Program

Marble goby is one of high price commercial freshwater fish. For its aquaculture, low growth rate and survival rate have being particular problem in nursery. This study investigated efficacy of garlic extract, which well known as high benefit for digestive system, as feed additive in marble goby juvenile. The fishes with size of 3.12±0.72 g were fed with natural food (blood worm, N), commercial diet (control feed, C), commercial diet mixed garlic aqueous extract (GAE) and ethanol extract (GEE) at concentration 0.5% (w/w) for 12 weeks. Growth parameters, digestive enzymes activities and histology were analyzed at 4, 8 and 12 weeks. For growth parameters, weight gain (WG), specific growth rate (SGR) and feed efficiency (FE), were calculated. For digestive enzyme activities, amylase, lipase, trypsin and chymotrypsin activities, which play major roles as sources of metabolic energy for growth, were determined. For histology, major digestive organs (esophagus, stomach, midgut and hindgut) were collected, prepared for H&E staining permanent slide and observed by compound microscope. It results that amylase activity showed significant different (p < 0.05) at 4 and 8 weeks from N and C groups. Whereas lipase activity of GAE showed significant different (p < 0.05) at 12 weeks. For histology analysis, GAE and GEE showed that villi heights were longer and tissue structure were more complete than N and C groups. Midgut villi, the important role for nutrient absorption, were patently increased in GAE and GEE. Interestingly, all growth parameters of GAE and GEE were significantly different (p ˂ 0.05). We concluded that garlic extracts could be effective for protein digestive enzymes, nutrition absorption, and growth performance in marble goby juvenile. Our research will be high useful to develop artificial diet formula for marble goby nursery that expect to helpful for increasing production.

Fifteen-week feeding trials were conducted to determine the optimum inclusion level of dietary cowpea or mung bean as novel protein sources for milkfish, Chanos chanos. Experimental diets containing either cowpea or mung bean at 0, 4, 8, 12, 16, or 20% of the diet equivalent to 0%, 3%, 7%, 10%, 13%, and 17% of the total dietary protein, respectively were formulated. Two groups of juvenile milkfish (ABW=7 and 9g) were stocked at 10 fish each in triplicate 250L capacity tanks supplied with well-aerated seawater in a flow-through system. One group was fed with the mung bean-based diets and the other with the cowpea-based diets. The fish were fed 3 times daily and growth was monitored every 3 weeks. Growth, protein efficiency ratio (PER) and feed conversion ratio (FCR) were not adversely affected by the inclusion of either mung bean or cowpea at any level in the diet. Likewise, nutrient compositions of the carcass and hepatosomatic index (HSI) were similar in all groups. The maximum inclusion level of dietary mung bean or cowpea was determined at 20% of the diet or 17% of the total dietary protein. To further evaluate the 2 diets, feeding trials in grow-out ponds and cages were carried out to compare their performance against a commercial milkfish feed. Better growth and FCR were observed in the cowpea- and mung bean- fed fish compared to those fed the control commercial milkfish feed in both pond and cage culture systems. Better feed efficiency resulting in better growth implies shorter culture period for the fish fed the formulated diets, which can also reduce water pollution due to feeding.

Fishmeal-free diets were tested for Nile tilapia (Oreochromis niloticus) in freshwater with varying degrees of success. However, tilapia farming is extending to saline waters. Salinity affect on the activities of hormones and enzymes which are biologically and physiologically important for growth and other functions. Beside growth, salinity appear to modify certain other aspects such as, feed intake, feed conversion efficiency, enzyme activity and hormones involved in the growth .Our objectives here, therefore, were to evaluate the combined effects of total fishmeal replacement and salinity on growth performances and relative expression of growth related genes.
A fishmeal (FM) diet and a non-fishmeal (NFM) diet were formulated (32% protein). Duplicated group of fish (initial weight : 6 g) reared in four salinity levels (0ppt, 4ppt, 8ppt and 12ppt) were fed one of the two diets to satiety twice a day for 12 weeks. At the end of the experiment, % specific growth rate (%SGR), mean feed intake (MFI), survival, food conversion ratio (FCR) and relative gene expression of hepatic IGF-I, IGF-II and growth hormone receptor were statistically compared.
The %SGR, MFI and FCR were not affected by the diets while effects were significant for salinity. The highest % SGR and MFI was observed in 4ppt while the lowest FCR was recorded in the 0ppt. Survival was 100 % in all treatments. Relative expression of hepatic IGF-I and IGF-II gene of Nile tilapia showed that its expression is regulated by the salinity. There were no dietary or dietary salinity interactive effects on IGF expression in liver. Gene expression of growth hormone receptor was not affected either by diet or salinity. Present finding provide evidence for the possibility of total fishmeal replacement in saline waters (0‰ to 12‰) without compromising growth, food consumption and relative expression of growth hormone receptor gene of Nile tilapia.

