An Evaluation of Chlorella (Chlorella pyrenoidosa) in the Feed of Juvenile Tiger Puffer (Takifugu rubripes)

Abstract

Microalgal protein demonstrates considerable potential for reducing dependence on fishmeal in aquafeeds. However, limited research has been conducted on the effects of Chlorella pyrenoidosa meal (CM) on carnivorous fish species. This study evaluated the impact of replacing 0% to 40% of fishmeal with CM within isonitrogenous and isolipidic diets (designated as CM0, CM5, CM10, CM20, and CM40) on juvenile tiger puffer (initial weight: 11.34 ± 0.01 g) over a 56-day feeding trial. Three replicate tanks were established for each experimental group, with 30 fish per tank. The experimental data were statistically analyzed using one-way analysis of variance (ANOVA) and regression analysis. The results showed that no significant differences were observed in final body weight (FBW: CM0, 55.45 ± 1.87 g; CM40, 45.6 ± 2.09 g) or specific growth rate (SGR: CM0, 2.83 ± 0.06%/d; CM40, 2.48 ± 0.08%/d) among the dietary groups; however, the growth linearly decreased with increasing CM levels. The CM40 group exhibited a significantly higher feed conversion ratio (FCR: CM0, 1.17 ± 0.02; CM40, 1.28 ± 0.03). Muscle texture parameters (hardness, adhesiveness, cohesiveness, springiness, gumminess, and chewiness) and proximate composition were not significantly affected by dietary treatments. In contrast, the CM40 diet significantly decreased the n-3/n-6 polyunsaturated fatty acid (PUFA) ratio in the muscle, while increasing the contents of 18:2n-6 and total n-6 PUFAs. These results indicate that substituting up to 5% of fishmeal with CM did not produce significant adverse effects on growth or body composition, although linear trends indicated a progressive decline in performance at higher inclusion levels. This investigation provides valuable insights into the application of sustainable protein alternatives in feeds for marine carnivorous fish.

1. Introduction

As the aquaculture industry continues to expand, the demand for fishmeal has risen correspondingly. Nevertheless, limited global fishmeal production and volatile market prices have intensified the need to identify sustainable, high-quality alternative protein sources [1]. Current strategies for fishmeal substitution in commercial aquafeeds primarily involve three categories: plant-derived proteins (predominantly soybean proteins and cereal by-products), animal-based proteins (including meals from livestock, poultry, and aquatic processing waste, as well as insect proteins), and single-cell proteins (such as those from bacteria, yeast, and microalgae) [1,2,3]. Although plant and animal proteins are relatively inexpensive and widely available, they exhibit several drawbacks, including high levels of antinutritional factors, low digestibility, imbalanced amino acid profiles, and potential adverse effects on farmed fish health [4,5]. In contrast, single-cell proteins—a novel resource derived mainly from fungi, bacteria, and microalgae—present notable advantages. Microorganisms exhibit rapid metabolic rates and high biomass accumulation efficiency, yielding substantially greater protein per unit volume than conventional crops. Moreover, microbial proteins generally contain 50–80% crude protein (dry matter basis) with a well-balanced amino acid profile [6], underscoring their significant potential as alternative ingredients in aquafeeds.

Chlorella (Chlorella spp., Chlorophyceae, Chlorophyta) can be cultivated at scale for protein production using organic nitrogen sources, achieving protein contents up to 60% on a dry weight basis, which qualifies it as a high-quality single-cell algal protein [7]. Furthermore, it contains a favorable amino acid profile, valuable n-3 polyunsaturated fatty acids, and bioactive compounds such as polysaccharides and natural pigments [8], all of which contribute positively to the growth and health of cultured fish. Notably, studies investigating the substitution of fishmeal with Chlorella meal have been conducted in several aquatic species. For instance, in rainbow trout (Oncorhynchus mykiss) [9] and crucian carp (Carassius auratus) [10], complete replacement of fishmeal with Chlorella meal did not compromise the growth performance and was even associated with enhanced outcomes. In gilthead seabream (Sparus aurata) [11], dietary inclusion of Chlorella meal successfully replaced up to 30% of fishmeal without adversely affecting the growth or feed utilization efficiency. However, in studies on Atlantic cod (Gadus morhua) [12], Barramundi (Lates calcarifer) [13], largemouth bass (Micropterus salmoides) [14] and Atlantic salmon (Salmo salar) [15], the optimal replacement ratio of algal protein for fishmeal ranges from 5% to 15%, indicating that algal protein still poses certain challenges for use in diets of carnivorous fish. Considering the above information, replacement ratios of 5% to 40% were selected in this study.

Tiger puffer (Takifugu rubripes) is an economically important carnivorous marine fish species in Asia. Previous studies indicate that its formulated feeds require a protein content of at least 45%, typically supplied by inclusion levels of fishmeal exceeding 40% [16,17,18]. This high reliance on fishmeal renders it a suitable model species for evaluating alternative protein sources. However, whether Chlorella pyrenoidosa, which possesses high protein content and a relatively thick polysaccharide cell wall, can replace fishmeal in the diet of juvenile tiger puffer remains unknown. In the present study, an 8-week feeding trial was conducted to investigate the effects of replacing fishmeal with incubated Chlorella pyrenoidosa meal on the growth performance, textural properties, proximate composition, fatty acid profiles, and amino acid compositions of tiger puffer. The results are expected to elucidate the suitable level of fishmeal replacement by Chlorella pyrenoidosa, and consequently provide valuable insights for the feasibility of microalgal proteins application in tiger puffer feeds and contribute to the development of sustainable algal-based ingredients for aquafeeds.

