【作者】 迟淑艳；

【导师】 谭北平；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2009， 博士

【摘要】 本文比较研究了低鱼粉饲料中添加微胶囊蛋氨酸或晶体蛋氨酸对军曹鱼和凡纳滨对虾生长性能的影响,揭示军曹鱼和凡纳滨对虾在摄食低鱼粉饲料的条件下对不同形式的蛋氨酸的利用效果。实验内容主要分为三部分:1)微胶囊蛋氨酸制备技术相关工艺参数的筛选及性能检测;2)微胶囊或晶体蛋氨酸在军曹鱼低鱼粉饲料中应用效果;3)微胶囊或晶体蛋氨酸在凡纳滨对虾低鱼粉饲料中的应用效果。实验结果如下:1)应用化学复相乳化法和物理流化床法对蛋氨酸进行微胶囊工艺参数的筛选和制备。经均匀设计实验表明,复相乳化法制备的明胶微胶囊蛋氨酸粒径较大,最高载药量为46.94%;流化床法中以邻苯二甲酸醋酸纤维素(CAP)、棕榈酸三酰甘油酯(TPA)和丙烯酸树脂(RES)为主要壁材,分别进行微胶囊蛋氨酸的制备,通过对进出风温度、蠕动泵流速、包被时间和雾化压力等指标的调整控制,得出载药量在60-70%之间、颗粒大小均匀、外表光滑的微胶囊产品。以载药量为依据,选取流化床法制备的微胶囊进行产品溶失性能的评估。配制五组等氮等能饲料,蛋氨酸的添加形式分别为:晶体蛋氨酸(MET)、羟基蛋氨酸(MHA)、邻苯二甲酸醋酸纤维素微胶囊蛋氨酸(CAP)、树脂微胶囊蛋氨酸(RES)和棕榈酸甘油酯微胶囊蛋氨酸(TPA)。将各处理的饲料投入海水后,分别于10min、20min、30min、60min及90min测定饲料蛋氨酸的溶失率和海水pH值的变化。结果表明,饲料中蛋氨酸的溶失率随着浸泡时间的延长呈上升趋势,RES组的溶失率显著低于MET组,海水pH值随着浸泡时间的增加呈下降趋势,CAP和RES组下降缓慢,蛋氨酸溶失率与海水pH值直接呈明显的负相关。上述研究表明,RES微胶囊蛋氨酸能够有效减缓蛋氨酸的溶失。2)将鱼粉组和低鱼粉后蛋氨酸缺乏组分别设为正、负对照组,试验组饲料在负对照组的基础上分别添加晶体蛋氨酸(MET)、羟基蛋氨酸(MHA)、邻苯二甲酸醋酸纤维素微胶囊蛋氨酸(CAP)、丙烯酸树脂微胶囊蛋氨酸(RES)和棕榈酸甘油酯微胶囊蛋氨酸(TPA),共七组等氮等能饲料,比较研究军曹鱼幼鱼对微胶囊蛋氨酸和晶体蛋氨酸的利用效率。每个处理设三个重复,每个重复放养20尾鱼(初重5.40±0.07g),流水养殖8周,水温为29-31℃。结果表明RES组的增重率和特定生长率在各蛋氨酸添加组中最高,分别显著高出负对照组23.64%和7.99% (P < 0.05), MET组蛋白质效率显著高于负对照组和其他几个蛋氨酸组,而饲料系数显著低于其他组(P < 0.05)。蛋氨酸组的肥满度和正对照组相比差异不显著(P>0.05);微胶囊蛋氨酸和晶体蛋氨酸组的肌肉粗蛋白含量以及蛋氨酸与必需氨基酸(A/E)的比值与正对照组相比差异不显著(P>0.05);微胶囊蛋氨酸组的军曹鱼肠道胰蛋白酶活性显著高于正对照组和MET组(P<0.05),RES组胃蛋白酶和正对照无显著差异,但是显著高于其他组(P<0.05);MET组军曹鱼幼鱼摄食后0.5h肠道Na+,K+-ATP酶活性显著高于其他各组(P<0.05),摄食3h后正对照组和RES组Na+,K+-ATP酶活性上升显著高于CAP和MHA组(P<0.05);摄食8h后正对照组和微胶囊蛋氨酸组显著高于MHA和MET组(P<0.05)。为进一步验证军曹鱼幼鱼对蛋氨酸的吸收速度,探讨晶体蛋氨酸、RES微胶囊蛋氨酸和蛋白结合态氨基酸(鱼粉对照组)在吸收上的差异,设置日投饲频率分别为2(0800和1600 hours)、3 (0800, 1200和1600 hours)、4 (0800, 1100, 1400和1600 hours)和5次(0800, 1000, 1200, 1400和1600 hours)的处理组,其中鱼粉组只投喂2次,每个处理设三个重复,每个重复放养20尾鱼(初重5.40±0.07 g),流水养殖4周,水温为29-31℃。结果表明,随投喂次数的增加各试验组增重率有增加的趋势,投喂5次的RES组增重率显著高于其他组(P<0.05),投喂3次以上各组和投喂2次的RES组的肥满度与对照组差异不显著(P>0.05);投喂3次的MET组肝脏总蛋白酶活性显著高于其他各组(P<0.05),投喂3次的RES组肠道淀粉酶活性显著高于其他各组,投喂2次和5次时,MET组胃蛋白酶活性显著高于RES组(P<0.05),随投喂次数的增加MET组的GOT/GPT显著升高,投喂4次的RES组GOT/GPT显著高于其它各处理(P<0.05)。3)设置五组低鱼粉蛋氨酸缺乏的等氮等能饲料,分别添加晶体蛋氨酸(MET)、羟基蛋氨酸(MHA)、邻苯二甲酸醋酸纤维素微胶囊蛋氨酸(CAP)、树脂微胶囊蛋氨酸(RES)和棕榈酸甘油酯微胶囊蛋氨酸(TPA),饲喂凡纳滨对虾养殖7周,测定其在养殖第28d和50d时的生长性能,饲料利用率以及第14、28和50d时的消化酶活性。五个处理各设三个重复,每个重复放养40尾虾(初重0.81±0.01g)。溶失率实验表明RES能够更有效的减缓饲料中晶体蛋氨酸的溶出。第28d时,MET组的饲料系数显著低于CAP组和MHA组,而增重率、蛋白质效率(PER)和特定生长率(SGR)则显著高于CAP组和MHA组(P<0.05)。MET数值上高出RES组;第50d时,MET和RES的增重率显著高于其他组(P<0.05),RES组的增重率、PER和SGR分别高出MET组3.56%、8.63%和2.19%,但无显著差异(P>0.05),RES组的FCR显著低于其余各组(P<0.05);试验第28d和50d时,RES组全虾粗蛋白含量显著高于MET组(P<0.05),RES组50d时的对虾肌肉蛋氨酸含量显著高于MET组(P<0.05);第28d时,摄食后0.5h时CAP组的肝胰腺MAT活性最高,TPA组在0.5h和1h时保持较低水平,3h时开始上升,MET组在0.5h、3h和6h时MAT活性均较低但是在12h时骤然升高,第50d时,摄食1h时三个微胶囊组的MAT活性与0.5h相比有所上升,RES组上升较缓慢而MET组下降,3h时RES组MAT活力达最大;与第28d相比第50d时总蛋白酶活力均有所回升,TPA组显著高于RES组(P<0.05),MET组脂肪酶活力均显著高于其它处理组(P<0.05),淀粉酶活力在数值上均低于微胶囊组(P<0.05);第28d时,MET组的凡纳滨对虾对饲料蛋白质的表观消化率显著高出其它各组(P<0.05),在第50d时低于RES组,但是差异不显著(P>0.05),在第28d和50d时RES组粗脂肪的消化率显著高于MET组(P<0.05)。