Enrichment of Artemia nauplii with squid and catfish oil by-products enhances survival, growth, and stress resistance in Thai mahseer (Tor tambroides) larvae

O enriquecimento de náuplios de Artemia com subprodutos de óleo de lula e peixe-gato aumenta a sobrevivência, o crescimento e a resistência ao estresse em larvas de mahseer tailandês (Tor tambroides)

Abstract

The potential of enriched live feeds, such as Artemia nauplii, to enhance the growth performance of Thai mahseer (Tor tambroides) larvae during the larviculture phase is not yet fully understood. This study evaluated the effects of Artemia enriched with squid oil (FSO) and Pangasius catfish oil (FCO) emulsions- on proximate and fatty acid composition of Artemia salina nauplii and larval, larval survival, growth, and tolerance to air exposure stress. Six enrichment treatments with varying FSO and FCO proportions were prepared: P0 (control), P1, P2, P3, P4, and P5. Feeding trials were conducted for 21 days using standardized larval densities, and fatty acid profiles were analyzed via gas chromatography. Results showed that Artemia salina nauplii enriched with P2 had the highest eicosapentaenoic acid (EPA, C20:5n-3), P1 had the highest docosahexaenoic acid (DHA, C22:6n-3), and P5 contained the highest oleic acid (C18:1n-9) and linoleic acid (C18:2n-6). Among all treatments, P1 enrichment most effectively improved larval survival, growth, and stress tolerance. Larvae fed P1 Artemia salina nauplii also exhibited higher DHA and highly unsaturated fatty acids (HUFA) levels compared to other treatments. Under these experimental conditions, P1 produced a DHA to EPA ratio of 2.89 in Artemia salina nauplii, making it the most suitable option for early feeding of Thai mahseer larvae.

Keywords:
live feed enrichment; lipid metabolism; aquaculture sustainability; stress tolerance; larval performance

Resumo

O potencial de alimentos vivos enriquecidos, como náuplios de Artemia, para melhorar o desempenho de crescimento das larvas de mahseer tailandês (Tor tambroides) durante a fase de larvicultura ainda não é totalmente compreendido. Este estudo avaliou os efeitos dos náuplios de Artemia salina enriquecidos com emulsões de óleo de lula (FSO) e óleo de bagre Pangasius (FCO) sobre a composição de proximate e ácidos graxos dos náuplios de Artemia salina e das larvas, bem como sobre a sobrevivência, o crescimento e a tolerância ao estresse por exposição ao ar. Foram preparadas seis formulações de enriquecimento com proporções variadas de FSO e FCO: P0 (controle), P1, P2, P3, P4 e P5. Ensaios de alimentação foram conduzidos por 21 dias utilizando densidades larvais padronizadas, e os perfis de ácidos graxos foram analisados por cromatografia gasosa. Os resultados mostraram que os náuplios de Artemia salina enriquecidos com P2 apresentaram o maior teor de ácido eicosapentaenoico (EPA, C20:5n-3); com a formulação P1, apresentaram o maior teor de ácido docosahexaenoico (DHA, C22:6n-3), e na P5, continham os maiores teores de ácido oleico (C18:1n-9) e ácido linoleico (C18:2n-6). Entre todos os tratamentos, o enriquecimento P1 foi o mais eficaz para melhorar a sobrevivência das larvas, o seu crescimento e a sua tolerância ao estresse. Larvas alimentadas com náuplios de Artemia salina P1 também apresentaram níveis mais elevados de DHA e ácidos graxos altamente insaturados (HUFA) em comparação com os outros tratamentos. Nestas condições experimentais, a proporção P1 produziu uma razão DHA/EPA de 2,89 nos náuplios de Artemia salina, tornando-a a opção mais adequada para a alimentação inicial das larvas de mahseer tailandês.

Palavras-chave:
enriquecimento de ração viva; metabolismo lipídico; sustentabilidade da aquicultura; tolerância ao estresse; desempenho larval

1. Introduction

Artemia is a microscopic organism inhabiting saline waters and has long been used as a primary live feed in the commercial hatchery production of fish and crustaceans (Wei et al., 2020; Choi et al., 2021; Zhang et al., 2025). Artemia has also been increasingly applied in freshwater fish culture due to its nutritional value in supporting larval survival and growth (Prusinska et al., 2020; Wang et al., 2022). However, newly hatched Artemia nauplii contain very low levels of ω-3 long-chain polyunsaturated fatty acids (HUFAs), particularly docosahexaenoic acid (DHA), which is essential for nervous system development and overall larval physiology (Choi et al., 2021; Morshedi et al., 2022).

