In vitro and in vivo antifungal activity of Satureja cuneifolia ten essential oil on Saprolegnia parasitica strains isolated from rainbow trout (Oncorhynchus mykiss,Walbaum) eggs

In the present study, the chemical composition and the antifungal properties against Saprolegnia parasitica (in vitro and in vivo) of the essential oils of thyme (Satureja cuneifolia) from Mediterranean region of Turkey were evaluated for the first time. The composition of oils was analysed using gas chromatography/mass spectrometry (GC/MS). The major constituents of oil of S. cuneifolia were cavracrol (46,84%) and cymene (16.90%). Antifungal effects of S. cuneifolia essential oil against S. parasitica strains (A1 and E1) were detected by disc diffusion and tube dilution assays. The antifungal effect of S. cuneifolia was determined to be stronger against S. parasitica E1 isolate (MIC 50 μL mL^?1, MLC 250 μL mL^?1) compared with S. parasitica A1 isolate (MIC 50 μL mL^?1, MLC 500 μL mL^?1). Following in vitro assays, effective doses of S. cuneifolia for disease control in rainbow trout eggs experimentally infected with S. parasitica were investigated. For this aim, infected eggs were treated with the essential oil (0, 5, 10, 20 and 50 ppm) during incubation period (21 days) after fertilization. Formalin (5 mL L^?1) was used as positive control. Hatching rate of eggs at the end of incubation period were calculated. The highest hatching rates were recorded in S. parasitica E1 strain at 5 and 10 ppm concentrations of S. cuneifolia and in S. parasitica A1 strain at 10 and 20 ppm (P < 0.05).     

The Efficacy of Copper Sulfate in Controlling Infection of Saprolegnia parasitica

Saprolegnia parasitica is a severe fish pathogen that causes important economic losses worldwide. Copper is an important additive in the aquaculture industry for control of algal growth, ectoparasites, and fungal disease. However, at present no data is available on the specific interaction of copper sulfate with oomycete. In our study, the efficacy of copper sulfate on the mycelium and zoospore of S. parasitica was assessed in vitro, and S. parasitica infection experiment was conducted to assess its performance in vivo. The results indicated that copper sulfate at ≥0.5 mg/L inhibited the growth of mycelium, no primary zoospores were released at ≥1.0 mg/L. Additionally, 0.5 mg/L copper sulfate could reduce the infection rate of S. parasitica in the grass carp, Ctenopharyngodon idellus. This study demonstrates the good efficacy of copper sulfate on the control of S. parasitica infection in grass carps, and suggests that copper sulfate could be used as a drug additive to control the S. parasitica infection in the aquaculture industry.     

Saprolegnia diclina IIIA and S. parasitica employ different infection strategies when colonizing eggs of Atlantic salmon,Salmo salar L.

Here, we address the morphological changes of eyed eggs of Atlantic salmon, Salmo salar L. infected with Saprolegnia from a commercial hatchery and after experimental infection. Eyed eggs infected with Saprolegnia spp. from 10 Atlantic salmon females were obtained. Egg pathology was investigated by light and scanning electron microscopy. Eggs from six of ten females were infected with S. parasitica, and two females had infections with S. diclina clade IIIA; two Saprolegnia isolates remained unidentified. Light microscopy showed S. diclina infection resulted in the chorion in some areas being completely destroyed, whereas eggs infected with S. parasitica had an apparently intact chorion with hyphae growing within or beneath the chorion. The same contrasting pathology was found in experimentally infected eggs. Scanning electron microscopy revealed that S. parasitica grew on the egg surface and hyphae were found penetrating the chorion of the egg, and re‐emerging on the surface away from the infection site. The two Saprolegnia species employ different infection strategies when colonizing salmon eggs. Saprolegnia diclina infection results in chorion destruction, while S. parasitica penetrates intact chorion. We discuss the possibility these infection mechanisms representing a necrotrophic (S. diclina) vs. a facultative biotrophic strategy (S. parasitica).     