为进一步验证凡纳滨对虾对蛋氨酸的吸收速度,探讨晶体蛋氨酸、RES微胶囊蛋氨酸和蛋白结合态氨基酸在吸收上的差异,设置日投饲频率分别为2 (0700和2100 hours), 4 (0700, 1200, 1800和2100 hours),和6次(0700, 0930, 1200, 1500, 1800和2100 hours)的实验,其中正对照组(鱼粉组)和负对照组(蛋氨酸缺乏)只投喂4次。每个处理设三个重复,每个重复放养30尾虾(初重1.27±0.01 g),养殖5周,水温为29-30℃。结果表明,投喂次数相同时, RES组增重率、特定生长率和蛋白质效率比在数值上均高于MET组,摄食率和饲料系数则均低于MET组,但是未见显著性差异(P>0.05);RES组在投喂6次时增重率和正对照组差异不显著(P>0.05);RES组在投喂4次时总蛋白酶活性达最高,与正对照组差异不显著(P>0.05)。综上所述,添加蛋氨酸有助于改善军曹鱼幼鱼和凡纳滨对虾对饲料中豆粕和肉骨粉的利用率,促进生长。以增重率为参考指标,显示军曹鱼幼鱼和凡纳滨对虾在短期的养殖试验中均能够利用晶体蛋氨酸用于生长,而蛋白质代谢酶指标则显示RES微胶囊在降低溶失率的同时在消化道内有较好的缓释作用,可以提高蛋氨酸的利用率,促进蛋白质的合成。今后研究中,有必要进一步改善RES微胶囊工艺,选择适宜的鱼粉和其它蛋白源的替代比例以满足动物最大生长。更多还原

【Abstract】 The present study was conducted to compare the effects of microcapsule methionine with crystalline methionine in diets with low fish meal on growth performance, protein metabolism and digestive enzyme activities of cobia (Rachycentron canadum) and Pacific white shrimp (Litopenaeus vannamei). The study contained three sections: 1. Processing and technique for microcapsule methionine. 2. Effects of supplementation microcapsule or crystalline methionine on growth performance in diets with low fish meal of cobia. 3. Effects of supplementation microcapsule or crystalline methionine on growth performanc of Pacific white shrimp in dietw with low fish meal. The results were presented as follows:1. The methionine microcapsules were prepared with emulsification and fluid bed (Wurster). Uniform design experimentation was used to filtration the correlative parameters of emulsification method. Drug loading of gelatin microcapsule by this method was 46.94% and the pellets diameter was bigger. Cellulose-acetate- phthalate (CAP), acrylic resin (RES) and tripalmitin (TPA), as the main wall materials, respectively, were used in fluid bed (Wurster) method. Drug loading of the microcapsules with smooth wall and uniformity diameter was between 60-70% by regulating air temperature of input and output, velocity of peristaltic pump, coating time and pulverization press and so on.According to drug loading, methionine microcapsules made by fluid bed were evaluated for leaching loss. One experiment was conducted to investigate the leaching loss of diets supplied coated methionine and crystalline methionine and soaked in seawater and pH value of seawater at 10min, 20min, 30min, 60min and 90min, respectively. Five experiment diets were designed with methionine supplementation in the forms of L-methionine (MET, as control), Methionine hydroxy analogue (MHA), cellulose-acetate-phthalate (CAP), tripalmitin-polyvinyl alcohol (TPA) and acrylic resin (RES) coated L-methionine, respectively. The results showed leaching loss of methionine increased with increasing soaking time. Leaching loss of methionine of RES group was significantly lower than that of MET group. The pH value descended with increasing soaking time. CAP and RES groups dropped slowly in pH value. Leaching loss of methionine had negative correlation with pH value of