To address this limitation, various enrichment techniques have been developed, including the use of commercial fish oils, microalgae, and other bioactive compounds (Monroig et al., 2006; El-Dahhar et al., 2024). The success of enrichment depends on several factors such as the lipid source, environmental conditions, and the Artemia strain (Han et al., 2000; Azimirad and Meshkini 2017). Innovative approaches reported include combining selenium with DHA (Pham et al. 2023), bioencapsulation with Chlorella vulgaris extract (Saputra et al., 2025), and the use of microalgae and cyanobacteria derived from industrial waste (Bertini et al., 2024), different dietary lipid sources (Ren et al., 2020). Nevertheless, studies utilizing combinations of locally available fishery by-products as lipid sources for Artemia enrichment remain scarce.

Among potential lipid sources, catfish oil (Pangasius spp.) and squid oil provide complementary fatty acid profiles. Catfish oil is rich in saturated fatty acids such as palmitic acid (C16:0) and monounsaturated fatty acids (MUFA), which serve as energy sources for Artemia metabolism (Aryani et al., 2023), but it contains relatively low levels of EPA and DHA. In contrast, squid oil is high in long-chain polyunsaturated fatty acids, particularly DHA (Hossain et al., 2024a). Therefore, combining these two oils may yield a more balanced fatty acid composition and improve the nutritional quality of enriched Artemia. To date, no study has investigated the use of squid oil and catfish oil, derived from local fish processing by-products, as enrichment ingredients.

Thai mahseer (Tor tambroides) is a high-value freshwater fish species cultured in Indonesia and Malaysia, but its aquaculture faces challenges due to the limited availability of high-quality fry (Akmal et al., 2022; Azrita et al., 2025). During the larval stage, survival and growth are strongly influenced by nutritionally rich live feeds such as Artemia, Moina, and Daphnia (Asaduzzaman et al. 2016; Joshua et al., 2024). This study aims to evaluate the effectiveness of enriching Artemia nauplii with a combination of squid oil (FSO) and catfish oil (FCO) derived from local fish processing by-products as live feed for Thai mahseer larvae. The evaluation focuses on improvements in HUFA content of Artemia and their subsequent effects on proximate composition and lipid deposition in the larvae. The working hypothesis is that the FSO–FCO combination will enhance HUFA levels in Artemia, improve overall nutritional quality, and promote better growth and lipid deposition in Thai mahseer larvae.

2. Materials and Methods

2.1. Oil sources

Squid oil was extracted from processing waste (residual meat trimmings) using the indirect heating technique (wet rendering). Approximately 1 kg of raw material was mixed with water at a ratio of 1:2 (w/v) and heated at 60-70 °C for 45 minutes. The oil phase was then separated by gravimetric settling for 30 minutes, following the procedure (Ackman, 1989) in Fish Oils and Lipids in the Seafood Industry. The average oil yield obtained was about 8–10% (w/w) of the wet raw material.

Oil from Pangasius catfish was extracted from mesenteric organs using a modified method of Aryani et al. (2023). About 500 g of fresh mesenteric tissue was homogenized with distilled water (1:3 w/v), then heated at 60 °C for 40 minutes. The released oil was separated by decantation, yielding approximately 12–15% (w/w) of the initial tissue weight. The fatty acid composition of both squid oil (FSO) and pangasius catfish oil (FCO), expressed as fatty acid methyl esters (FAMEs), is presented in Table 1.

Table 1
Fatty acid composition (% of total fatty acids) of FSO and FCO.

Fatty acids	FSO	FCO
16:0	30.45	2.98
18:0	1.45	5.65
18:1n-9c	2.55	30.52
C18:1n-9t	0.0	16.31
18:2n-6 (LA)	1.2	18.08
20:2n-6	0.56	7.31
C20:4n-6 (ARA)	1.10	0.63
C20:5n-3 (EPA)	36.65	0.16
C22:6n-3 (DHA)	9.75	0.18
Others1	16.29	18.18
Total	100	100

• ¹
  Others include minor fatty acids not individually identified in this analysis.

2.2. Hatching of Artemia cysts

Artemia sp. cysts (Supreme Plus, Golden West Artemia, INVE Aquaculture, Belgium) were incubated for 24 hours in 25 L conical–cylindrical tanks filled with seawater (27 g/L) under strong aeration at 28°C. Newly hatched nauplii were harvested, rinsed, and transferred to 4 L containers (one for each emulsion treatment) filled with clean seawater. Moderate aeration and an automatic oxygen control system were used to maintain dissolved oxygen at 6.0 ± 0.2 ppm and a constant temperature of 28°C.

2.3. Experiment 1: Evaluation of emulsions containing FSO and FCO at various ratios

Enrichment was performed by mixing 1.1 mL of the emulsion from each treatment into 1000 mL of saline water (27 g/L), followed by homogenization for 1 minute. This mixture was then added to individual enrichment containers (beakers) containing Artemia. Oxygen supply was automatically regulated to maintain a dissolved oxygen level of 6.0 ± 0.2 ppm, while the temperature was kept constant at 28°C. All enrichment treatments were conducted under standardized conditions, including an 18-hour enrichment duration, a nauplii density of 500,000 individuals per liter, and a fixed emulsion volume, based on a modified method from Roo et al. (2023)

Six treatments were designed to evaluate the effects of combining squid oil (FSO) and pangasius catfish oil (FCO) in the enrichment of Artemia salina nauplii for 18 hours, as follows (Table 2).

