【作者】 陈伟；

【导师】 麦康森；

【作者基本信息】 中国海洋大学 ， 水生生物学， 2009， 博士

【摘要】 本文选择我国典型的肉食性鱼类牙鲆（Paralichthys olivaceus）为研究对象,采用传统养殖实验、体外细胞培养和胃内灌喂三种不同的实验模式比较研究影响牙鲆利用大豆蛋白的因素,阐明水生动物不能有效利用植物性蛋白源的原因。研究内容包括:（1）大豆皂甙对牙鲆摄食、生长和组织学的影响;（2）大豆异黄酮对牙鲆摄食、生长和组织学的影响;（3）三种大豆抗营养因子对牙鲆肠上皮细胞形态、存活率、增殖、膜完整性和功能的影响;（4）等摄食条件下用豆粕替代鱼粉对牙鲆生长、饲料效率和表观消化率的影响。主要研究结果如下:（1）以鱼粉为主要蛋白源,鱼油为脂肪源,小麦粉为糖源,以三氧化二钇为外源性指示剂,制成4种等氮、等能（粗蛋白49.1%,总能20.1 KJ/g）的实验饲料,使饲料中大豆皂甙实际含量分别达到0 （对照组）、0.08%、0.32%和0.64%,研究不同大豆皂甙含量对牙鲆（2.58±0.01 g）摄食、生长、饲料效率、表观消化率和组织学的影响。实验在室内循环海水养殖系统中进行,每个处理设置三个重复,每天投喂2次,达饱食水平。分别在养殖实验开始、28 d和结束时对牙鲆进行称重。实验结果表明,随着饲料中大豆皂甙水平的升高,牙鲆摄食率、平均体重、鱼体粗脂肪含量和饲料粗蛋白表观消化率均显著下降（P < 0.05）。在实验前28 d,随着饲料中大豆皂甙含量的提高,牙鲆的摄食率呈线性下降趋势（r =– 0.869,P < 0.001）,但试验结束时各组间摄食率相比较差异不显著（P > 0.05）。与对照组相比较,无论在28 d还是试验结束时,饲料中添加0.64%大豆皂甙都显著降低了牙鲆的平均体重（P < 0.001）。添加0.32%的大豆皂甙在28 d时的平均体重显著低于对照组（P < 0.05）,但是试验结束时平均体重与对照组没有显著差异（P > 0.05）。高浓度的大豆皂甙（0.64%）还造成了牙鲆肝脏和后肠组织的病理性变化。通过分析认为,高含量的大豆皂甙显著抑制了牙鲆的摄食和生长性能,因此在以大豆制品来替代鱼粉饲喂牙鲆时不应该忽略大豆皂甙的抗营养作用。（2）以鱼粉为主要蛋白源,鱼油为脂肪源,小麦粉为糖源,以三氧化二钇为外源性指示剂,制成4种等氮、等能（粗蛋白49.1%,总能20.1 KJ/g）实验饲料,使饲料中大豆异黄酮实际含量分别达到0 （对照组）、0.1%、0.4%和0.8%,研究不同大豆异黄酮含量对牙鲆（2.58±0.01 g）摄食、生长、饲料效率、表观消化率和组织学的影响。实验在室内循环海水养殖系统中进行,每个处理设置三个重复,每天投喂2次,达饱食水平。实验结果表明,在饲料中添加0.1%和0.4%的大豆异黄酮对牙鲆摄食、生长、饲料效率、鱼体组成、表观消化率和组织学结构均没有显著影响（P > 0.05）。而与对照组相比较,饲料中添加0.8%大豆异黄酮显著降低了牙鲆的平均体重、鱼体的粗脂肪含量和饲料表观消化率（P < 0.05）,并造成了牙鲆后肠组织的病理性变化。通过分析认为,如果饲料中大豆异黄酮的含量不超过0.4%,它对牙鲆的抗营养作用不明显。由于一般大豆制品中大豆异黄酮的含量在0.1⁰.3%,因此本文建议在使用大豆制品来替代鱼粉饲喂牙鲆时可以不用考虑大豆异黄酮的抗营养作用。