【作者】 张乐；

【导师】 张美玲；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2023， 硕士

【摘要】 豆粕是杂食性鱼类重要的饲料蛋白质来源。随着水产养殖业的蓬勃发展,豆粕需求量持续上涨。近年来,豆粕价格的飙升也极大地增加了水产养殖的生产成本。饲料中豆粕的大量使用还存在与人争粮的问题,威胁国家粮食安全。因此,寻找适宜的替代性蛋白源,实现豆粕的减量替代,对于水产养殖的高质量稳定发展具有重要意义。黄粉虫作为一种新型的昆虫蛋白源,具有营养价值高,环境效益好,不与人争粮的特点。黄粉虫蛋白在水产养殖中替代鱼粉的研究已经受到广泛关注,但是关于黄粉虫蛋白是否可以有效替代豆粕的研究还相对较少。基于此,本实验以杂食性鱼类尼罗罗非鱼为研究对象,使用黄粉虫蛋白粉分别替代饲料中0%、15%、30%和45%的豆粕,对初始重量为70±0.12g的尼罗罗非鱼进行为期10周的养殖,从鱼体生长、肝肠健康和肌肉品质的角度评估了黄粉虫蛋白替代豆粕的可行性,并探讨了其产生影响的可能原因。本研究的主要结果如下:1.黄粉虫蛋白替代豆粕对尼罗罗非鱼生长性能和全鱼体成分的影响尼罗罗非鱼的存活率、终末体重、增重率、特定生长率、饲料转化率、日摄食率、肥满度、脏体比和肝体比在四个处理组之间未呈现显著性差异。躯壳比和腹脂比分别在YM30、YM30和YM45组显著升高。全鱼的水分、粗蛋白和灰分含量没有发生显著变化,而粗脂肪含量在YM45组显著高于其他三组。从生长的角度考虑,黄粉虫蛋白替代豆粕的比例可以达到45%,不会对尼罗罗非鱼的生长性能产生显著不良影响。2.黄粉虫蛋白高比例替代豆粕对尼罗罗非鱼肝肠健康的影响从肝脏健康的角度,黄粉虫蛋白能够替代尼罗罗非鱼饲料中30%的豆粕。当替代比例增加至45%,血清谷丙转氨酶活力(ALT)显著增加,肝组织切片呈现细胞边缘模糊、脂肪空泡增多、脂滴和糖原积累,同时生化分析结果显示肝脏总脂、甘油三酯(TG)、游离脂肪酸(NEFA)和糖原含量显著增加,糖原合成酶活力显著升高,总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)活力和谷胱甘肽过氧化物酶(GSH-PX)活力显著下降,活性氧(ROS)和丙二醛(MDA)显著积累,呈现明显的氧化应激。从肠道健康的角度,黄粉虫蛋白能够替代尼罗罗非鱼饲料中15%的豆粕。当替代比例增加至30%,肠道组织学形态开始发生改变,表现为肠道肌层变薄,肠绒毛变短、变宽,肠道跨上皮电阻显著下降,肠道通透性增加,紧密连接蛋白相关基因的相对表达显著上调。当替代比例达到45%,肠道损伤进一步加剧,肠道抗氧化状态发生紊乱。对差异最为明显的YM0和YM45组进行肝脏和肠道的转录组测序分析,结果表明肝脏和肠道的共同差异基因是与氧化应激密切相关的klf9(Kruppfel-like factor 9),同时其上游与氧化应激相关的nrf2(NF-E2-related transcription factor 2)基因在YM45组肝脏和肠道中的表达量均显著增加,说明黄粉虫蛋白高比例替代豆粕引起的肝肠损伤可能与氧化应激密切相关。这些结果表明,黄粉虫蛋白能够替代饲料中15%的豆粕而不影响尼罗罗非鱼的肝脏和肠道健康,当替代比例增加至30%时,会引起尼罗罗非鱼肠道屏障受损,增加至45%时,肠道损伤进一步加剧,同时会引起肝脏受损。3.黄粉虫蛋白高比例替代豆粕对尼罗罗非鱼肌肉品质的影响肌肉品质决定着消费者的认可度和商品价值,因此我们分析了黄粉虫蛋白替代豆粕对尼罗罗非鱼肌肉品质的影响。结果显示,肌肉粗蛋白在YM30和YM45组显著减少,粗脂肪含量在YM45组显著下降。必需氨基酸和风味氨基酸的含量没有受到黄粉虫蛋白替代豆粕的影响,而肌肉饱和脂肪酸(SFA)含量与对照组YM0相比,在YM30和YM45组显著降低。肌肉质地方面,YM45组尼罗罗非鱼肌肉硬度、胶着性显著升高。对肌肉进行组织学分析,发现YM45组尼罗罗非鱼肌肉中小直径肌纤维占比显著高于其他三组。进一步分析发现,在YM45组肌肉中,肌肉凋亡和萎缩相关基因的表达显著上调,肌肉的抗氧化能力显著下降。这些结果说明黄粉虫蛋白能够替代饲料中30%的豆粕而不显著改变尼罗罗非鱼肌肉品质,当替代比例达到45%会使得鱼肉质地发生改变。4.黄粉虫蛋白高比例替代豆粕引起肝肠损伤、肌肉质地改变的可能原因分析为了探究黄粉虫蛋白高比例替代豆粕引起尼罗罗非鱼肝肠受损、肌肉质地改变的原因,本研究对文献报道的几种可能原因进行分析检测。结果显示:(1)虽然黄粉虫蛋白替代豆粕降低了饲料中一些必需氨基酸的含量,但是仍然能够满足尼罗罗非鱼的需要。(2)对饲料脂肪酸含量进行分析,发现饲料脂肪酸组成的变化尚不足以引起鱼体肝肠损伤、肌肉质地改变。(3)对饲料中的果糖含量进行检测,发现饲料中的果糖含量随黄粉虫蛋白替代豆粕比例的增加逐渐降低,并不足以影响肝脏和肠道健康。(4)由于黄粉虫的多不饱和脂肪酸(PUFA)含量丰富,其脂肪容易发生氧化变质,因此,在养殖实验前后对饲料酸价进行分析。结果显示,养殖实验开始时,四个处理组间饲料酸价没有显著差异,而养殖结束时YM45组的饲料酸价显著高于其他三组。这说明黄粉虫蛋白以较高比例替代豆粕时,饲料容易发生酸败变质。我们推测饲料脂肪酸败可能是黄粉虫蛋白高比例替代豆粕引起尼罗罗非鱼肝肠损伤和肌肉质地改变的原因之一。综上,本研究选取尼罗罗非鱼为实验对象,探究了黄粉虫蛋白替代饲料中的豆粕对其生长性能、肝肠健康以及肌肉品质的影响,发现黄粉虫蛋白高比例替代豆粕引发的负面影响可能与饲料脂肪酸败有关。这提示我们在实际生产中使用黄粉虫蛋白作为饲料蛋白源时,需要格外注意储存和加工条件,以避免饲料酸败的发生。更多还原

【Abstract】 Soybean meal is an essential feed protein source for omnivorous fish.Due to the rising demand for soybean meal in aquaculture,the price of soybean meal has soared,which has greatly increased the production costs of aquaculture.The massive use of soybean meal in the aquatic feed also has the food-feed competition problem,threatening national food security.Therefore,it is urgent to find suitable alternative protein sources to reduce the use of soybean meal,which is important for the highquality and stable development of aquaculture.Yellow mealworm(Tenebrio molitor)is a novel insect protein source with abundant nutritional value and environmental benefits.It is suitable for large-scale farming and does not compete with humans for food.In recent years,research on the replacement of fishmeal with yellow mealworm meal has received lots of attention in aquaculture,but whether yellow mealworm meal could effectively replace soybean meal remains unclear.Hence,the present study was conducted by using yellow mealworm meal to replace 0%,15%,30%,and 45%soybean meal to feed Nile tilapia with an initial weight of 70±0.12 g for ten weeks.The feasibility was evaluated from the perspective of growth performance,liver health,intestinal