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1.
通过二代测序、软件拼接获得中国黄海海域黄条鰤(Seriola aureovittata)线粒体基因组全序列。其序列全长为16609 bp,碱基组成分别为A(26.68%)、G(17.84%)、C(30.12%)和T(25.36%);共有13条蛋白编码基因, 22个tRNA基因, 2个rRNA基因,除NAD6、trnQ、trnA、trnN、trnC、trnY、trnS、trnE、trnP外,其余基因均在H链上编码。黄条鰤线粒体基因组全序列与蛋白编码基因的A+T含量分别为52.05%和51.085%,具有明显的AT偏好性。线粒体基因中存在2个散在重复序列,分别位于NAD1基因序列正义链的中游和COX2基因序列反义链的上游。在其22个tRNA基因中,除了tRNAGly外,均具有典型的三叶草二级结构。黄条鰤线粒体基因组的蛋白编码基因起止位点与密码子除COX1、NAD5外,均与日本海域出产的黄条鰤(Seriola lalandi)完全吻合,且COX1、NAD5基因皆短于日本黄条鰤;两者间存在一定的遗传差异。基于18种隶属于13属的鲹科鱼类线粒体基因组全序列构建系统发生树,可知小甘鲹(Seriolina nigrofasciata)、黄条鰤、五条鰤(Seriola quinqueradiata)、高体鰤(Seriola dumerili)、长鳍鰤(Seriola rivoliana)同属一近支,且黄条鰤与五条鰤亲缘关系最近,与小甘鲹进化距离较远。  相似文献   

2.
为阐明黄条鰤幼鱼摄食消化特性,构建最佳胃排空数学模型,确立最适摄食投喂间隔,实验检测了黄条鰤幼鱼[(63.96±5.63) g]胃排空过程中内容物质量、肝脏和肠道中消化酶活性变化,分析了垂体中摄食调控相关基因的表达,比较了线性模型、平方根模型、立方模型3种数学模型对胃排空曲线的拟合程度。结果显示,黄条鰤在摄食后瞬时胃内容物湿重呈阶段性降低,18 h后降为0,属于直线下降型胃排空类型。胃排空过程中,肝脏淀粉酶、脂肪酶和糜蛋白酶活性呈先上升后下降、随后又上升接着下降的“M”型变化趋势,淀粉酶活性在摄食后0~6 h显著上升,脂肪酶和糜蛋白酶活性在0~9 h显著上升。三种酶活性在9~12 h均呈显著下降趋势,在15~18 h显著上升,且在18 h活性达到最高;肠道中的淀粉酶在0~6 h显著上升,随后下降,9~12 h呈上升趋势,12 h活性达到最高,随后逐渐下降,其脂肪酶和糜蛋白酶活性则是在摄食后0~12 h显著上升,12 h活性达到最高,随后逐渐下降。神经肽Y (npy)和食欲素(ore) mRNA表达水平呈先上升后下降趋势,npy在摄食后12~15 h内表达水平显著上升,ore在9~15 ...  相似文献   

3.
胰岛素样生长因子结合蛋白(insulin-like growth factor binding proteins, IGFBPs)在调节胰岛素样生长因子的生物学活性和生长调控中起着至关重要的生理作用。本研究克隆了黄条鰤(Seriola aureovittata) igfbp-1、igfbp-2a和igfbp-2b等3个基因cDNA编码的开放阅读框(ORF)序列,分析了其组织分布特征,并检测了工厂化不同养殖密度下黄条鰤肝脏中5个基因igfbp-1、igfbp-2a、igfbp-2b、igf-1和igf-2对生长的调控作用。结果显示,igfbp-1的ORF长为741 bp,共编码246个氨基酸;igfbp-2a的ORF长度为882 bp,共编码293个氨基酸;igfbp-2b的ORF长度为810 bp,共编码269个氨基酸。它们均具有广泛的组织表达特性,其中在肝脏中显著高表达,且具有性别二态性表达特性。工厂化养殖条件下,低密度组实验鱼生长速度最快且igfbp-1、igfbp-2a、igfbp-2b、igf-1和igf-2表达量均最高,与中、高密度组有显著性差异(P<0.05),且与血清IGF-1、GH表达趋势相同,与皮质醇含量及葡萄糖浓度的趋势相反;而中、高密度组实验鱼的生长及igfbp-1、igfbp-2a、igfbp-2b、igf-1和igf-2表达量均无显著差异。本研究表明,igfbp-1、igfbp-2a和igfbp-2b参与了不同养殖密度下黄条鰤生长的调控过程,且与igf-1、igf-2对生长的表达调控存在正向协同效应。研究结果为阐释黄条鰤生长的分子机制以及工厂化条件下适宜养殖密度的调控提供了理论依据。  相似文献   

