with C/L1.98 can maintain healthy skeleton growth, but those fed diets with C/L5.07 displayed skeletal malformations [31]. A previous study suggested that abalone Haliotis discus hannai fed formulated feeds with C/L10.06 showed significantly greater daily increments in shell length than those with C/L12.94 and C/L4.78 [32]. Furthermore, extremely high C/L could result in an insufficient intake of docohexaenic acid and α-tocopherol, which increases the occurrence possibility of axial and cranial skeletal deformities of fish by delaying the process of early biomineralization [33].
Thus, this study was performed to investigate the effects of different C/L on feed intake, somatic growth, and test characteristics of S. intermedius juveniles. It was expected to precisely quantify the optimal C/L for achieving the fastest and most coordinated test growth of juvenile S. intermedius. These findings can provide a reference for formulating feeds suitable for the effective and efficient production of juvenile S. intermedius with healthy body shapes, reflected by coordinated test height/test diameter, especially under intensive farming patterns.

2. Materials and Methods

2.1. Ethics Statement

The Institution Ethics Committee deemed it unnecessary to grant authorization for this research on S. intermedius because it is classified as an invertebrate. Nevertheless, all necessary precautions were taken to minimize potential harm to the animals and acknowledge their essential contribution to this research.

2.2. Experimental Diets and Feeding Experiment

Fresh Saccharina japonica (control diet) (crude protein 8.16%, crude lipid 0.4%) and five formulated feeds were prepared. The kelp was collected from March to June, and the age was 4–7 months. As described in Table 1, the five formulated feeds contained different C/L values of 1.04, 2.00, 4.01, 8.04, and 16.10. All solid ingredients were pulverized and sifted through a 200 μm sieve. Next, the powders in each formulated feed were blended evenly. Subsequently, oil was added and blended with the mixture above. After 35% water was added, the feedstuffs were extruded with a machine (DES-TS1280, Dingrun, Jinan, China) to make pellets. Finally, the feeds were dried (50 °C), cooled, sealed, and stored in a freezer (−20 °C).
S. intermedius juveniles were obtained from a local breeding farm. Following a 9-day domestication period, 270 healthy S. intermedius juveniles of similar size (initial weight: 1.19 ± 0.01 g, initial test height: 6.65 ± 0.03 mm, initial test diameter: 13.96 ± 0.05 mm) were selected and randomly allocated to 18 floating cages (height: 38 cm, diameter: 20 cm). All the cages were placed in a tank (500 L), and each cage held 15 S. intermedius juveniles. Petri dishes were used to prevent food from falling out of the cages. Each diet was randomly dispensed into three cages of juvenile S. intermedius.
S. intermedius juveniles were hand-fed until they showed obvious satiation. The feeding frequency was twice (10:00 and 19:30) daily. Every two days, 60% of the volume of seawater in the tank was changed. During the 90 d feeding experiment, the water temperature was 15.7 ± 4.1 °C, the pH was 8.1 ± 0.1, the salinity was 31 ± 0.8‰, and the DO concentration was maintained above 7.8 mg L−1 using an aerator.

2.3. Sampling

After each feeding, the residual feeds were collected, dried, and weighed to calculate the feed intake (FI) and feed conversion ratio (FCR). Then, the digestive tracts and gonads of nine sea urchins in each cage were individually dissected and weighed to calculate the digestive tract index (DTI) and GSI, respectively. The average values of the DTI and GSI of sea urchins from each cage were calculated for subsequent statistical analysis. After that, an appropriate amount of the gonads was put into Bonn’s reagent for 24 h before they were used for subsequent histological analysis.
The shells and lanterns of nine S. intermedius juveniles in each cage were individually weighed and recorded to calculate shell index (SI) and lantern index (LI), respectively. After that, the average values of SI and LI of sea urchins from each cage were calculated for subsequent statistical analysis. Three out of nine sea urchin shells were randomly selected and submersed in 1% NaClO solution for 12 h. Then, the spines on the primary tubercles were collected for the measurement of spine length, and the sea urchin shells were gently rubbed to make the other spines fall off. After that, the tests were cleaned and resubjected to 1% NaClO solution for 12 h. The tests were flushed with distilled water after they were removed from the NaClO solution. Next, the tests were dried in an oven (70 °C). An interambulacral plate was used to measure the test thickness. The equatorial part of the interambulacral plate was taken for scanning electron microscopy observation. Similarly, three of the remaining six sea urchin shells were randomly selected and processed into tests, which were subsequently dried, ground to a fine powder, and then stored in sealed bags for subsequent assays of calcium and magnesium contents.

2.4. Gonad Histological Analysis

Since Di et al. [1] described the detailed procedures, we just briefly outline the operation steps. After the gonads of S. intermedius were fixed, dehydrated, and embedded in paraffin, 4 μm slices were taken with a microtome (RM2016, Leica, Nussloch, Germany). Subsequently, the dewaxed sections were stained using a dye (hematoxylin and eosin). Finally, the microstructure of the gonads was observed under a microscope. The maturity stages (stages I–VI) of gonads were identified according to Zhang et al. [13]. Stage I is the recovery stage characterized by primary gametes and nutritive phagocytes; stage II is the growing stage characterized by large quantities of primary gametes and nutritive phagocytes; stage III is the premature stage characterized by a reduced number of nutritive phagocytes and increased gametes at all developmental stages; stage IV is the mature stage characterized by few nutritive phagocytes and clusters of mature gametes; stage V is the spawning stage characterized by depletion of nutritive phagocytes and loosely packed gametes; and stage VI is the spent stage characterized by no gametes.

2.5. Measurement of Spine Length and Test Thickness

The six longest spines of each sea urchin were selected, measured with a digital calliper (MarCal 16 EWR, Marh GmbH, Esslingen, Germany), and averaged to record the spine length.
Six points were taken to divide the interambulacral plate into five equally spaced segments from the mouth end to the back end. The thickness of each sea urchin was measured at six points with a digital caliper to calculate the average thickness (Figure 1).

