This study aimed to determine the anesthetic therapeutic window of Thymus vulgaris L. essential oil (TVEO) in juvenile Colossoma macropomum (tambaqui), through the integrated analysis of behavioral parameters, electrocardiographic recordings, opercular activity during the induction, and recovery periods of anesthesia, as well as the measurement of blood glucose levels in the animals after the anesthetic procedure. The fish were divided into the control, vehicle, and TVEO-treated groups at concentrations of 18.6 mg. L⁻1, 27.9 mg. L⁻1, 37.2 mg. L⁻1, and 46.5 mg. L⁻1, administered by immersion baths. Anesthetic efficacy was evaluated by means of induction time, loss of righting response, and recovery time. Electrocardiographic recordings were used to monitor heart rate and rhythm, allowing the analysis of possible pharmacological effects on cardiac electrical conduction. A concentration of 18.6 mg. L⁻1 promoted mild anesthesia without loss of postural response or relevant cardiovascular changes. Concentrations of 27.9 mg. L⁻1 and 37.2 mg. L⁻1 induced effective anesthesia, with rapid induction, adequate recovery, and electrocardiographic stability, characterizing the main therapeutic window. The concentration of 46.5 mg. L⁻1 resulted in transient cardiovascular depression, evidenced by a reduction in heart rate, suggesting autonomic modulation and possible interaction with cardiac ion channels. Thymus vulgaris essential oil exhibits concentration-dependent anesthetic action in C. macropomum, with concentrations of 27.9, 37.2, and 46.5 mg. L⁻1 representing the window for anesthesia in moderate to deep planes. The findings indicate the involvement of central and cardiovascular pharmacological mechanisms, reinforcing the potential of TVEO as a natural alternative to synthetic anesthetics in fish.
Arsenic (As) pollution in aquatic ecosystems presents a significant risk to fish health and to the viability of aquaculture. Although extensive documentation of the effects of arsenic poisoning has been made in several fish organs, its effects on the most important edible portion, skeletal muscle, remain relatively poorly understood. The purpose of this study was to evaluate the protective effect of zinc (Zn) and to determine how arsenic (As) causes muscle injury in common carp (Cyprinus carpio). Common carp were exposed to arsenic alone and in combination with zinc for 30 days. To explore the underlying mechanisms, histopathological investigation, reverse transcription quantitative polymerase chain reaction (RT-qPCR), Western blotting, and network toxicology were used. At the same time, arsenic exposure induced significant structural damage in the muscle of carp along with several other effects such as oxidative stress, inflammatory responses, endoplasmic reticulum (ER) stress, autophagy, and apoptosis. Arsenic particularly affected the antioxidant defense by the down-regulation of genes related to antioxidant defense, and by affecting the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. In addition, arsenic enhanced the inflammatory responses through nuclear factor kappa B (NF-κB) and promoted autophagy and apoptotic signaling. Zinc supplementation proved to be of great value and significantly reduced these pathogenic changes by restoring antioxidant capacity, blocking inflammatory signaling, and regulating autophagy and apoptosis-related pathways. A network toxicology analysis revealed that networks associated with oxidative stress, such as the phosphoinositide 3-kinase-protein kinase B (PI3K–Akt) signaling pathway and forkhead box O (FoxO) signaling pathway, were affected by arsenic and that zinc alleviated arsenic-induced muscle toxicity. These data together suggest that zinc is effective in protecting the muscle of carp against the damage caused by arsenic by regulating redox-related signaling pathways. These results contribute to the understanding of the mechanism of action of the toxicity of heavy metals in fish muscle and the potential of zinc as a protective agent against arsenic toxicity in aquaculture fish.