Table 2
Enrichment treatments of Artemia nauplii with varying ratios of FSO and FCO.

Experiment	Combination of FSO and FCO
P0	Artemia salina nauplii were not enriched with FSO or FCO after hatching.
P1	Artemia salina nauplii were maintained in 1000 mL of saline water containing 100% FSO, with the addition of 0.1 mL FSO and 0.1 mL egg yolk.
P2	Artemia salina nauplii were added to 1000 mL of saline water containing a mixture of 75% FSO and 25% FCO (0.075 mL FSO and 0.025 mL FCO), supplemented with 0.1 mL of egg yolk
P3	Artemia salina nauplii were added to 1000 mL of saline water containing a mixture of 50% FSO and 50% FCO (0.050 mL FSO and 0.050 mL FCO), supplemented with 0.1 mL of egg yolk
P4	Artemia salina nauplii were added to 1000 mL of saline water containing a mixture of 25% FSO and 75% FCO (0.025 mL FSO and 0.075 mL FCO), supplemented with 0.1 mL of egg yolk
P5	Artemia salina nauplii were added to 1000 mL of saline water containing 100% FCO, supplemented with 0.1 mL of FCO and 0.1 mL of egg yolk.

2.4. Larval rearing

Broodstock of Thai mahseer maintained at the Wet Laboratory of the Faculty of Fisheries and Marine Science, Bung Hatta University, Padang, West Sumatra, Indonesia, were used as the source of eggs and sperm. Artificial fertilization and egg incubation were conducted in glass aquaria using standard protocols commonly applied to mahseer species (Azrita et al., 2025). Hatched larvae (3 days post-hatch) were reared in 54-L glass aquaria containing 25 L of freshwater. Treated and filtered water was maintained at 28 ± 1 °C with a dissolved oxygen level of 6.0 ± 0.50 mg/L, and a stocking density of 10 individuals per litre (250 ind /glass aquarium. Half of the water volume was replaced daily.

During the rearing period from 7 to 27 days post-hatching (dph) (21 days), larvae were provided with Artemia salina nauplii enriched with different combinations of squid oil (FSO) and pangasius catfish oil (FCO) at a density of 1,300 individuals/mL. Larvae were stocked at a density of 10 individuals/L (250 individuals per aquarium). From 7 to 14 days post-hatch (dph), larvae were fed 1.0 mL of enriched Artemia salina nauplii (1,300 individuals/mL) every six hours.. Between 14 and 21 dph, each larva received 1.5 mL of enriched Artemia (approximately 1,950 individuals) at six-hour intervals. From 21 to 27 dph, the feeding volume was increased to 2.0 mL (around 2,600 individuals) per feeding, also given every six hours.

2.5. Larval growth and survival measurement

Dead larvae were monitored daily by siphoning the bottom of the aquarium. Survival rate was calculated as the ratio of the number of surviving larvae to the initial number of larvae at the beginning of the feeding trial. To evaluate the effect of Artemia nauplii nauplii enrichment treatments on the growth of Tor tambroides larvae were taken. A random sample of 50 larvae was collected from each aquarium for this purpose. The final body weight was measured with an accuracy of 1 mg mm using a Analytical Balance FUJITSU (Jepang). After measurement, the larvae were homogenized and stored in a freezer at −70°C until further chemical analysis.

2.6. Air exposure test

At the conclusion of the feeding trial (21 days of rearing), an acute air exposure challenge was conducted using 50 larvae from each aquarium. The larvae were gently collected with a fine-mesh net and transferred to a tank (40 × 30 × 20 cm) without water for 60 seconds. After exposure, they were returned to their respective tanks supplied with aerated freshwater. Survival was recorded 24 hours later, following a procedure modified from (Choi et al., 2021).

2.7. Chemical analysis

Triplicate samples of the experimental emulsions and enriched Nauplii of Artemia salina nauplii were collected for chemical analysis. The proximate composition of the emulsions, enriched Artemia salina nauplii, and the pooled whole bodies of Thai mahseer larvae from each treatment group were analyzed using standard procedures outlined by the Association of Official Analytical Chemists (AOAC, 1990). Crude lipid content was determined using a Soxtec extractor (ST 243 Soxtec; FOSS, Hillerod, Denmark). Moisture content was measured by drying the samples in an oven at 105°C until a constant weight was achieved. For fatty acid composition analysis, total lipids from the experimental emulsions, enriched Artemia salina nauplii, and whole larval bodies were extracted based on the method described by Folch et al. (1957), using a chloroform: methanol mixture (2:1, v/v). The extracted lipids were then subjected to acid-catalyzed transmethylation using BF₃–MeOH (Sigma, St. Louis, MO, USA) to obtain fatty acid methyl esters. Fatty acid profiles were analyzed by gas chromatography using a PerkinElmer Clarus 600 system (Shelton, CT, USA) equipped with a flame ionization detector and an SP-2560 capillary column (100 m length × 0.25 mm internal diameter, 0.2 μm film thickness; Supelco, Bellefonte, PA, USA), following the procedure outlined by Xue et al. (2006).