2. Materials and Methods

2.1. Experimental Diets

In this study, a control feed was formulated using fish oil (2.5%) and poultry oil (2%) as lipid sources. On the basis of the control formulation, partial replacement of fishmeal was performed by supplementing with varying proportions of Chlorella pyrenoidosa meal (CM), corresponding to 5%, 10%, 20%, and 40% replacement of fishmeal. Five isonitrogenous and isolipidic experimental feeds were thus prepared and labeled as CM0, CM5, CM10, CM20, and CM40, respectively. The cell wall of Chlorella pyrenoidosa was unbroken in this study. The CM contained 57.90% crude protein and 1.76% lipid (dry matter). After incubation, the Chlorella pyrenoidosa was concentrated with centrifuge. Then, the cell wall-breaking was conducted via a high-pressure homogenization at 100 bar for one time, after which the algae were spray-dried into meal. Approximately 20% of the cells were broken. All feed ingredients were ground and sieved through a 60-mesh screen before thorough mixing. Moisture was adjusted by adding approximately 30% water to facilitate pelleting. Pellets with a diameter of 2.5 mm were produced using a laboratory-scale pellet mill, then dried at 55 °C in a forced-air oven until the moisture content reached approximately 6%. After cooling, the pellets were packaged in double-layer plastic bags, sealed, and stored at −20 °C until use. The formulation and proximate composition of the experimental diets are summarized in

Table 1. Formulation and proximate composition of five experimental diets (% dry matter basis).

Ingredient	CM0	CM5	CM10	CM20	CM40
Fish meal 1	42	39.9	37.8	33.6	25.2
Soybean meal 1	15	15	15	15	15
Corn gluten meal 2	5	5	5	5	5
Chlorella pyrenoidosa meal	0.0	2.8	5.6	11.3	22.7
Wheat flour 1	28.4	27.6	26.8	25.1	21.6
Mineral premix 4	0.5	0.5	0.5	0.5	0.5
Vitamin premix 5	1	1	1	1	1
Monocalcium phosphate	1	1	1	1	1
Vitamin C	0.2	0.2	0.2	0.2	0.2
Choline chloride	0.2	0.2	0.2	0.2	0.2
Ethoxyquin	0.02	0.02	0.02	0.02	0.02
Yttrium oxide	0.1	0.1	0.1	0.1	0.1
Calcium propionate	0.1	0.1	0.1	0.1	0.1
Fish oil 1	2.5	2.6	2.7	2.9	3.4
Poultry oil 3	2	2	2	2	2
Soybean lecithin 1	2	2	2	2	2
Total	100	100	100	100	100
Proximate composition
Crude protein	44.60	44.58	44.43	44.47	44.43
Crude lipid	6.12	5.52	5.74	6.37	5.83
Moisture	6.10	5.47	6.33	6.76	6.90
Ash	10.60	10.39	10.11	9.81	9.03
Gross energy MJ/kg	23.06	23.07	22.92	23.22	23.39

¹ Fish meal, soybean meal, wheat flour, fish oil, soybean lecithin, were procured from Qingdao Surgreen Ocean Biological Feed Co., Ltd. (Qingdao, China), in which the purity of soybean lecithin was 50%. ² Corn gluten meal was purchased from Zhucheng Haotian Pharmaceutical Co., Ltd. (Weifang, China). ³ Poultry oil from Shandong Haiding Agriculture and Animal Husbandry Co., Ltd. (Jinan, China). ⁴ Purchased from Qingdao Master Biotechnology Co., Ltd. (Qingdao, China). The 0.5% marine fish trace element premix contained (expressed as content per kilogram of product): ferrous ≥ 37.14 g; zinc ≥ 16.43 g; manganese ≥ 3.03 g; copper ≥ 0.95 g; cobalt ≥ 0.1 g; selenium ≥ 0.04 g; iodine ≥ 0.1 g; moisture ≤ 10%. ⁵ Purchased from Qingdao Master Biotechnology Co., Ltd. The 1% seawater carnivorous fish vitamin premix contained (expressed as content per kilogram of product): vitamin A acetate ≥ 1,140,000 IU; vitamin D3 ≥ 180,000 IU; DL-α-tocopherol acetate ≥ 7.6 g; menadione nicotinamide bisulfite ≥ 1.2 g; thiamin nitrate ≥ 0.93 g; riboflavin ≥ 1.35 g; pyridoxine hydrochloride ≥ 1.10 g; cyanocobalamin ≥ 0.0075 g; D-calcium pantothenate ≥ 4.5 g; niacinamide ≥ 6.75 g; folic acid ≥ 0.465 g; D-biotin ≥ 0.0475 g; L-ascorbate-2-phosphate ≥ 16.7 g; inositol ≥ 10 g; moisture ≤ 10%.

. The fatty acid and amino acid profiles of the diets and key ingredients are presented in

Table 2. Fatty acids composition of the experimental diets and ingredients (% total fatty acids).

