Evaluation of Zebrafish Danio rerio as a Model for Enteric Septicemia of Catfish (ESC)

Abstract

Zebrafish (also known as zebra danio) Danio rerio were injected intramuscularly with Edwardsiella ictaluri at doses of 6 × 10³, 6 × 10⁴, or 6 × 10⁵ colony-forming units per gram (CFU/g) or sterile phosphate-buffered saline (sham) or were not injected. Mortality occurred from 2 to 5 d postinjection (dpi) at rates of 0, 76.6, and 81.3% for the low, medium, and high doses, respectively, and E. ictaluri was isolated from dead fish. Survivors were sampled at 10 dpi and E. ictaluri was not isolated. Sham-injected and noninjected controls did not suffer mortality. Histopathology trials were performed in which zebrafish were injected with 1 × 10⁴ CFU/g or sham-injected and sampled at 12, 24, 48, 72, and 96 h postinjection for histological interpretation. Collectively, these zebrafish demonstrated increasing severity of splenic, hepatic, cardiac, and renal interstitial necrosis over time. To evaluate the progression of chronic infection, zebrafish were injected with 1 × 10² CFU/g and held for 1 month postinjection. Beginning at 12 dpi and continuing for an additional 2 weeks, zebrafish demonstrated abnormal spiraling and circling swimming behaviors. Histopathology demonstrated necrotizing encephalitis. In immersion trials, zebrafish were exposed to low, medium, and high doses (averaging 1.16 × 10⁵, 1.16 × 10⁶, and 1.16 × 10⁷ CFU/mL of tank water) of E. ictaluri for 2 h. Mortality occurred from 5 to 9 d postexposure at rates of 0, 3.3, and 13.3% for the low, medium, and high doses, respectively; E. ictaluri was isolated from dead fish. Channel catfish Ictalurus punctatus exposed to the medium doses suffered 100% mortality, and E. ictaluri was isolated from these fish. This study demonstrates the potential use of zebrafish as a model for E. ictaluri pathogenesis.     

Protective effects of phlorotannins against ultraviolet B radiation in zebrafish (Danio rerio)

Exposure to ultraviolet B (UV-B) radiation has been associated with a variety of adverse effects in all forms of life, including micro-organisms, plants, animals and humans. Ultraviolet B induces cell damage at the molecular level and consequently organisms must employ strategies to protect themselves from sunlight and to repair UV-B-induced cellular damage. In this study, the UV-B protective effects of four different phlorotannins isolated from a brown alga (Ecklonia cava) were determined using zebrafish (Danio rerio) as an in vivo model. Zebrafish embryos were pretreated with phlorotannins and exposed to UV-B (50 mJ/cm(2)). The heart rate, generation of reactive oxygen species and nitric oxide, cell death and hyperpigmentation were assessed in order to evaluate UV-B-induced photo-damage. Treatment of the embryos with the algal phorotannins reduced UV-B-induced reactive oxygen species and nitric oxide levels, protected against UV-B-induced cell death and significantly reduced hyperpigmentation. We therefore suggest that phlorotannins isolated from E. cava can protect against UV-B radiation. Editor Note. Readers of the journal may be unfamiliar with the use of zebrafish embryos in research studies. There is no indication in this article of an ethical review of the study. This is because the use of fish embryos in research, at least in the UK, is not subject to a licensing procedure if they are less than 5 days post fertilization (dpf). In this study the embryos were 2 dpf.     

Comparison of two formulations of alfaxalone for immersion anaesthesia in laboratory zebrafish (Danio rerio)

ObjectiveTo compare two commercial formulations of alfaxalone for immersion anaesthesia in laboratory zebrafish.Study designProspective, blinded, randomized study.AnimalsA total of 20 adult Danio rerio (Tuebingen strain).MethodsZebrafish were divided into two groups of 10 (five female, five male) and placed in individual immersion baths containing 10 mg L^–1 of unpreserved alfaxalone (group 1) or preserved alfaxalone (group 2). Anaesthetists blinded to treatment used a composite score scale (CSS) (range 0–12) to assess fish every 30 seconds until induction of anaesthesia. Anaesthetic induction occurred when equilibrium and response to stimulus were lost. Fish were then placed in a clean water bath and scored every 60 seconds. Recovery from anaesthesia was defined as a CSS of ≤ 1. Time variables recorded were anaesthetic induction time (AIT), anaesthetic recovery time (ART) and total procedure time (TPT). Fish were observed for evidence of roupgross external pathology during the procedure. Following anaesthesia, four fish from each group were randomly chosen and euthanized for gill histopathology analysis immediately after recovery criteria were met. Data are presented as mean ± standard deviation. An independent t test was used to compare the difference in average anaesthetic time variables between groups (α = 0.05).ResultsThere were no statistical differences between groups in reported variables. TPT, AIT and ART were 10.2 ± 1.2, 1.9 ± 0.9 and 8.3 ± 1.2 minutes for group 1 and 10.8 ± 2.9, 2.4 ± 1.2 and 8.4 ± 2.7 minutes for group 2. No gross external pathology was evident, and no fish died during the experimental period. Histopathology showed normal gill pathology and no difference between the groups.Conclusions and clinical relevanceImmersion anaesthesia using 10 mg L^–1 of either formulation of alfaxalone resulted in anaesthesia of similar quality and duration.     

