Alternative protein sources in diets for Japanese flounder Paralichthys olivaceus (Temminck and Schlegel): II. Effects on nutrient digestibility and digestive enzyme activity

An 8‐week feeding trial was conducted to compare the effects of dietary protein sources on nutrient digestibility and digestive enzyme activity. Four experimental diets were formulated to contain one of the following as the sole protein source: fish meal (FM), fish protein concentrate (FPC), soy protein concentrate (SPC) and soy protein isolate (SPI). Each diet was randomly assigned to triplicate aquaria stocked with 25 Japanese flounder (Paralichthys olivaceus) each. The dry matter, crude protein and energy digestibility and individual amino acid availability of the SPC‐based diet were significantly lower than those of the other diets. The crude lipid digestibility of soy protein‐based diets was significantly lower than that of the FM‐based diet. The pepsin/protease activity was significantly higher in fish fed fish protein‐based diets compared with fish fed soy protein‐based diets. The lipase activity in fish fed the SPI‐based diet was the highest among the dietary treatments. These results indicate that P. olivaceus can effectively digest the protein from FPC and SPI (but not SPC) as well as FM. The low protein digestibility and amino acid availability of the SPC‐based diet may be related to the non‐protein compounds present in SPC, whereas the low‐lipid digestibility of soy protein‐based diets may contribute to the undigested soy protein fractions and/or the alcohol‐soluble components.     

Cytological studies on induced meiogynogenesis in Japanese flounder Paralichthys olivaceus (Temminck et Schlegel)

The cytological process of induced gynogenetic development and subsequent chromosome duplication by a cold shock treatment was observed in Japanese flounder Paralichthys olivaceus (Temminck et Schlegel). Mature eggs were at the metaphase of the second meiosis when inseminated with ultraviolet (UV)-irradiated sperm of red sea bream Pagrus major . After the beginning of cold shock treatment, the previously visible spindle became invisible, probably due to the side effect caused by cold shock treatment. The chromosomes at the centre of the metaphase plate were condensed. This condition continued during the duration of the cold shock treatment and several minutes after it. The release of the second polar body was blocked and it developed into a female-like pronucleus. Then, it fused with the female pronucleus to generate a diploid zygotic nucleus, and the egg exhibited the first mitosis. Consequently, the haploid female chromosome set of the egg was doubled by the inhibition of the second polar body release. There was a significant delay in developmental time in the gynogenetic eggs when compared with that in the normal eggs. From the time of insemination to early cleavage, the UV-irradiated heterospecific sperm nucleus remained condensed.     

Occurrence of Listonella anguillarum in seed production environments of Japanese flounder Paralichthys olivaceus (Temminck et Schlegel)

The present study was undertaken to investigate the distribution of Listonella anguillarum in the rearing water, fish and diets (rotifers) of Japanese flounder (Paralichthys olivaceus). A total of 793 isolates were obtained from the seed production environment of Japanese flounder and 175 out of them were identified as L. anguillarum by biochemical characterization, polymerase chain reaction (PCR) detection for VAH1 haemolysin gene and phylogenetic analysis of 16S ribosomal deoxyribonucleic acids (rDNA) sequences. These results strongly suggested that L. anguillarum is rapidly and accurately identified by the combination of incubation on thiosulphate–citrate–bile salt–sucrose agar at 35°C overnight and PCR detection for the VAH1 haemolysin gene. All flounder specimens and all rotifer samples harboured L. anguillarum at high densities of 6.9 × 10³–6.3 × 10⁵ colony forming units (CFU) g^?1 and 1.5 × 10⁴–2.3 × 10⁶ CFU g^?1, respectively, while as low as 5.0 × 10⁰–2.0 × 10¹ CFU mL^?1 of L. aguillarum were detected in only two of 11 seawater samples, even though no vibriosis occurred in larval and juvenile flounder of tanks. This fact strongly suggests that L. anguillarum is an inhabitant in the seed production environments of Japanese flounder.     