2.8. Survival rate, growth variables

An air exposure stress test was performed 24 h after the feeding trial. Survival rate (SR), initial body weight (W₀), final body weight (Wₜ), initial length (L₀) and final total length (L_t) were calculated using the following Equations 1, 2, 3:

$$\begin{array}{c}\text{Survival rates}\left(\text{SR}\text{, \%}\right)\text{=}\\ \text{100 × final larval number /initial larval number}\end{array}$$ (1)

$$\text{Weight gain}\left(\text{WG}\text{, mg}\right)\text{= Wt – Wo}$$ (2)

$$\text{Length gain}\left(\text{LG}\text{, mm}\right)\text{=}\left({\text{L}}_{\text{t}}{\text{– L}}_{\text{0}}\right)$$ (3)

2.9. Statistical analysis

All data were statistically analysed using SPSS software version 15.0 (SPSS Inc., Chicago, IL, USA). Results are presented as means ± standard deviation. The effects of dietary combination treatment levels on proximate composition, fatty acid profiles, survival, and growth were evaluated. The influence of dietary EPA levels was assessed using regression analysis and one-way analysis of variance (ANOVA) at a 5% significance level. Prior to analysis, data were tested for normality and homogeneity of variance using the Kolmogorov–Smirnov test and Levene’s test, respectively, as described by (Sokal and Rohlf, 1995).

3. Results and Discussions

3.1. Proximate composition and fatty acid profiles of enriched Artemia nauplii

The proximate and fatty acid composition of Artemia salina nauplii diets enriched with a combination of FSO and FCO corresponded to the composition of the experimental emulsions applied (Table 3). In P0 (control), the proportions of SFA (12.90%) and MUFA (19.96%) were relatively balanced, while PUFA remained low (4.46%). In P1 and P2, with enrichment predominantly from FSO, PUFA levels increased significantly (12.76% and 11.46%) compared to the control, consistent with the high PUFA content of FSO, although MUFA also showed a moderate rise (22.29–23.58%). The results of this study are consistent with previous reports indicating that the type and proportion of enrichment oils strongly influence the fatty acid profile of Artemia salina nauplii, particularly the levels of SFA, MUFA, PUFA, and the EPA/DHA ratio. In line with this, Haque et al. (2024) reported that squid oil contains approximately 45.8% PUFA, 26.4% MUFA, and 25.0% SFA. In this study, the palmitic acid (C16:0) content was higher in Artemia salina nauplii enriched with P1 (75% FSO:25% CFO), with a declining trend observed as the proportion of squid oil decreased from P2 to P5. According to Hossain et al. (2024a), squid oil contains 32.60% palmitic acid. This indicates that the palmitic acid content in Artemia salina nauplii is strongly influenced by the proportion of squid oil in the enrichment emulsion. Palmitic acid plays an important role as an energy source and as a structural component of cell membranes; however, excessive levels may disrupt the balance of essential fatty acids such as EPA and DHA.

Table 3
Proximate composition (%, wet weight basis) and fatty acid profile (% of total fatty acids) of Artemia nauplii following 24 h enrichment with different emulsions.