Fatty Acid	Diet					Ingredient
	CM0	CM5	CM10	CM20	CM40	FM	CM	PO
12:0	0.04	0.04	0.05	0.04	0.07	0.04	0.08	0.06
14:0	3.71	3.58	3.58	3.37	3.12	5.49	0.28	0.71
16:0	23.13	22.96	23.22	23.12	22.98	22.73	17.91	19.66
18:0	5.00	4.80	4.61	4.34	3.90	5.88	0.07	4.26
20:0	0.14	0.13	0.12	0.12	0.11	0.12	0.11	0.08
SFA	32.02	31.51	31.58	30.99	30.18	34.25	18.45	24.78
16:1n-7	4.14	4.01	3.94	3.83	3.46	5.45	3.78	3.01
18:1n-9	18.85	18.57	18.40	17.88	16.81	9.83	13.97	47.36
20:1n-9	0.79	0.75	0.75	0.72	0.65	1.15	0.04	0.01
22:1n-9	0.74	0.65	0.61	0.56	0.47	1.26	0.08	0.30
24:1n-9	0.06	0.08	0.06	0.07	0.06	0.08	0.00	0.14
MUFA	24.57	24.06	23.77	23.05	21.45	17.77	17.87	50.82
18:2n-6	16.01	16.72	16.95	17.57	18.80	2.72	26.99	21.60
20:2n-6	0.11	0.11	0.11	0.10	0.10	0.09	0.11	0.09
20:4n-6	0.28	0.26	0.24	0.24	0.21	0.49	0.03	0.00
n-6 PUFA	16.40	17.09	17.30	17.90	19.11	3.30	27.13	21.70
18:3n-3	1.62	2.11	2.58	3.45	5.73	0.95	25.13	0.80
20:5n-3	5.67	5.48	5.26	4.86	4.20	11.28	0.29	0.02
22:5n-3	0.87	0.86	0.79	0.78	0.60	1.90	0.07	0.04
22:6n-3	8.34	8.21	7.83	7.26	6.29	16.93	0.89	0.02
n-3 PUFA	16.49	16.66	16.46	16.34	16.81	31.05	26.39	0.88
n-3/n-6	1.01	0.97	0.95	0.91	0.88	9.40	0.97	0.04

SFA: saturated fatty acids; MUFA: mono-unsaturated fatty acids; n-6 PUFA: n-6 poly-unsaturated fatty acids n-6; n-3 PUFA: n-3 poly-unsaturated fatty acids.

and

Table 3. Amino acid composition of the experimental diets and ingredients (mg/g, dry matter).

Amino Acid	Diet					Ingredient
	CM0	CM5	CM10	CM20	CM40	FM	CM
Arg	24.79	23.83	23.96	24.55	24.63	41.15	33.16
His	13.61	12.79	12.59	12.61	11.26	24.05	9.65
Ile	18.55	17.40	17.89	17.80	16.45	28.78	15.80
Leu	34.15	32.07	32.76	33.36	32.09	47.75	34.25
Lys	29.73	28.11	28.66	28.71	27.27	56.03	33.30
Met	6.93	6.82	6.99	6.31	5.82	16.69	5.37
Phe	20.09	18.51	18.61	19.60	19.16	27.21	21.01
Thr	15.15	14.23	14.44	15.09	14.26	25.18	16.49
Val	19.94	18.73	19.60	19.89	19.30	31.18	24.08
EAA	182.95	172.49	175.51	177.92	170.25	298.02	193.11
Ala	22.99	21.99	22.58	23.87	24.40	38.34	37.60
Asp	35.06	32.30	33.34	33.80	32.19	55.38	33.04
Cys	7.69	6.88	9.71	8.50	9.51	9.76	8.50
Glu	67.54	62.45	63.41	64.16	59.84	81.74	49.24
Gly	23.16	21.98	22.10	22.18	21.12	42.32	25.20
Pro	24.19	22.33	21.80	21.87	21.38	28.49	21.03
Ser	15.21	14.17	14.43	14.89	14.33	22.35	13.95
Tau	7.40	5.55	6.06	6.03	4.51	8.44	3.22
Tyr	13.95	13.41	13.83	12.97	12.88	20.47	13.04
NEAA	209.80	195.51	201.19	202.24	195.65	298.84	201.60
TAA	392.75	368.01	376.70	380.16	365.90	596.86	394.71

EAA: essential amino acids; NEAA: non-essential amino acids; TAA: total amino acids.

, respectively.

2.2. Experimental Fish and Feeding Management

The experimental juvenile tiger puffers (Takifugu rubripes) were sourced from Tangshan Haidu Aquatic Products Co., Ltd. (Tangshan, China) and transported to the Langya Base of the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences. Prior to the formal trial, the fish were acclimatized to laboratory conditions for 30 days while being fed a commercial diet. To prevent cannibalism, these fish were lightly anesthetized with MS-222, and their teeth were carefully clipped. Subsequently, they were placed in a holding tank to recover. Following acclimation, a total of 450 healthy juveniles (initial average body weight: 11.34 ± 0.01 g) of uniform size were randomly distributed into 15 indoor aquaria (300 L each), with three replicate tanks per dietary treatment and 30 fish per tank. The feeding trial was conducted over 56 days in a recirculating seawater system. The fish were hand-fed to apparent satiation twice daily (07:30 and 18:00). Any uneaten feed was collected by siphoning approximately one hour after feeding, and the amount was recorded to calculate accurate feed consumption based on the average pellet weight. Water quality parameters, including temperature (maintained between 23–25 °C), salinity (22–29), pH (7.5–8.0), and dissolved oxygen (7 mg/L), were maintained consistent with levels reported by Wei et al. [16] throughout the experimental period. All animal handling and experimental procedures were reviewed and approved by the Animal Care and Use Committee of the Yellow Sea Fisheries Research Institute.