4.
热休克蛋白(heat shock proteins, HSPs)在鱼类的应激与免疫反应中发挥重要的生理调控作用,HSP70是该家族的重要成员。为探讨热休克蛋白在大洋性经济鱼类黄条鰤 (Seriola aureovittata)生长发育中的生理作用,本研究克隆获得了黄条鰤hsp70基因的全长cDNA序列,并采用定量PCR技术测定了其组织分布及在早期生长发育过程中的表达特征。结果显示,黄条鰤 hsp70基因的cDNA序列全长为2 332 bp,其中,5′-UTR长度为187 bp,ORF长度为1 920 bp,3′-UTR长度为225 bp,编码639个氨基酸,蛋白质分子量为70.1 kDa,等电点为5.16。黄条鰤 hsp70 mRNA的组织表达具有性别二态性差异,其中,在雌性鳃、心、脾脏和卵巢组织中显著高表达(P<0.05),且以卵巢中表达量最高;雄性垂体、鳃、头肾和精巢组织显著高表达(P<0.05),且以鳃中表达量最高。胚胎发育过程的表达检测显示,在卵裂前的受精卵中可检测到hsp70的表达,表明其具有亲本遗传的特性。同时,在胚胎发育过程的各个时期都可检测到hsp70 mRNA的表达,且在低囊胚期之前的各发育阶段一直保持较低表达水平,在原肠前期开始显著上调表达(P<0.05),其后保持相对较高表达水平,至胚胎孵化出膜期达峰值。在仔稚幼鱼中,hsp70 mRNA在初孵仔鱼和1 d仔鱼中高表达,其后在4 d仔鱼中显著降低(P<0.05),其后显著上调表达,至15 d仔鱼达峰值,其后在20 d仔鱼显著下降,并在25 d后稚鱼和幼鱼中保持相对较低表达水平。研究结果可为深入认识黄条鰤hsp70基因的结构特征、发生发育及其早期生长发育阶段的表达调控功能提供依据。  相似文献   

5.
孙盛明  苏艳莉  潘方艳  戈贤平 《水产学报》2020,44(11):1777-1791
为研究刺鼠相关蛋白(AGRP)和神经肽Y(NPY)基因在团头鲂幼鱼应答饥饿与再摄食过程中所起的调控作用,本研究采用RACE PCR技术首次克隆了团头鲂AGRP和NPY基因全长cDNA序列,通过设置正常对照组(control,体质量3%持续投喂4周)、饥饿再投喂组(F/refeeding,饥饿2周+3%再投喂2周)、饥饿过量投喂组(F/excessive refeeding,饥饿2周+8%再投喂2周)和饥饿组(fasted,持续饥饿4周)4个处理组,采用qRT-PCR技术分析团头鲂在脑和肠道组织中这两种基因在饥饿再投喂过程中的表达变化特征。结果显示,(1)组织学分析表明,持续饥饿组肠道黏膜下层出现明显的白细胞浸润现象。(2)团头鲂AGRP和NPY基因cDNA序列全长分别为770 和557 bp,其中有5’非编码区77和101 bp以及3’非编码区315和120 bp,开放阅读框为378和336 bp,共编码了125个氨基酸和111个氨基酸。系统进化树分析表明,团头鲂的AGRP和NPY基因分别与其他鲤科鱼类聚类一支,具有最近的亲缘关系。(3)qRT-PCR分析显示,AGRP和NPY基因在所有检测组织中均有表达,二者均在脑组织中表达量最高,其次是肝脏和肠道。(4)饥饿再投喂处理对团头鲂脑和肠道中AGRP和NPY基因表达影响显著,饥饿组团头鲂脑和肠道组织AGRP基因表达量均呈先升后降趋势,第28天,饥饿再投喂组能够显著上调团头鲂AGRP基因在脑组织中的表达,但饥饿过量投喂组能够显著上调AGRP基因在肠道中表达量。持续饥饿组团头鲂脑和肠道组织NPY基因表达呈先上升后下降趋势,且饥饿组脑和肠道组织中NPY表达量在28 d均显著高于其余3组。上述结果表明,AGRP和NPY基因在团头鲂摄食调控中发挥重要作用,且在脑和肠道组织具有不同表达模式。  相似文献   