Effects of feeding frequency and dietary energy level on growth and body composition of juvenile flounder, Paralichthys olivaceus (Temminck & Schlegel)

A two (dietary energy levels; low energy, LE and high energy, HE) × 4 (feeding frequency; one feeding in 2 days, one feeding daily, two times daily and three times daily) factorial experiment was performed to determine the effects of dietary energy level and/or feeding frequency on the growth and body composition of juvenile flounder Paralichthys olivaceus. The survival rate was not significantly different among treatments. Weight gain of fish fed the LE and HE diets significantly increased as feeding frequency increased. The weight gain of fish fed the LE diet was higher at each level of increasing feeding frequency. The weight gain of fish fed the HE diet was higher than that of fish fed the LE diet once in 2 days and once daily, but weight gain of fish fed the HE diet was lower than the LE diet three times daily. Daily feed intake was significantly influenced by feeding frequency, but not by dietary energy level. Feed efficiency of fish fed the HE diet once daily was significantly higher than that of fish fed the LE diet once in 2 days, but no significant difference in feed efficiency was observed among other groups of fish. The protein efficiency ratio of fish fed the LE diet once in 2 days was the lowest. Feeding frequency and dietary energy level had a significant effect on the body lipid content. A feeding frequency of two times or three times daily would be effective, depending on dietary energy level for maximum growth of juvenile flounder grown from 3.5 to 15 g.     

Efficacy of Vibrio anguillarum antigen administered by intraperitoneal injection route in Japanese flounder, Paralichthys olivaceus (Temminck et Schlegel)

Japanese flounder, Paralichthys olivaceus (Temminck et Schlegel), were immunized by the intraperitoneal (i. p.) injection route with formalin‐killed whole cells of Vibrio anguillarum that originated from a diseased fish. Fifty days later, a booster vaccination was given by the same route. Control fish were similarly treated with sterile phosphate‐buffered saline. The efficacy of vaccination was evaluated based on protection against two bacterial challenges and immune responses (both specific and non‐specific). The challenges were performed by i. p. injection with V. anguillarum or V. parahaemolyticus. The results indicated that the vaccinated fish showed higher non‐specific immune activity than the unvaccinated fish. The effects of vaccinations on the phagocytic activity of phagocyte, bactericidal and lysozyme activities were notable, especially on bactericidal activity. Determined by ELISA, antiserum of vaccinated fish displayed high antibody titres. The vaccination conferred protection against V. anguillarum challenge (81.25–93.75% relative percentage survival (RPS)). The RPS was 46.15–53.85% against V. parahaemolyticus challenge, indicating some degree of cross‐protective immunity.     

Effects of starvation on kidney melano-macrophage centre in olive flounder, Paralichthys olivaceus (Temminck and Schlegel)

We investigated the influence of nutritional conditions on histological changes in melano‐macrophage (MM) accumulation in the kidney caused by long‐term starvation. These data were used to determine nutritional indices for olive flounder, Paralichthys olivaceus (Temminck and Schlegel). Experimental groups were established: control, fed and starved. All fish were fed daily with commercial feed at 1–3% of their total body weight for 2 weeks before the start of the experiment (initial control). The control group received feed at 1–3% of their total body weight and the fed group received feed ad libitum, while the starved group fasted throughout the experiment. Ten fish were removed from the initial control at the start of the experiment and each of the three groups every 4 weeks during the 12 weeks of the experiment to examine to investigate kidney MM accumulation. Kidney MMs with dark brown pigment were randomly distributed in the kidneys of starved fish, increasing rapidly after week 4, while deposition levels remained low throughout the experiment in the control and fed groups. These results suggest that catabolic tissue breakdown is a major factor contributing to the formation of pigments within MMs. These results suggest that the degree of MMs deposition in the kidney can be used as alternative indicators to identify starvation in wild and cultured olive flounder.     