Although Nile tilapia is one of the most economically important freshwater fish, the cost of feed is the main obstacle factor to be considered for their cultivation. The culturing process should be improved by low-cost feed development from farming by-products. The grass fermentation in cow stomach is the low-cost raw material which could be used for feed ingredient. In this experiment, grass fermentation in cow stomach was added in the feed at a ratio percentage of 0 (control), 5 (GF5), 10(GF10), and 15(GF15). All feeds consisted of 30 percent proteins. The characteristics of digestive enzymes in the Nile Tilapia fed with the experimental feeds were also investigated. The results of growth were found that the final weight, the average increased weights, and average daily weight gain of fish in treatment GF10 were highest and significantly different (P<0.05). The results of digestive enzyme, the specific proteinase activity, was found to be highest among the extract enzyme from the intestine in the fish that was fed with GF10 at a pH value of 10 and 12. In conclusion, the diet containing 10 percentage of grass fermentation in cow stomach was suitable for culturing Nile tilapia.

Diets in which 50 % of the fish meal crude protein was replaced by soybean extract along with 60, 80 and 100 % of fish oil replacement by mixture of soy ,canola and linseed oils (1:1:1) were used in the study. Four isonitrogenous and isoenergetic diets were fed to juvenile Nile tilapia, Oreochromis niloticus (16,2 g AIWW) in triplicate (25 fish per tank) twice in equal portions at a fixed rate of 4 % BW.d-1 for 90 days. Fish doubled its initial weight and no statistical difference found among dietary treatments in terms of measured growth parameters. Diets supplemented with soybean extract and increasing amount of plant oil mixture proportionally increased the crude protein and significantly decreased (P<0,05) the crude ash levels in fish whole body samples compared to that of fish fed fish meal and oil control diet. Fish fed diets supplemented with soybean extract and increasing amount of plant oil mixture tended to increase the tissue essential and non-essential amino acid levels indicating that L-lysine and DL-methionine used in these diets were effectively used for growth in juvenile Nile tilapia. Whole body and tissue fatty acid compositions demonstrated that DHA was probably deposited whereas EPA was used for energy production in fish fed diets supplemented with soybean extract and increasing amount of plant oil mixture. Significantly higher (P<0,05) intermediate metabolites of the n-6 LC HUFA biosynthesis in whole body and tissue samples compared to that of fish fed the control diet also implicated that Nile tilapia might have activated the Δ5 and Δ6 elongation and desaturation enzymes. It was concluded that the combined replacement of dietary fishmeal and oil by soybean extract and plant oil mixture did not compromise growth rates, whole body and tissue essential and non-essential amino acid compositions, and n-3/n-6 ratios in this species.

Algae as dietary additives contribute to an increase in growth and feed utilization of cultured fish, also can strengthen the disease resistance. Chlorella is one of the microalgae used widely in aquaculture. It is a rich source of protein, lipid, vitamins, and other nutritional substances. This study was carried out to explore the effects of chlorella on growth performance, body composition, digestion, and disease resistance in rainbow trout.
Four diets containing 40% crude protein were formulated: 40% fish meal, designated as 40FM; 15% fish meal, 15FM; 15% fish meal supplemented with 2ppm chlorella, 15FM+2CL; 15% fish meal supplemented with 4ppm chlorella, 15FM+4CL. Four duplicate groups of fish with average body weight of 39.41±0.32g were fed one of four test diets for 12 weeks. Challenge test with the pathogenic bacterium Vibrio anguillarum was also conducted. Tissue samples of the anterior intestine (AI)) and distal intestine (DI) were collected for histological observation.
There was no significant difference in growth. However, feed conversion efficiency was lowest in 15FM and it was improved by supplementation with chlorella. Lipid content was lowest in 4CL, and it was improved by chlorella supplementation. There was no difference in histological features of the epithelial cells in fish. Survival rate following challenge test was significantly higher in 2CL than the others(p＜0.05).
The results might suggest that chlorella supplementation to the low fish meal diets can strengthen the disease resistance against the pathogenic bacterium Vibrio anguillarum in rainbow trout, and no negative effect on growth, body composition and digestion.