6.
通过同源克隆和染色体步移对光倒刺鲃(Spinibarbus hollandi)黑素皮质素受体-5 (melanocortin receptor-5,mc5r)基因进行了克隆,应用生物信息学方法对mc5r基因序列和推测的氨基酸序列进行分析,采用荧光定量PCR技术研究其m RNA的组织分布、日周期变化以及饥饿再投喂的表达情况。结果表明mc5r基因长度为1 947 bp,含有1个987 bp的外显子,编码329个氨基酸。通过序列同源比对及系统发育进化分析,光倒刺鲃mc5r的氨基酸序列与鲤(Cyprinus carpio)、犀角金线鲅(Sinocyclocheilus rhinocerous)的mc5r同源性达到98%。光倒刺鲃mc5r在大脑中显著表达,在间脑、心脏的表达量次之,在中脑、小脑表达量较少,在肾脏、胃、肌肉、脾、肠、性腺、肝脏、延脑、眼中微量表达。光倒刺鲃mc5r mRNA的日周期表达量随昼夜节律而变化,饥饿再投喂实验发现饥饿再投喂组鱼其脑中表达量会显著升高,随后会有下降趋势,表明mc5r可能与光倒刺鲃的摄食调控有关。  相似文献   

7.
为了探讨线粒体细胞色素C氧化酶亚基Ⅰ、Ⅱ(COⅠ、COⅡ)和16SrRNA基因在鰤属鱼类物种鉴定和群体划分中的适用性,在黄条鰤(Seriola lalandi)、高体鰤(Seriola dumerili)、五条鰤(Seriola quinqueradiata)等鰤属鱼类中克隆了 3种基因,并进行序列比对与系统进化分析....  相似文献   

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9.
黑素皮质素3型受体(MC3R)系统与动物摄食行为以及能量调控密切相关。为丰富鱼类MC3R相关基础研究,探索鱼类摄食行为与能量调控机制,本研究克隆了团头鲂(Megalobrama amblycephala)mc3r基因,采用分子生物信息学和相对荧光定量PCR方法分别对氨基酸序列保守结构域、组织表达分布和禁食条件下的表达变化等开展分析研究。结果显示,团头鲂mc3r基因编码区全长984 bp,编码327个氨基酸,与现有报道的MC3R氨基酸序列高度相似,具有典型的7次跨膜结构域。组织表达图谱分析表明,mc3rmRNA在下丘脑、垂体、肝脏和卵巢有相对较高的表达。禁食试验结果显示,下丘脑和垂体中的转录变化与周期性摄食信号相关,其中下丘脑mc3r mRNA在禁食72 h和14 d的表达显著上调(P0.05),垂体mc3r mRNA在禁食72 h和禁食72 h后恢复投喂6 h表达量显著升高(P0.05),而肝脏中mc3r mRNA在禁食48 h和14 d的表达量显著上调(P0.05)。此外,对血液皮质醇和血糖的监测结果显示,两者均在禁食14 d有显著变化,其中皮质醇水平显著高于对照组(P0.05),而血糖水平显著低于对照组(P0.05)。综合本研究结果表明,MC3R在鲤科鱼类中具有高度相似的氨基酸序列和结构域;在组织中的转录表达变化与食物摄取有明显相关性,对调控鱼体摄食行为和能量代谢具有重要作用。  相似文献   

