Characterization of digestive enzyme activity during larval development of gilthead seabream (Sparus aurata)

The evolution of the digestive enzyme equipment in seabream from hatching to 30 days old larvae was studied; there was a progressive increase in the activity of protease, amylase and acid and alkaline phosphatase from day 15 onwards. The use of specific inhibitors, and SDS-PAGE provided evidence to suggest that most of the proteases belonged to the serine group. A high -amylase activity was also denoted. Zymograms of larval extracts indicated that exogenous food has more a qualitative than a quantitative role in the secretion of digestive enzymes in this species.     

Optimum soybean lecithin contents in microdiets for gilthead seabream (Sparus aurata) larvae

The aim of the present study was to determine the optimum dietary levels of soybean lecithin (SBL) for seabream (Sparus aurata) larvae, and its influence on production performance and digestive enzymes activity. Larvae were fed five formulated microdiets with five levels of SBL. Complete replacement of live preys with the experimental microdiets for seabream larvae at 16 dph produced over 55% survival rates, particularly in fish fed with the highest levels of SBL. Moreover, increase in dietary SBL up to 80 g kg⁻¹ significantly improved larval growth, leading to high final total length and body weight. An increase in alkaline phosphatase activity with the elevation up to 80 g kg⁻¹ SBL was also found denoting a better maturation of the digestive system. Besides, there was a stimulatory effect of dietary SBL on PLA2 activity. Finally, increasing dietary SBL lead to better utilization of dietary highly unsaturated fatty acid, as it was reflected in their higher content in both neutral and polar lipid of the larvae. In summary, elevation of dietary SBL up to 80 g kg⁻¹ in microdiets for seabream significantly improved digestive enzymes activities, enterocyte maturation, utilization and deposition of dietary essential fatty acids and larval growth, as a consequence of a better digestion, absorption, transport and deposition of dietary nutrients.     

The use of squid protein hydrolysate as a protein source in microdiets for gilthead seabream Sparus aurata larvae

In the present study, the use of predigested proteins as an ingredient of microdiets offered to gilthead seabream larvae was tested. The protein source (freeze-dried squid powder) was hydrolysed with protease (trypsin and pancreatin). Different levels of raw squid protein and hydrolysate (100% protein, 50% protein/50% hydrolysate, 100% hydrolysate) were added to the microdiets to produce a dietary protein level of 65%. For comparison, cofeeding of Artemia nauplii and microdiet as well as microdiet supplemented with pancreatin were also offered to the larvae. The final average dry weights of 32-day-old larvae were 1.65 ± 0.04 mg, 1.38 ± 0.06 mg and 1.13 ± 0.1 mg, respectively, for larvae cofed 0%, 50% and 100% hydrolysate microdiets and Artemia nauplii. Survival of larvae was not affected by protein source. The survival of larvae cofed Artemia nauplii and microdiet was significantly higher than that of larvae fed exclusively on microdiet (68% and 80%, respectively). These results suggest that the use of hydrolysate (at 50% greater) as a protein source in diets for seabream larvae is not to be recommended.     

Effect of an experimental microparticulate diet on the growth, survival and fatty acid profile of gilthead seabream (Sparus aurata L.) larvae

In recent years, a great deal of interest has emerged in the development of microdiets as an economic alternative to live food, in the larval culture of marine fish species. The ability to grow Sparus aurata larvae on a prototype microparticulate diet was examined. To achieve this objective, four feeding regimes differing in the time when the microdiet was introduced (3, 7 or 12 days) and one based exclusively on an inert diet were tested, during the first 22 days of larval life. Significant differences in larval growth were found between the experimental feeding regimes and their corresponding controls (enriched rotifers during the whole experimental period); the larvae in the co-feeding regimes and with an exclusive microparticulate diet were always significantly smaller than larvae fed on rotifers alone. However, the difference was minimised by introducing the inert diet at a later date. A lower survival was found in larvae with a co-feeding regime, in comparison with the control treatments and the survival was significantly lower in larvae fed exclusively on a microparticulate diet. The fatty acid analysis revealed that the experimental microencapsulated diet and the rotifers enriched with Protein Selco® presented relatively similar fatty acid content. In spite of the slightly higher (n?3)/(n?6) and Docosahexaenoic acid (DHA)/Eicosapentaenoic acid (EPA) ratios and somewhat lower highly unsaturated fatty acid (HUFA) content found in the inert diet, the fatty acid composition of the diets cannot explain the differences found in larval performance. The results revealed that the complete replacement of live prey with the tested microparticulate diet is still not possible in S. aurata larval rearing. Nevertheless, better growth and survival results and a substantial reduction in the daily supply of live food can be achieved with a combination of microdiet and live prey.     