	P0	P1	P2	P3	P4	P5	P-values
Crude lipid	7.3±0.02	18.2±0.60	17.5±0.30	14.5±0.30	12.8±0.35	12.3±0.20	0.000
Moisture	72.6±0.81	72.5±0.62	75.80±0.62	72.4±1.64	68.8±0.31	67.5±0.30	0.000
Fatty acids*
C10:0	0.03±0.01a	0.04±0.00a	0.02±0.01a	0.03±0.00a	0.03±0.01a	0.02±0.01a	0.061
C12:0	0.10±0.01a	0.13±0.01b	0.11±0.02ac	0.10±0.01ad	0.17±0.05be	0.05±0.01cf	0.002
C14:0	0.75±0.01a	0.77±0.01b	0.73±0.02c	0.73±0.03ad	0.64±0.05e	0.55±0.02f	0.000
C15:0	0.25±0.01a	0.29±0.01b	0.27±0.01	0.27±0.01	0.24±0.01	0.25±0.01af	0.054
C16:0	10.53±0.01a	10.63±0.02b	9.33±0.02c	9.56±0.27d	9.36±0.06e	9.22±0.03f	0.000
C17:0	0.12±0.01	0.15±0.01	0.15±0.02	0.13±0.02	0.13±0.04	0.13±0.02	0.503
C18:0	0.48±0.02a	0.54±0.02b	0.96±0.02c	0.87±0.08d	0.91±0.04e	0.95±0.01f	0.000
C20:0	0.19±0.01a	0.26±0.02b	0.25±0.02c	0.35±0.09d	0.36±0.04e	0.35±0.02df	0.002
C21:0	0.03±0.00	0.04±0.01	0.03±0.00	0.05±0.02	0.03±0.02	0.02±0.01	0.183
C22:0	0.40±0.01a	0.44±0.01b	0.42±0.01c	0.39±0.02d	0.48±0.04e	0.50±0.03f	0.004
C24:0	0.03±0.00	0.04±0.01	0.04±0.00	0.05±0.02	0.05±0.02	0.05±0.03	0.556
C16:1n-7	2.77±0.02a	2.79±0.02b	2.77±0.04c	1.73±0.07d	1.21±0.04e	1.15±0.02f	0.000
C18:1n-7	4.53±0.02a	4.82±0.02b	4.33±0.01c	4.38±0.09d	2.93±0.05e	2.79±0.08f	0.000
C18:1n-9	12.53±0.09a	14.53±0.51b	16.36±0.09c	22.94±0.09d	24.54±0.05e	25.58±0.12f	0.000
C20:1n-9	0.07±0.01	0.07±0.01	0.07±0.01	0.08±0.02	0.06±0.04	0.04±0.02	0.290
C22:1n-9	0.06±0.00a	0.07±0.01b	0.05±0.01c	0.08±0.02d	0.06±0.02af	0.04±0.02f	0.021
C18:2n-6	1.35±0.02a	1.38±0.02b	1.34±0.02c	12.56±0.16d	15.51±0.04e	15.74±0.04f	0.000
C18:3n-3 (ALA)	1.41±0.03a	1.48±0.02b	1.42±0.03c	13.59±0.09d	14.84±0.04e	15.25±0.06f	0.000
C20:3n-3	0.11±0.01a	0.13±0.02ab	0.12±0.02ac	0.17±0.06d	0.14±0.06e	0.10±0.01f	0.224
C20:4n-6 (ARA)	0.45±0.03a	0.48±0.01b	0.46±0.02c	0.50±0.11d	0.55±0.03e	0.59±0.02f	0.003
C20:5n-3 (EPA)	0.88±0.03a	2.52±0.13b	2.77±0.09c	2.42±0.08d	1.58±0.08e	1.10±0.03f	0.000
C22:6n-3 (DHA)	0.25±0.03a	6.77±0.12b	5.34±0.31c	4.27±0.06d	1.85±0.04e	0.84±0.03f	0.000
SFA	12.90±0.06a	13.33±0.06b	12.30±0.08c	12.52±0.39d	12.42±0.19e	13.07±0.21f	0.000
MUFA	19.96±0.06a	22.29±0.55b	23.58±0.12c	29.10±0.21d	28.80±0.07e	29.60±0.25f	0.000
PUFA	4.46±0.07a	12.76±0.03b	11.46±0.43c	33.51±0.46d	34.47±0.20e	33.61±0.18f	0.000
n-3 FA	2.65±0.03a	10.30±0.02b	9.65±0.39c	20.45±0.25d	18.41±0.51e	21.29±0.12f	0.000
n-6 FA	1.81±0.04a	1.86±0.03b	1.80±0.06c	13.05±0.24d	16.06±0.01e	16.32±0.06f	0.000
n-3/n-6	1.47±0.02a	5.86±0.16b	5.35±0.11c	1.57±0.03d	1.15±0.01e	1.06±0.08f	0.000
DHA/EPA	0.29±0.04a	2.89±0.04b	1.92±0.02c	1.76±0.02d	1.17±0.01e	0.76±0.04f	0.000

• ^*
  Only major fatty acids are presented;
• Crude lipid ranged from 7.3% to 18.2% (WW), with corresponding moisture levels of 67.5%–75.8%. Values are presented as mean ± SD (n = 3). Different superscript letters in the same row indicate significant differences (P < 0.05).

The differences in SFA, MUFA, and PUFA levels among treatments were found to be highly significant (P < 0.05). In P3 (50% FSO + 50% CFO), MUFA content in Artemia salina nauplii increased to 29.10%, while PUFA remained high (33.51%), reflecting a balanced contribution from both oils. In P4 and P5, where CFO was more dominant, MUFA levels were higher (28.80–29.60%), although PUFA also remained considerable (34.47–33.61%). This pattern is consistent with the characteristics of CFO, which is rich in MUFA, particularly oleic acid, while still maintaining PUFA input from FSO. These findings are in agreement with Sattang et al. (2021) and Aryani et al. (2025), who also reported higher MUFA levels in diets supplemented with freshwater fish oil. It should be noted, however, that these results are limited to the fatty acid profile of enriched Artemia salina nauplii, and their implications for larval performance need to be further evaluated through direct feeding trials.