Saprolegnia molecular phylogeny among farmed teleosts in Nova Scotia,Canada

To identify the pathogens causing saprolegniosis among farmed fish in Nova Scotia, 172 infected tissues and 23 water samples were collected from six species of teleosts: Atlantic salmon (Salmo salar), brown trout (Salmo trutta), Arctic charr (Salvelinus alpinus), brook trout (Salvelinus fontinalis), striped bass (Morone saxatilis) and rainbow trout (Oncorhynchus mykiss) at nine facilities over a 600 km range. Following laboratory culture, 132 isolates were recovered. Six species of oomycetes were identified from analysis of the internal transcribed spacer (ITS) sequence of the nrDNA: Saprolegnia parasitica, Saprolegnia ferax, Saprolegnia diclina, Saprolegnia aenigmatica, Saprolegnia torulosa, Saprolegnia sp. and Pythiopsis cymosa. Further phylogenetic analyses of the ITS and cytochrome c oxidase subunit 1 (Cox1) regions revealed four strains of Saprolegnia parasitica (named here as S1, S2, S3 and S4), of which S1 and S2 were common (37% and 42% of the isolates), and two strains of S. ferax. Among S. parasitica, S2 and S3 are more closely related to each other than to S1 based on the phylogenetic analyses and predicted RNA secondary structure of the ITS region. Sexual structures with a similar morphology were formed by S1 and S3 in vitro, but were not formed by S2.     

Do fathead minnows,Pimephales promelas Rafinesque,alter their club cell investment in responses to variable risk of infection from Saprolegnia?

Fish in the Superorder Ostariophysi possess large epidermal club cells that release chemical cues warning nearby conspecifics of danger. Despite the long‐held assumption that such club cells evolved under the selective force of predation, recent studies demonstrated that predation has no effect on club cell investment. Rather, club cells have an immune function and cell production may be stimulated by skin‐penetrating pathogens and parasites. The current work investigates whether fathead minnows, Pimephales promelas, alter their club cell characteristics based on variation in infection risk. In a 2 × 3 design, we exposed minnows to infective cysts of two oomycete species (Saprolegnia ferax and S. parasitica) at three different concentrations (2, 20 or 200 cysts L^?1). Club cell characteristics (number and size) were quantified 12 days after exposure. Saprolegnia parasitica is thought to be more pathogenic than S. ferax, hence we predicted greater club cell investment and a larger turnover rate of cells by minnows exposed to S. parasitica than S. ferax. We also predicted that minnows exposed to higher numbers of cysts should invest more in club cells and have a higher turnover rate of cells. We found no difference in club cell density or size between fish exposed to the two Saprolegnia species; however, fish exposed to high concentrations of pathogens had smaller club cells than those exposed to low concentrations, indicating a higher rate of turnover of cells in the epidermis.     

Physiological characterization of Saprolegnia parasitica isolates from brown trout

Saprolegnia parasitica has caused large mortalities in brown trout, Salmo trutta, in Spain. Several strains of Saprolegnia parasitica have been isolated from these epizootics and characterized regarding their physiological adaptation and genetic diversity. These isolates exhibit similar physiological characteristics, i.e. radial growth, zoospore mobility and germination after encystment and differ substantially from other strains of the Saprolegnia diclina-parasitica complex tested. Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) of total DNA revealed a genetic similarity of about 85–100% between Spanish trout isolates whereas only 20–45% similarity to other strains of the Saprolegnia diclina-parasitica complex was found. The results suggest that the Spanish isolates are closely related strains and that they might have originated from one clone.     

Chemical composition and in vitro antifungal activity of Sambucus ebulus and Actinidia deliciosa on the fish pathogenic fungus,Saprolegnia parasitica

The aim of the present study was to determine the chemical composition of Sambucus ebulus and Actinidia deliciosa ethanolic extracts as well as their in vitro antifungal activity on the mycelial growth of the water mold, Saprolegnia parasitica. The preliminary minimum inhibition concentration (MIC) and minimum lethal concentration (MLC) were determined by the HeMP method and finally, concentrations of each extract ranging from 1 to 10% were prepared by an agar dilution method to assess the in vitro antifungal activity, quantitatively. Both herbal extracts inhibited growth of Saprolegnia hyphae in vitro. Complete in vitro growth inhibition was found at a concentration of ≥5% for S. ebulus, whereas it was not observed even at 10% concentration of A. deliciosa. Based on gas chromatography coupled with mass spectrometry (GC/MS) analysis, the main constituents of S. ebulus include monophthalate (66.17%), fatty acids (26.47%), phytol (4.25%), and acetic acid (2.11%). Using colorimetric assays, A. deliciosa contained phenolic contents at 162 mg gallic acid (GAE)/g DW and flavonoid contents at 2.31 mg quercetin (QE)/g DW. In conclusion, the results of this study indicated that S. ebulus and A. deliciosa showed some antifungal activities against S. parasitica with formerly exhibiting stronger activity (p < 0.05).