Evaluation of weaning performance of California halibut (Paralichthys californicus) larvae using growth,survival and digestive proteolytic activity

This study evaluated weaning success of California halibut, Paralichthys californicus, larvae onto a microdiet at various stages of development utilizing growth, survival and digestive enzyme activity. Weaning onto a microdiet was evaluated at 16, 26, 36 and 46 days posthatch (dph). Alkaline and acid proteases and leucine aminopeptidase activities were measured after weaning and compared between the weaned treatment and Artemia‐fed controls. Survival was significantly lower in the microdiet‐fed treatments compared to the control groups. Growth was significantly reduced in all weaning treatments compared to the control, except for the 46 dph group. No differences in enzyme activities were detected between treatment and diet at 16 and 26 dph; however, activities were higher for the microdiet‐fed larvae at 36 and 46 dph. This study demonstrates that California halibut larvae possess a differentiated and effective digestive system early in development and can be weaned with relative success (>40% survival) before completion of the metamorphosis (i.e., 36 dph). The lack of weaning success at an early date cannot be entirely because of the absence of a functional stomach but could be related to, among other factors, the low‐microdiet ingestion rates observed and higher leaching of smaller microdiets.     

Enhancement of gilthead seabream (Sparus aurata) larval growth by dietary vitamin E in relation to two different levels of essential fatty acids

The objective of this study was to determine the effect of dietary vitamin E on gilthead seabream (Sparus aurata) growth and survival, at two different highly unsaturated fatty acids (HUFAs) levels. Eighteen days old gilthead seabream larvae were fed four formulated experimental diets combining two different dietary levels of HUFAs (M: medium 2.5 + 1.5, DHA + EPA, H: high 5 + 2.5 DHA + EPA g per 100 g) with two different levels of vitamin E (M: medium 540 mg kg^?1, H: high 2900 mg kg^?1): MM, MH, HM, HH (HUFA/vitamin E). After 2‐week feeding trial, the average survival rate was 52.6% and there were no significant differences found among treatments. Increase in vitamin E up to high level markedly improved larval growth, particularly when dietary HUFA levels were lower, suggesting a higher protection value when these fatty acids are more limiting. At medium dietary HUFA levels, increase in vitamin E from medium to high level enhanced larval growth performance in terms of total length. Moreover, increase in vit E enhanced HUFAs content in the larval polar lipids denoting the anti‐oxidative effect of vitamin E.     

The effect of food competition and relative size of group members on growth of juvenile gilthead sea bream, Sparus aurata

The effects of food competition and relative size of group members on growth of sea bream, Sparus aurata were tested in groups composed of four fish. Each group consisted of a medium sized test fish (approximately 250 mg) and three fish larger or smaller than the test fish. In the first experiment food competition was addressed directly by contrasting its presence with its absence. Neither food competition nor relative size of school members had a significant effect on growth. However, there was a highly significant interaction between these two main effects. Under conditions of food competition the medium sized test fish had a three times higher specific growth rate when surrounded by small vs large fish. Under conditions of no food competition when the test fish were separated during feeding a similar but reverse pattern occurred due to differences in food conversion efficiency. In the second experiment, the effects of food quantity and relative size of group members were tested. Both main effects and their interaction were highly significant. Under conditions of low food competition (i.e., surplus food supply) fish grew again better when surrounded by larger fish as compared to their growth rate when surrounded by smaller ones. Size matching via a differential growth rate in a schooling species such as sea bream was suggested to increase the confusion effect by reducing the ability of a predator to focus on an odd sized fish. The implications of direct competition for food for the efficient culture of this species are discussed.     