The statistical differences observed (P < 0.05) indicate that the type of enrichment oil strongly influences the MUFA composition of enriched Artemia salina nauplii. In particular, the proportion of oleic acid (C18:1n-9) varied significantly between Artemia enriched with FSO and those enriched with CFO. Enrichment with FSO resulted in lower C18:1n-9 levels (12.53–16.36%), whereas enrichment with CFO markedly increased its proportion (22.94–25.58%). These variations are consistent with the inherent fatty acid profiles of the respective oils, where squid oil is typically dominated by long-chain n-3 PUFA such as EPA and DHA (Hossain et al., 2024b), while pangasius catfish oil is characterized by a higher MUFA content, particularly oleic acid (Sattang et al., 2021; Aryani et al., 2023).

The levels of EPA (C20:5n-3) and DHA (C22:6n-3) in Artemia salina nauplii varied significantly among treatments, with statistical analysis indicating highly significant differences (p < 0.05). In P0 (control), EPA (0.88%) and DHA (0.25%) were detected at very low levels. Enrichment with squid oil (FSO) in P1 and P2 significantly increased EPA (2.52–2.77%) and DHA (5.34–6.77%) compared to the control, confirming the role of FSO as a major source of long-chain n-3 PUFA (Hossain et al., 2024a). In P3 (50% FSO and 50% CFO), EPA (2.42%) and DHA (4.27%) remained relatively high, reflecting the contribution of both oils. Conversely, in P4 and P5, where CFO was more dominant, EPA (1.58–1.10%) and DHA (1.85–0.84%) sharply declined, consistent with the low EPA and DHA content in CFO. According to Sattang et al. (2021), pangasius catfish oil contains only 0.10% EPA and 0.20% DHA, while Aryani et al. (2023) reported 0.10% EPA and 0.11% DHA. Similarly, catfish oil (Clarias gariepinus) has also been reported to contain relatively low EPA levels (0.05–0.11%), but with slightly higher DHA levels, ranging from 0.28% to 0.77% of total fatty acid methyl esters (FAME) (Ningrum et al., 2023).

The DHA/EPA ratio in Artemia differed significantly among treatments. In P0 (control), the ratio was very low (0.29), indicating that DHA was much lower than EPA. Enrichment with squid oil (FSO) in P1 and P2 significantly increased the ratio (2.89 and 1.92), demonstrating that FSO strongly contributed to DHA enrichment. In P3 (50% FSO and 50% CFO combination), the ratio remained relatively high (1.76), reflecting the balanced contribution of both oils. In contrast, in P4 and P5, where CFO was more dominant, the ratio decreased to 1.17 and 0.76, consistent with the lower DHA content of CFO. Statistical analysis confirmed that the differences in the DHA/EPA ratio across treatments were highly significant (P < 0.05).

3.2. Proximate composition and fatty acid profiles of larvae

Table 4 indicates that, by the end of the trial, Thai mahseer larvae exhibited crude lipid contents ranging from 9.8% to 15.5% and moisture levels between 68.5% and 75.8%, reflecting the effects of live feed enrichment with Artemia nauplii. These values are in close agreement with those previously reported for mandarin fish larvae (Siniperca chuatsi) by Wang et al. (2022) and Pacific cod larvae (Gadus macrocephalus) by Choi et al. (2021).

Table 4
Proximate composition (% wet weight basis) and fatty acid composition (% of total fatty acids) of larval Thai mahseer fed Artemia salina nauplii enriched with different emulsions.

