Production of Tiger Puffer <Emphasis Type="Italic">Takifugu rubripes</Emphasis> Offspring from Triploid Grass Puffer <Emphasis Type="Italic">Takifugu niphobles</Emphasis> Parents

The tiger puffer Takifugu rubripes is one of the most popular aquacultural fish; however, there are two major obstacles to selective breeding. First, they have a long generation time of 2 or 3 years until maturation. Second, the parental tiger puffer has a body size (2–5 kg) much larger than average market size (0.6–1.0 kg). The grass puffer Takifugu niphobles is closely related to the tiger puffer and matures in half the time. Furthermore, grass puffer can be reared in small areas since their maturation weight is about 1/150 that of mature tiger puffer. Therefore, to overcome the obstacles of maturation size and generation time of tiger puffer, we generated surrogate grass puffer that can produce tiger puffer gametes through germ cell transplantation. Approximately 5000 tiger puffer testicular cells were transplanted into the peritoneal cavity of triploid grass puffer larvae at 1 day post hatching. When the recipient fish matured, both males and females produced donor-derived gametes. Through their insemination, we successfully produced donor-derived tiger puffer offspring presenting the same body surface dot pattern, number of dorsal fin rays, and DNA fingerprint as those of the donor tiger puffer, suggesting that the recipient grass puffer produced functional eggs and sperm derived from the donor tiger puffer. Although fine tunings are still needed to improve efficiencies, surrogate grass puffer are expected to accelerate the breeding process of tiger puffer because of their short generation time and small body size.     

Chromosomal‐level assembly of Takifugu obscurus (Abe, 1949) genome using third‐generation DNA sequencing and Hi‐C analysis

The Tetraodontidae family are known to have relatively small and compact genomes compared to other vertebrates. The obscure puffer fish Takifugu obscurus is an anadromous species that migrates to freshwater from the sea for spawning. Thus the euryhaline characteristics of T. obscurus have been investigated to gain understanding of their survival ability, osmoregulation, and other homeostatic mechanisms in both freshwater and seawater. In this study, a high quality chromosome‐level reference genome for T. obscurus was constructed using long‐read Pacific Biosciences (PacBio) Sequel sequencing and a Hi‐C‐based chromatin contact map platform. The final genome assembly of T. obscurus is 381 Mb, with a contig N50 length of 3,296 kb and longest length of 10.7 Mb, from a total of 62 Gb of raw reads generated using single‐molecule real‐time sequencing technology from a PacBio Sequel platform. The PacBio data were further clustered into chromosome‐scale scaffolds using a Hi‐C approach, resulting in a 373 Mb genome assembly with a contig N50 length of 15.2 Mb and and longest length of 28 Mb. When we directly compared the 22 longest scaffolds of T. obscurus to the 22 chromosomes of the tiger puffer Takifugu rubripes, a clear one‐to‐one orthologous relationship was observed between the two species, supporting the chromosome‐level assembly of T. obscurus. This genome assembly can serve as a valuable genetic resource for exploring fugu‐specific compact genome characteristics, and will provide essential genomic information for understanding molecular adaptations to salinity fluctuations and the evolution of osmoregulatory mechanisms.     

Puffer smells tetrodotoxin

Behavioral observation was conducted to test whether olfaction is functional to detect tetrodotoxin (TTX) in tiger puffer Takifugu rubripes using Y-maze. We placed either agarose carrier or one agarose and one agarose containing TTX (200 MU) at each head of the channel of Y-maze. Then three non-toxic hatchery-reared juveniles (body length, 5.6 ± 0.4 cm; n = 18) were released into the Y-maze and pecking behavior to carrier was observed for 3 h. The same procedure was tested for olfactory-ablated juveniles and for juveniles taht received sham operation. Juveniles showed significant selectivity to TTX, except for olfactory-ablated juveniles. These results indicate that pufferfish detects TTX by olfactory organ.     