Zebrafish (Danio rerio) is widely used as a vertebrate model to study intestinal inflammation due to structural and functional similarities of its gastrointestinal tract to mammals. Oxytetracycline (OTC), a commonly used antibiotic in aquaculture, can accumulate in aquatic organisms and disrupt gut homeostasis, inducing oxidative stress and inflammatory responses. This study evaluated the protective effects of hydroalcoholic Quercus brantii fruit extract against OTC-induced intestinal injury in zebrafish larvae. A total of 144 three-day-old larvae were randomly assigned to four experimental groups (n = 36 per group, three replicates of 12 larvae): control (untreated), OTC (10 mg/L for five days), OTC + Quercus brantii fruit extract 10 g/kg diet, and OTC + Quercus brantii fruit extract 20 g/kg diet. Histopathological analysis revealed that OTC exposure caused partial epithelial disruption, reduced mucosal fold height, decreased goblet cell density, and increased infiltration of inflammatory cells. Cotreatment with 10 g/kg Quercus brantii fruit extract partially ameliorated these changes, whereas 20 g/kg significantly preserved epithelial integrity, increased mucosal fold height, and reduced inflammatory cell infiltration. Gene expression analysis showed that OTC significantly upregulated NF-κB, TNF-α, and IL-6 levels. Administration of 10 g/kg Quercus brantii fruit extract reduced NF-κB and TNF-α expression but did not significantly affect IL-6, while 20 g/kg extract significantly decreased all three cytokines. These results indicate that hydroalcoholic Quercus brantii fruit extract exerts protective effects against OTC-induced intestinal injury, likely mediated through NF-κB inhibition.
Global climate change and rising temperatures pose a serious threat to the survival of aquatic ectotherms. To analyze the physiological response of black rockfish (Sebastes schlegelii) to acute heat stress, this study investigated its comprehensive adaptation mechanisms under thermal challenge. The levels of biochemical parameters in plasma and liver were assessed, while histological morphology was analyzed via hematoxylin–eosin staining. Cell apoptosis was assessed by TUNEL assay and the expression of related genes was detected by using qPCR. Results showed that exposure to high temperature significantly elevated red blood cell counts (from 1.46 ± 0.10 to 2.50 ± 0.15 × 1012/L, P < 0.05), white blood cell counts (from 191.80 ± 5.46 to 219.97 ± 4.58 × 10⁹/L, P < 0.05), hemoglobin (from 92.00 ± 6.48 to 119.67 ± 11.15 g/L, P < 0.05), hematocrit (from 30.83 ± 1.11 to 38.87 ± 1.61
The growth and development of fish rely heavily on skeletal muscle. This study systematically characterized the morphological and developmental features of skeletal muscle in Megalobrama amblycephala during early development (0–90 days post-hatching, dph). It also determined the stage-specific contributions of hyperplasia and hypertrophy, and integrated transcriptomic and 16S rRNA sequencing analyses to preliminarily uncover the molecular regulatory network and gut microbiota adaptations associated with the rapid growth window from 15 to 20 dph. Morphological observations revealed that the length, diameter, number, and area of both red and white muscle fibers continuously increased with developmental age, and these changes were significantly synchronized with those in body length and weight. Myofiber hypertrophy predominated during juvenile growth, whereas mosaic hyperplasia was markedly enhanced at 15–20 dph, suggesting that this period represents a critical window for determining subsequent growth potential. Transcriptomic analysis revealed that upregulated KEGG pathways at 20 dph were significantly enriched in growth-related pathways, including FoxO, insulin, and AMPK signaling. Moreover, fast-twitch-specific genes (e.g., myl1, mylpfa, tnnc2.2, and tnni2a.2) and the myogenic regulatory factor myog were significantly upregulated, indicating active molecular programming toward a fast-twitch phenotype. Gut microbiota analysis showed a marked shift in microbial composition at 20 dph, with decreased Proteobacteria abundance and increased Actinobacteriota abundance. Spearman correlation analysis further revealed that 15 dph-dominant genera (such as Pseudomonas and Sphingomonas) were negatively correlated with fast-muscle genes, whereas 20 dph-enriched genera (such as Rubellimicrobium) showed positive correlations with myogenic marker genes. Functional prediction indicated enhanced metabolism of energy, amino acids, and carbohydrates at 20 dph, with reduced lipid metabolism, which closely mirrored transcriptomic trends. Collectively, the gut microbiota at 15–20 dph was highly synchronized with the activation of muscle developmental genes, constituting a key ecological factor supporting rapid growth. This study provides new insights into the multi-dimensional regulatory mechanisms of early muscle development in fish and offers a theoretical basis for strategies to enhance growth performance through microecological modulation in M. amblycephala aquaculture.