The Impact of Reproductive Technologies on Stallion Mitochondrial Function

The traditional assessment of stallion sperm comprises evaluation of sperm motility and membrane integrity and identification of abnormal morphology of the spermatozoa. More recently, the progressive introduction of flow cytometry is increasing the number of tests available. However, compared with other sperm structures and functions, the evaluation of mitochondria has received less attention in stallion andrology. Recent research indicates that sperm mitochondria are key structures in sperm function suffering major changes during biotechnological procedures such as cryopreservation. In this paper, mitochondrial structure and function will be reviewed in the stallion, when possible specific stallion studies will be discussed, and general findings on mammalian mitochondrial function will be argued when relevant. Especial emphasis will be put on their role as source of reactive oxygen species and in their role regulating sperm lifespan, a possible target to investigate with the aim to improve the quality of frozen–thawed stallion sperm. Later on, the impact of current sperm technologies, principally cryopreservation, on mitochondrial function will be discussed pointing out novel areas of research interest with high potential to improve current sperm technologies.     

Evaluation of zebrafish Danio rerio as a model for enteric septicemia of catfish (ESC)

Zebrafish (also known as zebra danio) Danio rerio were injected intramuscularly with Edwardsiella ictaluri at doses of 6 x 10(3), 6 x 10(4), or 6 x 10(5) colony-forming units per gram (CFU/g) or sterile phosphate-buffered saline (sham) or were not injected. Mortality occurred from 2 to 5 d postinjection (dpi) at rates of 0, 76.6, and 81.3% for the low, medium, and high doses, respectively, and E. ictaluri was isolated from dead fish. Survivors were sampled at 10 dpi and E. ictaluri was not isolated. Sham-injected and noninjected controls did not suffer mortality. Histopathology trials were performed in which zebrafish were injected with 1 x 10(4) CFU/g or sham-injected and sampled at 12, 24, 48, 72, and 96 h postinjection for histological interpretation. Collectively, these zebrafish demonstrated increasing severity of splenic, hepatic, cardiac, and renal interstitial necrosis over time. To evaluate the progression of chronic infection, zebrafish were injected with 1 x 10(2) CFU/g and held for 1 month postinjection. Beginning at 12 dpi and continuing for an additional 2 weeks, zebrafish demonstrated abnormal spiraling and circling swimming behaviors. Histopathology demonstrated necrotizing encephalitis. In immersion trials, zebrafish were exposed to low, medium, and high doses (averaging 1.16 x 10(5), 1.16 x 10(6), and 1.16 x 10(7) CFU/mL of tank water) of E. ictaluri for 2 h. Mortality occurred from 5 to 9 d postexposure at rates of 0, 3.3, and 13.3% for the low, medium, and high doses, respectively; E. ictaluri was isolated from dead fish. Channel catfish Ictalurus punctatus exposed to the medium doses suffered 100% mortality, and E. ictaluri was isolated from these fish. This study demonstrates the potential use of zebrafish as a model for E. ictaluri pathogenesis.     

Assessment of diclofenac LC50 reference values in juvenile and embryonic stages of the zebrafish (Danio rerio)

The aim of the study was to compare the acute toxicity of diclofenac to juvenile and embryonic stages of the zebrafish (Danio rerio). Acute toxicity tests were performed on the aquarium fish Danio rerio, which is one of the model organisms most commonly used in toxicity testing. The tests were performed using a semi-static method according to OECD guideline No. 203 (Fish, acute toxicity test). Embryo toxicity tests were performed in zebrafish embryos (Danio rerio) in compliance with OECD No. 212 methodology (Fish, short-term toxicity test on embryo and sac-fry stages). The results were subjected to a probit analysis using the EKO-TOX 5.2 programme to determine 96hLC50 and 144hLC50 (median lethal concentration, 50% mortality after a 96 h or 144 h interval, respectively) values of diclofenac. The statistical significance of the difference between LC50 values in juvenile and embryonic stages of Danio rerio was tested using the Mann-Whitney non-parametric test implemented in the Unistat 5.1 programme. The LC50 mean value of diclofenac was 166.6 +/- 9.8 mg/L in juvenile Danio rerio, and 6.11 +/- 2.48 mg/L in embryonic stages of Danio rerio. The study demonstrated a statistically higher sensitivity to diclofenac (P < 0.05) in embryonic stages compared to the juvenile fish.     