Our previous experiment suggests that rainbow trout fed diet supplemented with arginine shows higher plasma ornithine (a precursor of polyamine), and lower plasma citrulline after 18 hours post-prandial. However, supplemental arginine is also reported to increase arginine degradation through urea cycle and consequently reduce arginine availability. Thus, to avoid excessive arginine degradation and to better understand the role of citrulline, this research was aimed to evaluate the effect of dietary supplementation of ornithine and citrulline on resistance of rainbow trout, Oncorhynchus mykiss against Vibrio anguillarum.
Juvenile rainbow trout was reared in 60 L aquaria with a recirculating system at 15^o C. Dietary treatment was consisted of control diet (CTRL) which was formulated to have 47 % protein and 15 % lipid level, while treatment diets were made by supplementing control diet with 2% l-arginine (ARG), l-ornithine (ORN), and l-citrulline (CIT) at the expense of cellulose. Fish fed with designated diet twice a day, 6 days a week, for 30 days feeding period. After 30 days, fish were then injected peritoneally with Vibrio anguillarum diluted with phosphate-buffered saline at 3.0 x 106 CFU per fish. At 1 day post injection, RNA of 5 fish per tanks was extracted for quantitative-real-time PCR analysis.
A better growth performance was found in ARG compared to CIT, while there is no significant difference found between CTRL with other treatments. In the gene expression analysis, it was found that higher expression level of interleukin-1-beta was found in ORN and CIT treatments. However, a better survival respond was only observed in CIT treatment. Thus, based on the results of this study, it can be concluded that citrulline supplementation improves rainbow trout’s resistance against Vibrio anguilarum.

This series of studies were aimed to investigate whether enzyme-treated fish meal (EFM) can be replaced by soybean products to sustain the fast growth of juvenile Pacific bluefin tuna (PBT) and to reduce dietary cost.
Three trials were carried out with different soybean products. In trial 1, 20% soybean meal (SM), soy protein concentrate (SPC) or full fat soybean meal (FFS) was included in the diets. In trial 2, 10 or 20% SPC were included in the diets with or without phytase supplementation. In trial 3, 10 or 20% soy protein (SP) or enzyme treated SP (ESP) made from soy milk with reduced soybean trypsin inhibitor (SBTI) were included in the diets. PBT juveniles of 25 to 28 days old were used and the trials were carried out at the indoor rearing tanks. For digestibility analysis, 0.5% chromic oxide was used as an inert marker.
In trial 1, when 20% soybean products were included, all soybean diets showed significantly lower growth performance but the survival rate was similar among the treatments, except FFS. In trial 2, the inclusion of 10 or 20% SPC with or without phytase supplementation remarkably reduced the growth performance, nutrients digestibility and retention efficiency. However, phosphorus digestibility was remarkably higher in SPC, which resulted in lower phosphorus discharge to the environment. In trial 3, both 10 or 20% inclusion of ESP significantly reduced the growth performance. However, the PBT juveniles could maintain the growth performance and nutrients retention efficiency until 20% inclusion of SP in diets. The inclusion of SP also helped to reduce phosphorus discharge to the environment. The results suggest that PBT juveniles have less ability to utilize soybean products; however, when SP is made from soy milk with reduced SBTI, until 20% can be included without compromising the growth performance.

Production of fish that are better adapted to grow fast when fed diets with low fishmeal (FM) and fish oil (FO) represents an advantage for the further development of sustainable aquaculture. In mammals, nutrition during peri-conception and early development modulate metabolic routes to prepare the offspring for adverse conditions. Since marine fish have a very low bio-conversion ability to produce omega 3 highly-unsaturated fatty acids (n-3HUFA), the lack of these essential fatty acids in plant oils restrain their use as dietary ingredients. Nevertheless, plant oils are high in n-3HUFA precursors. Diet and certain environmental factors may regulate the expression of genes of enzymes bio-converting these precursors, although not to such extend to cover the essential fatty acid requirements. In mammals, dietary fats have been recognised as potent epigenetic factors that exerted at a critical developmental stage early in life may have long-term consequences to the physiological functions later in life. This presentation will review a series of studies conducted in seabream to determine the potential of fatty acids as modulators of early nutritional programming to promote fish genome adaptation for a better utilization of diets with low FM and FO. The effect of nutritional epigenetic factors on reproduction and larval, juvenile and adult performance were studied. The results demonstrated the feasibility of the early programming of fish through broodstock feeding for a better use of vegetable meals and oils and the very long-term effects resulting from parental epigenetic control of embryogenesis, even along the whole life cycle of the fish. A strong transgenerational effect of broodstock feeding on the offspring metabolism and growth performance was observed even in the 1.5 years-old offspring. Feeding broodstock with low n-3HUFA and linolenic acid improved growth and feed utilization of offspring fed only 5%FM and 3%FO diets in comparison to offspring whose parents were fed high n-3HUFA diets.