2.6. Analysis of the Test Microstructure

The microstructure of the sea urchins was observed using a field emission scanning electron microscope (Nova Nano SEM 650, FEI, Hillsboro, OR, USA). Due to the poor conductivity of the sample, an ion-sputtering instrument (ETD-800, Vision Precision Instruments, Beijing, China) was used to coat the sample with platinum, and then the micromorphology of the outer and inner surfaces of the sample was observed (Figure 1). After microstructure images were captured, twenty holes were randomly chosen for the measurement of hole diameter and trabecula length and diameter (Figure 2), which were measured using an image analysis software (‘Fiji is Just ImageJ’ software, ver. 1.53t, http://fiji.sc/Fiji, accessed on 1 July 2024).

2.7. Analysis of Test Calcium and Magnesium Concentrations

Single-element standard solutions of calcium and magnesium (1000 µg mL−1) were quantitatively diluted with 5% nitric acid and 2% nitric acid to prepare a series of standard solutions with concentrations of 1, 5, 10, 50, 100, and 200 µg L−1. The series of standard solutions were tested by inductively coupled plasma-optical emission spectrometry (ICP-OES) (Optima 8000, Perkin Elmer, Waltham, MA, USA) to construct standard curves.
Subsequently, the weighed samples were put into a digestion tank with 5 mL of 5% nitric acid. Then, the digestion tank was put into a microwave digestion apparatus (TOPEX, Yiyao, Shanghai, China). After that, the samples were assayed by ICP-OES following the instructions. Finally, the calcium and magnesium contents of the samples were quantified based on the standard curves.

2.8. Calculations and Statistical Analysis

Survival rate (SR, %) = final sea urchin number/initial sea urchin number × 100.
FI (mg ind−1 day−1) = daily food consumption of sea urchins/final sea urchin number.
FCR = total food consumption/(final sea urchin wet weight − initial sea urchin wet weight).
WGR (%) = (final sea urchin wet weight − initial sea urchin wet weight)/initial sea urchin wet weight × 100.
Test diameter gain rate (TDGR, %) = (final sea urchin test diameter − initial sea urchin test diameter)/initial sea urchin test diameter × 100.
Test height gain rate (THGR, %) = (final sea urchin test height − initial sea urchin test height)/initial sea urchin test height × 100.
GSI (%) = final gonad wet weight/final sea urchin wet weight × 100.
DTI (%) = final digestive tract wet weight/final sea urchin wet weight × 100.
SI (%) = final shell wet weight/final sea urchin wet weight × 100.
LI (%) = final lantern wet weight/final sea urchin wet weight × 100.
After testing for normality and heterogeneity, all data were analyzed using SPSS 22.0 software, and a one-way analysis of variance was used to analyze the significance (p < 0.05) of differences among dietary groups. When a significant difference was found, differences between dietary groups were compared using the Tukey method. The statistical results were denoted as means ± S.E.M.

3. Results

3.1. Survival, Feed Intake and Feed Conversion Ratio

On average, the survival rates ranged from 88.89% to 95.55%, showing no marked differences among the dietary groups (Table 2).
The FCR (10–15) of juvenile S. intermedius fed kelp was markedly higher than that of individuals fed formulated feeds (1–3) at each sampling point during the whole experimental period (p < 0.05). The FCR increased with the increase in sea urchin body weight in the dietary groups. The FI and FCR of S. intermedius showed no marked differences among all formulated feed groups (Table 2).

3.2. Weight Growth Rate

WGR30 (df = 5.12, F = 6.41, p = 0.004) and WGR60 (df = 5.12, F = 10.50, p = 0.000) of S. intermedius in C/L4 group were only markedly greater than that in C/L1. WGR90 (df = 5.12, F = 12.46, p = 0.000) of S. intermedius in C/L4 was comparable to that in kelp, C/L2, and C/L8, but was markedly greater than that in C/L1 and C/L16 (Figure 3).

3.3. Gonad Growth

As diet C/L increased from 1 to 4, the GSI increased from 16.35% to 19.66% and then decreased to 17.99% with a further increase in C/L. The GSI of juvenile S. intermedius fed kelp was markedly lower than that of juveniles fed dry feeds (p < 0.05). The GSI of S. intermedius in C/L4 was only markedly greater than that in kelp and C/L1 (p < 0.05) (Table 3).
The gonads of S. intermedius in the kelp group all stayed at stage II with no gametes observed in both male and female individuals. Some premature gonads of S. intermedius in each feed group entered stage III, with gametes observed in both male and female individuals. Notably, the ovum and spermatozoa of S. intermedius in the C/L2 and C/L4 groups were fewer than those in the other feed groups (Figure 4).

3.4. Test Growth Performance

S. intermedius fed kelp showed markedly longer spines but thinner test than that fed dry feeds (p < 0.05). No marked differences in spine length and test thickness were found among the dry feed groups (Table 4).
The TH/TD30 of juvenile S. intermedius fed dry feeds was comparable to that of individuals fed kelp. However, as feeding time prolonged, S. intermedius juveniles fed kelp exhibited markedly greater TH/TD60 and TH/TD90 than those fed diets with moderate or relatively higher C/L (4–16) (p < 0.05) (Table 5).
The TDGR30 (df = 5.12, F = 7.07, p = 0.003) of juvenile S. intermedius in C/L4 was only markedly greater than that in C/L1 and C/L16. As feeding time was prolonged, the TDGR60 (df = 5.12, F = 20.09, p = 0.000) and TDGR90 (df = 5.12, F = 5.90, p = 0.006) of juvenile S. intermedius in C/L4 were markedly greater than those in other dietary groups, except for kelp and C/L2 (Figure 5).
The THGRs of juvenile S. intermedius fed formulated feeds were markedly lower than those of individuals fed kelp at all sampling timepoints (p < 0.05). The THGR30 (df = 5.12, F = 17.64, p = 0.000) of S. intermedius juveniles in C/L2 was only markedly greater than that in C/L16. The THGR60 (df = 5.12, F = 24.19, p = 0.000) and THGR90 (df = 5.12, F = 29.94, p = 0.000) of S. intermedius juveniles in C/L4 were markedly greater than those in the other feed groups except for C/L2 (Figure 6).