10.
姚晓丽  赵金良 《中国水产科学》2022,29(12):1728-1737
为探讨饲料代替活饵投喂对鳜(Siniperca chuatsi)胃饥饿素(ghrelin)的调控作用, 本研究通过转录组测序和基因组测序数据匹配, 首次获得了鳜(Siniperca chuatsi)胃饥饿素前体基因 DNA 序列, 对其胃中分泌细胞进行定位。 诱食试验模拟鳜进食前看到食物但不能摄食, 进食试验模拟鳜正常摄食。研究检测了诱食、进食后胃饥饿素基因和蛋白表达水平变化。结果表明, 鳜胃饥饿素前体由信号肽、成熟肽(胃饥饿素)和 C-端肽 3 部分构成;前体基因含有 4 个外显子和 3 个内含子, 属于Ⅱ型基因型; 编码 107 个氨基酸, 成熟肽胃饥饿素由 20 个氨基酸构成, 活性中心为 GSSF。免疫组化显示胃饥饿素阳性细胞位于胃腺中。诱食试验中, 活饵组胃饥饿素 mRNA 和蛋白表达水平均显著升高; 而饲料组胃饥饿素基因 mRNA 和蛋白表达水平未发生显著变化, 且显著低于活饵组(P<0.05)。进食实验中, 活饵组进食后 0 h, 胃饥饿素 mRNA 和蛋白表达水平显著上升(P<0.05), 进食后 2 h 下降至摄食前水平 (P<0.05), 后续无显著变化(P>0.05); 饲料组进食后 0 h 胃饥饿素 mRNA 表达水平未出现显著变化(P>0.05), 蛋白水平呈波动变化, 进食后 2 h 及后续胃排空时间内皆无显著变化(P>0.05)。综上, 鳜胃饥饿素细胞位于胃腺位置, 参与调节摄食活动, 进食前上升, 进食后下降, 而饲料投喂下 ghrelin 对摄食调节作用明显减弱, 研究结果可为鳜饲料养殖提供科学参考。  相似文献   

11.
Neuropeptide Y (NPY) and cholecystokinin (CCK) play important roles in regulating appetite in vertebrates, including mammals and fish. Understanding the appetite mechanism is important in aquaculture to improve production performance. The yellowtail Seriola quinqueradiata is one of the most cultured fish in Japan, but little is known about its appetite hormones. In the present study, complementary DNA encoding for NPY was cloned in yellowtail and consists of 604 bp, in which deduced amino acid sequences show high identity to those of other teleosts. In tissue distribution, the npy and cck mRNAs were detected in all examined tissues (whole brain, telencephalon, optic tectum, hypothalamus, cerebellum, pituitary, retina, stomach, pyloric caeca, anterior intestine, liver, and kidney). In the fasting experiment, only npy mRNA expression in the hypothalamus responded to fasting, showing a significantly high value compared with that in control fish. The expression of cck mRNA in the examined tissues did not change with fasting. The npy mRNA expression in the hypothalamus might be involved in feeding regulation in yellowtail.  相似文献   

12.
聂国兴  邓大鹏  闫潇 《水产学报》2020,44(11):1926-1934
鱼类通过摄食活动获得能量和营养物质,而食欲是影响鱼类摄食的重要因素之一。食欲调控是一个复杂的网络,大脑整合机体能量和摄食信号,通过食欲调节因子(包括促进食欲因子和抑制食欲因子)调控鱼类摄食行为。Apelin作为一种新型内分泌因子,在体内发挥广泛的生物学作用。其中,apelin在摄食调节方面的作用正在引起学者的关注。本文以哺乳动物为参照,综述了apelin的组织分布、饥饿和营养物质对apelin表达的影响、apelin处理对鱼类摄食的影响及其可能的机制和信号通路,旨在为促进鱼类摄食和水产养殖提供参考。  相似文献   

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Ghrelin, a non-amidated peptide hormone, is a potent anorectic neuropeptide implicated in feeding regulation in mammals and non-mammalian vertebrates. However, the involvement of ghrelin in the feeding behavior of teleosts has not been well understood. To better understand the role of ghrelin in the regulation of appetite in fish, in this study, we cloned the cDNAs encoding ghrelin and investigated their mRNA distributions in gibel carp tissues. We also assessed the effects of different nutritional status on ghrelin mRNA abundance. Ghrelin mRNAs were ubiquitously expressed in ten tissues (intestine, liver, brain, mesonephron, head kidney, spleen, skin, heart, muscle, gill and pituitary gland), and relatively high expression levels were detected in the gut. Postprandial studies analysis revealed a significant postprandial decrease in ghrelin mRNA expression in the gut (1 and 3 h after the regular feeding time). In addition, ghrelin mRNA expression in the gut significantly increased at day 7 after fasting and declined sharply after refeeding, which suggested that ghrelin might be involved in the regulation of appetite in gibel carp. Overall, our result provides basis for further investigation into the regulation of feeding in gibel carp.  相似文献   

