Ultrastructural changes associated with carbon tetrachloride hepatotoxicity in channel catfish, Ictalurus punctatus Rafinesque

Abstract. Ultrastructural changes occurring in juvenile channel catfish, Ictalurus punctatus Ratinesque, hepatocytes were examined following exposure to carbon tetrachloride (CCl₄). Juvenile catfish were intraperitoneally injected with a sublethal dose of undiluted CCl₄and controls, used for comparisons, received physiological saline. The rough endoplasmic rcticulum (RER) was slightly dilated by 1 h and was significantly dilated by 4 and 24 h post-CCl₄-exposure. At 4 h, the RER became degranulated, mitochondrial eristae indistinct with electron dense flocculant matrieal deposits, and nuclear membranes indented. Autophagic vacuoles were present in some hepatocytes. Within 48 h post-exposure, the hepatocytes were indistinguishable from control hepatocytes. CCl₄ can induce reversible or irreversible cell damage which appears dose dependent.     

Cloning and seasonal changes in ovarian expression of a TSH receptor in the channel catfish, Ictalurus punctatus

The cDNA encoding the thyroid stimulating hormone (TSH) receptor was isolated from the ventral aorta (thyroid follicles) and in both gonads of the channel catfish. The seasonal changes in ovarian expression of this receptor seem to be correlated to reproduction. TSH, but not LH or FSH, activated the recombinant TSH receptor expressed in COS cells. These results indicate that the isolated cDNA encodes a functional and highly specific TSH receptor. The seasonally regulated expression in the catfish ovary suggests a role for TSH in gametegenesis.     

黄颡鱼促黄体激素受体基因克隆及在雌鱼繁殖周期中的表达

通过简并引物扩增及SmartTM Race技术,首次克隆了黄颡鱼促黄体激素受体(LHR)基因cDNA全长序列.该基因全长2 524 bp,编码701aa,含有属于糖蛋白激素受体(GpHR)家族的典型跨膜螺旋结构区域(TM helix).9个富含亮氨酸的重复性序列(LRRs);4个潜在的N-端糖基化位点:29NFTC,82 NVSR,203 NGSR,548NLTV;24个Ser,6个Thr和5个Tyr磷酸化位点.同时400S为潜在的PKC位点.通过RT-PCR分析组织表达,卵巢繁殖周期中卵巢和脑的表达水平并结合血浆中E2含量的变化,发现LHR在卵巢中大量表达,其次是脑、肾脏、心脏、肝脏和肠存在少量或微量的表达;在卵巢繁殖周期中,卵巢LHR表达从Ⅲ期-V期处于较高水平,V期达到最高峰,随后下降到最低值;而脑的表达高峰出现在Ⅳ期,与血浆中E2变化相一致.推测卵巢和脑中LHR基因参与卵黄的生成,调控卵母细胞的成熟和排卵.     

Insulin and IGF-I receptors and tyrosine kinase activity in carp ovaries: changes with reproductive cycle

Insulin and insulin-like growth factor I (IGF-I) receptors from carp ovaries were semipurified with wheat germ agglutinin at different moments of the reproductive cycle and their binding characteristics and tyrosine kinase activity were studied. Specific receptors for insulin and IGF-I were found. IGF-I receptors presented higher binding (23.8 ± 1.5%), number of receptors (965 ± 20fm/mg) and affinity (K_D 0.24 ± 0.03nM) than those shown for insulin receptors (4.1 ± 1%, 530 ± 85fm/mg and 0.85 ± 0.1nM, respectively). Insulin and IGF-I receptors have a tyrosine kinase activity which is not different from that found in muscle of the same species. Seasonal changes were found in binding, with maximum values for insulin and IGF-I reached at the end of pre-spawning period (June). However, while IGF-I binding was observed in all stages, insulin binding decreased in autumn and disappeared in winter, which suggests a different role for the two peptides in ovarian physiology.     