     

Protective effects of a synbiotic against experimental Saprolegnia parasitica infection in rainbow trout (Oncorhynchus mykiss)

A combination of probiotics and prebiotics as synbiotics allows assessing their synergistic effects. This study evaluated the effects of a synbiotic supplement on growth performance, haematological parameters and resistance to Saprolegnia parasitica in rainbow trout, Oncorhynchus mykiss (Walbaum) fingerlings. Fish fed a dietary synbiotic in three levels of 0.5, 1.0 and 1.5 g kg^?1 thrice a day. The fingerlings were challenged with Saprolegnia parasitica after 60 days post feeding and their mortalities recorded up to 15 days. The fingerlings at all three experimental treatments showed significant (P < 0.05) increases in final mean weights and specific growth rates (SGR). The best feed conversion ratio (FCR), feed conversion efficiency (FCE) and maximum survival rate were also obtained by the fish fed 1.0 g synbiotic kg^?1 diet. Furthermore, supplementation with synbiotic significantly increased blood factors at all treatments. After challenges with Saprolegnia parasitica, the synbiotic‐fed groups showed significantly higher survival rates compared with the control group. These results reveal that a dietary synbiotic of 1.0 g kg^?1 fed for 60 days leads to increased growth performance and survival rate as well as improved feeding efficiency in rainbow trout fingerling, rendering them more resistant against infection by Saprolegnia parasitica.     

Biocontrol of saprolegniosis in rainbow trout (Oncorhynchus mykiss Walbaum) using two bacterial isolates (LE89 and LE141) of Pseudomonas fluorescens

The probiotic activity of 15 bacterial isolates that inhibit Saprolegnia parasitica in vitro was tested for the biocontrol of saprolegniosis in rainbow trout (Oncorhynchus mykiss Walbaum), adding the bacteria to tank water for 14 days at a concentration of 10⁶ bacteria ml^?1 water. Pseudomonas fluorescens LE89 and Pseudomonas fluorescens LE141 were effective in controlling experimental infection with S. parasitica since of the fish treated with LE89, 24.5% ± 16.27% (p < 0.05) became infected, as did 42.8% ± 8.41% (p < 0.05) of those treated with LE141. Given their protective effect when administered in water, their effect was also studied when administered in feed before and after experimental infection. Both bacterial isolates survived low pH levels and the action of bile, grew in skin and intestinal mucus, were resistant to several antibiotics and survived in feed; however, neither of the two isolates prevented S. parasitica infection when administered in feed.     

In vitro passages impact on virulence of Saprolegnia parasitica to Atlantic salmon,Salmo salar L. parr

The effect of serial in vitro subculturing on three pathogenic strains of Saprolegnia parasitica was investigated. The isolates were passed through Atlantic salmon, Salmo salar L. parr, and then re‐isolated as single spore colonies. All strains caused infection. The isolate obtained from diseased fish served as a virulent reference culture and was designated ‘AP’ (‘activated through passage’). Successive subculturing was made by obtaining an inoculum from AP to produce the 2nd subculture and then passaged to the 3rd subculture (from the 2nd), until the 15th passage was obtained. Spores used to produce storage cultures were collected at passages 5, 10 and 15. The different passages of each strain were used to artificially infect Atlantic salmon parr. Morphological characterization of growth patterns was performed to observe differences occurring due to serial in vitro subculturing. Two of the strains declined in virulence after 15 successive in vitro subcultures, whereas one did not. This study is the first to investigate attenuation of virulence in Saprolegnia and whether or not isolates of S. parasitica should be passed through the fish host prior to challenge experiments. It reveals that some strains degenerate more rapidly than others when subjected to successive in vitro subculturing on glucose–yeast extract.     