Food enrichment is a prominent feeding strategy used to enhance the welfare and physiological performance of captive aquatic animals; however, its effects on the brain remain poorly understood. In this study, three feeding regimes with similar nutritional levels—designated as the Control group (commercial diet), Group 1 [90
Myostatin (Mstn), a secretory protein from the transforming growth factor-β (TGF-β) superfamily, is mostly present in skeletal muscle across vertebrates. The mstn gene exhibits a conserved genomic structure consisting of three exons and two introns in both teleost fish and mammals. Unlike mammals which typically possess a single mstn copy, most fish species harbor two or more copies derived from whole-genome duplication events, and these paralogs show broader tissue expression profiles including muscle, brain, liver, kidney, and gonads. The function and mechanism of action in fish appear generally similar to those of other animals, although fish-specific gene duplication and tissue distribution patterns suggest possible functional divergence. Mstn primarily exerts its growth-inhibitory effect by suppressing muscle satellite cell activation, inhibiting myoblast proliferation, and downregulating myogenic regulatory factor (MRF) family expression. These molecular actions collectively restrict both hyperplastic and hypertrophic muscle growth. Disruptive mutations, including nucleotide deletions or substitutions, in the coding sequence of the fish mstn gene lead to a loss of Mstn function, which in turn stimulates myoblast proliferation and differentiation, potentially leading to pronounced muscle hyperplasia and hypertrophy. Emerging evidence further indicates that fish Mstn also plays pleiotropic roles in lipid metabolism regulation and immune modulation. In this paper, we evaluate the sequence structure, tissue distribution, function, and mechanism of mstn action in fish, with a focus on mstn's role and molecular mechanism in controlling fish muscle growth, to provide fundamental information for fish quality improvement.
The effects of dietary vitamin E level and source on the reproductive performance of female red hybrid tilapia, Oreochromis sp., was investigated. Broodstock diets were formulated with added α-tocopheryl acetate (α-ToAc) at 0 (T0), 60 (T60) or 600 (T600) mg per kg diet, which represented sub-optimal, at optimal or ten times above optimal levels, respectively, of vitamin E requirement reported for maximum growth of hybrid tilapia. A fourth diet was supplemented with a palm oil–derived tocotrienols-rich fraction (TRF) designed to contribute α-tocopherol (α-T) at 60 mg per kg diet (PalmE60). TRF consisted of a mixture of co-existing α-T with tocotrienols (T3; 75.5
This study aimed to assess the protective role of dietary hesperidin (HEP) in Nile tilapia (Oreochromis niloticus) under ammonia stress conditions. The investigation focuses on its capacity to alleviate ammonia-induced metabolic disorders, oxidative stress, and inflammatory responses. For 60 days, 160 Nile tilapia (26.46 ± 0.17 g) were split into four equal groups (control, HEP, AMM, and AMM + HEP). The control and AMM groups were administered a basal diet, whereas the HEP and AMM + HEP groups received a basal diet containing 150 mg HEP/kg. The AMM and AMM + HEP groups were subjected to 0.21 mg/L of un-ionized ammonia. Prolonged exposure to ammonia induced oxidative stress by increasing the malondialdehyde (386.67
Heat stress is a growing constraint in aquaculture, yet effective nutritional strategies for newly domesticated stream-dwelling fish remain poorly understood. Here, we investigated whether dietary lipid-source supplementation of a commercial feed could modulate chronic heat stress responses in the stream-dwelling fish Acrossocheilus fasciatus. Fish were reared for 60 days under two temperature regimes (24 °C and 32 °C) and fed a commercial basal diet supplemented with graded fish oil (F) to corn oil (C) ratios: F0:C4, F1:C3, F2:C2, and F4:C0. Growth performance, survival, brain cortisol, liver histology, and hepatic expression of genes involved in lipid remodeling, inflammation, and glycolytic metabolism were evaluated. High temperature significantly depressed growth and increased cortisol concentration, hepatic vacuolization, and the expression of stress-responsive metabolic and inflammatory genes. These adverse effects were most evident in the F0:C4 group, which showed the lowest growth, the highest cortisol level, strong induction of fads2, elovl5, tnfα, il6, eno1, and aldoa, and pronounced liver damage under 32 °C. Moderate fish oil supplementation, particularly the F1:C3 diet, alleviated several heat-induced alterations, preserving liver integrity and reducing inflammatory and glycolytic responses. By contrast, the F4:C0 diet improved growth at 24 °C but did not provide comparable protection against hepatic and inflammatory disturbances under 32 °C. These results indicate that dietary lipid source can modulate heat stress responses in A. fasciatus and that diets supporting growth under optimal temperature may not necessarily provide the most favorable physiological response under warming conditions. Overall, a moderate fish oil-to-corn oil ratio showed the most balanced physiological response under chronic heat stress.