2.3. Collection of Samples

Upon conclusion of the 56-day feeding trial, all fish were fasted for 24 h. The total number and final body weight of surviving fish in each tank were recorded. Seven fish were then randomly sampled from each aquarium for analysis. After anesthetization with eugenol (diluted 1:10,000 in water), two fish per tank were used for the determination of whole-body proximate composition. The remaining five fish were used for tissue collection. Blood samples were drawn from the caudal vein, allowed to clot at 4 °C for 3–4 h, and subsequently centrifuged (3500 rpm, 10 min, 4 °C) to obtain serum. Liver, muscle, and midgut tissues were excised following dissection. All tissue and serum samples were immediately flash-frozen in liquid nitrogen and stored at −80 °C for subsequent analysis. Additionally, two fish per tank were randomly selected and preserved at −20 °C as backup specimens.

2.4. Proximate Composition and Muscle Texture Analysis

The proximate composition (crude protein, crude lipid, moisture, and ash) of the experimental diets and fish tissue samples was determined in accordance with the standard methods of AOAC (2005). Specifically, moisture content was quantified by drying samples to a constant weight at 105 °C; crude protein was determined by the Kjeldahl method using an automated nitrogen analyzer (FOSS Soxtec 2050, FOSS A/S, Hillerød, Denmark); crude lipid was extracted by the Soxhlet method using the same instrument; and ash content was measured by combustion in a muffle furnace (SX2-4-10, Longkou Electric Furnace Manufacturing Factory, Yantai, China) at 550 °C for 8 h. Muscle textural properties, including hardness, adhesiveness, cohesiveness, gumminess, springiness, and chewiness, were assessed using a texture analyzer (TMS-Pro, FTC, Texture Technologies Corp., Hamilton, MA, USA) fitted with a 25 N load cell. The analytical procedures and settings were consistent with those described by Wei et al. [16].

2.5. Analysis of Fatty Acid and Amino Acid Profiles

Fatty acid profiles were analyzed by gas chromatography (GC-2010 Pro, Shimadzu Corporation, Kyoto, Japan). Total lipids were first extracted from samples using a chloroform-methanol mixture. Fatty acids were then converted to fatty acid methyl esters (FAMEs) via saponification with KOH-methanol followed by methylation with boron trifluoride-methanol (BF₃-methanol). Separation was performed on a fused silica capillary column (SH-RT-2560, 100 m × 0.25 mm × 0.20 μm, Shimadzu Corporation, Kyoto, Japan) with a flame ionization detector. The oven temperature was programmed to increase from 150 °C to 200 °C at 15 °C/min, then from 200 °C to 250 °C at 2 °C/min. Individual fatty acids were identified by comparison with standards and expressed as a percentage of total fatty acids [19]. Amino acid composition was determined using an L-8900 automatic amino acid analyzer (Hitachi Ltd., Tokyo, Japan).

2.6. Statistical Analyses

All data were subjected to tests for homogeneity of variance (Levene’s test) and normality (Shapiro–Wilk test) prior to statistical analysis. A one-way analysis of variance (ANOVA) was performed using SPSS 16.0 software (IBM, Chicago, IL, USA) to evaluate treatment effects. Where significant differences were detected (p < 0.05), Tukey’s honestly significant difference (HSD) post hoc test was applied for multiple comparisons. Data are presented as mean ± standard error (SE). Regression models were selected primarily based on statistical significance (p < 0.05), with the coefficient of determination (R²) serving as a secondary criterion when comparing models of equivalent significance.

3. Results

3.1. Growth Performance and Somatic Indicators

The growth performance and body condition indices of tiger puffer are summarized in

Table 4. Effects of dietary C. pyrenoidosa meal on growth performance of juvenile tiger puffer.