In vitro Antifungal activity of essential oils from Tymbra,Origanum, Satureja species and some pure compounds on the fish pathogenic fungus,Saprolegnia parasitica

In the present study, the chemical composition and antifungal activity of the essential oils of the three Lamiaceae species, black tyme (Thymbra spicata L.), oregano (Origanum onites L.) and savory (Satureja tymbra L.) against Saprolegnia parasitica Coker strains (3) were evaluated. The composition of oils was analysed by gas chromatography/mass spectrometry (GC/MS). The major constituent of the oils was carvacrol (82.68% in Thymbra spicata, 82.24% in Origanum onites and 52.38% in Satureja tymbra). The antifungal activity of essential oils was determined by a tube dilution and disc diffusion method. All essential oils inhibited fungal strains at concentration of 100 μL mL^?1. The essential oil of O. onites and T. spicata was the most active (MIC 10 and 50 μL mL^?1, respectively). Thymol did not show inhibiting activity. The essential oils exhibited stronger inhibition than pure carvacrol. Essential oil derived from Tymbra, Origanum and Satureja species has the potential to be used as health control of rainbow trout (Oncorhyncus mykiss) against S. parasitica infection. This study describes the antifungal activity of three essential oils for developing alternative and native control methods against Saprolegniasis.     

Reduction of in vitro growth in Flavobacterium columnare and Saprolegnia parasitica by products containing peracetic acid

Commercial products containing peracetic acid (PAA) are strong disinfectants with a wide spectrum of antimicrobial activity and have been suggested as potential therapeutic agents in aquaculture. The aim of this study was to compare the in vitro reduction of growth on two fish pathogens, Flavobacterium columnare and Saprolegnia parasitica, by seven commercial PAA‐containing products. Flavobacterium columnare was exposed to 1, 2, 4, 6, 8 and 10 mg L^?1 PAA and S. parasitica was exposed to 0.5, 1, 2, 4, 6, 8 and 10 mg L^?1 PAA in petri dishes for 24 h incubation. The reduction of growth was measured in comparison to a PAA‐free control. A reduction of the growth was observed for both pathogens with increasing PAA concentration. Hydrogen peroxide (H₂O₂) possibly has a role in the effectiveness of the products, since products with lower PAA concentrations had a higher concentration of H₂O₂. The commercial products with a low concentration of PAA and a low PAA:H₂O₂‐ratio were generally more effective against pathogens. The practical application of the products with low PAA concentration should be prioritized.     

Susceptibility by amberjack (Seriola dumerili), yellowtail (S. quinqueradiata) and Japanese flounder (Paralichthys olivaceus) to Neobenedenia girellae (Monogenea) infection and their acquired protection

This study investigated: 1) susceptibility differences to infection by Neobenedenia girellae (Capsalidae) between amberjack Seriola dumerili (Carangidae), yellowtail S. quinqueradiata and Japanese flounder Paralichthys olivaceus (Paralichthyidae); 2) growth and egg production of N. girellae on each fish species; 3) acquired protection of each fish species against this parasite. The number of N. girellae on S. dumerili was significantly higher than on S. quinqueradiata and P. olivaceus when these fishes were exposed to oncomiracidia in the same aquarium. Neobenedenia girellae growth on S. dumerili was fastest and, thus the number of eggs laid by parasites on S. dumerili was greater than on the other two species. Seriola dumerili and P. olivaceus, which were previously infected with N. girellae and treated by freshwater bath, acquired partial protection against re-infection by N. girellae. The relative re-infection of three S. dumerili individuals out of eleven individuals was markedly low compared with the initial infection, and the relative initial infection and re-infection on two P. olivaceus out of eleven individuals was markedly low. The results of this study could be useful to control N. girellae infections when cultivating S. dumerili, S. quinqueradiata and P. olivaceus.     

不同地理种群瓦氏马尾藻ITS序列特征及其系统进化分析

为研究不同地理种群瓦氏马尾藻ITS的序列变异,实验采用PCR扩增和序列测定方法,获得了3个不同地理种群15株瓦氏马尾藻的ITS全长序列,并进行序列分析。结果显示,15个个体共出现4种不同的ITS序列,它们相应的ITS1、5.8S、ITS2长度相同,均为762、158和507 bp,共有3个位点发生碱基变异。结合从GenBank中下载的马尾藻科3个属24种马尾藻的ITS序列,以羊栖菜属的羊栖菜,喇叭藻属的拟小叶喇叭藻和下延喇叭藻作为3个外群,采用邻位相连法构建分子系统进化树,结果显示,瓦氏马尾藻的ITS序列优先聚在一起然后以较高的置信度与南海马尾藻和球囊马尾藻聚为一支,在系统发生上显示出更近的亲缘关系。在选取的马尾藻中瓦氏马尾藻与南海马尾藻遗传距离最近为0.004,与拟小叶喇叭藻遗传距离最远为0.422。     