斑马鱼成纤维细胞生长因子(FGFs)信号肽的预测分析

斑马鱼(Danio rerio)是鲤科短担尼尔属的一种热带观赏鱼[1].斑马鱼作为一种新型的脊椎模式生物,可以进行大规模的正向基因饱和突变与筛选[2].随着斑马鱼基因组测序工程的完成,接着需要进行的就是斑马鱼的基因功能组、转录组、蛋白质组及代谢组学的研究.较准确地注释斑马鱼基因组以及从整体上了解基因家族的结构特点和功能对积极推动基因功能研究具有重要意义.     

Exogenous expression of rat aquaporin-3 enhances permeability to water and cryoprotectants of immature oocytes in the zebrafish (Danio rerio)

Movement of water and cryoprotectants through the plasma membrane needs to be accelerated for successful cryopreservation of zebrafish oocytes/embryos, which are much larger than their mammalian counterparts. Aquaporin-3 is a water/solute channel that can transport not only water but also various cryoprotectants. In this study, we attempted to increase the permeability of immature zebrafish oocytes at stage III to water and cryoprotectants by exogenous expression of rat aquaporin-3. Immature zebrafish oocytes were injected with rat aquaporin-3 cRNA and cultured for 5-12 h. Permeability to water and cryoprotectants was then determined based on changes in the volumes of the oocytes in a hypertonic sucrose solution and various cryoprotectant solutions at 25 C. The permeability to water of the aquaporin-3 cRNA-injected oocytes was three times higher than that of intact and water-injected oocytes. The permeability of the aquaporin-3 cRNA-injected oocytes to ethylene glycol, glycerol, propylene glycol, and DMSO was also 2-4 times higher than that of intact oocytes. Thus, the permeability of immature zebrafish oocytes to water and cryoprotectants was enhanced by exogenous expression of aquaporin-3. Cryopreservation of teleost oocytes may be realized through a further increase in permeability.     

调控动物生殖功能的lncRNAs研究进展

长链非编码RNA(long noncoding RNA,lncRNA)是一类转录本长度大于200个核苷酸(nt)的非编码RNA,其可被转录但通常没有编码潜能,不能翻译成为蛋白质,故早期被认为是基因组转录过程中形成的副产物.近年在生殖领域随着二代测序技术的发展,已发现lncRNA可在转录、转录后修饰、翻译、翻译后修饰及表...     

饲粮中添加β-羟基-β-甲基丁酸对母猪繁殖性能及免疫机能的影响

本试验旨在研究妊娠后期及哺乳期母猪饲粮中添加β-羟基-β-甲基丁酸(HMB)对母猪繁殖性能及免疫机能的影响。试验选取24头分娩日期相近的3~6胎妊娠后期母猪(长×大),随机分成2组,每组12个重复。对照组饲喂基础日粮,试验组在基础日粮中添加0.2%HMB。试验期为母猪妊娠84天到仔猪21日龄断奶。结果表明:与对照组相比,试验组仔猪初生窝重和初生均重分别提高10.70%和3.95%,7日龄窝重和均重分别提高6.12%和5.58%,21日龄断奶窝重和均重提高6.70%和5.45%,仔猪初生至21日龄断奶窝增重和每头日增重提高8.08%和7.55%(P0.05)。试验组母猪血清中IgG含量显著高于对照组(P0.01)。由此可见,母猪妊娠84天到哺乳期饲粮中添加0.2%的HMB,有提高仔猪初生重和哺乳期增重的趋势,显著增强母猪免疫机能。     

Reproductive response of fat‐tailed Barbarine ewes subjected to short‐term nutritional treatments including spineless cactus (Opuntia ficus‐indica f. inermis) cladodes