Parameters	CM0	CM5	CM10	CM20	CM40	Regression
						Model	R2	p
IBW (g)	11.34 ± 0.01	11.34 ± 0.01	11.34 ± 0.04	11.34 ± 0.03	11.34 ± 0.01
FBW (g)	55.45 ± 1.87	55.04 ± 1.94	51.75 ± 4.77	50.74 ± 1.62	45.6 ± 2.09	linear	0.419	0.009
SR (%)	97.78 ± 1.11	94.44 ± 4.01	96.67 ± 0.00	97.78 ± 1.11	96.67 ± 1.92	quadratic	0.134	0.647
WGR (%)	389.11 ± 16.68	385.3 ± 16.68	356.11 ± 40.97	347.5 ± 15.09	302.06 ± 18.26	linear	0.426	0.008
PDR (%)	31.32 ± 1.30	31.21 ± 0.22	30.47 ± 1.15	30.54 ± 0.53	28.95 ± 0.54	linear	0.270	0.047
PER	1.92 ± 0.04	1.91 ± 0.01	1.88 ± 0.04	1.89 ± 0.05	1.75 ± 0.04	quadratic	0.534	0.033
FI (%)	81.35 ± 1.83	79.54 ± 1.35	79.51 ± 1.43	79.9 ± 0.9	80.33 ± 1.36	quadratic	0.105	0.737
FCR	1.17 ± 0.02 a	1.18 ± 0.00 ab	1.20 ± 0.02 ab	1.19 ± 0.03 ab	1.28 ± 0.03 b	quadratic	0.563	0.023
SGR	2.83 ± 0.06	2.82 ± 0.06	2.70 ± 0.15	2.67 ± 0.06	2.48 ± 0.08	linear	0.439	0.007
HSI (%)	8.24 ± 0.16	9.23 ± 0.27	8.85 ± 0.37	8.45 ± 0.44	8.63 ± 0.54	quadratic	0.162	0.198
VSI (%)	12.91 ± 0.42	13.6 ± 0.35	13.84 ± 0.27	12.86 ± 0.56	13.43 ± 0.68	quadratic	0.085	0.502
CF (g/cm3)	2.96 ± 0.15	2.85 ± 0.11	2.96 ± 0.10	2.93 ± 0.13	2.88 ± 0.19	quadratic	0.078	0.544

IBW: Initial weight; FBW: Final weight; SR: Survival rate; WGR: Weight gain ratio; PDR: Protein deposition rate; PER: Protein efficiency ratio; FI: Feed intake; FCR: Feed conversion ratio; SGR: Specific growth rate; HSI: Hepatosomatic index; VSI: Viscerosomatic index; CF: Condition factor. Data in the same row not sharing the same superscript were significantly different (p < 0.05).

. The feed conversion ratio (FCR) increased progressively with increasing dietary CM levels, and the CM40 group exhibited a significantly (p < 0.05) higher FCR than the control group (Table 4). This trend was further supported by a significant quadratic regression (p < 0.05). One-way ANOVA did not detect significant differences in final body weight, weight gain rate, protein deposition rate, protein digestibility, or specific growth rate among groups, while subsequent regression analysis revealed significant linear or quadratic relationships (p < 0.05) for these parameters. Survival rate, feeding rate, hepatosomatic index, viscerosomatic index, and condition factor were not significantly affected by dietary treatment.

3.2. Whole-Body and Tissue Proximate Composition

One-way ANOVA indicated no significant differences in the crude protein, crude lipid, moisture, or ash content of the whole body (

Table 5. Effects of dietary C. pyrenoidosa meal on the proximate composition of whole tiger puffer, liver and muscle (% wet weight).

Ingredient	Treatment					Regression
	CM0	CM5	CM10	CM20	CM40	Model	R2	p
Whole fish
Crude protein	15.5 ± 0.15	15.23 ± 0.26	15.25 ± 0.16	15.39 ± 0.14	15.4 ± 0.23	linear	0.001	0.903
Crude Lipid	5.11 ± 0.19	5.81 ± 0.07	5.21 ± 0.34	5.46 ± 0.51	5.69 ± 0.32	linear	0.055	0.400
Moisture	75.86 ± 0.14	74.93 ± 0.32	75.68 ± 0.26	74.99 ± 0.34	75.07 ± 0.13	linear	0.138	0.172
Ash	2.51 ± 0.05	2.55 ± 0.01	2.60 ± 0.04	2.72 ± 0.04	2.68 ± 0.08	linear	0.345	0.021
Muscle
Crude protein	18.32 ± 0.06	17.85 ± 0.19	18.27 ± 0.05	18.08 ± 0.12	18.1 ± 0.23	linear	0.009	0.744
Crude Lipid	1.93 ± 0.07	1.93 ± 0.08	2.02 ± 0.06	2.00 ± 0.06	2.04 ± 0.02	linear	0.156	0.145
Moisture	78.55 ± 0.02	79.01 ± 0.29	78.57 ± 0.10	78.84 ± 0.12	78.69 ± 0.29	linear	0.000	0.955
Liver
Crude protein	5.77 ± 0.01	5.61 ± 0.09	5.42 ± 0.04	5.30 ± 0.02	5.44 ± 0.22	quadratic	0.512	0.028
Crude Lipid	57.64 ± 0.57	54.87 ± 3.15	55.85 ± 1.37	54.77 ± 1.60	55.52 ± 1.40	linear	0.051	0.460
Moisture	30.93 ± 2.86	32.46 ± 0.31	33.20 ± 4.00	31.81 ± 0.91	33.32 ± 2.44	linear	0.021	0.604

). However, linear regression analysis showed that whole-body ash content was positively correlated with the dietary CM level (p < 0.05). In the liver, quadratic regression analysis indicated a significant effect on crude protein content (p < 0.05), which initially decreased and then increased with increasing CM inclusion.

3.3. Muscle Textural Properties

Neither one-way ANOVA nor regression analysis detected any significant (p > 0.05) differences in hardness, adhesiveness, cohesiveness, springiness, gumminess, or chewiness across dietary treatments (

Table 6. Effects of dietary C. pyrenoidosa meal on the muscle texture of juvenile tiger puffer.