Use of Onion (Allium cepa) and Garlic (Allium sativum) Wastes for the Prevention of Fungal Disease (Saprolegnia parasitica) on Eggs of Rainbow Trout (Oncorhynchus mykiss)

This study was designed to prevent fungal disease (Saprolegnia parasitica) that occurs on rainbow trout eggs (Oncorhynchus mykiss) by using wastes of onion (Allium cepa) and garlic (Allium sativum) plants. For this purpose, fertilized rainbow trout eggs were exposed to garlic skin, garlic stem and onion skin aqueous methanolic extracts by bathing in concentrations of 0.4, 0.8, 1.6 and 3.2 g/L, whereas the control group was left untreated. The larvae in all groups were monitored until they become free-swimming larvae, the number of eggs died due to fungus was recorded, and the data obtained from experimental groups and control group were compared. As a result, it was determined that onion skin had no effect on the number of eggs that died due to fungus (p > .05), while garlic skin and garlic stem extracts significantly reduced the number of fungal infestations without affecting the number of live larvae or the embryological development of the eggs (p < .05). In light of these data, we conclude that garlic skin (0.4, 0.8, and 1.6 g/L) and garlic stem (0.8 and 1.6 g/L) aqueous methanolic extracts are effective at preventing Saprolegnia parasitica infestation on rainbow trout eggs and may be used in aquaculture.     

巨须裂腹鱼水霉菌的分离鉴定及其对脾脏转录组的影响

为分析感染水霉菌对巨须裂腹鱼脾脏转录组的影响，探索水霉菌感染巨须裂腹鱼的分子机制，实验随机选取生活在同一水域中的5尾健康和5尾感染水霉菌的巨须裂腹鱼，采用PDA琼脂培养基和分子生物学方法对巨须裂腹鱼水霉菌进行分离鉴定，并采用Illumina HiSeqTM 2000 高通量测序平台，对健康和感染水霉菌的巨须裂腹鱼脾脏组织进行转录组测序，并对测序数据进行拼接、注释和差异表达基因分析。结果显示，从巨须裂腹鱼皮肤上分离鉴定得到3株水霉菌；转录组数据显示，健康组和感染水霉病组分别获得46 619 504 条和43 912 876 条数据，与健康组相比，感染水霉菌组共有1 889 个基因发生差异表达，其中1 414 个基因上调，475 个基因下调，随机选取6个差异表达基因进行实时荧光定量PCR (RT-qPCR)验证，验证结果与转录组测序一致。对健康组和感染水霉菌组的差异表达基因进行GO功能富集发现，上调基因主要富集于241个功能中，下调基因主要富集于60个功能中，上述基因主要涉及分子功能类、细胞组分类和生物过程类等生理功能；KEGG通路富集分析发现，差异表达基因主要富集在免疫疾病、内分泌和代谢疾病、病毒感染性疾病、消化系统及排泄系统等。研究表明，感染水霉菌会影响巨须裂腹鱼脾脏组织多种基因的表达量，实验结果为进一步探索巨须裂腹鱼水霉菌的感染机制奠定了基础。     

肖四海湖五种渔具的鳜渔获结构特征及其对鳜资源的影响

为评估不同渔具对鳜资源的影响,于2007年5月和12月对长江中游浅水湖泊肖四海湖刺网、延绳钓、网簖、电拖网和电捕仪5种渔具捕获的鳜渔获物结构特征进行了调查分析.结果发现,共采集鳜样本359尾,全长分布范围为92 ～600 mm,优势全长集中于251～350 mm;体质量分布范围为10～3 380 g,优势体质量集中于300 ～500 g.种群由5个年龄组构成,2～3龄为优势龄组,占总数的74.6％.刺网、延绳钓和网簖对鳜有较强的捕捞选择性,网目大小为80 mm和100 mm刺网的鳜渔获物中2龄及以上成熟个体占总数的93.3％,个体平均体质量466 g,“标鳜”(0.4 ～0.75 kg)个体占总数量的57.0％;延绳钓捕获的鳜渔获物中2龄及以上成熟个体占总数的86.9％,“标鳜”个体占总数量的43.5％;网簖捕获的鳜渔获物中90％以上为1龄的未成熟个体.电拖网和电捕仪捕获的鳜全长范围明显较大,其渔获物以1龄和2龄个体为主.综合分析表明,刺网适于作为鳜捕捞的主要渔具,延绳钓可以作为一种鳜捕捞的辅助渔具.网簖对鳜补充群体有较大危害,不适于作为鳜的捕捞网具.电拖网和电捕仪均属于违法渔具,对鳜资源危害巨大,应该加大监管力度,严禁使用.     