	P0	P1	P2	P3	P4	P5	P-values
Crude lipid	9.8±0.02	15.5±0.50	14.2±0.20	14.5±0.30	10.8±0.45	10.3±0.20	0.000
Moisture	73.4±0.25	71.2±0.62	75.80±0.62	71.4±1.25	69.5±0.11	68.5±0.30	0.000
Fatty acid*
C10:0	0.03±0.01	0.03±0.01	0.02±0.00	0.02±0.00	0.02±0.01	0.02±0.01	0.000
C12:0	0.55±0.01	0.45±0.02a	0.09±0.01	0.08±0.01	0.14±0.01	0.03±0.01	0.000
C14:0	0.38±0.01	0.47±0.01	0.62±0.01	0.64±0.01	0.53±0.01	0.47±0.01	0.001
C15:0	0.22±0.01	0.27±0.01	0.23±0.01	0.21±0.01	0.24±0.01	0.21±0.01	0.000
C16:0	9.47±0.02	8.40±0.02	8.51±0.02	7.91±0.02	7.49±0.07	7.83±0.02	0.000
C17:0	0.12±0.01	0.13±0.01	0.10±0.01	0.12±0.02	0.10±0.01	0.11±0.01	0.034
C18:0	0.45±0.01	0.46±0.01	0.84±0.02	0.74±0.02	0.72±0.01	0.81±0.02	0.000
C20:0	0.18±0.01	0.23±0.02	0.23±0.01	0.27±0.02	0.31±0.01	0.30±0.01	0.000
C21:0	0.03±0.01	0.34±0.01	0.34±0.01	0.05±0.00	0.01±0.01	0.02±0.01	0.000
C22:0	0.35±0.01	0.36±0.01	0.36±0.01	0.34±0.01	0.41±0.01	0.45±0.00	0.000
C24:0	0.03±0.00	0.04±0.00	0.04±0.00	0.05±0.01	0.03±0.01	0.04±0.01	0.033
C16:1n-7	2.95±0.04	3.09±0.01	3.33±0.01	2.16±0.02	1.43±0.02	1.26±0.01	0.000
C17:1n-7	4.16±0.02	5.94±0.59	5.38±0.19	5.63±0.02	3.24±0.01	3.07±0.02	0.000
C18:1n-9	13.55±0.04	16.21±0.12	18.83±0.10	29.74±0.09	26.95±0.04	27.64±0.01	0.000
C20:1n-9	0.07±0.01	0.09±0.01	0.09±0.01	0.09±0.01	0.07±0.01	0.04±0.01	0.000
C22:1n-9	0.05±0.01	0.19±0.01	0.06±0.01	0.09±0.01	0.06±0.01	0.04±0.01	0.000
C18:2n-6	1.76±0.02	2.41±0.05	1.55±0.02	16.45±0.11	19.95±0.09	19.69±0.02	0.000
C18:3n-3	1.79±0.02	2.67±0.01	1.84±0.02	17.87±0.06	19.85±0.09	19.04±0.02	0.000
C20:3n-3	0.15±0.01	0.25±0.01	0.16±0.02	0.75±0.02	0.18±0.01	0.14±0.01	0.000
C20:4n-6 (ARA)	0.59±0.01	0.92±0.01	0.64±0.02	0.62±0.01	0.67±0.01	0.76±0.01	0.000
C20:5n-3 (EPA)	1.19±0.02	4.52±0.01	3.72±0.27	3.22±0.06	2.14±0.01	1.55±0.01	0.000
C22:6n-3 (DHA)	0.36±0.02	12.38±0.47	7.46±0.02	5.69±0.10	2.50±0.01	1.23±0.02	0.000
SFA	12.09±0.05	11.18±0.03	11.22±0.06	10.44±0.03	10.00±0.05	10.29±0.05	0.000
MUFA	20.78±0.06	25.52±0.70	27.68±0.28	37.71±0.09	31.74±0.06	32.06±0.01	0.000
PUFA	5.84±0.05	23.15±0.42	15.37±0.31	44.60±0.29	45.30±0.30	42.41±0.02	0.000
n-3 FA	3.49±0.05	19.82±0.06	18.18±0.31	27.58±0.19	24.67±0.10	21.96±0.01	0.000
n-6 FA	2.35±0.04	3.33±0.06	2.19±0.01	17.07±0.12	20.63±0.21	20.45±0.02	0.000
n-3/n-6	1.48±0.02	5.95±0.22	6.02±0.14	1.62±0.01	1.20±0.01	1.07±0.00	0.000
DHA/EPA	0.31±0.01	2.73±0.11	2.00±0.15	1.77±0.00	1.17±0.01	0.80±0.01	0.000
FA	38.71±0.05	59.85±0.84	54.27±0.61	92.74±0.38	87.04±0.31	84.75±0.04	0.000

• ^*
  Only major fatty acids are presented.

At the end of the feeding trial, larvae from the P2 treatment (75% FSO and 25% FCO) exhibited significantly higher concentrations of palmitic acid (C16:0) and palmitoleic acid (C16:1n-7) than those from all other treatments (P < 0.05). Conversely, the lauric acid (C12:0) content of P2 larvae was markedly lower compared with P0 and P1 (P < 0.05). In addition, linoleic acid (C18:2n-6) levels in larvae fed P1- or P2-enriched Artemia nauplii were significantly reduced relative to those in P3 (50% FSO and 50% FCO) and P4 (25% FSO and 75% FCO) (P < 0.05). Arachidonic acid (ARA, C20:4n-6), however, was highest in the P1 group, differing significantly from all other treatments (P < 0.05). These findings support earlier reports identifying squid oil as a rich source of long-chain polyunsaturated fatty acids (LC-PUFA), particularly EPA and DHA, which play key roles in growth, neural development, and stress tolerance in fish larvae (Zhang et al., 2023; Hossain et al., 2024b; Dadras et al., 2024).