15.
Zhang  Yanpeng  Zhang  Zhen  Liang  Xu-Fang  He  Shan  Xu  Jing 《Aquaculture International》2021,29(6):2619-2634

Neuropeptide Y receptor Y8 (NPY8R) is a fish-specific receptor, whose role in feeding regulation remains poorly understood. In the present study, two subtypes of NPY8R (NPY8Ra and NPY8Rb) were identified in Chinese perch, and their amino acid sequences were highly conserved in fish. The predicted tertiary protein structures of human NPY1R, Chinese perch NPY8Ra, and NPY8Rb tended to be conserved as a whole. However, the predicted tertiary protein structures of sc-NPY8Ra and sc-NPY8Rb were different at several sites. Chinese perch NPY8Ra and NPY8Rb were mainly expressed in brain. Intracerebroventricular administration of siRNA-NPY8Rb significantly decreased the mRNA expression levels of NPY8Rb and POMC (appetite-suppressive gene) and significantly increased the food intake of Chinese perch at 2 h and 8 h after injection. Similar result has not been observed in the siRNA-NPY8Ra administration group. The above results suggested that NPY8Ra might not play a role in appetite regulation and NPY8Rb might be an effector in the negative feedback regulation of feeding in Chinese perch.

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16.
神经肽Y系统在硬骨鱼类摄食调控中的作用的研究进展   总被引:1,自引:0,他引:1  
对硬骨鱼摄食调节机制的研究可以为鱼类养殖中优化饲料配方和养殖方式提供重要的科学指导。在众多参与摄食调节的内分泌因子中,神经肽Y(neuropeptide Y,NPY)家族多肽由于同时参与脑和胃肠道对食欲的调节,备受研究者关注。哺乳动物中存在3种类型的NPY家族多肽,其中NPY是脑中的促摄食因子,而肽YY(peptide YY,PYY)和胰多肽(pancreatic polypeptide,PP)是胃肠道中的抑摄食因子。这3种多肽与其共同的受体——NPY家族受体结合发挥其生理作用。NPY家族多肽和受体被共同称为NPY系统。硬骨鱼经历了第3次基因组复制,其NPY系统的组成更为复杂。目前对于硬骨鱼NPY系统在摄食调节中具体作用的研究还很不完善,尤其是对NPY家族受体的研究尚处于起步阶段。本文论述了硬骨鱼中NPY系统的组成、受体与配体的结合能力以及NPY家族多肽和受体在摄食调节中的作用,以期为该领域未来的研究提供参考。  相似文献   

17.
为了解黄条(Seriola aureovittata)早期发育阶段的消化生理特性,测定了黄条胚胎、仔稚幼鱼阶段脂肪酶、淀粉酶、胰蛋白酶和碱性磷酸酶活性变化。结果显示,在黄条仔鱼出膜前胚胎阶段,即能检测到脂肪酶、淀粉酶和碱性磷酸酶活性;初孵仔鱼体内(1 d)初次检测出胰蛋白酶的活性。脂肪酶和碱性磷酸酶比活力在仔鱼孵化后迅速增强(P<0.05),在4 d开口时,2种酶比活力达最高值;淀粉酶比活力在7 d时达最大值;胰蛋白酶比活力在仔鱼阶段缓慢上升,15 d时比活力最大。稚鱼阶段内脏团中脂肪酶、碱性磷酸酶和胰蛋白酶活性基本维持稳定,幼鱼阶段内脏团脂肪酶、碱性磷酸酶和胰蛋白酶活性都呈现上升趋势;稚鱼和幼鱼阶段内脏团中淀粉酶活性下降并基本稳定于较低水平。研究表明,黄条仔稚幼鱼发育过程中,各种消化酶活性变化明显,且与其发育阶段和食性密切相关。在尚未摄食饵料的早期仔鱼体内已存在消化酶,认为其是母源传递而来,不是由外源性饵料所致;幼鱼阶段内脏团脂肪酶、碱性磷酸酶和胰蛋白酶比活力明显提高,这反映出随苗种生长发育,其肠道结构和消化机能逐渐完善,并且对脂肪、蛋白质的需求逐渐增强。  相似文献   