乳酸菌对牙鲆稚鱼养殖水体和肠道菌群的影响

在牙鲆稚鱼中投喂添加由单一鼠李糖乳杆菌P15制备的微生态制剂和黄霉素,在60d的投喂期内,采用平板计数法检测好氧性异养菌的总数、总弧菌数和乳酸菌的数量。结果表明,在投喂菌液和冻干菌粉后,养殖水体和牙鲆肠道的乳酸菌数均呈上升趋势,在30d后乳酸菌数量达到稳定并在肠道内定植。同时,由于乳酸菌的抑制作用,弧菌的数量下降,以肠道中的弧菌最明显。乳酸菌对养殖水体和牙鲆肠道的好氧性异养菌没任何影响。乳酸菌对养殖水体和肠道菌群的影响与抗生素具有相近的效果,表明乳酸菌作为饲料添加剂可以取代抗生素应用在牙鲆的养殖中。     

Pathogenicity of Edwardsiella tarda to olive flounder, Paralichthys olivaceus (Temminck & Schlegel)

The LD50 and cytotoxic and enzymatic activities of both cells and extracellular products (ECPs) of eight Edwardsiella tarda strains were determined and their bacterial superoxide dismutase gene (sodB) and catalase gene (katB) were sequenced. Strains were also examined for their ability to resist the immune responses of olive flounder, Paralichthys olivaceus. LD50 values of strains (FSW910410, KE1, 2, 3, 4, 5 and 6) in olive flounder ranged between 10(2.5) and 10(5.3) cfu (colony forming units) per fish. Unlike the avirulent strain SU100 (LD50>or=10(7)), all pathogenic strains were able to survive in flounder serum and head kidney leucocytes (except for KE2). The virulent strains possessed type I sodB and katB, whereas SU100 had type II sodB but not katB. However, there was no difference between avirulent and virulent strains in haemolytic and cytotoxic activities. The results of this study demonstrated that the ability of E. tarda to resist complement activity and phagocytosis is conferred by its superoxide dismutase and catalase, which thus play an essential role in the pathogenicity of this bacterium. In addition genotyping of sodB and kat B proved to be a very useful tool to distinguish virulent from avirulent strains.     

Effects of formalin on haematological and blood chemistry in olive flounder, Paralichthys olivaceus (Temminck et Schlegel)

Erythrocytes of olive flounder, Paralichthys olivaceus (Temminck et Schlegel), were treated with serial concentrations of formalin (37% formaldehyde) to investigate in vitro haemolysis and methaemoglobin formation. In addition, the short‐term toxicity of formalin concentrations of 0, 100, 212 and 300 ppm was also studied by clinical tests in which fish were subjected to 3‐h bath exposure. There was no haemolysis of fish erythrocytes exposed to formalin concentrations ranging from 31.3 to 2000 ppm. Methaemoglobin formation, however, was induced at concentrations greater than 500 ppm. Red blood cell count, haemoglobin, haematocrit, mean corpuscular haemoglobin concentration and percentage of immature erythrocytes were also markedly elevated in all formalin‐exposed groups (P<0.05). Formalin exposure also caused significant increases in alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase, potassium, chloride, magnesium and inorganic phosphorus (P<0.05). However, total protein decreased significantly in the formalin‐exposed groups (P<0.05). No significant differences in white blood cell count, mean corpuscular volume, mean corpuscular haemoglobin, albumin, glucose, total cholesterol, high‐density lipoprotein cholesterol, free cholesterol, alanine aminotransferase, calcium, creatinine and total bilirubin were observed in the formalin‐exposed groups (P>0.05).     

Optimum dietary protein level for maximum growth of juvenile olive flounder Paralichthys olivaceus (Temminck et Schlegel)