（3）通过分离和原代培养牙鲆肠道上皮细胞,并以其为细胞模型,研究大豆中三种热稳定性抗营养因子对其形态、存活率、增殖、膜完整性和功能的影响。本实验研究的三种抗营养因子为大豆皂甙、植酸和棉子糖,其在培养液中的浓度分别为2、4和8 g L^-1。结果表明:与其他三个处理组相比（对照、植酸和棉子糖）,在培养液中添加2 g L^-1的大豆皂甙显著抑制牙鲆肠道上皮细胞的存活、增殖和细胞碱性磷酸酶活力（P < 0.001）,并严重破坏了牙鲆肠上皮细胞细胞膜的完整性,改变了细胞的形态结构;与对照组相比,在培养液中添加4 g L^-1的植酸显著改变牙鲆肠上皮细胞的形态结构,并抑制细胞的存活、增殖、细胞膜完整性和细胞碱性磷酸酶活力（P < 0.05）;与对照组相比,在培养液中添加8 g L^-1的棉子糖对牙鲆肠道上皮细胞的形态结构、存活、增殖、细胞膜完整性和细胞碱性磷酸酶活力均没有显著影响（P > 0.05）。本实验添加的浓度参考了这三种抗营养因子在一般大豆制品中的含量,也是当牙鲆摄食含有50%豆粕的饲料后它的肠上皮细胞所在环境的抗营养因子含量,比其他以细胞为研究模型的报道要高。通过分析认为,在使用大豆制品来替代鱼粉饲喂牙鲆时应重视大豆皂甙和植酸对鱼类肠道的损害作用,并设法去除,不过可以不用考虑棉子糖的抗营养作用。（4）以鱼粉和豆粕为蛋白源,配制了4种等氮等能的实验饲料（粗蛋白50.0%,总能20.5 KJ/g）。采用长时间胃内灌喂法（被动摄食）投喂牙鲆（60.0±0.66 g）,在摄食量相同的情况下,研究以鱼粉为蛋白源的饲料中添加多种抗营养因子（0.2%大豆胰蛋白酶抑制因子、0.2%大豆皂甙、0.2%大豆异黄酮、0.4%植酸钠和0.5%棉子糖）和以45%豆粕替代鱼粉为蛋白源的饲料中添加晶体氨基酸（0.75%蛋氨酸和0.25%赖氨酸）对牙鲆生长、存活、饲料效率和表观消化率的影响。实验在室内循环海水养殖系统中进行,每桶5尾鱼,每个处理设置三个重复,每天人工灌喂饲料1次,每尾鱼1ml （干重0.54 g）。研究结果表明:采用灌喂法养殖的牙鲆生长和存活状况良好,对照组饲料效率达到1.22,且实验期间没有出现鱼体死亡现象;在摄食量相同的情况下,用豆粕替代45%的鱼粉显著降低了牙鲆的体增重、饲料效率和表观消化率（P < 0.05）,且平均体增重还不到鱼粉组的一半（从15.68g到7.73g,P < 0.05）;在摄食量相同的情况下,饲料中添加多种抗营养因子在实验开始的前四天显著降低了牙鲆的体增重和饲料效率（P < 0.05）,但随着实验周期的延长,在饲料中添加多种抗营养因子对牙鲆的体增重、饲料效率和表观消化率均没有显著影响（P > 0.05）;在摄食量相同的情况下,与豆粕组相比较,在豆粕饲料中添加蛋氨酸和赖氨酸对牙鲆的体增重、饲料效率和表观消化率均没有显著影响（P > 0.05）。通过分析认为,在用豆粕替代45%鱼粉的实验中,抗营养因子不是导致牙鲆生长下降的主要因素;在替代45%鱼粉的豆粕饲料中添加蛋氨酸和赖氨酸并不能有效改变豆粕的替代效果;由于本实验采用的是人工灌喂的投喂模式,不会存在诱食性差的因素。因此本文认为可能还存在影响牙鲆利用大豆蛋白源的其他因素,并提出了以下三个易被忽略的因素:①豆粕中存在大量不能被鱼类利用的能量物质;②大豆蛋白本身的抗营养作用;③大豆蛋白和鱼粉蛋白水解生成的小肽混合物组成不同。更多还原

【Abstract】 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.更多还原