Gonadal Development and Fertility of Triploid Grass Puffer Takifugu niphobles Induced by Cold Shock Treatment

Tiger puffer Takifugu rubripes is one of the most valuable fish species in Japan; however, there has not been much progress in their selective breeding until recently despite their potential in aquaculture. Their long generation time and the large body size of their broodstock make breeding difficult. Recently, we made a surrogate broodstock, which produced gametes of different species in salmonids. Therefore, by using closely related recipients, which have small body sizes and short generation times, it is possible to accelerate breeding of the tiger puffer. Thus, we considered the grass puffer Takifugu niphobles, which has a short generation time and a small maturation size, as a potential recipient for gamete production of the tiger puffer. Furthermore, if sterile triploid individuals are used as recipients, the resulting surrogate broodstock would produce only donor-derived gametes. Therefore, we examined conditions for inducing triploidy by suppressing meiosis II to retain the second polar body in grass puffer. We found that cold shock treatment, which is 5°C for 30 min starting from 5 min after fertilization, is optimal to obtain high triploidization and hatching rates. Although the resulting triploid grass puffers produced small amounts of gametes in both sexes, the offspring derived from the gametes could not live for over 3 days. Furthermore, we found that triploid grass puffer showed normal plasma sex steroid levels compared with diploids. These are important characteristics of triploid grass puffer as surrogate recipients used for germ cell transplantation.     

Intestinal histomorphology,autochthonous microbiota and growth performance of the oscar (Astronotus ocellatus Agassiz, 1831) following dietary administration of xylooligosaccharide

The present study investigates the changes in intestinal histomorphology, autochthonous microbiota and growth performance of the oscar, Astronotus ocellatus, following dietary administration of different levels of xylooligosaccharide (XOS). One hundred forty‐four oscars (8.88 ± 0.23 g; n = 144) were randomly stocked in 12 aquaria (100‐L) assigned to four treatments repeated in triplicate. Fish were fed a commercial diet, Biomar, supplemented with different levels (0 [control], 0.5, 1, 2%) of XOS for 8 weeks. Treatments were investigated under static aerated water conditions with a 70% daily water exchange. Evaluation of intestinal histomorphology (villus height, enterocytes height and thickness of the tunica muscularis) revealed no significant differences between XOS‐fed groups and the control treatment (P > 0.05). However, administration of XOS in the oscar diet increased the total autochthonous intestinal heterotrophic bacteria significantly (P < 0.05). Autochthonous lactic acid bacteria levels were also significantly elevated in XOS‐fed groups (P < 0.05). Furthermore, dietary XOS remarkably increased growth performance (control: 22.76 ± 2.79, 2% XOS: 29.13 ± 2. 8; n = 12) parameters of the oscar (P < 0.05). These results demonstrated the beneficial effects of XOS on the growth performance and intestinal microbiota of A. ocellatus.     

Growth,body composition and digestive enzyme responses of Caspian Kutum,Rutilus frisii (Kamenskii, 1901), juveniles fed different levels of carbohydrates

The study evaluated the effects of dietary carbohydrate levels on growth performance and digestive enzyme activities in juveniles Caspian Kutum, Rutilus frisii (Kamenskii, 1901). Fish with an initial average weight of 0.8 ± 0.2 g (means ± SD) were fed five isonitrogenous and isoenergic formulated diets with different carbohydrate levels of 15%, 20%, 25%, 30% and 35% in triplicate groups for 10 weeks (fifteen 45‐L aquaria with 30 fish per tank and water temperature of 21.5–23.5°C). Based on the results, growth performance of Caspian Kutum was significantly improved by increasing carbohydrate levels from 15% to 35% (p < .05). A result of body composition analysis showed that the protein content was elevated with increasing carbohydrate levels (p < .05). Digestive enzyme activities were significantly influenced by dietary carbohydrate levels (p < .05). Trypsin, chymotrypsin, lipase and amylase specific activities increased significantly with rising carbohydrate levels from 15% to 35%. Alkaline phosphatase and N amino peptidase activities increased with carbohydrate levels increasing from 15% to 25%, but decreased with a further rise in the carbohydrate level from 25% to 35%. The results indicate that increasing carbohydrate levels from 15% to 35% have positive effects on growth parameters. However, carbohydrate amounts at levels higher than 35% and their effects on growth performance and digestive enzymes are not obvious, hence, more widespread studies on this nutrient are recommended.     