3.5. Test Calcium Content and Magnesium Contents

The contents of magnesium (Mg) and calcium (Ca), as well as Mg/Ca, of S. intermedius juveniles fed kelp were highest. The Mg content of juvenile S. intermedius in kelp was markedly greater than that in feed groups except for C/L2 (p < 0.05). No marked differences were found in the Ca content of S. intermedius among the different dietary groups. The Mg/Ca of S. intermedius juveniles in kelp was only markedly greater than that in C/L16 (p < 0.05) (Table 6).

3.6. Test Surface Microstructure

3.6.1. Outer Surface Microstructure of Test

The test microstructure arrangement of juvenile S. intermedius fed kelp was more regular than that of individuals fed feeds except for C/L2 (Figure 7).
The trabecula diameter of S. intermedius juveniles in C/L2 was only markedly lower than that in C/L16 and kelp (p < 0.05). The trabecula length of S. intermedius juveniles in kelp was only markedly greater than that in C/L1 (p < 0.05). The trabecula length/trabecula diameter of S. intermedius in the C/L2 group was markedly greater than that of individuals in C/L1 and C/L16 except for kelp, C/L4, and C/L8 (p < 0.05) (Table 7).

3.6.2. Inner Surface Microstructure of Test

The juvenile S. intermedius fed kelp showed a significantly greater degree of inner surface resorption than those fed dry feeds. The juveniles fed C/L2 showed a greater degree of inner surface resorption than those fed other feeds (Figure 7).
The hole diameter of S. intermedius in kelp was markedly greater than that of individuals in formulated feeds (p < 0.05). No marked differences were found in the hole diameter of S. intermedius among the different formulated feeds. The trabecula diameter, trabecula length, and trabecula length/trabecula diameter of S. intermedius in kelp were highest but were comparable to those of individuals in dry feed groups (Table 7).