Chronic exposure of channel catfish, Ictalurus punctatus, to ammonia: Effects on growth and survival

The growth of juvenile channel catfish (Ictalurus punctatus) was reduced in a linear manner during a 31 day growth trial when exposed to concentrations of ammcnia ranging from 48 to 989 μg/l NH₃N (0.31 to 5.71 mg/l NH⁺₄N). On a wet weight basis, growth was reduced by 50% at 517 μg/l NH₃N and the no growth occured at 967 μg/l. The no growth level was 60% of 96-h LC50 value. Mortality was increased significantly at 989 μg/l NH₃N and above. The sublethal effects of ammonia may depend, in part, on the concentrations of NH₄⁺ and and Na⁺ in solution.     

Changes in tissue lipid levels in the freshwater catfishClarias batrachus associated with the reproductive cycle

The levels of free fatty acid (FFA), monoglyceride (MG), diglyceride (DG), triglyceride (TG), phospholipid (PL), free cholesterol (CF) and esterified cholesterol (CE) were measured in the liver, plasma, ovary, abdominal fat and muscle during different phases of the annual reproductive cycle in femaleClarias batrachus. During the preparatory phase, hepatic lipogenic activity predominated over mobilization and consumption. In the prespawning season, an increased hepatic lipogenic activity was maintained, but lipids were transferred from the liver to the ovary. In the spawning phase, the diminished food intake, and enhanced caloric demand for spawning behaviour and activity limited hepatic lipogenic activity, and TG lypolysis was increased as was the production of more FFA. Maximum accumulation of vitellogenin, as reflected by maximum rise in ovarian PL titre was characteristic of this phase. Marked reductions in ovarian lipid occurred during the postspawning phase. In the resting phase, there was a recovery of lipogenic activity, but PL synthesis was still inhibited. In contrast to other investigated teleosts, there were extremely high level of FFA in liver, plasma, ovary and muscle throughout the annual reproductive cycle inC. batrachus. FFA appears to be the main lipid metabolite which had a very high turnover. As evidenced by the high TG content, abdominal fat seems to be the main fat depot, not the liver and muscle.Correspondent author     

黄颡鱼促卵泡激素受体基因的克隆及其在雌鱼生殖周期中的表达

为研究黄颡鱼促卵泡激素受体(FSHR)基因的克隆以及该基因在卵巢发育周期中的表达情况,实验首先采用SmartTMRace技术,获得了黄颡鱼促卵泡激素受体基因cDNA全长序列,该基因全长2 340 bp,编码661aa,具有G蛋白偶联受体超家族(G protein-coupled receptor,GPCR)的7个跨膜螺旋区(transmembrane domain)。经分析发现,得到的FSHR基因序列具有9个富含亮氨酸的重复性序列(LRRs);5个潜在的N-端糖基化位点;蛋白激酶C(proteinkinase C,PKC)磷酸化位点(403Y)位于第一个包外环,C-末端(C-terminal domain)G蛋白偶联区域,存在3个蛋白激酶C磷酸化位点(634S,643S,632T)。这些区域可能与FSHR的功能有密切关系。RT-PCR发现,FSHR基因在卵巢和脑中大量表达,在其他组织中均存在少量或微量的表达;在繁殖周期中,卵巢FSHR基因表达从Ⅲ期～Ⅳ期处于较高水平,在Ⅵ期时下降。研究推测,卵巢中FSHR基因参与黄颡鱼卵母细胞成熟及卵黄蛋白的积累过程。     

Lesions associated with natural and experimental infections of Aeromonas hydrophila in channel catfish, Ictalurus punctatus (Rafinesque)

Abstract. Histologic differences were observed in channel catfish, Ictalurus punctatus (Rafinesque), with naturally occurring cutaneous (bacteria isolated only from lesions of skin and superficial muscle) and systemic Aeromonas hydrophila infections. Systemic infections were characterized by diffuse necrosis in several internal organs and the presence of melanin-containing macrophages in the blood. Fish with only cutaneous infections had several types of concealed lesions including increased amounts of lipofuscin and haemosiderin in the liver and spleen; however, most visceral organs were not necrotic. The average condition factor of fish with cutaneous infections was lower than for fish with systemic infections. Early histologic lesions in channel catfish experimentally infected by immersion in a suspension of A. hydrophila were similar to lesions observed in naturally occurring systemic infections and to lesions previously reported in channel catfish injected intraperitoneally with A. hydrophila . In experimentally infected fish, all lesions healed in fish that did not die, and prolonged infections limited to skin and muscle did not occur.     