Reproductive outputs in fat‐tailed Barbarine sheep in central Tunisia are often low because of feed shortage and the low nutritive value of diets. Supplementation with conventional concentrates is economically unsuitable in central Tunisia, so more cost‐effective and sustainable alternative feeding strategies need to be developed. We tested effects of short‐term nutritional treatments including cactus cladodes during the induction of ‘male effect’ on fertility and prolificacy parameters (follicular growth, ovulatory response and early embryo losses). One hundred and twenty ewes were distributed in 4 equal groups balanced for live weight grazed natural pastures and were supplemented for 21 days, starting day 10 after introduction of rams, with cactus cladodes (CA), cactus cladodes and soybean meal (CAS), concentrate (CC) or only soybean meal (S). Nutritional treatment did not affect live weight in this experiment. Ewes receiving cactus had higher number of large pre‐ovulatory follicles (≥6 mm; 1.08 ± 0.05), between days 14 and 19 after introduction of rams, than females in the CC and S ewes (0.64 ± 0.06; p < 0.05). However, there were no differences in the onset of oestrous behaviour in response to ‘male effect’ or in the number of corpora lutea. Average ovulation rates were 1.42 ± 0.16 for CC, 1.47 ± 0.13 for CAS, 1.31 ± 0.15 for CA and 1.31 ± 0.13 for S groups respectively. Finally, reproductive wastages at day 35 after mating were not different between groups being 0.33 ± 0.19 for CC, 0.60 ± 0.17 for CAS, 0.43 ± 0.16 for CA and 0.31 ± 0.15 for S groups respectively. It is concluded that Barbarine ewes fed nutritional treatments including cactus performed similarly to those receiving diets including conventional concentrate feeds.     

一氧化氮对动物生殖机能的影响

一氧化氮(NO)是动物体内发挥多种生物学效应的自由基,是一种重要的生物信使,它几乎对哺乳动物的各个系统都会产生一定的作用。近几年的研究结果也表明,NO对动物生殖机能的许多方面都有很大的影响。文章着重介绍了NO对动物排卵前LH峰、排卵、胚胎附植和发育、妊娠、分娩以及NO对动物精子获能、精子活力、阴茎勃起等生殖过程的影响。     

封面封底

线粒体是一种广泛存在于真核细胞并为机体提供能量的细胞器,其自噬对于清除功能障碍的线粒体,维持线粒体的动态平衡和细胞稳态具有重要意义。近年来,线粒体自噬对雌性动物生殖的影响愈发受到关注。本文在线粒体自噬生理功能基础上,从雌性动物主要生殖器官与组织、卵母细胞、颗粒细胞及胚胎等方面综述了线粒体自噬对雌性动物生殖功能的影响。     

Effects of Dietary Contamination by Zearalenone and Its Metabolites on Serum Anti‐Müllerian Hormone: Impact on the Reproductive Performance of Breeding Cows

We investigated the effects of in vivo exposure to low zearalenone levels on the anti‐Müllerian hormone endocrine levels and the reproductive performance of cattle. Urine and blood samples and reproductive records were collected from two Japanese Black breeding female cattle herds with dietary zearalenone contamination below the threshold levels (<1 ppm) at 30 days after calving. Urinary zearalenone, α‐zearalenol and β‐zearalenol concentrations were measured by chromatography‐tandem mass spectrometry, and serum anti‐Müllerian hormone concentrations were determined along with serum biochemical parameters. Urinary concentrations of α‐zearalenol were significantly higher (p < 0.05) in cattle in Herd 1 than in cattle in Herd 2, reflecting the different amounts of zearalenone in the diet of the two herds. Although the number of 5‐mm and 10‐mm follicles of the herds and their fertility after artificial insemination were similar, the serum anti‐Müllerian hormone concentrations in herds 1 and 2 were 438.9 ± 48.6 pg/ml and 618.9 ± 80.0 pg/ml, respectively, with a trend towards a significant difference (p = 0.053), which may indicate differences in the antral follicle populations between herds. Thus, zearalenone intake from dietary feed, even when below the threshold zearalenone contamination level permitted in Japan, may affect the ovarian antral follicle populations, but not the fertility, of post‐partum cows.     

Localization of Insulin‐Like Growth Factor‐I (IGF‐I) and IGF‐I Receptor (IGF‐IR) in Equine Testes