4. Discussion

Protein is an essential macronutrient that supports growth, maintenance, and reproduction by supplying the necessary amino acids to sea urchins [34,35,36]. However, proteins, as one of the most expensive nutrients in formulated feeds, should be mainly used for body growth instead of energy supply [37]. Lipids and carbohydrates are generally utilized as inexpensive non-protein energy substances for the preparation of feeds [19,20]. Appropriate C/L values could improve feed utilization, while low or high dietary C/L may depress growth performance and feed utilization. The WGR is traditionally used as an indicator of sea urchin somatic growth in sea urchins and other aquatic animals. In this study, juvenile S. intermedius fed C/L4 showed higher WGR than those fed low or high C/L groups. This optimal diet C/L (4) was slightly higher than that (approximately 3) reported for juvenile and adult L. variegatus [21,36]. These inconsistent findings could be attributed to different sizes and growth stages of sea urchins. However, previous researchers also found that the optimal diet C/L value was approximately 4 for blunt snout bream (Megalobrama amblycephala) and redclaw crayfish (Cherax quadricarinatus) [37,38]. The optimal diet C/L was below 2 for yellowfin seabream (Sparus latus), yellow croaker (Larmichthys crocea), orange-spotted grouper (Epinephelus coioides), and tilapia (Oreochromis niloticus) [39,40,41,42]. However, the optimal diet C/L was above 4 for grass carp (Ctenopharyngodon idella), turbot (Scophthalmus maximus), and H. discus hannai [32,43,44]. However, the differences in the optimal diet C/L could be attributed to differences in the diet habits of these aquatic animals. It is well known that carnivorous fish exhibit a superior capability to utilize lipids and have poorer tolerance to high-carbohydrate diets than herbivorous and omnivorous fish. As an omnivorous invertebrate species, S. intermedius can readily accept starch and cellulose-rich foods such as kelp, pumpkin, and maize and need a certain amount of lipid-rich foods such as fish and shellfish [23,24].
In this study, the downregulated growth of juvenile S. intermedius fed high C/L could be due to lipid deficiency. An insufficient supply of essential fatty acids leads to the slow growth of sea urchins [45]. In low-C/L formulated feed groups, excessively high lipid content can result in low feed utilization efficiency, which restricts the intake of protein and other nutrients required for maximum growth [46]. A high-lipid diet can lead to lipid deposition and lipid peroxidation, which can cause a slowdown in body growth [47,48]. Low dietary carbohydrate levels also appeared to be inadequate for the growth of L. variegatus in the low-C/L formulated feed groups [21,36]. The energy provided by low carbohydrates in the formulated feed is insufficient to sustain the normal growth of sea urchins, and some proteins and lipids will be consumed for energy supply instead of tissue growth. This can not only lead to the waste of proteins and lipids but can also induce the metabolic burden of proteins and lipids, which is not conducive to the somatic growth of sea urchins. Previous studies considered that the energy supplied by dietary carbohydrates is prone to use for test growth rather than for gonad growth in Psammechinus miliaris [49,50]. However, it should be noted that the mass of gonads was not excluded when calculating the WGR of juvenile sea urchins in previous studies. Thus, the optimal C/L requirement was usually underestimated by using WGR to assess the somatic growth of sea urchin juveniles.
Sea urchin gonads are both the nutrient storage organ and the sex organ responsible for gametogenesis [51]. In general, sea urchins allocate fewer nutrients and less energy to somatic growth when they enter the gametogenesis stage [52]. In the current research, S. intermedius juveniles fed diets with C/L4 had the highest GSI. Similarly, a previous study found that juvenile L. variegatus fed diets with C/L3 had higher gonad weight than those with C/L6 [22]. Chinese sturgeon (Acipenser sinensis) fed high lipid levels were observed to develop their ovaries from stage II to stage III or IV in low-C/L formulated feed groups [38,53]. In high-C/L formulated feed groups, the higher proportion of diet carbohydrates can arouse the sexual maturation of rainbow trout (Oncorhynchus mykiss) [54]. In this research, the gonads of juvenile S. intermedius fed kelp remained at stage II, but some of the gonads of juvenile S. intermedius fed formulated feeds with different C/L ratios entered stage III. There were fewer ova and spermatozoa in moderate C/L (2 and 4) groups than in low or high C/L (1, 8, and 16) groups. Under the circumstances of sexual maturation, more energy will be allocated to gonad development and gametes production than somatic growth. Thus, moderate C/L (2 and 4) can, to some extent, hinder early sexual maturation, reduce the substances and energy used for gametes production, and better promote the somatic growth of sea urchin juveniles.
Coordinated and rapid growth is important for producing high-quality sea urchin seeds. The test diameter was commonly used to assess the somatic growth of sea urchins [24,55,56], but the height of sea urchin test was generally neglected. Lately, the uncoordinated test growth in height and diameter of juvenile S. intermedius has drawn increased attention. Juvenile L. variegatus fed dry feeds with 33% proteins exhibited slightly lower TH/TD than those of individuals fed 9% and 15% proteins [57]. In this experiment, the TH/TD and test microstructure of S. intermedius juveniles fed natural food kelp were regarded as the standard and normal values. Generally, the TH/TD tended to decrease as the C/L increased. This indicated that juvenile S. intermedius fed diets with extremely high C/L showed signs of skeletal malformations. This was supported by the findings of Tan et al. [31], which showed that skeletal malformations of L. longirostris occurred in the groups with C/L5.07 rather than C/L1.98 [31]. This could be due to the insufficient intake of essential fatty acids and lipid-soluble vitamins such as α-tocopherol, which have been found to be critical for biomineralization and thus decrease the occurrence of axial and cranial skeletal deformities [33]. Most sea urchin species have natural cryptic behavior, and kelp can provide shelter for sea urchins. However, based on our observations, S. intermedius juveniles fed dry feeds tend to be more sensitive and often seek refuge under Petri dishes. This behavior may inhibit the growth in test height of juveniles. Therefore, special culture systems with more shelters should be designed for the coordinated growth of sea urchin juveniles fed dry feeds. In this study, the juvenile S. intermedius fed kelp showed higher WGR and test growth rates than those fed formulated feeds. Sea urchins showed a stronger preference for natural foods like kelp and achieved better growth performances. Therefore, it is crucial to improve the palatability and attraction of formulated feeds in the future.
In this experiment, juvenile S. intermedius fed formulated feeds had shorter spines and thicker tests than those fed kelp. Among the formulated feed groups, juvenile S. intermedius fed diets with high C/L (8 and 16) showed slightly thicker test than those with C/L1. This could be due to the different shell biomineralization of juvenile S. intermedius. A previous study showed that juvenile L. variegatus fed a high-protein diet had a thicker test than that fed a low-protein diet [57]. In this experiment, more carbohydrates in the high-C/L formulated feeds were used for energy support of juvenile sea urchins, and proteins were saved for thickening the tests. The growth of sea urchin test is characterized by the resorption on the inner surface, the expansion of existing inorganic networks, and the formation of new networks [58]. Elements from test resorption are reutilized for calcification in other parts. The test microstructure exhibited numerous trabeculae and holes [59,60]. The diameter of the hole and trabecula shows a positive correlation with the longitudinal growth of the equatorial plates, which are eventually reflected in the test height of sea urchins [61,62]. This study suggested that S. intermedius juveniles fed formulated feeds with a moderate level of C/L (2 and 4) had larger holes and longer but thinner trabeculae on the inner and outer surfaces than those fed relatively low or high C/L (1, 8, and 16). A larger hole diameter and thinner test will increase the efficiency of test construction. In this study, dietary C/L had no marked impact on the content of Ca deposited in the test of juvenile S. intermedius, but the content of Mg deposited in the test of juveniles fed diets with C/L2 was greater than that of individuals fed other formulated feeds. Due to the instability of magnesium calcite, there will be more active calcite deposition and resorption in the test of juvenile S. intermedius fed C/L2, which can accelerate the growth of the test [63,64]. A thinner but larger test suggests faster outer surface expansion and inner surface resorption. In this experiment, S. intermedius juveniles fed kelp exhibited higher test height and diameter and more obvious resorption on the inner surface than those of individuals fed dry feeds. Similarly, a study indicated that sea urchins with greater growth rates exhibited more pronounced resorption and thinner tests [63]. Moreover, fast growth can result in a limited supply of minerals required for forming trabeculae in sea urchins, which could drive the formation of thinner tests with thinner trabeculae and larger holes.

5. Conclusions

In conclusion, juvenile S. intermedius fed a C/L4 formulated feed had the greatest WGR and test growth rate in the formulated feed groups. Juvenile S. intermedius fed C/L1 and C/L2 showed the most coordinated test growth in terms of TD/TH among the formulated feed groups. However, the WGR of juvenile S. intermedius fed C/L1 was significantly lower than those fed C/L4. Juvenile S. intermedius fed C/L2 formulated feeds had larger holes and longer and thinner trabeculae than those fed the other feeds from the perspective of test microstructure, which could account for the most coordinated test growth of juvenile S. intermedius. Thus, it was recommended that the optimal C/L should be above 2 but more than 4 to achieve rapid and coordinated test growth of S. intermedius juveniles.