Cloning and sequencing of FSH-β and LH-β subunits and changes in their expression during the reproductive cycle in the three-spined stickleback, Gasterosteus aculeatus

To achieve a better understanding of the role of gonadotropins (GTHs) in the stickleback we have cloned the full-length cDNAs of the β-subunits of follicle stimulating hormone (FSH) and luteinizing hormone (LH), and analysed the expression during the seasonal cycle. In females, LH-β levels were low during winter and early spring, increased to a peak in late May and declined to low levels again in July. FSH-β expression peaked earlier, in January and declined spring. In males, LH-β expression peaked in May. During June–September, when spermatogenesis occurs, LH-β levels were very low. FSH-β expression peaked earlier, in January, and reached the lowest levels in July. Thus, when spermatogenesis starts, the expression of both GTH-β mRNAs display their lowest levels.     

Brain steroid contents in the catfish Heteropneustes fossilis: sex and gonad stage-specific changes

Neurosteroids are those which are synthesized in the central nervous system independently of supply by peripheral endocrine glands. In the present study, brain contents of the steroid hormones, estradiol-17β (E(2)), testosterone (T), corticosteroids, and progestins were investigated in both male and female catfish Heteropneustes fossilis in prespawning (vitellogenic) and spawning (post-vitellogenic) phases using ELISA or HPLC. The data show that the measured steroid hormones showed both stage-specific and sex-related variations. Brain E(2) was significantly higher in males in the prespawning phase and in females in the spawning phase. Testosterone was significantly higher in males in comparison with females in the prespawning phase. Cortisol was significantly higher in the prespawning and spawning phases in males than in females. Corticosterone level was low in the brain. 21-deoxycortisol and deoxycorticosterone were significantly higher in the prespawning phase than in the spawning phase. Male brain recorded the highest concentration of deoxycorticosterone. Progesterone (P(4)) was high in the prespawning phase and low in the spawning phase in both sexes. Levels of 17-hydroxy-4-pregnene-3,20-dione and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-DP) and the metabolites of P(4) were the highest in females in the prespawning phase. The stage-specific and sexual differences in the content of the steroids suggest their biosynthesis in the brain, which may have implications in brain functions, in addition to reproductive regulation.     

Plasma sex steroid hormonal profile and gonad histology during the annual reproductive cycle of river catfish Hemibagrus nemurus (Valenciennes, 1840) in captivity

Plasma sex steroid hormonal profile and gonad histology were correlated to study the annual reproductive cycle of Hemibagrus nemurus. Hormones were measured by Enzyme Linked Immunosorbent Assay. Gonad tissues were observed by using light microscopy. The highest testosterone (T) value for male was observed in November and that of female was in October. 11-ketotestosterone (11-KT) and 17β-estradiol (E2) levels were highest in June and November, respectively. Hormonal profiles of T, 11-KT and E2 showed several peaks which indicated a non-seasonal pattern. There were significant differences (p < 0.05) in the monthly levels of T, 11-KT and E2. Gonadosomatic index of H. nemurus ranged from 1.14 ± 0.02 % to 7.06 ± 1.40 %, and high gonadosomatic indices were recorded in May, August and November. Gonad histology revealed that spermatozoa were always present in the testes which implied continuous spermatogenesis and asynchronous ovarian development pattern was observed in the ovaries. The annual reproductive cycle of H. nemurus did not show a seasonal pattern and this indicate that H. nemurus is a non-seasonal breeder with several spawning cycles and can be referred to as indeterminate batch spawner. The major significances of this study are annual sex steroid hormonal profile and asynchronous ovarian development of H. nemurus. This information will contribute to our knowledge of reproductive biology of H. nemurus.     