Effects of dietary polyunsaturated fatty acid/vitamin E (PUFA/tocopherol ratio on antioxidant defence mechanisms of juvenile gilthead sea bream (Sparus aurata L., Osteichthyes, Sparidae)

Lipid peroxidation, specifically polyunsaturated fatty acid (PUFA) oxidation is highly deleterious, resulting in damage to cellular biomembranes, and may be a principal cause of several diseases in fish including jaundice and nutritional muscular dystrophy. Tissue lipid PUFA content and composition are critical factors in lipid peroxidation, as is the level of endogenous antioxidant molecules such as vitamin E. The primary objective of the present study was the characterization of antioxidant systems in a cultured juvenile marine fish, gilthead sea bream (Sparus aurata) with the underlying aim to understand how to avoid oxidation problems that may cause pathologies and disease and so to enhance growth and quality of early ongrowing stages. Juvenile sea bream were fed diets having either high or low levels of fish oil and supplemented or basal levels of vitamin E with PUFA/vitamin E ratios ranging from 117±12 in the diet with low PUFA supplemented with vitamin E to 745±48 in the diet with high PUFA with no additional vitamin E. None of the diets had serious deleterious effects on growth or survival of the fish, but the different dietary regimes were effective in significantly altering the PUFA/vitamin E ratios in the fish livers with values ranging from 5.7±0.4 in fish fed the diet with low PUFA supplemented with vitamin E to 91.1±13.2 in fish fed the diet with high PUFA with no additional vitamin E. This had effects on the peroxidation status of the fish as indicated by the significantly altered levels of in vivo lipid peroxidation products measured in liver, with fish fed the diet rich in PUFA and low in vitamin E showing significantly higher values of thiobarbituric acid reactive substances (TBARS) and isoprostanes. The isoprostane levels generally followed the same pattern as the TBARS levels supporting its value as an indicator of in vivo oxidative stress in fish, as it is in mammals. However, few significant effects on antioxidant enzyme activities were observed suggesting that more severe conditions may be required to affect these activities such as increasing the PUFA/vitamin E ratio or by increasing peroxidative stress through the feeding of oxidized oils.     

Characterization and functional properties of digestive proteases in two sparids; gilthead seabream (Sparus aurata) and common dentex (Dentex dentex)

Digestive proteases present in two sparids, seabream (Sparus aurata) and common dentex (Dentex dentex), have been characterized using both biochemical and electrophoretic techniques. Although optimum pH and temperature for maximum activity of both acid and alkaline proteases were similar in the two species, important differences in total activity, as well as in thermal and pH stability were found. Specific inhibitors and SDS-PAGE zymograms were used to clarify such differences. Evidences support the existence of a more active and complex protease set in dentex. Results are discussed from the perspective of their application to the formulation of feeds for each species.     

The pseudo-green water technique for intensive rearing of sea bream (Sparus aurata) larvae

The pseudo green water technology for sea bream(Sparus aurata) larval rearing was evaluated by analyzingresults of a 2-year study, performed in a pilot scale system. Thetechnology is characterized by the daily addition of phytoplankton in therearing tanks during the first month of rearing. Effects of egg origin,spawningseason and initial larval density on the larval rearing were investigated. Fishreared in pseudo green water systems for 60 days, presented highbiological performance in terms of survival (56 ± 16%), meanweight (62 ± 12 mg), total length (20 ± 1mm), conformity with wild standard (88 ± 9%), andfunctional swim bladder (93 ± 4%). The results present homogenitybetween the categories (origin of eggs, time of spawning, initial egg density)studied, proving the stability of the technology.     

The bioactivity of gonadotropin releasing hormones and its regulation in the gilthead seabream,Sparus aurata: in vivo andin vitro studies