Author Contributions

Conceptualization, P.G. and R.Z.; methodology, H.L.; validation, D.G.; formal analysis, W.D.; resources, J.C.; data curation, P.G., H.L., D.G., W.D. and J.C.; writing—original draft preparation, P.G. and R.Z.; writing—review and editing, J.D. and Y.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by grants from the Dalian Science and Technology Bureau (2023JJ13SN064), the Natural Foundation of Liaoning Province (2023-MSLH-015), the Natural Foundation of Education Department of Liaoning Province (LJKMZ20221096), the Xingliao Talent Plan for Top Young Talents (XLYC2203036), and the High-Level Talent Support Grant for Innovation in Dalian (2022RJ14).

Institutional Review Board Statement

Because S. intermedius are invertebrates, the Ethics Committee of Dalian Ocean University did not require this research to be authorized.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data of this study can be provided by the corresponding author upon request.

Acknowledgments

The authors thank Rujian Xu for assisting in the completion of the measurement of spine length and test thickness.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
C/LCarbohydrates to lipids ratio
TDTest diameter
THTest height
IBWInitial body weight
WGRWeight gain rate
GSIGonadosomatic index
FCRFeed conversion ratio
FIFeed intake
DTIDigestive tract index
SIShell index
LILantern index
TDGRTest diameter gain rate
THGRTest height gain rate