斑点叉尾鮰TICAM在细菌和病毒感染后的基因表达特征

采用实时定量RT-PCR方法研究斑点叉尾在不同病原(链球菌、嗜水气单胞菌、迟钝爱德华菌、斑点叉尾病毒)感染后TICAM基因在mRNA水平的组织和时空表达特征。感染嗜水气单胞菌可引起TICAM基因在肝脏和脾脏中的上调表达,在注射后24h和12h后上调最大分别为2.3倍和1.9倍;而在头肾和后肠中的表达则下调到感染后48h的0.15倍和24h的0.53倍;感染链球菌后则导致在肝脏、脾脏、后肠和头肾中TICAM基因表达的强烈上调,最大上调幅度为感染后7d肝脏中表达提高了23倍,其次,在脾脏和头肾中基因表达最大可上调到感染前的10倍左右;在感染迟钝爱德华菌后,TICAM基因在肝脏、脾脏、后肠和头肾中表达上调。其中,感染后7d脾脏中的表达提高到感染前的23.1倍。而感染叉尾病毒后,TICAM基因在肝脏、头肾和后肠的表达上调但幅度不大,基因表达在4种组织内表达变化量在1.5～3.7倍范围内波动,而在脾脏中TICAM表达下调,在感染24h后达到最低为感染前的0.13倍。以上实验结果显示,斑点叉尾TICAM基因表达变化与病原感染密切相关,暗示TICAM基因在斑点叉尾天然免疫中起重要作用。     

Metabolic enzyme activities in larvae of the African catfish,Clarias gariepinus: changes in relation to age and nutrition

The influence of ontogeny and nutrition on metabolic enzyme activities in larvae of the African catfish, Clarias gariepinus, was studied. After start of exogenous feeding, the larvae were reared for 10 days under three different nutritional conditions: Artemia nauplii, a dry starter diet, and starvation. The live feed gave the best growth (96 mg within 10 days) whereas the dry diet resulted in low growth (33 mg). This growth difference was reflected in larval RNA and DNA concentrations, but not in the levels of soluble protein. Enzymes representing the following aspects of metabolism have been analysed: NADPH generation (G6PDH, ME), glycolysis (PFK, PK), gluconeogenesis (FDPase), amino acid catabolism (GOT, GPT) and oxidative catabolism (CS). All enzymes were present from the start of exogenous feeding onwards, but their maximum specific activities displayed different developmental patterns. In catfish larvae fed on Artemia, G6PDH and ME activities steadily increased with age and weight of the larvae. CS levels remained, after an immediate enhancement upon onset of exogenous feeding, on a rather stable plateau. The amino acid-degrading enzymes GOT and GPT showed maximum levels at days 3–5 of feeding or at a body weight of 10–20 mg, but decreased thereafter. Activities of PFK, PK and FDPase showed low initial levels, and increased significantly with age and size. Based on the ontogenetic patterns of metabolic enzymes, in C. gariepinus larvae an early and a late developmental phase can be distinguished. During the early phase, the glycolytic and gluconeogenetic capacities are low, whereas they are enforced during the later phase. The oxidative capacity is high both during the early and the late phase. The metabolic changes in catfish development coincide with other major ontogenetic events, e.g., alterations of muscle organization, gill morphology, respiration and stomach structure and function. Rearing catfish larvae on a dry diet instead of Artemia partly altered the developmental pattern described: The ontogenetic elevation of CS, PFK and FDPase was delayed and the early peak in GOT and GPT activities was not realized. Particularly during the early developmental phase, the enzyme behaviour of the larvae fed on dry food was similar to that of starved larvae.Abbreviations CS citrate synthase - FDPase fructose-1,6-diphosphatase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase - G6PDH glucose-6-phosphate dehydrogenase - ME malic enzyme - PFK phosphofructokinase - PK pyruvate kinase     

Proteases of the Aeromonas hydrophila complex: identification, characterization and relation to virulence in channel catfish, Ictalurus punctatus (Rafinesque)

Abstract. Traditional biochemieal techniques and a stain to detect proteases in polyacrylamide gels were used to identify and partially characterize three proteases, P1, P2 and P3, produced by Aeromonas hydrophila strain Ah 22. P1 was found to be a heat-labile serine protease with an optimum pH of 7·5, while P2 is a heat-stable metalloprotease with an optimum pH of 8·0, and P3 is a moderately heat-stable metalloprotease with peak activity beween pH 7 and 11. A comparison of 17 other strains of the A. hydrophila complex indicated that four produced P1, P2 and P3. Two strains produced just P1 and P3; one produced only P3; six produced two different serine proteases, P2a and P2b; and two produced a number of uncharacterized proteases. Virulence studies in age-0 + channel catfish indicated no correlation between either quantitative or qualitative protease production and virulence.     