The Asian sea bass is one of the most economically important food fish cultured in the tropical and subtropical regions in the Asia–Pacific. Because of its relatively high market value, it has become an attractive commodity of both large to small-scale aquaculture enterprises. The development of suitable feed is required for this fish to make sure if its ingredients are properly digested. In addition, the digestive enzymes play a role in the growth and survival of aquatic animals. Trypsin and chymotrypsin are the 2 proteases that play the essential role in protein digestion and involving in growth rate. In Lates calcarifer, these two enzymes are indicated as growth parameters by feed. Therefore, enzyme assay at optimal condition is significant for its accuracy activity determination. Digestive enzyme distribution and activity in the digestive tracts of the Lates calcarifer. Characterization of digestive enzymes from digestive tracts of Lates calcarifer showed that Trypsin had optimum conditions at pH 8.0 temperature 60 °C while Chymotrypsin had optimum conditions at pH 5.0 temperature 60 °C.

[Introduction] Previous study suggested that lipid rich microalgae Schizochytrium sp. was able to replace fish oil in fish feed and showed possibility of suitable lipid source of non-fish oil and non-fish meal diet. There is necessity of higher level of study on replacement level of microalgae, the objective of this study was further study on non-fish meal and non-fish oil diet for red seabream, using microalgae Schizochytrium sp. which is rich in DHA (22:6n3).
[Method] Six iso-nitrogenous and iso-lipidic experimental diets were prepared (CP 44.6±0.6%, CL 13.2±1.2%). Control diet contained both fish meal (40%) and fish oil (6%). In second diet, fish meal was replaced by plant meals (soy protein concentrate, soybean meal, corn gluten meal) [NFM+FO]. In third diet, fish meal and fish oil was replaced by plant proteins and rape seed oil [NFM+NFO]. In the other three diets, fish meal and fish oil was replaced by algae meal (Schizochytrium sp. powder) respectively (5%, 10%, 15%), and plant proteins [AM5, AM10, AM15]. Triplicated groups of juvenile red seabream (4.5g±1.1) were fed the experimental diets for 12 weeks to near satiation.
[Results] The growth of the fish fed NFM+FO showed lowest growth, as elevation of algae meal, the growth was improved and showed no significance with control and NFM+FO. Feed efficiency showed same trend. Lipid content of NFM+NFO group was significantly lower than the other group. The results might suggest that plant protein diet with microalgae can be prospective material against skyrocketing prices of fish meal and fish oil.

A 12-week feeding trial was conducted to evaluate effects of high-level replacement of dietary fish meal (FM) with plant protein in diet on growth performance, histology and gene expression of inflammatory cytokines of juvenile red sea bream. Three isonitrogenous (44.7% crude protein) and isolipidic (12.8% crude lipid) practical diets were formulated to contain 50% FM, designated as FM; 20% fish meal, designated LFM; LFM diet supplemented with 0.5% taurine and 0.05% enzyme complex, designated as TELFM, respectively. Duplicate groups of 20 fish were fed one of the three diets to apparent satiation twice daily.
Fish fed with LFM have significantly lower weight gain and higher feed conversion ratio than FM groups. Supplementation with taurine and enzyme complex to LFM improved the growth. Typical pathological change (neutrophil infiltration and blood congestion) mainly appeared in intestine submucosa of fish fed LFM. In TELFM group, no significant morphological change was observed in intestine, as well as in the liver. However, in LFM groups, pathological alterations such as atrophy, hydropic degeneration and nuclear dislocation were observed in hepatocyte. Inflammatory cytokine genes showed different expression level among dietary treatments. The expression of interleukin-1β and interleukin-10 in intestine of LFM group were significantly higher than the FM and TELFM groups. The expression of intestinal interleukin-8, tumor necrosis factor-α and transforming growth factor-β1 were also up-regulated in fish fed with LFM. In LFM group, interleukin-8, tumor necrosis factor-α and transforming growth factor-β1 in liver were significantly higher than other two groups. These results suggest that inclusion of plant protein induces growth retardation and pathological changes in intestinal submucosa and hepatocyte of red sea bream and dietary supplementation with taurine and enzyme complex improve the negative impact.