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Figure 1. The area of test plates chosen for the microstructure scanning in S. intermedius. (A) Overall appearance of inorganic test of S. intermedius. (B) Measurement points (black dots) for test thickness of S. intermedius. (C) Microstructure of test outer surface of S. intermedius. a: the equatorial part of the interambulacral plate chosen for microstructure scanning. b: the relatively regular area chosen for quantifying the hole and trabecula.
Figure 1. The area of test plates chosen for the microstructure scanning in S. intermedius. (A) Overall appearance of inorganic test of S. intermedius. (B) Measurement points (black dots) for test thickness of S. intermedius. (C) Microstructure of test outer surface of S. intermedius. a: the equatorial part of the interambulacral plate chosen for microstructure scanning. b: the relatively regular area chosen for quantifying the hole and trabecula.
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Figure 2. Hole diameter, trabecula diameter, and trabecula length in the test outer surface microstructure of S. intermedius.
Figure 2. Hole diameter, trabecula diameter, and trabecula length in the test outer surface microstructure of S. intermedius.
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Figure 3. Effects of different dietary carbohydrate-to-lipid ratios on weight gain rate of juvenile sea urchin (Strongylocentrotus intermedius). Mean bars of the same cluster bearing different letters indicate that they are significantly different at p < 0.05.
Figure 3. Effects of different dietary carbohydrate-to-lipid ratios on weight gain rate of juvenile sea urchin (Strongylocentrotus intermedius). Mean bars of the same cluster bearing different letters indicate that they are significantly different at p < 0.05.
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Figure 4. Effects of different dietary carbohydrate-to-lipid ratios on the gonad histology of juvenile sea urchin (Strongylocentrotus intermedius). (A) C/L1, male, stage III; (B) C/L1, female, stage III; (C) C/L2, male, stage III; (D) C/L2, female, stage III; (E) C/L4, male, stage III; (F) C/L4, female, stage III; (G) C/L8, male, stage III; (H) C/L8, female, stage III; (I) C/L16, male, stage III; (J) C/L16, female, stage III; (K) Kelp, male, stage II; (L) Kelp, female, stage II. NP: nutritive phagocyte. SP: spermatocyte. S: spermatozoa. EV: early vitellogenic oocyte. VO: vitellogenic oocyte. O: ovum.
Figure 4. Effects of different dietary carbohydrate-to-lipid ratios on the gonad histology of juvenile sea urchin (Strongylocentrotus intermedius). (A) C/L1, male, stage III; (B) C/L1, female, stage III; (C) C/L2, male, stage III; (D) C/L2, female, stage III; (E) C/L4, male, stage III; (F) C/L4, female, stage III; (G) C/L8, male, stage III; (H) C/L8, female, stage III; (I) C/L16, male, stage III; (J) C/L16, female, stage III; (K) Kelp, male, stage II; (L) Kelp, female, stage II. NP: nutritive phagocyte. SP: spermatocyte. S: spermatozoa. EV: early vitellogenic oocyte. VO: vitellogenic oocyte. O: ovum.
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Figure 5. Effects of different dietary carbohydrate-to-lipid ratios on test diameter gain rate of juvenile sea urchin (Strongylocentrotus intermedius). Mean bars of the same cluster bearing different letters indicate that they are significantly different at p < 0.05.
Figure 5. Effects of different dietary carbohydrate-to-lipid ratios on test diameter gain rate of juvenile sea urchin (Strongylocentrotus intermedius). Mean bars of the same cluster bearing different letters indicate that they are significantly different at p < 0.05.
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Figure 6. Effects of different dietary carbohydrate-to-lipid ratios on test height gain rate of juvenile sea urchin (Strongylocentrotus intermedius). Mean bars of the same cluster bearing different letters indicate that they are significantly different at p < 0.05.
Figure 6. Effects of different dietary carbohydrate-to-lipid ratios on test height gain rate of juvenile sea urchin (Strongylocentrotus intermedius). Mean bars of the same cluster bearing different letters indicate that they are significantly different at p < 0.05.
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Figure 7. Effects of different dietary carbohydrate-to-lipid ratios on stereom structure in the test of juvenile sea urchin (Strongylocentrotus intermedius). (A) C/L1, outer surface; (B) C/L2, outer surface; (C) C/L4, outer surface; (D) C/L8, outer surface; (E) C/L16, outer surface; (F) Kelp, outer surface. (G) C/L1, inner surface; (H) C/L2, inner surface; (I) C/L4, inner surface; (J) C/L8, inner surface; (K) C/L16, inner surface; (L) Kelp, inner surface. M: irregular calcite network. N: the resorption of calcite.
Figure 7. Effects of different dietary carbohydrate-to-lipid ratios on stereom structure in the test of juvenile sea urchin (Strongylocentrotus intermedius). (A) C/L1, outer surface; (B) C/L2, outer surface; (C) C/L4, outer surface; (D) C/L8, outer surface; (E) C/L16, outer surface; (F) Kelp, outer surface. (G) C/L1, inner surface; (H) C/L2, inner surface; (I) C/L4, inner surface; (J) C/L8, inner surface; (K) C/L16, inner surface; (L) Kelp, inner surface. M: irregular calcite network. N: the resorption of calcite.
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Table 1. Formulation and proximate composition of the formulated feeds (% dry weight).
Table 1. Formulation and proximate composition of the formulated feeds (% dry weight).