Thein vivo andin vitro potency of native and modified forms of gonadotropin releasing hormone (GnRH) to release gonadotropin (GtH) was studied inSparus aurata and correlated with their relative susceptibility to degradation by cytosolic-bound enzymes of the pituitary, kidney, and liver. Salmon (s) GnRH and luteinizing hormone-releasing hormone (LHRH) are equipotent whereas analogs of these peptides ((D-Arg⁶-Pro⁹NET)-sGnRH, (D-Ala⁶-Pro⁹NET)-LHRH, (D-Trp⁶)-LHRH) are superactive in inducingin vivo GtH release (at 10 μg/kg body weight). In anin vitro superfusion system of pituitary fragments all analogs are equipotent to the native peptides (at 10⁻¹⁰ to 2.5 × 10⁻⁷M). sGnRH and LHRH are rapidly degraded by cytosolic peptidases of the pituitary, liver, and kidney. The preferred site of cleavage is the Tyr⁵-Gly⁶ bond. Substitution of the position 6 glycine by D-amino acids renders the 5–6 bond resistant to degradation and shifts the main site of cleavage to the Pro⁹-Gly¹⁰NH₂ bond. Substitution at position 6 (as above) and at position 10 with Pro⁹NET results in analogs that are resistant to degradation. We propose that enzymatic cleavage terminates GnRH bioactivityin vivo and thus increased resistance to degradation is a major determinant of GnRH analog superactivity.     

罗氏沼虾幼体及成虾消化酶活性

罗氏沼虾幼体及成虾消化酶活性魏华，赵维信（上海水产大学，上海２０００９０）关键词罗氏沼虾，幼体，消化酶ＴＨＥＡＣＴＩＶＩＴＩＥＳＯＦＤＩＧＥＳＴＩＶＥＥＮＺＹＭＥＳＯＦＬＡＲＶＡＥＡＮＤＡＤＵＬＴＩＮＭＡＣＲＯＢＲＡＣＨＩＵＭＲＯＳＥＮＢＥＲＧＩＩ￥...     

Lactobacillus spp. bacteria as probiotics in gilthead sea bream (Sparus aurata, L.) larvae: Effects on growth performance and digestive enzyme activities

In this study, the influence of commercial probiotic, Lactobacillus spp., supplementation was investigated on growth parameters and digestive enzyme activities in gilthead sea bream, Sparus aurata, during larval development. All experiments were triplicated and designed in three different administrations of probiotic from 3 days after hatching (DAH) concurrently with starting of exogenous feeding. In the first group, probiotic was added to live food (rotifer and Artemia). In the second group, probiotic was supplemented directly to both live food and water. In the third group, probiotic was added directly to water. Also, no probiotic treatment was maintained in control group. Total bacterial counts among probiotic probiotic-supplemented groups were significantly different from total bacterial counts in controls in water and digestive tract of larvae (p < 0.05). The mean of total bacterial counts in control was approximately 4 × 10⁴-fold increased from the experimental groups in the sea water (p < 0.05). Besides, mean digestive enzyme activities of all probiotics treatment groups were significantly different (p < 0.05) with that of the control. Except probiotic water supplementation group, in all treatments, the specific activities of pancreatic and intestinal enzymes were significantly higher (p < 0.05) in larvae to which probiotic had been supplemented by live food and live food with water. Also, S. aurata larvae that had probiotic administered by live food with water demonstrated significant (p < 0.05) increases in both survival (13–105% higher) and specific growth rate (2–9% higher) as compared to controls. As a result, supplementation of probiotic to directly tank water could not significantly increase growth parameters and digestive enzyme activities and therefore, administration of probiotics by this method would not be effective in terms of husbandry parameters and nutritional condition.     

Effect of diet composition on nutrient digestibility and digestive enzyme levels of gilthead sea bream (Sparus aurata L.)

An experiment was conducted to evaluate the effects of dietary composition on apparent digestibility coefficients of protein, fat and carbohydrate of gilthead sea bream. In addition α‐amylase and proteolytic activities were measured in the digestive tract of fish, held at 20±1°C. Six experimental diets were formulated containing approximately 40%, 45% and 50% protein, 11% and 21% fat and a starch level which fluctuated from 14% to 36%. Fish having an average weight of 100–130 g were used, fed at 1.5% of their body weight daily, and digestibility was measured using an indirect method. Enzyme activities were measured in the digestive tract of fish, fed diets containing a combination of 40%, 50% protein with 11%, 21% fat at 0.5, 5, 10, 24 h after a single meal. Starch digestibility was reduced with its level in the diet. It also negatively affected fat digestibility as well as protein digestibility, the last only at the high fat level. Dietary fat level had a negative effect on starch and protein digestibility. Fat affected also strongly α‐amylase levels in the digestive tract, while its effect on protease activity was of smaller magnitude. These results indicate that digestive enzyme activities and nutrient digestibility values are affected by dietary composition, carbohydrates and fat indicating the strongest effect.     