seawater. In conclusion, acrylic resin was the better wall material than the others to retard leaching loss of methionine.2. Seven iso-nitrogen and iso-energy diets, including fish meal (positive control), low fish meal (negative control) and five methionine supplemented with MET, MHA, CAP, RES and TPA, respectively, were prepared to investigate utilization of coated and crystalline methionine in intestine of juvenile cobia. Each treatment was randomly assigned to triplicate groups of 20 fish (initial weight 5.40±0.07 g) per aquarium. Fish were maintained in flow-through aquaria for 8 weeks at water temperature ranged from 29 to 31℃.The results showed that weight gain and specific growth rate of fish fed RES was the highest in methionine supplementation groups and significantly higher 23.64% and 7.99% than that of negative group (P<0.05). Protein efficiency ratio of fish fed MET was significantly higher than negative control and the other methionine supplementation groups (P<0.05), and that feed conversion ratio of fish fed MET was significantly lower than that of the others (P<0.05). Condition factor of fish fed methionine had no significant difference with positive control (P>0.05). Crude protein contents and rate of methionine to essential amino acid (A/E) in muscle of fish fed microcapsule and crystalline methionine groups had no significant difference with positive control (P>0.05). Trypsin activities of intestine of fish fed microcapsule methionine were significantly higher than that of fed crystalline methionine and the positive control (P<0.05). Compared to positive group, fish fed the diet with RES had no significant difference in pepsin activities, whereas significantly higher than other groups (P<0.05). After feeding 0.5h, Na+, K+-ATP activities in intestine of fish fed MET were significantly higher than that of the other groups (P<0.05). After feeding 3h, Na+, K+-ATP activities of fish fed RES and positive control ascended and was higher than that of fish fed CAP and MHA, and that activities of fish fed positive control and microcapsule methionine were significantly higher than that of MHA and MET after feeding 8h(P<0.05).One feeding frequency trial was conducted in order to validate the rates of absorption in intestine of juvenile cobia and explore the difference in absorption of crystalline methionine, RES microcapsule and amino acid of intact protein (Control). Cobia were fed a commercial pelleted feed in feeding frequency 2 (at 0800 and 1600 hours), 3 (at 0800, 1200 and 1600 hours), 4 (at 0800, 1100, 1400 and 1600 hours), and 5 times day-1 (at 0800, 1000, 1200, 1400 and 1600 hours), respectively, the control was fed 2 timesday-1 only. Each treatment was randomly assigned to triplicate groups of 20 fish (initial weight 5.40±0.07g) per aquarium. Fish were maintained in flow- through aquaria for 4 weeks at water temperature ranged from 