health,and flesh quality.The possible reasons for the effects were also explored.The main results of this study are as follows:1.Effects of yellow mealworm meal replacing soybean meal on the growth performance and whole body composition of Nile tilapiaFinal body weight(FBW),weight gain(WG),specific growth rate(SGR),feed conversion ratio(FCR),condition factor(CF),viscerosomatic index(VSI),and hepatosomatic index(HSI)of Nile tilapia did not show significant changes among the four treatments.The carcass ratio and mesenteric fat index of Nile tilapia increased significantly in YM30,YM30 and YM45 respectively.The moisture,crude protein,and ash contents of the whole body did not change significantly,while crude lipid content was significantly higher in the YM45 group than in the other three groups.From the perspective of growth performance,yellow mealworm meal could replace 45%soybean meal without significant adverse effects on the growth performance of Nile tilapia.2.Effects of yellow mealworm meal replacing high percentage of soybean meal on the hepatic health and intestinal health of Nile tilapiaFrom the perspective of hepatic health,yellow mealworm meal could replace 30%soybean meal in the feed of Nile tilapia.When the replacement ratio increased to 45%,serum alanine transaminase(ALT)activity increased significantly.Liver tissue showed blurred cell boundaries,increased lipid vacuoles,and accumulated lipid droplets and glycogen.Liver lipids,triglycerides(TG),nonesterified fatty acid(NEFA),glycogen content,and glycogen synthase activity increased significantly.Total antioxidant capacity(T-AOC),superoxide dismutase(SOD)activity,and glutathione peroxidase(GSH-PX)activity decreased significantly.Significant accumulation of reactive oxygen species(ROS)and malondialdehyde(MDA)were also found.The liver of Nile tilapia fed with YM45 diets showed significant oxidative stress.From the perspective of intestinal health,yellow mealworm meal could replace 15%soybean meal in the feed of Nile tilapia.When the replacement ratio increased to 30%,the histological morphology of the intestine was altered,as evidenced by the thinner muscular layer,and shorter and wider intestinal villi.A significant decrease in intestinal trans-epithelial resistance was also detected,which indicates an increase in intestinal permeability.The relative mRNA expression of tight junction proteins related genes was significantly up-regulated.When the replacement ratio reached 45%,the intestinal injury was further exacerbated and the intestinal antioxidant status was disturbed.Transcriptome sequencing analysis of the liver and intestine was conducted in YM0 and YM45 and the results showed that the common differential gene klf9(Kruppfel-like factor 9)in the liver and intestine was closely associated with oxidative stress.Meanwhile,the expression of its upstream nrf2(NF-E2-related transcription factor 2)gene,which is related to oxidative stress,was significantly upregulated in both liver and intestine in the YM45 group.It suggested that hepatic and intestinal injury caused by the high percentage of yellow mealworm meal replacing soybean meal might be closely related to oxidative stress.These results suggested that yellow mealworm meal was able to replace 15%soybean meal in the feed of Nile tilapia without affecting the hepatic and intestinal health,but when the replacement percentage reached 30%,it caused damage to the intestinal barrier.When the replacement ratio was up to 45%,the intestinal damage was further exacerbated,and hepatic injury happened.3.Effects of yellow mealworm meal replacing high percentage of soybean