Parameters	Treatment					Regression
	CM0	CM5	CM10	CM20	CM40	Model	R2	p
Hardness (N)	2.45 ± 0.29	2.93 ± 0.41	3.31 ± 0.47	3.27 ± 0.47	2.91 ± 0.28	quadratic	0.092	0.417
Adhesiveness (mJ)	0.06 ± 0.01	0.04 ± 0.00	0.05 ± 0.00	0.05 ± 0.01	0.04 ± 0.00	quadratic	0.120	0.287
Cohesiveness (Ratio)	0.39 ± 0.01	0.38 ± 0.02	0.42 ± 0.02	0.39 ± 0.02	0.37 ± 0.01	quadratic	0.102	0.365
Springiness (mm)	0.86 ± 0.08	1.07 ± 0.10	1.10 ± 0.10	1.13 ± 0.12	0.93 ± 0.09	quadratic	0.153	0.180
Gumminess (N)	0.93 ± 0.10	1.13 ± 0.19	1.41 ± 0.23	1.31 ± 0.21	1.11 ± 0.13	quadratic	0.120	0.287
Chewiness (mJ)	0.81 ± 0.14	1.25 ± 0.26	1.65 ± 0.37	1.59 ± 0.33	1.07 ± 0.21	quadratic	0.175	0.129

).

3.4. Muscle Fatty Acid Composition and Amino Acid Composition

Cubic regression analysis showed that dietary CM level significantly (p < 0.05) influenced the contents of total saturated fatty acids (SFA), total n-6 polyunsaturated fatty acids (n-6 PUFA), and the n-3/n-6 PUFA ratio (

Table 7. Effects of dietary C. pyrenoidosa meal on the muscle fatty acid composition of tiger puffer (% total fatty acids).

Fatty Acid	Treatment					Regression
	CM0	CM5	CM10	CM20	CM40	Model	R2	p
12:0	0.26 ± 0.02 b	0.20 ± 0.01 a	0.23 ± 0.01 ab	0.21 ± 0.01 a	0.20 ± 0.01 a	cubic	0.566	0.023
14:0	0.48 ± 0.02	0.54 ± 0.05	0.46 ± 0.07	0.49 ± 0.03	0.40 ± 0.03	cubic	0.235	0.380
16:0	23.78 ± 0.12	22.98 ± 0.25	23.64 ± 0.14	23.82 ± 0.30	23.76 ± 0.25	cubic	0.427	0.094
18:0	7.95 ± 0.14	7.76 ± 0.12	7.75 ± 0.13	8.26 ± 0.20	7.65 ± 0.12	cubic	0.404	0.115
20:0	0.11 ± 0.01	0.13 ± 0.00	0.12 ± 0.00	0.12 ± 0.00	0.12 ± 0.00	cubic	0.371	0.15
SFA	32.58 ± 0.10 ab	31.61 ± 0.19 a	32.20 ± 0.11 ab	32.90 ± 0.47 b	32.13 ± 0.31 ab	cubic	0.562	0.024
16:1n-7	0.85 ± 0.01	0.95 ± 0.06	0.87 ± 0.01	0.91 ± 0.05	0.86 ± 0.01	cubic	0.164	0.56
18:1n-9	14.55 ± 0.08	14.36 ± 0.14	14.31 ± 0.13	14.42 ± 0.28	14.21 ± 0.07	cubic	0.194	0.48
20:1n-9	0.06 ± 0.02	0.12 ± 0.04	0.11 ± 0.04	0.08 ± 0.03	0.06 ± 0.03	cubic	0.209	0.441
22:1n-9	2.02 ± 0.08 b	2.05 ± 0.07 b	1.95 ± 0.05 ab	1.83 ± 0.02 ab	1.73 ± 0.05 a	cubic	0.703	0.003
24:1n-9	0.17 ± 0.01	0.20 ± 0.02	0.15 ± 0.00	0.16 ± 0.01	0.15 ± 0.01	cubic	0.251	0.347
MUFA	17.66 ± 0.05	17.68 ± 0.05	17.38 ± 0.19	17.4 ± 0.31	17.01 ± 0.08	linear	0.422	0.009
18:2n-6	11.75 ± 0.10 a	11.98 ± 0.41 a	11.88 ± 0.29 a	12.25 ± 0.15 a	13.56 ± 0.12 b	linear	0.546	0.002
20:2n-6	0.48 ± 0.02	0.55 ± 0.00	0.50 ± 0.01	0.49 ± 0.02	0.51 ± 0.02	cubic	0.428	0.094
20:4n-6	0.12 ± 0.01	0.14 ± 0.01	0.14 ± 0.01	0.13 ± 0.01	0.11 ± 0.01	quadratic	0.394	0.049
n-6 PUFA	12.35 ± 0.11 a	12.67 ± 0.42 a	12.52 ± 0.29 a	12.87 ± 0.13 a	14.18 ± 0.12 b	cubic	0.779	0.001
18:3n-3	0.41 ± 0.03 a	0.55 ± 0.04 a	0.55 ± 0.02 a	0.70 ± 0.03 b	1.08 ± 0.03 c	cubic	0.96	0.000
20:5n-3	4.85 ± 0.04	4.79 ± 0.12	5.00 ± 0.13	4.76 ± 0.08	4.56 ± 0.11	cubic	0.394	0.125
22:5n-3	2.26 ± 0.02	2.36 ± 0.08	2.30 ± 0.05	2.18 ± 0.06	2.19 ± 0.02	cubic	0.468	0.065
22:6n-3	20.14 ± 0.26	19.93 ± 0.60	19.77 ± 0.60	19.26 ± 0.40	19.22 ± 0.26	linear	0.235	0.067
n-3 PUFA	27.66 ± 0.24	27.64 ± 0.52	27.62 ± 0.53	26.90 ± 0.43	27.05 ± 0.38	linear	0.161	0.138
n-3/n-6	2.24 ± 0.04 b	2.19 ± 0.11 ab	2.21 ± 0.04 b	2.09 ± 0.05 ab	1.91 ± 0.03 a	cubic	0.645	0.008