Citric acid as a feed additive in pacu Piaractus mesopotamicus (Holmberg, 1887) diets

The objective of this study was to evaluate citric acid (0.0, 1.0, 2.0 and 3.0%) in isonitrogenous (23% of digestible protein) and isoenergetic (13.38 MJ of digestible energy/kg) pacu Piaractus mesopotamicus (Holmberg, 1887) diets. A 90‐day feeding trial was conducted to evaluate the growth performance, haematological parameters and pH of the diets, stomach and gut, somatic indices, nitrogen retention and body composition of pacu juveniles. Fish (n = 160, 12.53 ± 0.17g) were distributed in 16 aquaria (300‐L) with a recirculating water system (4 L/min) and controlled temperature (25.26 ± 0.47°C) in an experimental design completely randomized with four treatments and four replicates. Posteriorly, apparent digestibility coefficients (ADC) were assessed with pacu (n = 96, 80.35 ± 5.12 g) fed experimental diets including 0.1% chromium oxide III. Diet pH decreased (p < .05) with graded levels of citric acid to reduce pH in the stomach and gut. Pacu fed with 2.0% citric acid showed superior (p < .05) final weight at 30 days, compared to control; however, this did not differ by 60 and 90 days where was no difference (p > .05) in the haematology, somatic indices, body composition, or digestibility among treatments. The data showed that dietary citric acid improved the growth of pacu at 30 days, but had no long‐term effects on the digestibility of nutrients or the availability of P or Ca in the experimental diets.     

Purification and characterization of tributyltin-binding protein of tiger puffer, Takifugu rubripes

We successfully purified Trub.TBT-bpα, a tributyltin (TBT) binding protein (bp) of the tiger puffer, Takifugu rubripes. Tiger puffer was injected intraperitoneally with TBT (1.0 mg/kg body weight) and Trub.TBT-bpα was purified from serum by ammonium sulfate fractionation, gel filtration chromatography and polyacrylamide gel electrophoresis. Gel electrophoresis revealed that the Trub.TBT-bpα has a molecular mass of approximately 48.5 kDa and contains at least 40% N-glycan. The deduced 212 amino acid sequence of the protein showed the highest identity (41%, 212 amino acid overlap and E-value: 9e−42) with TBT-binding protein type 1 (TBT-bp1) of Paralichthys olivaceus (Japanese flounder). Analysis of the gene structure of Trub.TBT-bpα suggests that this protein belongs to the lipocalin superfamily, which may be important in the accumulation and elimination of TBT. Phylogenetic analysis suggests that functionalization of TBT-bps has occurred during evolution, and that the functions of this group of proteins might be important for fish survival.     

Length‐weight relationships for three fish species from the coastal waters of the northern Yellow Sea,China

Length–weight relationships (LWRs) were determined for three fish species: Thamnaconus modestus(Günther, 1877), Callionymus beniteguri  ( Jordan & Snyder, 1900) and Takifugu rubripes (Temminck & Schlegel, 1850), collected from the coastal waters of the northern Yellow Sea (near Dalian City) using bottom trawl nets in October 2017 and set nets in April 2018. Parameter b values in the fitted LWRs were 3.367(T. modestus), 2.817(C. beniteguri) and 3.200 (T. rubripes), respectively.     

Toxicity of pufferfish Takifugu rubripes cultured in netcages at sea or aquaria on land

Marine pufferfish (family Tetraodontidae) are believed to accumulate tetrodotoxin (TTX) mainly in liver and ovary through the food chain by ingesting TTX-bearing organisms such as starfish, gastropods, crustacean, flatworms, ribbonworms, etc. Consequently, it is hypothesized that non-toxic pufferfish can be produced if they are cultured with TTX-free diets in netcages at sea or aquaria on land, where the invasion of TTX-bearing organisms is completely shut off. To confirm this hypothesis, more than 5000 specimens of the pufferfish (“torafugu”, Takifugu rubripes) cultured in such manners for 1–3 years were collected from several locations in Japan during 2001–2004, and toxicity of their livers and some other parts was examined according to the Japanese official mouse assay method for TTX. In addition, typical specimens were submitted to LC/MS analysis. The results showed that all the livers and other parts tested were ‘non-toxic’ in both of the mouse assay (less than 2 MU/g) and LC/MS analysis (less than 0.1 MU/g). Thus, it is undoubtedly confirmed that pufferfish are intoxicated through the food chain, and non-toxic pufferfish can be successfully produced by netcage or land culture. The livers from these fish can be used with safety as a Japanese traditional food “fugu-kimo” (puffer liver).     