18.
The changes in the specific growth rate (SGR), feed intake (FI), feed conversion efficiency (FCE), and digestive enzyme activities (protease, amylase, and lipase in the hepatopancreas) of Fenneropenaeus chinensis juveniles (0.753 ± 0.041 g, wet weight) exposed to different periods of food deprivation were investigated during the period of refeeding in a controlled laboratory experiment. The starvation-refeeding cycles consisted of the following seven regimes of 24-day duration: satiate feeding (control, SC); no feeding for 4 (S4), 8 (S8), 12 (S12), 16 (S16), 20 (S20), and 24 (S24) days; then feeding to apparent satiation for the next 20, 16, 12, 8, 4, and 0 days, respectively. The various starvation-refeeding cycles have significant influences on the growth of the test shrimps. The SGR of the shrimps exposed to S4 and S8 treatments exceeded the controls, and no significant differences in SGR (P > 0.05) were found between the SC and S12 or S16 treatments at the end of the refeeding. These changes may have resulted from the significant increase in FI and FCE. The protease activities of test shrimps exposed to different periods of food deprivation were significantly lower (P < 0.05) than those of shrimps under SC treatment at the end of food deprivation then showed a tendency to rapidly increase during refeeding. However, nearly the exact opposite occurred for amylase activities and lipase activities. Lipid and carbohydrate reserves in the hepatopancreas are preferentially mobilized with protein-sparing effect at the beginning of food deprivation. Proteins are the main nutritional store during the refeeding. The findings of the research will be beneficial in the design of feeding regimes and will improve our knowledge on some aspects of the nutrition physiology of F. chinensis related to their biology.  相似文献   

19.
为了解黄条鰤(Seriola aureovittata)早期发育阶段的消化生理特性,测定了黄条鰤胚胎、仔稚幼鱼阶段脂肪酶、淀粉酶、胰蛋白酶和碱性磷酸酶活性变化。结果显示,在黄条鰤仔鱼出膜前胚胎阶段,即能检测到脂肪酶、淀粉酶和碱性磷酸酶活性;初孵仔鱼体内(1 d)初次检测出胰蛋白酶的活性。脂肪酶和碱性磷酸酶比活力在仔鱼孵化后迅速增强(P<0.05),在4 d开口时,2种酶比活力达最高值;淀粉酶比活力在7 d时达最大值;胰蛋白酶比活力在仔鱼阶段缓慢上升,15 d时比活力最大。稚鱼阶段内脏团中脂肪酶、碱性磷酸酶和胰蛋白酶活性基本维持稳定,幼鱼阶段内脏团脂肪酶、碱性磷酸酶和胰蛋白酶活性都呈现上升趋势;稚鱼和幼鱼阶段内脏团中淀粉酶活性下降并基本稳定于较低水平。研究表明,黄条鰤仔稚幼鱼发育过程中,各种消化酶活性变化明显,且与其发育阶段和食性密切相关。在尚未摄食饵料的早期仔鱼体内已存在消化酶,认为其是母源传递而来,不是由外源性饵料所致;幼鱼阶段内脏团脂肪酶、碱性磷酸酶和胰蛋白酶比活力明显提高,这反映出随苗种生长发育,其肠道结构和消化机能逐渐完善,并且对脂肪、蛋白质的需求逐渐增强。  相似文献   

20.
Abstract The temporal dynamics of daily food consumption were examined in individually housed fish that experienced four cycles of 1 week of feed deprivation followed by 2 weeks of feeding to satiation. Four species were compared: European minnows Phoxinus phoxinus: Cyprinidae; three‐spined sticklebacks Gasteosteus aculeatus: Gasterosteidae: gibel carp Carassius auratus gibelio: Cyprinidae; and the longsnout catfish Leiocassis longirostris: Bagridae. The stickleback, carp and catfish showed significant compensatory increases in food intake following deprivation, with the response becoming clearer in successive cycles. The temporal pattern of consumption during the refeeding periods differed between the four species. In sticklebacks, daily intake over a refeeding period initially decreased, but then recovered. In minnows, intake tended to decline over a refeeding period. Gibel carp showed an increase in daily intake on refeeding, but this may have reflected an adverse response to weighing. Over a refeeding period, catfish had a weak tendency to show an initial decline, followed by an increase. These differences are discussed in relation to differences in experimental protocols and biological differences between the species.  相似文献   

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