Ingredients (%)Carbohydrate to Lipid Ratios
1.042.004.018.0416.10
Seaweed meal20.020.020.020.020.0
Kelp meal15.015.015.015.015.0
Casein14.414.414.414.414.4
Gelatin3.63.63.63.63.6
Fish meal77777
Mineral premix2.02.02.02.02.0
Vitamin premix2.02.02.02.02.0
Calcium propionate 0.180.180.180.180.18
Choline chloride0.10.10.10.10.1
Ethoxyquin0.010.010.010.010.01
Palm oil7.05.03.01.00.0
Soybean lecithin11111
Microcrystalline cellulose27.7125.0120.8118.6111.01
Cornstarch04.710.915.123.7
Proximate composition
Crude protein25.0525.0725.0825.1025.12
Crude lipid8.997.005.013.022.03
Carbohydrate9.3814.0120.1224.2532.74
Gross Energy (MJ/Kg)11.1711.1011.4911.4412.55
Seaweed powder: crude protein 14%, crude fat 0.4%. Kelp powder: crude protein 15%, crude fat 0.5%. Casein: crude protein 85%, crude fat 0.6%. Gelatin: crude protein 80%, crude fat 1%. Fish meal used in this study was manufactured by using wild anchovy (Engraulis ringens) captured in Peru. The crude protein and crude lipid of the fish meal were 68.7% and 9.7%, respectively. Mineral premix (mg or g/kg diet): CuSO4·5H2O, 10 mg; ZnSO4·H2O, 50 mg; FeSO4·H2O, 80 mg; MnSO4·H2O, 45 mg; CoCl2·6H2O (1%), 50 mg; NaSeSO3·5H2O (1%), 20 mg; Ca(IO3)2·6H2O (1%), 60 mg. MgSO4·7H2O, 1200 mg; zeolite, 18.35 g. Vitamin premix (mg or g/kg diet): vitamin D5mg; Vitamin K 10 mg; Vitamin B12, 10 mg; Vitamin B620 mg; folic acid 20 mg; Vitamin B 125 mg; Vitamin A 32 mg; Vitamin B 245 mg; Pantothenic acid 60 mg; Biotin 60 mg; niacin 200 mg; Tocopherol 240 mg; Inositol 800 mg; Ascorbic acid, 2000 mg; Microcrystalline cellulose, 16.47 g. Cornstarch: nitrogen-free extract 98.5%.
Table 2. Effects of different dietary carbohydrate-to-lipid ratios on survival rate, feed intake, and feed conversion ratio of juvenile sea urchin (Strongylocentrotus intermedius).
Table 2. Effects of different dietary carbohydrate-to-lipid ratios on survival rate, feed intake, and feed conversion ratio of juvenile sea urchin (Strongylocentrotus intermedius).
Formulated Feeds with Different Carbohydrate to Lipid RatiosKelpStatistical Analyses
124816dfFp
SR (%)91.11 ± 2.2295.55 ± 2.2291.11 ± 5.8893.33 ± 3.8588.89 ± 5.8893.33 ± 3.855.120.300.904
FI-30 (mg ind−1 day−1)48.48 ± 0.80 b48.86 ± 2.77 b46.56 ± 2.63 b48.03 ± 1.57 b45.73 ± 0.57 b509.13 ± 5.94 a5.123997.810.000
FI-60 (mg ind−1 day−1)63.31 ± 1.38 b60.95 ± 1.52 b57.65 ± 2.55 b59.99 ± 2.23 b55.05 ± 1.10 b567.23 ± 13.54 a5.121288.480.000
FI-90 (mg ind−1 day−1)84.25 ± 2.38 b75.36 ± 1.69 b69.07 ± 2.83 b67.19 ± 2.05 b60.23 ± 0.73 b641.79 ± 19.44 a5.12816.540.000
FCR-301.38 ± 0.12 b1.10 ± 0.04 b1.08 ± 0.06 b1.13 ± 0.05 b1.09 ± 0.06 b10.70 ± 0.14 a5.122017.630.000
FCR-601.82 ± 0.13 b1.57 ± 0.05 b1.41 ± 0.13 b1.54 ± 0.07 b1.45 ± 0.03 b13.24 ± 0.34 a5.12889.930.000
FCR-902.84 ± 0.21 b2.29 ± 0.16 b1.89 ± 0.08 b2.02 ± 0.07 b1.88 ± 0.06 b14.95 ± 0.71 a5.12279.540.000
Mean values with the different superscript letters within the same row are significantly different at p < 0.05. SR: survival rate; FI: feed intake; FI-30: daily feed intake of each sea urchin within 30 days; FI-60: daily feed intake of each sea urchin within 60 days; FI-90: daily feed intake of each sea urchin within 90 days; FCR: feed conversion ratio; FCR-30: feed conversion ratio at 30 days; FCR-60: feed conversion ratio at 60 days; FCR-90: feed conversion ratio at 90 days.
Table 3. Effects of different dietary carbohydrate to lipid ratios on gonad and digestive tract growth of juvenile sea urchin (Strongylocentrotus intermedius).
Table 3. Effects of different dietary carbohydrate to lipid ratios on gonad and digestive tract growth of juvenile sea urchin (Strongylocentrotus intermedius).
Formulated Feeds with Different Carbohydrate to Lipid RatiosKelpStatistical Analyses
124816dfFp
GW (g)0.65 ± 0.05 c0.73 ± 0.05 bc0.94 ± 0.05 a0.81 ± 0.03 ab0.76 ± 0.04 bc0.16 ± 0.03 d5.1233.730.000
GSI (%)16.35 ± 0.73 b18.00 ± 0.88 ab19.66 ± 0.66 a18.52 ± 0.59 ab17.99 ± 0.88 ab2.99 ± 0.48 c5.1257.290.000
DTW (g)0.22 ± 0.01 ab0.23 ± 0.01 ab0.27 ± 0.02 a0.20 ± 0.01 b0.19 ± 0.02 b0.28 ± 0.02 a5.125.600.007
DTI (%)5.47 ± 0.275.52 ± 0.265.72 ± 0.304.74 ± 0.284.64 ± 0.325.06 ± 0.385.121.110.404
Mean values with the different superscript letters within the same row are significantly different at p < 0.05. GW: gonad weight; GSI: gonadosomatic index; DTW: digestive tract weight; DTI: digestive tract index.
Table 4. Effects of different dietary carbohydrate-to-lipid ratios on shell parameters of juvenile sea urchin (Strongylocentrotus intermedius).
Table 4. Effects of different dietary carbohydrate-to-lipid ratios on shell parameters of juvenile sea urchin (Strongylocentrotus intermedius).
Formulated Feeds with Different Carbohydrate to Lipid RatiosKelpStatistical Analyses
124816dfFp
SW (g) 2.07 ± 0.07 c2.25 ± 0.07 bc2.45 ± 0.10 b2.22 ± 0.06 bc2.10 ± 0.07 c2.80 ± 0.05 a5.1212.090.000
SI (%)51.32 ± 1.0653.11 ± 0.8452.69 ± 0.9253.44 ± 0.8750.55 ± 1.1151.15 ± 0.935.121.120.399
LW (g) 0.24 ± 0.01 b0.24 ± 0.01 b0.27 ± 0.01 b0.24 ± 0.01 b0.24 ± 0.01 b0.35 ± 0.01 a5.1257.790.000
LI (%)5.99 ± 0.245.74 ± 0.155.79 ± 0.185.72 ± 0.185.92 ± 0.126.25 ± 0.185.121.1310.396
TT (mm)0.47 ± 0.02 ab0.50 ± 0.01 a0.52 ± 0.01 a0.54 ± 0.02 a0.53 ± 0.02 a0.44 ± 0.01 b5.127.030.003
SL (mm) 4.90 ± 0.19 b4.94 ± 0.22 b5.07 ± 0.22 b4.90 ± 0.12 b4.87 ± 0.19 b7.31 ± 0.11 a5.1228.050.000
Mean values with the different superscript letters within the same row are significantly different at p < 0.05. SW: shell weight; SI: shell index; LW: lantern weight; LI: lantern index; TT: test thickness; SL: spine length.