29 to 31℃. Weight gain was increased with increasing feeding frequency. Fish fed with RES 5 times day-1 was significantly higher than the others (P<0.05). Condition factor of fish fed 3 times day-1and fed RES 2 times day-1 were not different from the control (P>0.05). Total protease activities in liver of fish fed MET 3 times day-1 and amylase activities in intestine of fish fed RES 3 times day-1 were significantly higher than the others (P<0.05). Pepsin activities of fish fed MET were significantly higher than RES with 2 times day-1 and 5 timesday-1 (P<0.05). GOT/GPT of fish fed MET ascended with increasing feeding frequency, whereas GOT/GPT of fish fed RES 4 times day-1 was higher than the others (P<0.05).3. This study was conducted to evaluate the effects of methionine supplementation form in diets of Pacific white shrimp (Litopenaeus vannamei) on growth performance, feed utilization and digestive enzyme activities at day 14, 28 and 50. Five iso-nitrogen and iso-energy diets were prepared in which three diets were supplemented with cellulose-acetate-phthalate (CAP), tripalmitin-polyvinyl alcohol (TPA) and resin (RES) coated L-methionine, respectively. Hydroxyl-methionine calcium (MHA) was supplemented as the fourth diet and crystalline L-methionine diet (MET) was used as the control. Five triplicate groups of shrimp (0.81±0.01g in initial body weight) were fed each test diets for 7 weeks. Leaching test showed that RES coating had the better effects in reducing leaching loss of crystalline methionine in feed. Protein efficiency ratio, weight gain and specific growth rate of shrimp fed MET and RES were significantly higher than CAP and MHA (P<0.05) and MET was higher than RES without difference (P>0.05). Feed conversion ratio (FCR) of shrimp fed MET were significantly lower than CAP and MHA (P<0.05) at day 28. At day 50, weight gain of shrimp fed MET and RES were significantly higher than that of the others (P<0.05), while FCR of shrimp fed RES was significantly lower than the others (P<0.05). Weight gain, PER and SGR of shrimp fed RES were found.56%, 8.63% and 2.19% than without significant differences with the others. Crude protein of shrimp body in RES and TPA was markedly higher than the others (p<0.05). Methionine contents in muscle of RES and MHA groups were significantly higher than those of MET group, which had the lowest lysine contents (p<0.05). At day 28, after fed 0.5h, MAT activity in hepatopancrease of shrimp fed CAP was the highest, MAT activity of shrimp fed TPA was constantly low level after feeding 0.5h and 1h and rose after fed 3h. MAT activity of shrimp fed MET was low at 0.5h, 3h and 6h, but rose the highest at 12h. At day 50, MAT activities at 1h of shrimp fed microcapsule methionine climbed compared with 0.5h, at the same time, climb of MAT activity of shrimp fed RES was slowness, whereas MET dropped. After feeding 3h, MAT