Furthermore, EPA and DHA levels were significantly higher in larvae fed Artemia salina nauplii enriched with P1 (100% FSO) than in all other treatments (P < 0.05). The P0 group exhibited the lowest n-3 fatty acid content and n-3/n-6 ratio, both of which were significantly lower than those recorded in P1 and P3 (P < 0.05). In contrast, larvae from the P5 group (100% FCO) showed the lowest DHA/EPA ratio among all treatments (P < 0.05), reflecting the limited contribution of freshwater fish oil to n-3 PUFA deposition (Prusińska et al., 2020; Sattang et al., 2021; Aryani et al., 2023). Overall, these results underscore the crucial role of squid oil as an enrichment medium to improve the nutritional quality of live feed, thereby enhancing the growth and health performance of Thai mahseer larvae. Nevertheless, FCO can still be applied as a carrier oil to reduce production costs and may be more appropriate for later growth stages (juveniles), when DHA and EPA requirements are relatively lower than during the larval phase (Thiruvasagam et al., 2024).

Crude lipid ranged from 9.8% to 15.5% (WW), with corresponding moisture levels of 68.5%–75.8%. Values are presented as mean ± SD (n = 3). Different superscript letters in the same row indicate significant differences (P < 0.05).

3.3. Larva growth and survival

At the end of the 21-day feeding trial, Thai mahseer larvae in the P1 treatment exhibited significantly higher weight gain than all other treatments (P < 0.05; Figure 1). In terms of total length, no significant differences were observed among P1, P2, P3, and P4 (P > 0.05), whereas these groups differed significantly from P0 and P5 (P < 0.05; Figure 2). The survival rate of larvae was also significantly higher in the P1 group compared to all other treatments (P < 0.05; Figure 3). In addition, the air stress test conducted 24 hours after the feeding trial revealed significant differences among treatments, except between P0, P3, and P4 (P < 0.05; Figure 4). Overall, mortality in the air stress test ranged from 4% to 16%.

Figure 1
Final body weight (mg) of Thai mahseer larvae after feeding on Artemia nauplii enriched with various diets at the conclusion of the trial. Data are expressed as mean ± SD (n = 3). Columns marked with different letters denote statistically significant differences among treatments (P < 0.05).

Figure 2
Final total length (mm) of Thai mahseer larvae after feeding on Artemia nauplii enriched with various diets at the conclusion of the trial. Data are expressed as mean ± SD (n = 3). Columns marked with different letters denote statistically significant differences among treatments (P < 0.05).

Figure 3
Survival rate (%) of Thai mahseer larvae fed Artemia nauplii enriched with different diets at the end of the feeding trial. Values represent means ± SD (n = 3). Bars with different letters indicate significant differences (P < 0.05).

Figure 4
Survival (%) after air exposure of Thai mahseer larvae fed Artemia nauplii enriched with different diets at the end of the feeding trial. Values are presented as means ± SD (n = 3). Bars with different letters indicate significant differences among treatments (P < 0.05).

These results contrast with those reported for Pacific cod, where enrichment of Artemia nauplii with commercial emulsions at EPA/DHA ratios of 19.15, 21.55, and 15.75 led to mortality rates ranging from 10% to 85%, with the highest mortality observed at the greatest DHA/EPA ratio (Choi et al., 2021). According to Morshedi et al. (2022), the presence of n-3 HUFA, particularly DHA, in the diet plays a crucial role in enhancing stress resistance in marine fish larvae. A similar trend was observed in Asian sea bass (Lates calcarifer), where larvae fed Artemia without HUFA enrichment exhibited lower survival rates after air-exposure stress compared to those fed diets enriched with n-3 HUFA (Pham et al., 2023).

4. Conclusion

This study demonstrated that the highest DHA and EPA levels were obtained from Artemia salina nauplii enriched with 100% FSO and the 75% FSO: 25% FCO combination, which also improved survival, growth, and air-exposure stress resistance in Thai mahseer larvae. Nevertheless, FCO still holds potential as a cost-effective energy source, particularly when combined with PUFA-rich oils (e.g., algal oil or marine fish oil), serving as a carrier oil to reduce production costs. Furthermore, FCO is more suitable for later growth stages (juveniles), where the demand for DHA and EPA is lower than during the larval phase.

Acknowledgements

The authors sincerely thank the Ministry of Education, Culture, Research, and Technology of the Republic of Indonesia for providing financial support through grant number 001/LLX/DT.05.00/PL/2025. Gratitude is also extended to the students, Fairizal Oktavi Hidayat and Weni Septia, as well as to the fish farming partners, for their valuable contributions to the successful completion of this research. All experimental work was conducted in accordance with the Standard Operating Procedures (SOP) of the Aquaculture Laboratory at Bung Hatta University.

Data Availability Statement

The data that support the findings of this study are openly available in figshare at https://figshare.com/articles/dataset/_b_Enrichment_of_Artemia_nauplii_with_squid_and_catfish_oil_by-products_enhances_survival_growth_and_stress_resistance_in_Thai_mahseer_b_b_Tor_tambroides_b_b_larvae_b_/30968755, reference number https://doi.org/10.6084/m9.figshare.30968755.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  23 Mar 2026
• Date of issue
  2026

History

• Received
  10 Apr 2025
• Accepted
  29 Dec 2025