Immunofluorescence screening of Renibacterium salmoninarum in the tissues and eggs of farmed chinook salmon spawners

A simple and rapid on site-test method was used to screen farmed chinook salmon (Oncorhynchus tschawytscha) broodstock for bacterial kidney disease (BKD) at the time of spawning. Kidney tissue and ovarian fluid smears were examined, using the indirect fluorescent antibody technique (IFAT). To check the validity of our field screening procedure, intra-ovum fluid, taken from random samples of 300 surface-sterile, fertilized eggs, was examined in the laboratory for the presence or absence of Renibacterium salmoninarum, using the IFAT. Prior to sampling, eggs were incubated at 15°C for 40 days in enrichment KDM-2 broth. R. salmoninarum cells were found in one out of 10 samples of pooled contents of eggs from fish showing no R. salmoninarum in the kidney tissue (these fish were assumed to have R. salmoninarum-free ovarian fluid); three out of 10 pooled samples from fish in which R. salmoninarum was detected in the kidney but not the ovarian fluid; all of the 10 pooled samples from fish with R. salmoninarum in both the kidney tissue and the ovarian fluid. The number of R. salmoninarum in the pooled egg contents was low (2–23 per 100 fields). Using the IFAT technique it was not possible to determine whether the BKD-bacteria in the eggs were alive or dead. Methods of BKD treatment and screening are discussed as is the validity of using these techniques to prevent vertical transmission of BKD in farmed salmon.     

Morphological, physiological and biochemical parameters characterizing the over-ripening of rainbow trout eggs

In rainbow trout (Oncorhynchus mykiss) freshly ovulated eggs and over-ripened eggs which had been retained in the coelomic cavity for 7, 14 and 21 days were investigated in aspects of morphology, physiology and biochemistry. Egg viability was significantly reduced from 85.9±16.4% in freshly ovulated eggs to 25.1±21.9% in over-ripened eggs which had been retained in the coelomic cavity for 21 days. Further during over-ripening in the ovarian fluid the pH significantly decreased, while the levels of proteins, of esterified and non esterified fatty acids and the activities of aspartate aminotransferase and acid phosphatase significantly increased. Also egg parameters changed: the wet weight of the unhardened eggs increased, the weight increase during hardening and the levels of esterified and of non esterified fatty acids significantly decreased. In freshly ovulated eggs the yolk consisted of a homogenous mass and the perivitelline space was small, but in over-ripened eggs the yolk was non homogenous with numerous vesicular inclusions and the perivitelline space was enlarged. When freshly ovulated eggs were incubated in water the cortical reaction was detectable within 5 min, in over-ripened eggs hardly no extrusion of cortical vesicles was visible and the width of the perivitelline space was very irregular.For the investigated freshly ovulated and over-ripened samples the egg viability significantly correlated with ovarian fluid parameters (pH, protein, non esterified fatty acids, esterified fatty acids, aspartate aminotransferase, acid phosphatase) and egg parameters (weight increase during hardening, weight of the hardened eggs).     

Studies on cryopreservation of eggs from rainbow trout (Salmo gairdneri) and coho salmon (Oncorhynchus kisutch)

The effect of pre-freezing treatments as well as freezing of inseminated, not water-activated eggs from rainbow trout Salmo gairdneri, and coho salmon, Oncorhynchus kisutch, was investigated in relation to survival and further development.Effects above freezing temperatures included: the temperature at insemination, viability of inseminated and unactivated eggs after storage, suitability of an incubation medium and the tolerance of eggs to various levels of the cryoprotectant dimethylsulfoxide (DMSO). Freezing experiments included: investigating the action of DMSO (0, 1, 2 mole) and the tolerance of coho eggs to temperatures between ?4.6 to ?30°C. Insemination temperatures between 0.5°C and 9.8°C (coho eggs) as well as incubation in an artifical medium (1-0°C) for 80 min (rainbow trout eggs) and 170 min (coho eggs) did not influence subsequent fertility. Storage of inseminated and unactivated rainbow trout eggs for 135 min and beyond reduced egg fertility. DMSO at 2 and 4 mole was detrimental to coho eggs (1-0°C). One mole DMSO had no (coho) or reduced (rainbow trout) influence on egg fertility when it was added gradually.In the presence of 1 mole DMSO most eggs remained unfrozen (67–89%) when kept for 10 min in frozen artificial medium (?4.6%) and 27–32% subsequently reached the eyed stage (control = 100). Further cooling (0.3°C/min) to ?10°C was still tolerated (62% unfrozen, 22% eyed eggs) but not to ?20°C (6% unfrozen, no development) and ?30°C (no survival). Use of 2 mole DMSO did not improve the results.     