Survival of zebrafish, Brachydanio rerio (Hamilton-Buchanan), embryo after immersion in methanol and exposure to ultrasound with implications to cryopreservation

This study examined the viability of embryos after immersion in highly concentrated methanol solutions (40–60%) and exposing embryos to ultrasound to enhance efficient transport of the cryoprotectant. The exposure to ultrasound, methanol concentrations, duration of treatment and the stages of embryonic development was found to have measurable effects on embryo viability. The effect of ultrasound was more evident at high voltage (>440 V) settings and at early developmental stages (30 and 60% epiboly stage). Older embryos were more resistant to cryoprotectant toxicity and ultrasound‐induced mortality. The high concentration of methanol (60%) was more toxic to embryos than the low concentration (40%). When methanol treatment and ultrasound were applied simultaneously the optimum concentration was found to be 45% methanol (45% survival; P<0.05) in a 3 min treatment. Although there was no significant difference between the 2 and 3 min treatments, embryos treated for 4 min had a significantly lower survival rate (P<0.05). These findings provide initial results to select the developmental stage of the embryo, the concentration of methanol for the preparation of a vitrification solution and duration of ultrasound treatment for cryopreservation. Furthermore, it indicates the potential use of ultrasound to enhance the transport of methanol intracellularly with minimum mortality of the developing embryos.     

Pathological changes of the endocrine pancreas in dogs and cats in comparison with clinical data]

In a retrospective study concerning the functional morphology of the endocrine pancreas the files of the Institute for Veterinary Pathology, University of Munich, of the last 12 years were reviewed. Cases of diabetes mellitus as well as developmental disorders and tumors of the endocrine pancreas were examined by routine histological and immunohistological methods. Cases are discussed together with clinical data and the relevant literature. The forms of diabetes mellitus were categorized according to the classification used in human pathology.     

Modeling industry-wide sediment oxygen demand and estimation of the contribution of sediment to total respiration in commercial channel catfish ponds

Data collected from 45 commercial channel catfish, Ictalurus punctatus, ponds were used to develop empirical models predicting sediment oxygen demand (SOD). Seven acceptable models were combined with a Monte-Carlo sampling distribution to predict industry-wide sediment oxygen demand (SOD_i). The SOD_i values obtained from the best equation were used in simulations to assess the effect of diurnally varying water column dissolved oxygen (DO) concentrations on SOD and the effect of pond water depth on the contribution of SOD to overall pond respiration. Estimated SOD_i ranged from 62 to 962 mg m⁻² h⁻¹, with a mean of 478 mg m⁻² h⁻¹. There was a 95% probability of mean SOD_i being ≥700 mg m⁻² h⁻¹. The effects of diurnal variation in DO concentration in the water column on expression of SOD was modeled by combining maximum SOD_i, an empirical relationship between DO and SOD, and simulated pond DO concentrations. At DO concentrations >15 mg l⁻¹, diel SOD in catfish ponds exceeded 20 g O₂ m⁻² day⁻¹. But when average diel DO was <4 mg l⁻¹ and the range of DO concentration was 6–8 mg l⁻¹, SOD decreased to 13 g O₂ m⁻² day⁻¹ because DO availability limited the full expression of potential SOD. Respiration totals for sediment (average SOD_i), plankton, and fish respiration were calculated for pond water depths ranging from 0.25 to 4 m. Although whole-pond respiration increases as pond depth increases, the proportion of total respiration represented by sediment decreased from 48 to 10% by increasing water depth over this range. The results of these studies show that SOD is a major component of total pond respiration and that certain management practices can affect the impact of SOD on pond oxygen budgets. Mixing ponds during daylight hours, either mechanically or by orienting ponds for maximum wind fetch, will increase oxygen supply to sediments, thereby allowing maximum expression of SOD and maximum mineralization of sediment organic matter. Given a mixed condition caused by wind or other artificial means, the construction of deeper ponds increases the total mass of DO available for all respiration, causing nighttime DO concentrations to decline at a slower rate, reducing the need for supplemental aeration. Because a pond’s water volume decreases over time from sediment accumulation, annual aeration costs will increase with pond age. Constructing ponds with greater initial depth will therefore reduce long-term cost of aeration, allow more flexible management of pond water budget, and reduce the long-term expense associated with pond reconstruction.