Effect of dietary replacement of fish meal protein with crawfish shell meal (Procambarus clarkii) on the growth performance,digestive enzyme activity and anti-oxidative capacity of Oujiang color common carp (Cyprinus carpio var. color)

This study was designed to evaluate the effects of dietary crawfish shell meal (CSM) on the growth performance, digestive enzyme activity and anti-oxidative capacity of Oujiang color common carp. 180 juvenile fish (initial weight 3.0 ± 0.5 g) were assigned to the control group and the CSM25 group (fed with 25% substitution of fish meal by CSM) for 8 weeks. Results showed that significant improvement in the weight gain rate (WGR) in CSM25 group than the control (p < .05), whereas no significant change in feed conversion ratio, specific growth rate and feeding intake between the groups (p > .05). Also, no significant difference was obsevered between the groups in terms of serum biochemical index and muscle composition (p > .05). And liver superoxide dismutase, catalase, glutathione peroxidase (GPx), lysozyme (LYZ) and protease activities in CSM25 group were significantly higher (p < .05). Moreover, histological analysis indicated that width of mucosa and thickness of muscular coat in mid-intestine were significantly changed (p < .05). In conclusion, dietary supplement with CSM25 induced high oxidative stress, promote protease activities and had no negative effects on the growth performance of Oujiang color common carp. Hence, CSM may be a viable alternative protein source to reduce the demand for fish meal.     

Differences in reproductive strategies between obscure puffer Takifugu obscurus and ocellated puffer Takifugu ocellatus during their spawning migration

Reproductive strategies were compared between obscure puffer Takifugu obscurus and ocellated puffer Takifugu ocellatus captured in waters near Yangzhong Island in the lower reaches of the Yangtze River during the spawning migration season from February to June in two consecutive years (1995 and 1996). Results showed that obscure puffer and ocellated puffer have adopted different reproductive strategies, including different spawning times, different size at maturity, and different number and size of oocytes, resulting in two different larval sizes. In detail, the timing of the spawning migration and status of gonad development of obscure puffer was about 1 month earlier than that of ocellated puffer; the obscure puffer was obviously longer and heavier than ocellated puffer in both mature male and female fish; mean GSI of obscure puffer females (15.8%) was significantly higher than that of ocellated puffer females (14.6%); the average diameter of ocellated puffer eggs (1.49 ± 0.12 mm) was significantly larger than that of the obscure puffer (1.22 ± 0.08 mm); and obscure puffer females (320.8 oocytes mg^?1 somatic wet weight) had significantly higher relative fecundity than ocellated puffer females (125.2 oocytes mg^?1 somatic wet weight). These differences in reproductive strategies between two closely related species of the Takifugu genus indicate that both obscure puffer and ocellated puffer fit the r/K dichotomy. Obscure puffer shows K‐selected characters with maturity at relatively large size and r‐selected characters with relatively many and small offspring, whereas ocellated puffer shows r‐selected characters with maturity at a relatively small size and K‐selected characters with relatively few and large offspring.     

Brief migration of the grass puffer, Takifugu niphobles, to fresh water from salt water

The grass puffer, Takifugu niphobles, is a peripheral freshwater fish often seen in brackish water. In the Sai River, Miyazu, Japan, we found that the grass puffer migrate to fresh water from salt water in June and July. Most of the grass puffer stayed in fresh water for 3.6 h on average and returned to the sea during the day. A low salinity tolerance experiment indicated that the grass puffer can live in fresh water for 2 days but not for longer than 4 days. Based on these findings, we discuss the physiological effects and ecological implications of the migration.     

Effects of dietary zearalenone on the serum biochemistry,hepatic and intestinal histology,and intestinal microbiota of juvenile Dabry′s sturgeon (Acipenser dabryanus)