Fish meal (FM) made from wild-caught small fishes is a necessary feedstuff for aquaculture of carnivorous fishes. The demand for FM in the world has been increasing due to world aquaculture production expanding. Recently, insects have drawn attention as protein source with substitution for FM in fish diet. We have developed the method to remove an inhibitory fraction from insect larva or pupa (Japanese Patent Application: 2016-113379), and succeed in a marked substitution for FM with housefly (Musca domestica) larva for red seabream (Pargus major) and with black soldier fly (Hermetia illucens) larva for red spotted grouper (Epinephelus akaara).
In this study, we performed one month feeding trials with young red seabream (Pargus major) to evaluate influence of the diets containing yellow mealworm larva (Tenebrio molitor; MW) removed the inhibitory fraction. Test diets were formulated as 25% MW (FM content 40%), 40% MW (FM content 25%), 65% MW (complete replacement of FM) and 65% FM (without MW). Interestingly growth and feeding amount of red seabream was promoted depending on increase of MW content in the diets, and the growth rate of 65% MW group were highest among the groups. To further confirm influence of the inhibitory fraction removed from MW, we additionally set a test group in which the inhibitory fraction was supplemented to the 65% MW diets. Since significant growth reduction was confirmed in the fraction-supplemented group compared with 65% MW group, the fraction removed from MW was proved to inhibit the growth of red seabream. In conclusion, complete replacement of FM for MW was apparently achieved with by the removal of inhibitory fraction in red seabream. Besides, our results expect MW is not merely alternative feedstuff, but has a potential to be protein source to improve aquaculture productivity remarkably.

Exploration of alternative protein sources in place of fish meal (FM) has been an important issue in fishery industry, since drastic price fluctuation has been pressured on aquaculture industry operation. It is for this reason that alternative proteins sources of FM, including insect meals, has explored to stably supply aquatic food. The present study demonstrated potential of house fly larva meal (HFLM) as alternative protein source of FM by assessing contents in HFLM and evaluating growth performance when FM (70% in diet) is solely replaced by HFLM in feeding trial with young red seabream (Pagrus major).
Our results indicated that HFLM contained a constitution of proteinosic amino acids close to FM and acceptable component of minerals but a small amount of ω-3 fatty acid relative to FM. Following the comparison of Body Weight Gain on feeding trial, it emerged that the diet HFLM + Fly Larva Oil (FLO) showed a statistically significant slower growth compared with other diet In this diet group, total feed intake per fish was less than the other diet groups. Importantly, during the necropsy, we observed an apparent white chyle in the pyloric caeca and intestines of this diet group only which means that FLO is not digested, suggesting its responsibility for growth retardation. The improvement in growth and feed intake (close to that of FM diet) was observed when half amount of FLO is substituted by fish oil (FO). It is possible and particularly important to increase the growth performance by removing FLO and replacing it with FO (more ω-3 fatty acids). These corrections will position HFLM as a substitution of protein source for the diets of marine fishery fishes.

Fish meal supplies the largest portion of dietary protein in fish diets. The manufacturing process of heating fish meal during production affects protein digestibility and absorption of protein by fish. While non-heated manufacturing processes is less affected by thermal denaturation. However, few study examined utility of non-heated animal protein sources on growth performance of fish. We investigated the effect of heated and non-heated squid and krill meal on the diet of juvenile red sea bream (RSB).
Five test diets were formulated to contain heated squid meal (HS), non-heated squid meal (NHS), heated krill meal (HK), non-heated krill meal (NHK) and fish meal (FM) as the control. Fifty fish (initial mean weight = 3.5g) were randomly distributed into ten 100L circular experimental tanks at a stocking density of 50 individuals per tank. Fish were fed one of the five diets for 5 weeks, three times daily until satiation. The carcass was sampled at 1^st, 3^rd and 5^th week after the initial feeding.
In terms of growth performance, fish fed the krill meal diet showed better growth than that of squid meal diet during 1^st week of rearing period. However, the squid meal diet group showed better performance than krill meal diet group after 3^rd week. In the 5^th week, difference in growth increased among treatments. These results indicate that heating animal protein source prior to incorporation in the diet affects growth of fish and non-heated squid and krill meal gives good growth compared to heated squid and krill meal.
This suggests that krill meal is effective in early feeding of juvenile RSB, however, increased in growth also increased in utilization of squid meal in the diet.