Notes: SFA: saturated fatty acids; MUFA: mono-unsaturated fatty acids; n-6PUFA: n-6 poly-unsaturated fatty acids n-6; n-3 PUFA: n-3 poly-unsaturated fatty acids. Data in the same row not sharing the same superscript were significantly different (p < 0.05).

). The content of monounsaturated fatty acids (MUFAs) followed a quadratic pattern, initially increasing and then decreasing (p < 0.05). Both linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3) exhibited cubic trends, decreasing initially before increasing again at higher CM levels (p < 0.05).

With the exception of taurine, which decreased linearly with increasing CM inclusion (p < 0.05), the contents of all other detected amino acids remained unaffected by the dietary treatments (

Table 8. Effects of dietary C. pyrenoidosa meal on the muscle amino acid composition of tiger puffer (mg/g, dry matter).

Amino Acid	Treatment					Regression
	CM0	CM5	CM10	CM20	CM40	Model	R2	p
Arg	51.97 ± 0.16	51.45 ± 0.28	52.38 ± 1.00	53.00 ± 1.21	51.73 ± 0.97	quadratic	0.171	0.542
His	19.64 ± 0.17	20.00 ± 0.08	20.09 ± 0.49	20.08 ± 0.37	19.51 ± 0.07	cubic	0.243	0.188
Ile	38.37 ± 0.29	38.72 ± 0.25	38.57 ± 0.49	39.61 ± 0.74	38.06 ± 0.46	quadratic	0.254	0.339
Leu	62.14 ± 0.66	62.74 ± 0.39	62.22 ± 0.85	64.01 ± 0.73	61.75 ± 0.70	quadratic	0.222	0.412
Lys	79.31 ± 0.42	79.49 ± 0.35	79.57 ± 1.21	81.55 ± 1.11	78.61 ± 0.34	quadratic	0.335	0.197
Met	22.30 ± 0.08	18.75 ± 2.11	21.67 ± 0.57	20.76 ± 1.01	21.93 ± 0.16	quadratic	0.22	0.416
Phe	32.76 ± 0.05	32.10 ± 0.17	32.45 ± 0.86	32.94 ± 0.18	32.74 ± 0.59	quadratic	0.158	0.577
Thr	32.29 ± 0.39	32.66 ± 0.35	32.19 ± 0.74	33.67 ± 0.89	31.90 ± 0.48	quadratic	0.173	0.537
Val	39.40 ± 0.18	39.59 ± 0.16	39.70 ± 0.49	40.42 ± 0.80	39.10 ± 0.43	quadratic	0.252	0.343
EAA	378.18 ± 1.90	375.51 ± 2.63	378.82 ± 6.28	386.04 ± 4.77	375.33 ± 4.08	quadratic	0.275	0.297
Ala	42.98 ± 0.17	42.55 ± 0.12	43.82 ± 0.68	44.72 ± 0.71	43.32 ± 0.78	quadratic	0.463	0.068
Asp	72.31 ± 0.90	72.75 ± 0.54	72.71 ± 1.33	75.41 ± 1.69	72.38 ± 0.81	quadratic	0.247	0.355
Cys	11.12 ± 0.77	12.05 ± 0.94	13.80 ± 1.19	11.60 ± 0.93	12.13 ± 1.16	quadratic	0.156	0.584
Glu	113.36 ± 1.93	113.89 ± 1.15	113.05 ± 2.23	117.90 ± 3.33	112.73 ± 0.93	quadratic	0.17	0.543
Gly	39.66 ± 1.01	38.82 ± 0.99	41.38 ± 1.32	41.98 ± 1.56	41.05 ± 1.65	quadratic	0.257	0.333
Pro	28.97 ± 0.28	27.46 ± 0.38	29.02 ± 0.73	29.03 ± 0.55	27.51 ± 1.15	quadratic	0.327	0.209
Ser	27.40 ± 0.28	27.49 ± 0.19	27.43 ± 0.62	28.72 ± 0.68	27.11 ± 0.43	quadratic	0.282	0.285
Tau	17.12 ± 1.12 a	15.60 ± 0.19 ab	14.66 ± 0.63 ab	14.44 ± 0.25 ab	12.81 ± 0.52 b	linear	0.678	0.000
Tyr	26.90 ± 0.20	26.48 ± 0.68	26.48 ± 0.45	27.35 ± 0.26	27.28 ± 0.30	quadratic	0.263	0.321
NEAA	362.69 ± 3.26	361.49 ± 2.61	367.68 ± 5.82	376.7 ± 9.05	363.52 ± 5.21	quadratic	0.312	0.232
TAA	740.88 ± 5.16	737.00 ± 3.46	746.51 ± 11.98	762.74 ± 13.82	738.85 ± 9.29	quadratic	0.305	0.244

Note: EAA: essential amino acids; NEAA: non-essential amino acids; TAA: total amino acids. Data in the same row, not sharing the same superscript were significantly different (p < 0.05).