Zearalenone (ZEN) is a common contaminant in animal feed, but the adverse effects of ZEN on the intestinal microbiota of fish have not yet been investigated. To reveal the effects of ZEN on serum biochemistry, hepatic and intestinal histology, and intestinal microbiota of fish, Dabry′s sturgeon (Acipenser dabryanus) received feed containing 1030 μg kg⁻¹ ZEN (ZEN-treated group) for 7 weeks and were compared to a control feed that have not been fortified with ZEN (control group). The results showed that dietary supplementation with ZEN did not significantly affect the serum contents of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, high-density lipoprotein cholesterol (HDLC) and estradiol (E₂; p > .05). The serum contents of total protein, albumin, triglycerides, total cholesterol and low-density lipoprotein cholesterol in the ZEN-treated group were significant lower than the control group (p < .05). The serum contents of glucose in the ZEN-treated group was significant higher than the control group (p < .05). Intestinal histology was normal in the ZEN-treated group with comparsion to the control group. Compared to the control group, the appearance of nuclear pyknosis and vacuoles in the hepatic cell in the ZEN-treated group. The α-diversity index (Chao 1, Faith pd and Shannon diversity index) significant increased in the ZEN-treated group compared to the control group (p < .05). Simpson diversity index was not affected by the dietary ZEN-treated (p > .05). Principal coordinates analysis (PCA) showed different clustering of prokaryotic communities in the ZEN-treated group compared to the control group. Further Anosim (Analysis of similarities) found that significnat differences in species community structure composition (R > .44) between the control group and the ZEN-treated group (p < .05). The number of operational taxonomic units (OTUs) was decreased after Dabry's sturgeon were fed diets supplemented with ZEN. At the genus level, the differences in the relative abundances of the first 20 main microbiota genera were considerable. In summary, this study suggests that dietary containing 1030 μg kg⁻¹ ZEN may be interfere with physiological parameters, and also affect the intestinal microbiota α-diversity, the numbers of OTUs and the microbiota compostion at the genus level of the juvenile Dabry′s sturgeon.     

<Emphasis Type="Italic">Takifugu obscurus</Emphasis> is a euryhaline fugu species very close to <Emphasis Type="Italic">Takifugu rubripes</Emphasis> and suitable for studying osmoregulation

Background  

The genome sequence of the pufferfish Takifugu rubripes is an enormously useful tool in the molecular physiology of fish. Euryhaline fish that can survive both in freshwater (FW) and seawater (SW) are also very useful for studying fish physiology, especially osmoregulation. Recently we learned that there is a pufferfish, Takifugu obscurus, common name "mefugu" that migrates into FW to spawn. If T. obscurus is indeed a euryhaline fish and shares a high sequence homology with T. rubripes, it will become a superior animal model for studying the mechanism of osmoregulation. We have therefore determined its euryhalinity and phylogenetic relationship to the members of the Takifugu family.     

Growth performance and metabolic response of juvenile grouper Epinephelus moara (Temminck & Schlegel, 1842) fed low dietary protein and high lipid levels

Nine test diets were formulated at three low levels of crude protein (P; 35%, 40% or 45%) and three high levels of crude lipid (L; 9%, 12% or 15%) to investigate whether Epinephelus moara differs in the nutrient requirements from any of the other closely‐related grouper species, and if there are any indications that a further decrease in protein with increasing dietary lipid would induce a higher protein‐sparing effect. Fish (20.34 ± 0.82 g/ind) were distributed among 27 experimental cages (100 cm diameter, 80 cm depth), with 20 fish per cage and three replicates for each test diet. The experiment was conducted over an 8‐week period at a temperature of 23.9–25.7°C. Weight gain and specific growth rate (SGR) tended to increase either with an increase in dietary protein level (at the same lipid level) or with the increase in dietary lipid level (at the same protein level, except at the 45% protein level). The highest weight gain and SGR occurred in fish fed the diet P45/L12. The serum cholesterol, high‐density lipoprotein‐cholesterol (HDL‐C) and low‐density lipoprotein‐cholesterol (LDL‐C) contents in fish were significantly increased as the dietary lipid increased. The increase of dietary protein significantly decreased the activities of serum and hepatic alanine aminotransferase (ALT) as well as aspartate aminotransferase (AST). Hepatic fatty acid synthase (FAS) activity significantly increased with the decrease of dietary protein and lipid levels (p < .05). However, lipoprotein lipase (LPL) activity showed an inverse trend compared to FAS. Hepatic glutamate dehydrogenase (GDH) activity significantly increased with the increase in the dietary protein level. In conclusion, the results reveal that growth performance and metabolic responses of E. moara are dependent on dietary protein and lipid levels, and that protein‐sparing action can be induced by increasing the dietary lipid. To support the feeding and growth of juvenile E. moara under the present conditions, 45% protein and 12% lipid are adequate.     