meal on the flesh quality of Nile tilapiaFlesh quality determines the acceptance of consumers and the commercial value,thus the effects of yellow mealworm meal replacing soybean meal on the flesh quality of Nile tilapia was investigated.The results showed that the crude protein content of muscle decreased significantly in the YM30 and YM45 groups,and muscular crude lipid content decreased significantly in the YM45 group.The contents of essential amino acids,delicious amino acids,and flavor amino acids were not affected by the substitution.In terms of flesh texture,hardness and gumminess were significantly higher in the muscle of Nile tilapia in the YM45 group than in the YM0.Histological analysis of the muscle revealed that the percentage of small-diameter myofibers was significantly higher in the YM45 group than in the other three groups.Further analysis revealed that the expression of genes related to muscle apoptosis and atrophy was significantly upregulated in the YM45 group,and the muscular antioxidant capacity was significantly reduced in the YM45 group.These results indicated that yellow mealworm meal could replace 30%soybean meal in Nile tilapia diets without substantially altering the flesh quality.When the replacement ratio was increased up to 45%,the fish flesh texture was changed.4.Analysis of possible reasons for hepatic and intestinal injury and flesh texture changes caused by yellow mealworm meal replacing high percentage of soybean mealTo investigate the possible reasons for hepatic and intestinal injury and flesh texture changes in Nile tilapia caused by the high percentage of yellow mealworm meal substitution for soybean meal,several possible causes reported in the literature were analyzed and tested in the present study.The results showed that:(1)Although the replacement of soybean meal with yellow mealworm meal decreased the content of some essential amino acids in the feed,it can meet the needs of Nile tilapia.(2)Analysis of the fatty acid content in the feeds revealed that the changes in the fatty acid composition were not enough to cause hepatic and intestinal damage or change the flesh texture.(3)The fructose content of the feed was tested as well.It showed that the fructose content in the diets decreased gradually as the increase of replacement ratio and it is not the main reason for hepatic or intestinal injury.(4)Since yellow mealworms are rich in polyunsaturated fatty acids and prone to lipid oxidation and deterioration,the acid value of diets was analyzed at the start and the end of the feeding experiment.The results showed that there was no significant difference in feed acid value among the four treatments at the start of the feeding experiment,while the acid value of diets in YM45 was significantly higher than the other three groups after the feeding experiment.It indicated that feed rancidity and deterioration easily occurred when soybean meal was replaced by yellow mealworm meal at a high proportion.We speculate that the rancidity of diets may be the main reason for hepatic and intestinal injury and flesh texture changes caused by the high percentage of yellow mealworm meal replacing soybean meal.In conclusion,this study investigated the effects of replacing soybean meal with yellow mealworm meal on growth performance,hepatic and intestinal health,and flesh quality in Nile tilapia,and found that the negative effects triggered by high percentage substitution might be related to the rancidity of diets.It suggested that more attention should be paid to the storage and processing conditions when yellow mealworm meal was used as a feed protein source.更多还原