).

4. Discussion

The feasibility of Chlorella meal (CM) as a partial fishmeal substitute has been demonstrated in several teleost species, with studies reporting that 15.03% replacement can enhance growth performance in largemouth bass [14]. In contrast, the present study on tiger puffer revealed a different pattern. Although no significant differences in weight gain rate (WGR) were detected by one-way ANOVA, regression analyses indicated a significant linear decrease in both WGR and specific growth rate (SGR). coupled with a linear increase in feed conversion ratio (FCR), as dietary CM levels rose. This suggests an adverse effect on growth performance at higher levels.

This discrepancy may be attributed to two primary factors. First, the carnivorous nature and high trophic level (3.6) of tiger puffer likely play a critical role. Carnivorous fishes like tiger puffer typically possess low amylase activity compared to protease activity, limiting their capacity to digest carbohydrates such as starch present in alternative ingredients [16]. Similar growth reductions with plant-based substitutes have been observed in other carnivorous species, including large yellow croaker (Larimichthys crocea) [20], rainbow trout (Oncorhynchus mykiss) [21], and black seabream (Acanthopagrus schlegelii) [11]. Second, the integrity of the Chlorella cell wall in the CM used may be a limiting factor. Chlorella pyrenoidosa possesses a rigid, non-digestible cell wall that is resistant to enzymatic degradation in fish lacking cellulase [22]. The high-pressure homogenization (100 bar) applied in this study may have been insufficient for complete cell wall disruption, thereby reducing the bioavailability of intracellular nutrients. This is corroborated by the observed linear decrease in protein deposition rate and protein efficiency ratio. Supporting this notion, studies on Atlantic salmon have shown that using cell wall-disrupted Chlorella meal allows for higher inclusion levels without compromising amino acid digestibility compared to whole-cell meal [23]. Furthermore, supplementing diets with cellulase has been shown to improve the utilization of Chlorella meal in crucian carp [10].

Regarding whole-body composition, a significant linear increase in ash content was associated with higher CM inclusion. This may be explained by the retention of mineral-rich, unconsumed incubation medium in the spray-dried CM product. Additionally, the inherent mineral profile of C. pyrenoidosa differs considerably from that of fishmeal; notably, it has a much lower calcium content and a reversed calcium-to-phosphorus ratio (0.14 in C. pyrenoidosa vs. 1.3 in fishmeal) [24]. Notably, when the experimental fish samples were obtained, a marked increase in the caudal vertebral deformity ratio in fish was observed as the proportion of Chlorella meal replacing fishmeal increased. Unfortunately, this ratio was not specifically statistically recorded. These compositional differences likely contributed to the altered mineral deposition and may represent a latent factor affecting growth.

While one-way ANOVA indicated no significant differences in most muscle fatty acids and amino acids, regression analysis revealed significant correlations between dietary CM level and specific nutrients. Significant linear or polynomial trends were observed for certain fatty acids (SFA, MUFA, n-6 PUFA, 18:2n-6, and 18:3n-3). It is noteworthy that the taurine content in fish muscle decreased with increasing CM substitution levels, exhibiting a highly significant linear downward trend. This may be attributed to the lower taurine content in CM compared to fish meal. Previous studies have reported that dietary taurine deficiency can result in reduced growth performance [25,26], which may also be a non-negligible factor contributing to the decreased growth performance observed in the present study. In general, the muscle composition often reflects dietary profiles [27], a phenomenon documented in species such as Nile tilapia (Oreochromis niloticus) [28], turbot (Scophthalmus maximus) [29], and largemouth bass (Micropterus salmoides) [30].

Compared to other chlorella species, still little information has been available for the use of C. pyrenoidosa in fish feeds. This prevented an in-depth discussion of the present results. However, considering the high protein content and relatively balanced nutrient compositions, more future studies are indeed needed about the application of C. pyrenoidosa in fish feeds. C. pyrenoidosa with broken cell wall is highly recommended in future studies.

5. Conclusions

In conclusion, this study indicates that replacing no more than 5% of fishmeal with C. pyrenoidosa meal did not produce significant adverse effects on growth or body composition, although linear trends indicated a progressive decline in performance at higher inclusion levels. High replacement levels (for example, 40%) significantly decreased the feed efficiency.

The rigid cell wall of Chlorella is proposed to be a major constraint on nutrient bioavailability in this carnivorous species. Future research should prioritize optimizing physical disruption techniques (e.g., high-pressure homogenization) or employing enzymatic hydrolysis to break down the cell wall effectively. Such advancements are crucial for enhancing the digestibility of CM and unlocking its potential as a sustainable alternative protein source in feeds for carnivorous fish. Although one may face great difficulty in collection of high-quality feces, the analysis of the apparent digestibility coefficient is highly necessary in efficacy evaluation of dietary algae. Novel methods should be developed for feces collection in tiger puffer, and activity analysis of digestive enzymes can be used as an auxiliary evaluation method.