西伯利亚鲟海豚链球菌的分离鉴定及毒力基因检测

2013年8-9月,四川雅安汉源湖养殖的西伯利亚鲟发生一种以体表溃疡、内脏出血和心外膜囊肿为特征的传染病.为明确其病因,本研究从自然发病鱼的肝、脾和肾进行了病原菌的分离、人工感染、分离菌的表型特征和分子生物学特征的检测.结果从患病鱼体内分离到一株G+链状球菌(Ab130920),人工感染实验证实了其病原性,生理生化特性与海豚链球菌(ATCC29178)基本一致;16S rDNA序列(GenBank登录号:KJ162337)与GenBank中S.iniae 16S rDNA序列同源性最高,在以16S rDNA序列及其GenBank中同源性较高的相关序列构建的系统发育树上,分离菌与海豚链球菌聚为一支;同时,在基于S.iniae lctO基因的特异性PCR检测中,从分离株基因组DNA扩增出预期大小的870 bp条带,进而鉴定分离菌Ab130920为S.iniae.在对cpsD、simA、sagA、pdi和scpI等5种S.iniae毒力基因的多重PCR检测中,分离菌均扩增出相应大小的特异性片段,表明其为一毒力较强的菌株,与人工感染实验的高致病性结果相佐证;药物敏感性检测发现其对阿莫西林、强力霉素、氟苯尼考等抗菌药物敏感,但对新生霉素、利福平、氧氟沙星耐药.     

不同循环饥饿投喂模式对尼罗罗非鱼补偿生长的影响

为了探讨尼罗罗非鱼对不同循环饥饿投喂模式的补偿生长效应,本实验分别采用每天投喂(S0)及饥饿1 d+投喂3 d(S1F3)、饥饿1 d+投喂5 d(S1F5)、饥饿1 d+投喂7 d(S1F7)、饥饿2 d+投喂3 d(S2F3)、饥饿2 d+投喂5 d(S2F5)和饥饿2 d+投喂7 d(S2F7)6种不同的循环投喂模式,用含33%蛋白质和8%脂肪的饲料饲养尼罗罗非鱼(均重13.50 g),饲喂期为43 d。结果显示,S0组的增重率(806.74%)最高,与S1F3、S1F5和S1F7组差异不显著,但分别比S2F3、S2F5和S2F7组显著提高40.3%、33.6%和10.4%;S0组的特定生长率最低(5.36%),显著低于其他各组;与对照组相比,采用循环投喂模式没有改善饲料转化率和蛋白质效率,但却能明显提高日摄食率;各实验处理组肝体比、脏体比、肥满度、鱼体灰分含量、肌肉RNA/DNA比值及血清总胆固醇、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇和尿素氮含量与对照组差异不显著;S1F3、S1F5和S1F7组鱼体蛋白质和脂肪含量、血清甘油三酯含量及血清谷丙转氨酶和谷草转氨酶活性与S0组差异不显著,但S2F3、S2F5和S2F7组鱼体蛋白质和脂肪含量及血清甘油三酯含量显著低于S0组,血清谷丙转氨酶和谷草转氨酶活性显著高于S0组。研究表明,尼罗罗非鱼在S1F3、S1F5和S1F7模式下获得了完全补偿生长,而在S2F3、S2F5和S2F7模式下仅获得了部分补偿生长,且均是通过提高恢复投喂期间的摄食量来实现补偿生长。在获得完全补偿生长的3组中,S1F3组的实际投喂天数最短,仅为33 d,比每天投喂模式缩短了23.3%,因此在本实验条件下,饥饿1 d+投喂3 d是最佳的循环饥饿投喂模式。