Starvation before slaughtering as a tool to keep freshness attributes in gilthead sea bream (Sparus aurata)

Instrumental quality parameters related to freshness were evaluated for gilthead sea bream (Sparus aurata) after different storage periods on ice (2, 4, 7 and 11 days of storage) for one, four and eight days of pre-slaughter starvation. The study was carried out on 360 gilthead sea bream from five different groups reared on a commercial fish farm. Starvation period had a significant effect on fish texture: the force necessary to compress 4 mm of flesh after two days ice storage in 1 day starved fish (16.50 ± 0.59 Newtons) was less than that for four day (18.98 ± 0.63 Newtons) and eight day starved fish (20.43 ± 1.12 Newtons). Significant differences in flesh puncture and flesh compression were not detected, although these values did increase with increasing starvation period. A significant increase in pH and myofibrilar protein solubility was also detected for fish starved for one day.     

The effect of dietary protein and total energy content on digestive enzyme activities, growth and survival of Litopenaeus setiferus (Linnaeus 1767) postlarvae

The effect of dietary protein and energy content on the activity of digestive enzymes (total proteinases, trypsin, chymotrypsin α‐amylase and lipase), and growth and survival of Litopenaeus setiferus postlarvae was investigated under controlled conditions. There was a clear relationship between the diet fed to the postlarvae, growth and survival. Highest weight gain (2110 ± 96.7%) was obtained with a 400 g kg^?1 protein and low energy diet (13.9 kJ g^?1) (P < 0.05). The optimal protein to energy ratio (P/E) estimated was 28.8 mg of protein kJ^?1. Good survival was obtained with low energy diets containing between 200 and 400 g kg^?1 protein and with high energy diets containing 300–500 g kg^?1 protein. Higher values for total proteinases, trypsin and α‐amylase were obtained with the low energy, 400 g kg^?1 protein diet. Chymotryptic activity was considerably lower than that of other proteinases and lipase activity was too low to be reliably measured with the turbidimetric method employed. Total proteinase activity was significantly lower than in experimentally grown postlarvae. The α‐amylase activity was at least two orders of magnitude higher in wild postlarvae than in animals fed with the best experimental diet. Protein requirement was related to total energy content of the diet: best growth and digestive enzyme activity coincide with the low energy, 400 g kg^?1 protein diet. These results suggest that dietary carbohydrates cannot spare protein because growth rates obtained with diets containing 200–300 g kg^?1 protein (337 and 226 g kg^?1 dextrin content, respectively) were significantly lowered.     

Alteration of liver and muscle fatty acid composition in gilthead seabream (Sparus aurata) juveniles held at high stocking density and fed an essential fatty acid deficient diet

The aim of the present study was to determine the combined effect of both stress and EFA deficiency on several biological and biochemical parameters. Fish were fed during 15 weeks two isocaloric and isoproteic diets: a control diet based on fish oil and formulated to meet the n-3 HUFA requirements for this species (1.5% of n-3 HUFA) and a deficient diet containing beef tallow and formulated to be deficient in n-3 HUFA. Each experimental diet was evaluated both at high and low stocking densities (10 and 3.2 kg m^–3 of initial density, respectively).High stocking density produced a chronic stress situation with elevation of plasma cortisol levels. It also caused a reduction in hepatosomatic index and liver lipid contents, increasing the oleic acid/n-3 HUFA ratios in the polar lipids. Fish fed the EFA deficient diet at low stocking density showed common deficiency symptoms. High stocking density in fish fed the EFA deficient diet induced a higher degree of EFA deficiency symptoms leading to mortality, liver steatosis, liver lipid deposition, reduced muscle lipid and reduced n-3 HUFA contents, which particularly affected EPA, but not DHA, suggesting a preferential retention of the latter fatty acid, specially in the phosphoglycerides fraction.     