Artificial body tactile stimulation (TS) has been shown to improve fish welfare by reducing aggressive behavior and stress, thereby serving as a sensory enrichment. Here, we tested the effect of TS on aggressive behavior and stress response in the Siamese fighting fish (Betta splendens), an ornamental species reared worldwide and renowned for its high aggressiveness. Twenty isolated male fish were exposed either to TS (provided by a frame with vertical plastic sticks lined with silicone bristles, inserted in the middle of the tank) or to a control treatment (sticks without bristles) for 22 days. Individual aggressive behavior was evaluated before and after the treatment period using mirror tests and staged pair fights. We measured cortisol, brain serotonin (5-HT), and dopamine (DA) to infer stress and possible associations with aggressiveness and the reward system. Although fish actively interacted with the bristles, TS did not alter overt physical fighting but significantly increased frontal and lateral displays. Furthermore, TS elevated cortisol levels, decreased 5-HT, and increased the 5-HIAA/5-HT turnover ratio. DA levels remained unchanged, indicating no activation of neural reward pathways. Contrary to our expectations, TS did not mitigate aggressiveness in this species but rather seemed to prompt an increase in both behavioral and physiological reactivity (cortisol levels and 5-HT activity) to fighting. Our findings indicate that artificial selection for high aggression in B. splendens may alter sensory processing to touch, causing physical contact to act as a stressor rather than an enrichment.
MP contamination and biomarkers were investigated in the economic importance fish species (Luciobarbus esocinus Heckel, 1843, Arabibarbus grypus Heckel, 1843 and Cyprinus carpio, Linnaeus, 1758). When MP content was considered, the highest MP concentration was found in the digestive system, followed by the gills, liver, and muscle tissue, respectively. Although regional and species-specific variations were observed in fish tissues, the predominant colors were blue and black, the predominant sizes were 151–500 µm and 501–3,000 µm, the predominant shapes were fragments and fibers, and the predominant polymer type was polyethylene. Significant interspecific differences were observed in microplastic (MP) accumulation. Arabibarbus grypus from the Atatürk Dam reservoir exhibited the highest burden (91 MP), nearly doubling the levels found in both Luciobarbus esocinus from the Karakaya Dam (47 MP) and Cyprinus carpio from the Keban Dam (46 MP). At the reservoir level, sediment and fish samples from Keban contained noticeably higher MP concentrations than those from Atatürk or Karakaya. In water, sediment, and fish samples collected on a regional basis, the Keban Dam has been identified as an area with a higher concentration of MP. While changes (increases/decreases) in the activities of antioxidant enzymes (SOD, CAT, and GPX) in gill and liver tissues were observed in fish from the Keban Dam, where MP levels were high; overall, increases were observed in MDA and ROS levels, as well as in the immunological parameters IL-6 and TNF-α, the DNA damage index 8-OHdG, and the apoptosis marker Caspase-3 levels (p < 0.05). The samples taken from the Keban Dam stood out as the region at highest risk, showing the highest levels of oxidative stress (ROS, MDA), DNA damage (8-OHdG), and apoptosis (Caspase-3) across all species compared to other regions. Among the species, Cyprinus carpio has been identified as the most sensitive to pollution, particularly based on the highest damage parameters recorded in the gill tissue at the Keban Dam. Overall, these results highlight that microplastics not only accumulate in fish tissues but also trigger biochemical defense mechanisms, and underscore their potential to cause non-lethal physiological stress in freshwater fish populations.