The insulin‐like growth factor‐I (IGF‐I) is a key regulator of reproductive functions. IGF‐I actions are primarily mediated by IGF‐IR. The main objective of this research was to evaluate the presence of IGF‐I and IGF‐I Receptor (IGF‐IR) in stallion testicular tissue. The hypotheses of this study were (i) IGF‐I and IGF‐IR are present in stallion testicular cells including Leydig, Sertoli, and developing germ cells, and (ii) the immunolabelling of IGF‐I and IGF‐IR varies with age. Testicular tissues from groups of 4 stallions in different developmental ages were used. Rabbit anti‐human polyclonal antibodies against IGF‐I and IGF‐IR were used as primary antibodies for immunohistochemistry and Western blot. At the pre‐pubertal and pubertal stages, IGF‐I immunolabelling was present in spermatogonia and Leydig cells. At post‐pubertal, adult and aged stages, immunolabelling of IGF‐I was observed in spermatogenic cells (spermatogonia, spermatocyte, spermatid, and spermatozoa) and Leydig cells. Immunolabelling of IGF‐IR was observed in spermatogonia and Leydig cells at the pre‐pubertal stage. The immunolabelling becomes stronger as the age of animals advance through the post‐pubertal stage. Strong immunolabelling of IGF‐IR was observed in spermatogonia and Leydig cells at post‐puberty, adult and aged stallions; and faint labelling was seen in spermatocytes at these ages. Immunolabelling of IGF‐I and IGF‐IR was not observed in Sertoli cells. In conclusion, IGF‐I is localized in equine spermatogenic and Leydig cells, and IGF‐IR is present in spermatogonia, spermatocytes and Leydig cells, suggesting that the IGF‐I may be involved in equine spermatogenesis and Leydig cell function as a paracrine/autocrine factor.     

A 6‐bp Deletion Variant in a Novel Canine Glutathione‐S‐Transferase Gene (GSTT5) Leads to Loss of Enzyme Function

Objectives

Glutathione‐S‐transferases (GSTs) detoxify reactive xenobiotics, and defective GST gene polymorphisms increase cancer risk in humans. A low activity GST‐theta variant was previously found in research beagles. The purpose of our study was to determine the molecular basis for this phenotype and its allele frequency in pet dogs.

Methods

Banked livers from 45 dogs of various breeds were screened for low GST‐theta activity by the substrate 1,2‐dichloro‐4‐nitrobenzene (DCNB), and were genotyped for variants in a novel canine GST gene, GSTT5. Whole‐genome sequences from 266 dogs were genotyped at one discovered variant GSTT5 locus.

Results

Canine livers ranged 190‐fold in GST‐theta activities, and a GSTT5 exon coding variant 385_390delGACCAG (Asp129_Gln130del) was significantly associated with low activity (P < 0.0001) and a marked decrease in hepatic protein expression (P = 0.0026). Recombinant expression of variant GSTT5 led to a 92% decrease in V_max for DCNB (P = 0.0095). The minor allele frequency (MAF) for 385_390delGACCAG was 0.144 in 45 dog livers, but was significantly higher in beagles (0.444) versus nonbeagles (0.007; P = 0.0004). The homozygous genotype was significantly over‐represented in Pembroke Welsh corgis (P < 0.0001) based on available whole‐genome sequence data.

Conclusions

An Asp129_Gln130del variant in canine GSTT5 is responsible for marked loss of GST‐theta enzyme activity. This variant is significantly over‐represented in purpose‐bred laboratory beagles and in Pembroke Welsh corgis. Additional work will determine the prevalence of this variant among other purebred dogs, and will establish the substrate range of this polymorphic canine enzyme with respect to common environmental carcinogens.     

饲粮蛋氨酸水平对繁殖期蓝狐繁殖性能的影响

本试验旨在研究饲粮中蛋氨酸水平对繁殖期蓝狐繁殖性能的影响。选择健康雌性初产蓝狐150只,随机分为5组,每组30个重复,每个重复1只蓝狐,均为单笼饲养。Ⅰ、Ⅱ、Ⅲ、Ⅳ和Ⅴ组分别饲喂在基础饲粮中添加0.20%、0.45%、0.70%、0.95%和1.20%蛋氨酸的试验饲粮。试验期为2014年2月10日至2014年5月31日。结果表明:Ⅱ、Ⅲ和Ⅳ组蓝狐最先开始发情,也最早结束,Ⅰ和Ⅴ组蓝狐发情时间稍晚。受配率、受胎率均以Ⅱ组最高,Ⅱ组受配率分别比Ⅰ、Ⅲ、Ⅳ和Ⅴ组高出17%、3%、10%及14%。窝产仔数和初生个体重各组间差异不显著(P0.05);初生窝重随着饲粮蛋氨酸水平的升高呈现先升高后降低的趋势,并且Ⅰ和Ⅱ组显著高于Ⅴ组(P0.05);Ⅱ组初生成活率极显著高于Ⅴ组(P0.01)。由此得出,饲粮中添加0.45%蛋氨酸,即饲粮中蛋氨酸水平为0.98%时,可满足繁殖期蓝狐对蛋氨酸的需要。