胭脂鱼仔鱼的生长和消化酶活力变化

对胭脂鱼(Myxocyprinus asiaticus)孵化后1～37 d仔鱼的体长、体重、RNA/DNA比率、胰蛋白酶、脂肪酶和淀粉酶的酶活力变化进行了测定。结果显示:胭脂鱼仔鱼1～37 d体长稳定增长。体重增加呈阶段性,在1～21 d体重增长缓慢,21～29 d体重增加较快,29～37 d体重快速增加。仔鱼从7 d开始摄食,1～6 d,RNA/DNA比率变化很小,蛋白含量逐渐降低;7～21 d,RNA/DNA比率波动较大,蛋白含量增长缓慢;21～37 d,RNA/DNA比率和蛋白质均增长迅速。生长率(GR)与RNA/DNA比率的回归方程为:GR=1.01RNA/DNA+1.6077(R2=0.8516)。从2、5和7 d开始分别检测到明显的胰蛋白酶、脂肪酶和淀粉酶活力,之后,消化酶活力随着发育先快速地上升然后波动性地下降到一个稳定水平。结果表明:胭脂鱼仔鱼从内源性到外源性营养转化过程中,不同的消化酶能够检测到明显酶活力的时间不同,到仔鱼开始摄食时已经具备消化蛋白质、脂肪和淀粉的能力;RNA/DNA比率可以作为判定仔鱼生长率的可靠指标。     

Regulation of growth, fatty acid composition and delta 6 desaturase expression by dietary lipids in gilthead seabream larvae (Sparus aurata)

The Δ6 and Δ5 desaturases and elongases show only very limited activity in marine fish, and little is known of the possibility of enhancing Δ6 desaturase gene expression in these fish. The use of plant oils in marine fish diets is limited by their lack of n−3 highly unsaturated fatty acids (HUFA) despite an abundant content of the 18C fatty acid precursor linoleic and α-linolenic acids. The objective of the present study was to determine the ability of larval gilthead seabream to utilize vegetable oils and assess the nutritional regulation of Δ6 desaturase gene expression. Seventeen-day-old gilthead seabream larvae were fed during a 17-day period with one of four different microdiets formulated with either sardine fish oil (FO), soybean, rapeseed or linseed oils, respectively, or a fifth diet containing defatted squid meal and linseed oil. Good larval survival and growth, both in terms of total length and body weight, were obtained by feeding the larvae either rapeseed, soybean or linseed oils. The presence of vegetable oils in the diet increased the levels of 20:2n−9 and 20:2n−6, 18:2n−9, 18:3n−6, 20:3n−6 and 20:4n−6, in larvae fed rapeseed and soybean oils in comparison to those fed FO. In addition, a sixfold increase in the relative expression of Δ6 desaturase-like gene was found in larvae fed rapeseed and soybean oils, denoting the nutritional regulation of desaturase activity through its gene expression in this fish species. However, feeding linseed oil did not increase the expression of the Δ6 desaturase gene to such a high extent.     

Effect of fishmeal replacement by poultry by‐product meal on growth performance,proximate composition,digestive enzyme activity,haematological parameters and gene expression of gilthead seabream (Sparus aurata)

Two feeding trials examined the replacement of fishmeal (FM) with poultry by‐product meal (PBM) in the diet of juvenile Sparus aurata. In Feeding trial I (100 days), three diets were formulated, where FM protein was replaced by 50% (PBM50) and 100% (PBM100) PBM, while in Feeding trial II (110 days), four diets were formulated using the same FM control diet, but FM was replaced at lower levels: 25% (PBM25), and 25% (PBM25 + ) and 50% (PBM50 + ) with the supplementation of lysine and methionine amino acids. PBM protein can successfully replace 50% of FM protein in the diet of S. aurata without adverse effects on survival, feed intake, growth performance and feed utilization, given that the diet is balanced with lysine and methionine. The proximate composition of body and muscle was unaffected by the diet, but the total FM replacement resulted in reduced lipid and energy contents in fish. A fifty per cent FM replacement by PBM did not affect haematological parameters indicating a good fish health. Similarities in trypsin and chymotrypsin activities with FM‐fed fish suggest a high digestibility of PBM. High dietary levels of PBM reduced the liver gene expression of GH/IGF axis and of cathepsin D suppressing fish growth and modulating the protein turnover.