Dietary enzymatically-treated Artemisia annua L. supplementation could alleviate oxidative injury and improve reproductive performance of sows reared under high ambient temperature

The medicinal plant Artemisia annua L. is well known for its antimalarial compound artemisinin and the antioxidant capacity of its active ingredients. However, low bioavailability of Artemisia annua L. limits its therapeutic potential, fermentation of Artemisia annua L. can improve its bioavailability. This study was aimed to investigate the effects of dietary supplementation of enzymatically-treated Artemisia annua L. (EA) on reproductive performance, antioxidant status, milk composition of heat-stressed sows and intestinal barrier integrity of their preweaning offspring. 135 multiparous sows of average parity 4.65 (Landrace × large white) at day 85 of pregnancy were randomly distributed into 3 treatments. Sows in the control group were housed at control rooms (temperature: 27.12 ± 0.18 °C, temperature-humidity index (THI): 70.90 ± 0.80) and fed the basal diet. Sows in the HS, HS + EA groups were fed the basal diet supplemented with 0 or 1.0 g/kg EA respectively, and reared at heat stress rooms (temperature: 30.11 ± 0.16 °C, THI: 72.70 ± 0.60). Heat stress increased the malondialdehyde (MDA) content, reduced the activities of total antioxidant capacity (T-AOC) and total superoxide dismutase (T-SOD) of sows and piglets, and seriously compromised the antioxidant capacity of the sows and the intestinal integrity of their offspring. However, dietary supplementation of 1.0 g/kg EA reduced the MDA content, increased the activities of T-SOD and T-AOC in serum, colostrum, and milk of heat-stressed sows, and increased colostrum yield and 14-d milk fat content. EA supplementation also increased piglet weaning weight and the activities of T-SOD and T-AOC in serum. In addition, the abundances of intestinal tight junction proteins claudin-1 and occludin were up-regulated in piglets in EA-supplemented group. In conclusion, dietary EA supplementation at 1.0 g/kg can alleviate the oxidative stress in heat-stressed sows, improve the antioxidant capacity in both sows and their offspring, and promote the intestinal barrier integrity in their offspring. EA may be a potent dietary supplement that ameliorates oxidative stress in livestock production by improving the antioxidant capacity.     

Effects of dietary amino acid l‐lysine on survival,growth and haemato‐biochemical parameters in Indian catfish,Heteropneustes fossilis (Bloch, 1974), fingerlings

An 8‐week feeding experiment was conducted to estimate the dietary lysine requirement of fingerling Indian catfish, Heteropneustes fossilis (total length = 6.35 ± 1.10 cm; weight = 4.70 ± 0.65 g) by feeding six isonitrogenous (400 g/kg CP) and isocaloric diets (17.90 kJ/g) containing six supplemental lysine levels ranging from 11.0 to 23.5 g/kg (diets I to VI, in incremental steps of 2.5 g/kg). Triplicate groups with 20 fish each were stocked in 75‐L circular continuous flow‐through troughs and fed experimental diets at 4% BW/day twice daily (08:00 and 18:00 h). Maximum live weight gain (304%), best‐feed conversion ratio (FCR, 1.46) and protein efficiency ratio (PER, 1.71) were obtained at 18.5 g/kg dietary lysine, beyond which these values showed a slight declining tendency. Best values for somatic and hematological indices were also obtained at this level. Significantly (p < .05) higher body protein and low carcass moisture and intermediate fat contents were noted in fish fed diets containing 18.5 g/kg lysine. The quadratic regression analyses of live weight gain, FCR, PER and body protein deposition (BPD) data indicated lysine requirements at 19.3, 18.8, 18.6 and 18.6 g/kg of dry diet, respectively. Based on these results, it is recommended that the H. fossilis diet should contain lysine at a level of 19.0 g/kg of dry diet, corresponding to 47.5 g/kg of dietary protein, for optimum growth and efficient feed utilization.     

Dietary folic acid (FA) requirement of a genetically improved Nile tilapia Oreochromis niloticus (Linnaeus, 1758)

The present study was conducted to determine the dietary folic acid (FA) requirement of genetically improved Nile tilapia, Oreochromis niloticus. Experimental diets were supplemented with 0.04, 0.22, 0.48, 0.96, 2.25, or 4.07 mg/kg FA, respectively, and fed to three replicate groups of 15 tilapia (mean initial weight = 60.23 ± 2.60 g; mean ± SD) for 12 weeks. Results showed that the weight gain rate increased linearly with increasing dietary FA from 0.04 to 0.40 mg/kg, then remained stable with higher supplementation. Similarly, hepatic FA concentration, red blood cell count (RBC), hemoglobin concentration (HB) and hematocrit (HCT) all markedly increased in fish fed with 4.07 mg/kg FA compared to those fed a control diet (p < .05). Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities decreased with increasing dietary FA (p < .05). The FA requirement was estimated to be 0.4 mg/kg based on weight gain rate, and 0.7 mg/kg based on liver FA accumulation.