Effects Of Antinutritional Factors On The Utilization Of Soybean Proteins By Japanese Flounder, Paralichthys Olivaceus

Three experiment models (feeding trail, primary culture of intestinal epithelial cell in vitro and gastric infusion) were conducted to investigate the factors that influence the utilization of soybean meal by Japanese flounder (Paralichthys olivaceus). The objectives of the present study were to investigate why aquatic animal can not effectively utilize the plant protein sources, and how to increase the dosage of plant protein sources in aquatic feed by pretreatment and nutritional combination. The present studies include the following: (1) Effects of soybean saponins on feed intake, growth and histological structure of juvenile Japanese flounder; (2) Effects of soybean isoflavones on feed intake, growth and histological structure of juvenile Japanese flounder; (3) Effects of three soybean antinutritional factors on the morphology, livability, viability, membrane integrity and functions of intestinal epithelial cells of Japanese flounder; (4) Effects of fishmeal replacement by soybean meal on growth, feed efficiency ratio and apparent digestibility of Japanese flounder at equal feed intake. The results were summarized as follows:(1) Triplicate groups of juvenile Japanese flounder (2.58±0.01 g) were fed graded levels of soybean saponins for 56 days. The basal diet contained fish meal, fish oil and wheat flour, in which different levels of soybean saponins were added to obtain 0, 0.8, 3.2 and 6.4 g kg-1saponins (Diet 1 to 4). Feed intake, growth, feed efficiency ratio (FER), apparent digestibility and distal intestine structure were studied in fish fed different diets. Fish were weighed at day 28 and 56, and feed consumption was monitored daily. Results showed that feed intake, weight, whole-body crude lipid and apparent digestibility coefficients (ADC) of crude protein decreased significantly with increasing dietary soybean saponins levels (P < 0.05). Feed intake linearly decreased with increasing dietary saponins levels during the first four weeks (r =–0.869, P < 0.001), but no significant difference was detected among dietary treatmens at the end of the experiment (P > 0.05). Diet 4 significantly decreased the weight and FER of fish at day 28 and 56 compared to the other treatments (P < 0.05). The weight of fish fed Diet 3 was significantly depressed at day 28 but had no statistical difference at day 56 with the control diet. The integrity of histological structure of the distal intestine was gradually destroyed as soybean saponins levels increased. These results indicated that high level of dietary soybean saponins significantly depressed the feed intake and growth responses of Japanese flounder. It is suggested that the adverse effects of saponins in the soybean products can not be neglected when they were used as alternate fish feed ingredients.(2) Triplicate groups of juvenile Japanese flounder (2.58±0.01 g) were fed graded levels of soybean isoflavones for 56 days. The basal diet contained fish meal, fish oil and wheat flour, in which different levels of soybean isoflavones were added to obtain 0, 1.0, 4.0 and 8.0 g kg-1 isoflavones (Diet 1 to 4). Feed intake, growth, FER, apparent digestibility and distal intestine structure were studied in fish fed different diets. Results showed that 1.0 and 4.0 g kg-1 isoflavones supplementation (Diet 2 and Diet 3) did not significantly affect feed intake, weight, FER, whole-body composition and apparent digestibility coefficients (ADC) of the fish compared to the control group (P > 0.05). Diet 4 with 8.0 g kg-1 isoflavones significantly depressed the weight, FER, crude lipid content in fish body and ADC of flounder compared to the other groups (P < 0.05). The integrity of histological structure of the distal intestine was also destroyed in the fish fed Diet 4. These results indicat that soybean isoflavones are unlikely to affect fish growth performance at the level to be present in usual soybean products (1³ g kg-1).(3) Intestinal epithelial cells of Japanese flounder were isolated and cultured in vitro to evaluate and compare the effects of three heat stable soybean antinutritioanl factors on its morphologic characterization, livability, viability, membrane integrity and function. Four different treatments were tested in this experiment: Control, SS (contain 2 g L^-1soybean saponins), SP (contain 4 g L^-1sodium phytate) and SR (contain 8 g L^-1raffinose). Results showed that soybean saponins at the concentration of 2 g L^-1 in the culture medium significantly decreased the livability, viability and alkaline phosphatase (AKP) activity of the intestinal epithelial cells compared to the other three treatments (P < 0.001), the integrity of the membrane and cell morphology were also found severely damaged in the saponin-treated cells. Sodium phytate at the level of 4 g L^-1 in the culture medium significantly decreased the livability, viability and AKP activity of the intestinal epithelial cells compared to the control group (P < 0.05), integrity of the cell membrane and the cell morphology were also damaged in the sodium phytate-treated cells. Raffinose at the level of 8 g L^-1 in the culture medium did not significantly afftect the livability, viability and AKP activity of the intestinal epithelial cells compared to the control group (P > 0.05). These results indicat that the negative effects of soybean saponins and phytate in the soybean products can not be neglected when they used as alternative fish feed ingredients, while raffinose at levels to be present in fish diets containing commercially available plant-derived protein sources, are unlikely to affect fish growth performance.(4) Using fishmeal and soybean meal as protein sources, four isonitrogenous (crude protein 50.0%) and isocaloric (gross energy 20.5 KJ/g) semiliquid diets were formulated to meet the protein and energy requirements of Japanese flounder. The study was performed to evaluate the effect of supplementation of antinutritional factors (0.2% soybean trypsin inhibitor, 0.2% soybean saponins, 0.2% soybean isoflavones, 0.4% sodium phytate and 0.8% raffinose) and amino acids (0.75% methionine and 0.25% lysine) on growth performance and nutrient digestibility of Japanese flounder (60±0.66 g) fed diets containing either fishmeal or 45% fishmeal replacement by soybean meal as dietary protein. The fish of all treatments were force-fed by gastric tube at equal feed intake. Diet was assigned to triplicate groups of 5 fish per aquarium. Every fish were infused 1 ml semiliquid feed (0.54 g dry matter) to the stomach per day and weighed every four days. Results showed that force-feeding was a feasible method to rear flounder with high feed efficiency ratio (FER, 1.22 in control) and survival (100%). Although feed intake was equal, 45% fish meal replaced by soybean meal significantly decreased the weight gain, FER, and digestibility compared to the control treatment (P < 0.05). Supplementation with antinutritional factors significantly decreased the weight gain and FER of the fish compared to the control group only in the first four day (P < 0.05), but had no significant effect during the rest experiment period (P > 0.05). Weight gain, FER and digestibility were not affected by supplementation with amino acids compared to the soybean meal group (P > 0.05). It is suggested that there might be some other factors that restrict the utilization of soybean meal by Japanese flounder besides antinutritional factors and amino acids imbalance. The following three factors are hypothesized: some energy substances contained in soybean that can not be well utilized by fish; the negative effect of the soybean protein itself; different composition of hydrolyzed small peptides between fish meal and soybean meal.