Table 5. Effects of different dietary carbohydrate-to-lipid ratios on test parameters of juvenile sea urchin (Strongylocentrotus intermedius) at different sampling timepoints (0, 30th, 60th, and 90th day).
Table 5. Effects of different dietary carbohydrate-to-lipid ratios on test parameters of juvenile sea urchin (Strongylocentrotus intermedius) at different sampling timepoints (0, 30th, 60th, and 90th day).
Formulated Feeds with Different Carbohydrate to Lipid RatiosKelpStatistical Analyses
124816dfFp
TD-0 (mm)14.12 ± 0.1313.86 ± 0.1313.97 ± 0.1314.13 ± 0.1213.92 ± 0.1413.78 ± 0.125.120.560.729
TD-30 (mm)19.23 ± 0.22 c20.33 ± 0.19 a20.03 ± 0.21 ab19.53 ± 0.20 bc19.11 ± 0.19 c20.54 ± 0.18 a5.125.610.007
TD-60 (mm)21.17 ± 0.23 b21.64 ± 0.26 b22.46 ± 0.27 a21.47 ± 0.19 b20.93 ± 0.18 b22.76 ± 0.21 a5.125.650.007
TD-90 (mm)21.91 ± 0.29 b22.37 ± 0.23 b23.59 ± 0.27 a22.50 ± 0.19 b21.62 ± 0.22 b23.66 ± 0.32 a5.124.860.012
TH-0 (mm)6.78 ± 0.096.61 ± 0.096.55 ± 0.076.71 ± 0.076.69 ± 0.096.56 ± 0.105.120.960.482
TH-30 (mm)9.01 ± 0.11 bc9.41 ± 0.14 ab9.24 ± 0.13 bc8.94 ± 0.13 bc8.75 ± 0.13 c9.83 ± 0.12 a5.126.010.005
TH-60 (mm)9.85 ± 0.15 bc9.86 ± 0.13 bc10.11 ± 0.17 b9.55 ± 0.12 cd9.31 ± 0.13 d10.86 ± 0.11 a5.129.300.001
TH-90 (mm)9.98 ± 0.15 bc10.09 ± 0.13 b10.28 ± 0.21 b9.73 ± 0.14 bc9.32 ± 0.17 c11.28 ± 0.28 a5.125.930.005
TH/TD-00.48 ± 0.000.48 ± 0.000.47 ± 0.000.47 ± 0.000.48 ± 0.000.48 ± 0.015.120.230.944
TH/TD-300.47 ± 0.010.46 ± 0.010.46 ± 0.010.46 ± 0.010.46 ± 0.010.48 ± 0.015.122.190.124
TH/TD-600.47 ± 0.01 ab0.46 ± 0.00 ab0.45 ± 0.01 b0.45 ± 0.01 b0.45 ± 0.01 b0.48 ± 0.01 a5.123.490.035
TH/TD-900.46 ± 0.01 ab0.45 ± 0.01 ab0.44 ± 0.01 b0.43 ± 0.01 b0.43 ± 0.01 b0.48 ± 0.01 a5.124.050.022
Mean values with the different superscript letters within the same row are significantly different at p < 0.05. TD: test diameter; TH: test height.
Table 6. Effects of different dietary carbohydrate-to-lipid ratios on calcium and magnesium contents in the test of juvenile sea urchin (Strongylocentrotus intermedius).
Table 6. Effects of different dietary carbohydrate-to-lipid ratios on calcium and magnesium contents in the test of juvenile sea urchin (Strongylocentrotus intermedius).
Formulated Feeds with Different Carbohydrate to Lipid RatiosKelpStatistical Analyses
124816dfFp
Ca (mg/g)329.00 ± 1.15347.67 ± 11.92350.67 ± 1.86335.33 ± 7.17341.00 ± 10.50354.67 ± 6.895.121.600.233
Mg (mg/g)14.90 ± 0.12 b15.70 ± 0.36 ab15.17 ± 0.42 b14.17 ± 0.33 b13.87 ± 0.78 b16.93 ± 0.24 a5.127.450.002
Mg/Ca (mmol/mol)75.48 ± 0.32 ab75.33 ± 1.15 ab72.11 ± 2.38 ab70.48 ± 1.63 ab67.88 ± 4.04 b79.60 ± 0.78 a5.123.930.024
Mean values with the different superscript letters within the same row are significantly different at p < 0.05. Ca: calcium; Mg: magnesium.
Table 7. Effects of different dietary carbohydrate-to-lipid ratios on hole and trabecula of juvenile sea urchin (Strongylocentrotus intermedius).
Table 7. Effects of different dietary carbohydrate-to-lipid ratios on hole and trabecula of juvenile sea urchin (Strongylocentrotus intermedius).
Formulated Feeds with Different Carbohydrate to Lipid RatiosKelpStatistical Analyses
124816dfFp
OSHD (μm)10.13 ± 0.1010.75 ± 0.3210.97 ± 0.1410.80 ± 0.4510.55 ± 0.2711.69 ± 0.545.122.290.111
OSTD (μm)3.16 ± 0.10 ab2.83 ± 0.12 b3.11 ± 0.17 ab3.38 ± 0.04 ab3.75 ± 0.26 a3.65 ± 0.10 a5.125.520.007
OSTL (μm)7.42 ± 0.36 b8.34 ± 0.12 ab8.85 ± 0.31 a8.56 ± 0.09 ab8.27 ± 0.18 ab9.51 ± 0.42 a5.126.350.004
OSTL/OSTD2.41 ± 0.09 b3.04 ± 0.10 a2.95 ± 0.15 a2.62 ± 0.05 ab2.33 ± 0.14 b2.68 ± 0.04 ab5.127.620.002
ISHD (μm)15.35 ± 0.19 b15.66 ± 0.28 b15.15 ± 0.45 b13.98 ± 0.30 b13.80 ± 0.75 b18.39 ± 0.88 a5.129.370.001
ISTD (μm)9.20 ± 0.158.77 ± 0.378.86 ± 0.168.06 ± 0.279.21 ± 0.309.36 ± 0.195.120.800.572
ISTL (μm)12.69 ± 0.3912.93 ± 0.1312.65 ± 0.4511.99 ± 0.6011.85 ± 0.7614.00 ± 0.255.122.590.083
ISTL/ISTD1.46 ± 0.051.55 ± 0.081.47 ± 0.061.38 ± 0.081.33 ± 0.051.59 ± 0.065.122.200.122
Mean values with the different superscript letters within the same row are significantly different at p < 0.05. OSHD: outer surface hole diameter; OSTD: outer surface trabecula diameter; OSTL: outer surface trabecula length; ISHD: inner surface hole diameter; ISTD: inner surface trabecula diameter; ISTL: inner surface trabecula length.
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Gong, P.; Liu, H.; Gou, D.; Di, W.; Cao, J.; Ding, J.; Chang, Y.; Zuo, R. Optimal Dietary Carbohydrates to Lipids Ratio for Fast and Coordinated Test Growth of Juvenile Sea Urchin (Strongylocentrotus intermedius). Fishes 2025, 10, 57. https://doi.org/10.3390/fishes10020057

AMA Style

Gong P, Liu H, Gou D, Di W, Cao J, Ding J, Chang Y, Zuo R. Optimal Dietary Carbohydrates to Lipids Ratio for Fast and Coordinated Test Growth of Juvenile Sea Urchin (Strongylocentrotus intermedius). Fishes. 2025; 10(2):57. https://doi.org/10.3390/fishes10020057

Chicago/Turabian Style

Gong, Panke, Haijing Liu, Dan Gou, Weixiao Di, Jiahao Cao, Jun Ding, Yaqing Chang, and Rantao Zuo. 2025. "Optimal Dietary Carbohydrates to Lipids Ratio for Fast and Coordinated Test Growth of Juvenile Sea Urchin (Strongylocentrotus intermedius)" Fishes 10, no. 2: 57. https://doi.org/10.3390/fishes10020057

APA Style

Gong, P., Liu, H., Gou, D., Di, W., Cao, J., Ding, J., Chang, Y., & Zuo, R. (2025). Optimal Dietary Carbohydrates to Lipids Ratio for Fast and Coordinated Test Growth of Juvenile Sea Urchin (Strongylocentrotus intermedius). Fishes, 10(2), 57. https://doi.org/10.3390/fishes10020057

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