Elasmobranchs maintain osmotic balance through specialized renal mechanisms regulating ion transport, nitrogen conservation, and epithelial integrity. Although euryhaline species exhibit adaptive physiological responses to salinity variation, the molecular bases of these strategies remain poorly understood at the species level. Here, we performed comparative transcriptomic analysis of kidney tissues from two sympatric hammerhead sharks, Sphyrna lewini and Sphyrna zygaena, collected under comparable marine salinity conditions ( 26–28‰). RNA-seq identified 16,543 differentially expressed genes (DEGs), of which 11,614 were successfully annotated across multiple databases. Enrichment analysis highlighted pathways related to peroxisome function, amino sugar and nucleotide sugar metabolism, lysosomal activity, and fatty acid metabolism. S. lewini, a more dilution-tolerant species reported from coastal, estuarine, and occasionally riverine habitats, exhibited higher expression of genes related to mitochondrial respiration, ion transport (e.g., Na⁺/K⁺-ATPase, NKCC2, V-type H⁺-ATPase), redox regulation, and membrane remodeling, indicating an energetically demanding osmoregulatory strategy involving active ion transport and renal urea retention. In elasmobranchs, maintaining elevated urea concentrations is metabolically costly due to the need for continuous synthesis, reabsorption, and protection of cellular proteins from urea-induced destabilization. In contrast, S. zygaena, which is more commonly associated with shelf and offshore habitats, showed elevated expression of genes associated with extracellular matrix organization, tight junction structure (e.g., claudins), glycosaminoglycan modification, and endoplasmic reticulum ion buffering (e.g., TRIC-B), supporting a structurally reinforced kidney epithelial phenotype adapted to relatively stable marine salinities. These findings provide comparative molecular insights into species-specific osmoregulatory strategies and offer a framework for understanding physiological differentiation among closely related elasmobranchs.
Previous studies on artificial diet domestication in mandarin fish (Siniperca chuatsi) have mainly focused on feeding adaptation, digestive physiology, and basal metabolic regulation. However, whether artificial diet feeding modifies host responses to bacterial infection remains unclear. In this study, Aeromonas hydrophila was used as a bacterial challenge model to compare the responses of mandarin fish fed live bait or an artificial diet. Four groups were established: L, live bait-fed control; AL, live bait-fed infected; A, artificial diet-fed control; and AA, artificial diet-fed infected. Compared with their corresponding controls, both AL and AA showed increased lymphocyte-related indices, reduced neutrophil counts, elevated serum immune-antioxidant indices, and hepatic inflammatory cell infiltration. Compared with A, AA showed significant reductions in red blood cell percentage, mean corpuscular volume, and mean corpuscular hemoglobin. Histological observations further revealed indistinct hepatocyte boundaries and loosely arranged cytoplasm in AA compared with AL. Gene expression analysis showed that interleukin-1β was upregulated, whereas apolipoprotein B and interleukin-8 were downregulated in AA compared with A. Metabolomic analysis identified more differential metabolites in AA vs A (893) than in AL vs L (208). Shared differential metabolites were mainly enriched in nucleotide metabolism, pyrimidine metabolism, the pentose phosphate pathway, and glycerophospholipid metabolism. Metabolites specific to AL vs L were associated with amino acid metabolism, glutathione metabolism, and ABC transporters, whereas those specific to AA vs A were enriched in nucleotide metabolism, one-carbon metabolism, cofactor biosynthesis, and steroid hormone biosynthesis. Collectively, these findings demonstrate that mandarin fish fed different diets exhibit distinct immunophysiological and metabolic responses to A. hydrophila infection, with AA showing more pronounced hepatic injury and broader metabolic remodeling.