This experiment was conducted to determine the optimum dietary protein level for juvenile olive flounder Paralichthys olivaceus (Temminck et Schlegel) fed a white fish meal and casein‐based diets for 8 weeks. Olive flounder with an initial body weight of 4.1 ± 0.02 g (mean ± SD) were fed one of the six isocaloric diets containing 35%, 45%, 50%, 55% and 65% crude protein (CP) at a feeding rate of 4–5% of wet body weight on a dry‐matter basis to triplicate groups of 20 fish per aquarium. After 8 weeks of feeding, per cent weight gain (WG) and feed efficiency ratios of fish fed the 55% CP diet were not significantly higher than those from fish fed the 50% and 65% CP diets, but significantly higher than those from fish fed the 35% and 45% CP diets. Fish fed the 50%, 55% and 65% CP diets had significant higher specific growth rates than did fish fed the 35% and 45% CP diets; however, there was no significant difference among fish fed the 50%, 55% and 65% CP diets. The protein efficiency ratio was inversely related to the dietary protein level; that is, maximum efficiency occurred at the lowest dietary protein level. Broken‐line model analysis indicated that the optimum dietary protein level was 51.2 ± 1.8% for maximum weight gain in juvenile olive flounder. The second‐order polynomial regression analysis showed that the maximum WG occurred at 57.7% and it revealed that the minimum range of protein requirement was between 44.2% and 46.4%. These findings suggest that the optimum dietary protein level for maximum growth could be greater than 46.4%, but less than 51.2% CP in fish meal and casein‐based diets containing 17.0 kJ g^?1 energy for juvenile olive flounder.     

Dietary vitamin A requirements of juvenile Japanese flounder Paralichthys olivaceus

A 70‐day feeding experiment was conducted to assess the dietary vitamin A (VA) requirements of juvenile Japanese flounder (Paralichthys olivaceus). Six semi‐purified diets with VA supplementations of 0, 5000, 10 000, 15 000, 20 000 and 25 000 IU kg^?1 were fed twice a day to triplicate groups of 20 juveniles per tank with an initial weight of 1.59 ± 0.01 g (mean ± SE). Weight gain (WG) and specific growth rate (SGR) increased as dietary VA increased up to 10 000 IU kg^?1. Significantly lower WG and SGR were observed for the 0 IU kg^?1 treatment than for treatments of 5000, 10 000 and 15 000 IU kg^?1. Highest WG and SGR were observed in fish fed 10 000 IU kg^?1; slightly lower values were recorded in fish fed 15 000, 20 000 or 25 000 IU kg¹. No significant difference was observed in survival rate among treatments. Whole body total lipid was significantly higher in fish fed 0 and 5000 IU kg^?1 than for other levels. Reduced growth and small livers were observed as signs of VA deficiency in fish fed 0 IU kg^?1. Slightly reduced growth and pale fragile livers were observed as effects of VA excess in fish fed 25 000 IU kg^?1. Total retinol contents in liver and eyes increased with increasing levels of dietary VA. No retinol was detected in livers, and significantly lower total retinol content was observed in eyes, of fish fed 0 IU kg^?1. WG analysed by the broken line method indicated that an optimum dietary VA requirement of 9000 IU kg^?1.     

Effects of arachidonic acid on pigmentation and fatty acid composition of Japanese flounder, Paralichthys olivaceus (Temminck and Schlegel)

The dietary effect of arachidonic acid (20:4n-6, AA) on the composition of the head and the body, and on the pigmentation of Japanese flounder, Paralichthys olivaceus (T. & S.), was studied by means of microbound diets. Increasing levels of this essential fatty acid produced higher growth and pigmentation success in juveniles after 45 days of feeding. The role played by AA in the composition of phosphatidylinositol and eicosanoid synthesis is discussed in relation to the normal function of the neural system and the production of hormones involved in the metamorphosis and pigmentation of flatfish.     

Evaluation of optimum dietary protein-to-energy ratio in juvenile olive flounder Paralichthys olivaceus (Temminck et Schlegel)