activity of shrimp fed RES was the highest. Total protease activity was increased at day 50 compared with day 28, and shrimp fed TPA was significantly higher than RES (p<0.05). Shrimp fed MET had significantly higher lipase activity than the others (p<0.05) and lower amylase than microcapsule methionine groups without difference (P>0.05). Apparently digestibility for protein of shrimp fed MET was significantly higher than the others at day 28, however, lower than RES without difference. Apparently digestibility for lipid of shrimp fed RES was significantly higher than that of shrimp fed MET (P<0.05).One feeding frequency trial was conduted in order to validate the rates of absorption in hepatopancrease of Pacific white shrimp and explore the difference in absorption of crystalline methionine, RES microcapsule and amino acid of intact protein (positive control, fish meal group and negative control, methionine deficiency). Shrimp were fed a commercial pelleted feed, fed in feeding frequency 2 (at 0700 and 2100 hours), 4 (at 0700, 1200, 1800 and 2100 hours), and 6 timesday-1 (at 0700, 0930, 1200, 1500, 1800 and 2100 hours), the positive control and negative control were fed 4 times day-1 only. Each treatment was randomly assigned to triplicate groups of 30 shrimps (initial weight 1.27±0.01 g) per aquarium. Shrimps were maintained in aquaria for 5 weeks at water temperature ranged from 29 to 30℃. Shrimp fed MET or RES had no different differencs in weight gain, SGR and PER with the same feeding frequency. Weight gain of shrimp fed RES 6 times day-1 had no difference with positive control (P>0.05). FR and FCR of shrimp fed RES was lower than MET (P>0.05) at the same feeding frequency. Crude protein contents of shrimp body of shrimp fed MET 6 times day-1 were significantly higher than that 4 times day-1 (P<0.05). Total protease activities of shrimp fed RES 4 times day-1 were the highest, which was no difference with positive control (P>0.05). GOT activities of shrimp fed MET 2 times day-1were significantly higher than 4 and 6 times day-1, and that significantly lower than positive control (P<0.05).In conclusion, supplementation methionine in diets with low fish meal could improve utilization of soybean meal and meat and bone meal and promot growth of juvenile cobia and Pacific white shrimp. On the basis of weight gain, free methionine could be used for growth for juvenile cobia and Pacific white shrimp in short term, however, on the basis of metabolize enzyme activities, RES microcapsule not only had the better delay release in alimentary tract but also reduced leaching loss in seawater, which enhanced methionine utilization and protein synthesis. It is necessary to improve processing and technique of RES microcapsule further and choose feasible substituted ratio between fish meal and other protein sources for the best growth of fish and shrimp.更多还原