Understanding ontogenetic shifts in bimodal respiration is essential for elucidating how air-breathing fishes optimise physiological performance, yet whether individuals at different life stages sustain aerobic function without aerial access is poorly understood. This study investigated ontogenetic shifts in reliance on aquatic respiration and hypoxia tolerance in African catfish (Clarias gariepinus) across five size classes, fry (< 1 g), fingerlings (1–5 g), juveniles (20–40 g), and sub-adults (100 g, 300 g), under submergence at normoxia ( 17 kPa), moderate hypoxia (8.7 ± 0.8 kPa), and extreme hypoxia (2.2 ± 0.1 kPa). Ventilation frequency (fV) and time to loss of equilibrium (LOE) served as indices of respiratory effort and tolerance. All size classes maintained equilibrium for 48 h under normoxic submergence, confirming facultative air-breathing capacity, though the accompanying fV rise was uniform across sizes. Under hypoxia, fV responses were strongly size-dependent: fry upregulated fV by 90
Global warming presents a substantial threat to aquatic ectotherms, including the economically significant Atlantic salmon (Salmo salar). Although warm acclimation can increase heat tolerance, little is known about the dynamic processes and cellular responses associated with the acquisition and loss of this increased tolerance. This study investigated changes in critical thermal maximum (CTmax), liver structure, hepatic antioxidant activity, and heat shock protein (HSP) expression in Atlantic salmon during prolonged warm acclimation followed by cold acclimation back to baseline temperature. CTmax increased by approximately 1.7 °C during warm acclimation, with no further significant increase observed after 7 days of warming. During subsequent cold acclimation, the acquired CTmax declined slowly, remaining elevated for over 20 days. Long-term warm acclimation induced liver damage, with partial recovery observed during the one-month cold acclimation period. Molecularly, hepatic antioxidant responses were transiently activated during warm acclimation and increased again at later stages of subsequent cold acclimation, with GSH remaining elevated throughout both phases. For heat shock responses, hsp70 and hsp90 expressions increased during warm acclimation and returned to baseline during early cold acclimation, whereas HSP70 and HSP90 protein levels remained elevated. Furthermore, the expression patterns of the paralogous genes serpinh1b-1 and serpinh1b-2 differed, likely indicating functional divergence. This study provides a comprehensive temporal profile of thermal acclimation dynamics in Atlantic salmon, highlighting the asymmetric rates of thermal tolerance acquisition and loss, as well as the regulation of molecular defense mechanisms. These findings provide new temporal insight into thermal plasticity and associated cellular defense responses in the context of climate-related warming.
Embryonic stem (ES) cell-based platforms in non-model teleosts remain scarce, which may constrain in vitro studies in functional genomics and developmental biology in aquaculture-relevant species. Here, we report the derivation and characterisation of ATES1, an ES-like cell line derived from Anabas testudineus, a commercially important freshwater fish. ATES1 has been maintained for over 250 passages (> 1500 days) under feeder-free conditions in Leibovitz-15 medium supplemented with foetal bovine serum, fish serum, embryo extract, and human basic fibroblast growth factor (hbFGF). Proliferation was optimal at 28 °C, with both fish serum and hbFGF significantly enhancing cell growth. The cell line exhibited multiple characteristics associated with ES-like cells in vitro during early passages (approximately up to passages 25–35), including ES cell–like morphology, high self-renewal capacity (5-bromo-2′-deoxyuridine incorporation > 90
This study evaluated growth indices, survival, digestive enzyme activities, and genes related to aggressiveness in tropical gar (Atractosteus tropicus) larvae fed with diets supplemented with a commercial product rich in lipids (CPRL) with a high docosahexaenoic acid (DHA) concentration. Experimental diets for A. tropicus larvae were supplemented with 20, 30, and 40 g/Kg of CPRL, compared to a control diet without CPRL supplementation. The four treatments were defined by the final percentage of DHA based on total fatty acids (FA) as follows: 3.2
With advances in biotechnology and tissue engineering, the development of fish muscle cell lines from aquatic resources has gained increasing attention. However, most studies rely on immediate tissue processing after sampling, and few studies have investigated the potential of establishing cell lines from stored samples. In this study, we evaluated the potential for establishing skeletal muscle cell lines from black sea bream (Acanthopagrus schlegelii) tissue stored at 4 ℃ for 0, 2, 5, 10, 20, and 30 days. Primary culture was conducted using two methods: explant culture and dissociated culture. Cell line development was assessed in relation to cold storage duration, and established cell lines were characterized for their growth and differentiation capacities at early (passage 5) and late (passage 20) stages. To further investigate the effects of cold storage, we examined cell yield and viability after tissue dissociation, as well as histological and ultrastructural changes in the tissue. Results demonstrated that explant culture allowed cell line development from tissues stored up to 10 days, whereas dissociated culture was effective only up to 5 days. All cell lines established via dissociated culture retained the ability to differentiate into myotubes. Tissue-level analysis indicated that cell viability remained above 80
Aquaculture in saline environments is expanding worldwide, but elevated salinity can alter growth performance and physiological homeostasis in cultured fish. Nile tilapia (Oreochromis niloticus) is a euryhaline freshwater species widely used in salinity tolerance research and aquaculture production. Here, this study evaluated whether dietary mannitol improves salinity adaptation in tilapia. Fish (1.86 ± 0.04 g) were fed isonitrogenous and isoenergetic diets containing 0, 1, 2, 5 or 10