An 8‐week feeding trial was conducted to estimate the optimum dietary protein to energy (P/E) ratio in juvenile olive flounder Paralichthys olivaceus. Eight experimental diets were formulated with two energy levels and four protein levels at each energy level. Two energy levels of 12.5 and 16.7 kJ g^?1 diets were included at crude protein (CP) levels of 25%, 30%, 35% and 45% with 12.5 kJ g^?1, and CP levels of 35%, 45%, 50% and 60% with 16.7 kJ g^?1. After 1 week of the conditioning period, fish initially averaging 8.1±0.08 g (mean±SD) were randomly distributed into the aquarium as groups of 15 fish. Each diet was fed on a dry‐matter basis to fish in three randomly selected aquariums at a rate of 3–5% of total wet body weight per day for 8 weeks. After 8 weeks of the feeding trial, weight gain (WG), feed efficiency ratio and specific growth rate of fish fed 45% CP with 16.7 kJ g^?1 energy diet were significantly higher than those from the other dietary treatments (P<0.05). WG of fish fed 12.5 kJ g^?1 energy diets increased with the increase of dietary protein levels. However, WG of fish fed 16.7 kJ g^?1 energy diets increased with the increase of dietary protein levels up to 45% CP and then decreased when fish fed 50% and 60% CP diets. Both dietary protein and energy affected protein retention efficiency and energy retention efficiency. Haemoglobin (Hb) of fish fed 35% and 45% CP diets with 12.5 kJ g^?1 energy were significantly high and not different from Hb of fish fed 45% and 50% CP diets with 16.7 kJ g^?1 energy. Haematocrit of fish fed 45% CP diet with 16.7 kJ g^?1 energy was significantly higher than those from fish fed 25% and 30% CP diets with 12.5 kJ g^?1 energy (P< 0.05). Based on the results of this experiment, we concluded that the optimum dietary P/E ratio was 27.5 mg protein kJ^?1 with diet containing 45% CP and 16.7 kJ g^?1 energy in juvenile olive flounder.     

Genetic relationship between broodstocks of olive flounder, Paralichthys olivaceus (Temminck and Schlegel) using microsatellite markers

For the first generation of a selective breeding programme, it is important to minimize the possibility of inbreeding. This mostly occurs by mating between closely related individuals, while proper mating can provide an opportunity to establish the base families with wide genetic variation from which selection for subsequent generations can be more effective. Genotyping with microsatellite‐based DNA markers can help us determine the genetic distances between the base populations. The genetic markers further facilitate the identification of the correct parents of the offspring (parentage assignments) reared together with many other families after hatching. We established a genetic analysis system with microsatellite DNA markers and analysed the genetic distances of three farmed stocks and a group of fish collected from wild populations using eight microsatellite markers. The averaged heterozygosity of the farming stocks was 0.826 and that of the wild population was 0.868. The hatchery strains had an average of 8.6 alleles per marker, which was less than a wild population that carried an average of 14.3 alleles per marker. Significant Hardy–Weinberg disequilibrium (HWDE) was observed in two farming stocks (P<0.05). Despite relatively low inbreeding coefficiency of the hatchery populations, the frequency of a few alleles was highly represented over others. It suggests that the hatchery stocks to some extent have experienced inbreeding or they originated from closely related individuals. We will develop a selective program using the DNA markers and will widen the usage of the DNA‐based genetic analysis system to other fish species.     

Effects of dietary arginine and lysine levels on growth performance and biochemical parameters of juvenile Japanese flounder Paralichthys olivaceus

ABSTRACT: To investigate the effects of disproportionate levels of dietary arginine and lysine on juvenile Japanese flounder Paralichthys olivaceus , growth performance and biochemical parameters were evaluated by feeding five test diets, comprising different levels of arginine and lysine, to triplicate groups of juveniles (initial bodyweight 1.85 g) for 40 days. Crystalline amino acids were supplemented to test diets to correspond to the amino acid pattern found in the whole body protein of the Japanese flounder, except for arginine and lysine. After the feeding trials, plasma arginine, lysine and urea levels, excreted ammonia-N, and liver arginase activity were analysed. Survival, specific growth rate, feed conversion efficiency, and apparent protein retention were adversely affected ( P  <   0.05) by the deficiency in dietary arginine or lysine concentrations. An excess of either lysine or arginine in the diet did not depress growth when the diets contained adequate levels of either arginine or lysine, proving that there is no evidence for an arginine–lysine antagonism. Results for plasma arginine, lysine and urea levels, excreted ammonia, and liver arginase activity also demonstrated that Japanese flounder juveniles are not sensitive to excess dietary levels of lysine and arginine.     

牙鲆亲本对子代贡献率的实验研究

利用18对高度多态性微卫星标记, 对13尾牙鲆(Paralichthys olivaceus)亲鱼的188尾后代进行亲子鉴定, 计算其非亲排除率、鉴定准确率以及亲本对子代的贡献率。使用4个微卫星标记进行亲子鉴定, 排除率≥0.999 9, 准确率为92.02%; 用6个标记的排除率≥0.999 999, 准确率为96.81%; 用8个标记的排除率≥0.999 999 999, 准确率为97.87%; 用10个标记的准确率为99.47%; 用12～18个标记的准确率为100%。亲子鉴定准确率随非亲排除率和微卫星标记数目的增多而升高。检验所有子代全部是实验亲本的后代, 即13个亲本对子代均有贡献, 但不同亲本的贡献率不同, 最高为47.34%, 最低为0.53%。实验表明, 基于多标记的非亲排除率检测方法可以用于牙鲆亲子鉴定, 其鉴定准确率可以达到100%; 不同亲本对子代贡献率不同, 差异很大, 产生差异的原因有待进一步研究。

     

Optimal dietary taurine level for growth of juvenile Japanese flounder Paralichthys olivaceus

The requirement for taurine in juvenile Japanese flounder Paralichthys olivaceus was determined by feeding diets containing various levels of taurine and cystine. Test diets supplemented with 0.5, 1.0 and 1.5% of taurine or with 0.5, 1.0 and 1.5% of L -cystine were prepared. The basal diet contained 55% protein from white fish meal. These diets were fed to juvenile Japanese flounder with an initial mean bodyweight of 0.9 g (total length (TL) 48 mm) for 5 weeks. Approximately 1.4% taurine content in the diet was required for optimum growth of juvenile flounder. A positive linear relationship was noted between the content of taurine accumulated in the muscle, liver and brain and the level of taurine in the diet. However, there was no increased taurine content in tissues of fish fed the cystine-supplemented diet. In contrast, the fish fed control and cystine-supplemented diets showed higher contents of cystathionine in the tissues. The concentration of cystathionine in tissues rapidly decreased with an increase of taurine in the diet. It was also observed that for each of the dietary groups, a trace amount of taurine was excreted. These results suggest that the taurine content in the diet affects the sulfur amino acid metabolism of juvenile Japanese flounder, and indicate that juvenile flounder are unable to biosynthesize taurine from cystine.     

复合诱食剂对牙鲆摄食生长的影响

以初始体质量为(3.01±0.04)g的牙鲆(Paralichthys olivaceus)为实验对象,进行为期70 d的摄食生长实验。共配制5种等氮等能饲料,其中以鱼粉和豆粕为蛋白源(豆粕蛋白替代45%的鱼粉蛋白)配制出基础饲料,分别在基础饲料中添加0.0%、0.5%、1.0%和1.5%的复合诱食剂配制成4种豆粕取代饲料,同时以全鱼粉饲料为对照饲料,研究复合诱食剂对牙鲆摄食生长的影响。结果表明,当豆粕取代饲料中诱食剂添加量为1.0%时,牙鲆的摄食率(P<0.05)、饲料效率(P<0.05)、鱼体脂肪含量(P<0.01)以及肝脏、肠胰蛋白酶活力和肠氨肽酶活力(P<0.01)显著高于不添加诱食剂组;当豆粕取代饲料中诱食剂的添加量为0.0%和0.5%时,牙鲆的特定生长率显著低于全鱼粉组(P<0.05);而当添加量为1.0%和1.5%时,牙鲆的特定生长率与全鱼粉组比较没有出现显著差异(P>0.05)。结论认为,当以豆粕蛋白替代45%的鱼粉蛋白时,添加1.0%的复合诱食剂能够显著提高牙鲆幼鱼对饲料的摄食率和生长率。