Fertility of rainbow trout (Salmo gairdneri) gametes: Gamete viability in artificial media

A series of experiments was performed to test the effect of potassium ions (K⁺) on the storage of rainbow trout (Salmo gairdneri) sperm cells and eggs. Storage of eggs for up to 10 min in saline solutions, extender with K⁺ concentrations of ≤3.4 mM and coelomic fluid did not affect fertility. Fish Extender #6 had a detrimental effect on eggs within 15 s of application and reduced fertility to near zero within 10 min. Variation in the K⁺ concentration indicated that high (≥6.8 mM) concentrations decreased egg fertility while low (≤3.4 mM) concentrations had no effect on fertility. Insemination of eggs before the addition of Fish Extender #6 allowed storage for 14 h. Semen diluted (1:10) in coelomic fluid maintained fertility for 45 s, whereas semen diluted (1:1) in coelomic fluid maintained fertility for 60 min. Fish Extender #6 at K⁺ concentrations between 0 mM and 68 mM had no effect on fertility of semen diluted 1:2.5 after 1 h of storage.     

Comparison of iodine and glutaraldehyde as surface disinfectants for red porgy (Pagrus pagrus) and white sea bream (Diplodus sargus sargus) eggs

The efficacy of iodine and glutaraldehyde as fish egg surface disinfectants were assessed in red porgy (Pagrus pagrus) and white sea bream (Diplodus sargus sargus) eggs, two species of interest for Mediterranean aquaculture. Iodine was effective in reducing the bacterial load of the 1-day-old eggs when applied at 50 mg L⁻¹ for 5 min. The same concentration did not cause any significant change in hatching success or survival of the larvae for the first 5 days. Glutaraldehyde failed to reduce the bacterial load of the fish eggs at concentrations that were safe for the eggs (100 mg L⁻¹ for 5 min), as it had a significant effect in preventing hatching of the developed embryo. Disinfecting 0-day-old eggs with iodine resulted in a significant reduction of hatching percentage, while larval survival thereafter was unaffected. The results of the present study suggest that iodine may be an appropriate egg disinfectant for both red porgy and white sea bream.     

Changes in thyroid hormone levels in eggs and larvae and in iodide uptake by eggs of coho and chinook salmon,Oncorhynchus kisutsch andO. tschawytscha

Developmental profiles of thyroxin (T₄), triiodothyronine (T₃) and radioactive iodide uptake were established for eggs and T₄ and T₃ profiles were established for larvae (whole-body, yolk-only and body-only) of coho and chinook salmon. T₄ and T₃ were consistently present in all samples. In eggs, hormone levels remained fairly constant in all cohorst for at least the first three weeks of incubation, but then fluctuated in both directions in some sample groups. Large increases in T₄ (from 9 ng/g to 245 ng/g) were seen in 1985 chinook eggs 28 days after fertilization. Radioactive iodide uptake (which was used as a possible indicator of thyroxinogenesis) increased at least 10-fold in both 1986 coho and chinook eggs from 23–30 days after fertilization. T₄ (62 ng/g) and T₃ (393 ng/g) were found in the bodies of 28-day-old 1986 chinook embryos. In whole larvae, hormone levels varied depending upon the cohort studied. In general, initial body-only concentrations of both T₄ and T₃ decreased as body weight increased, but before yolksac resorption was completed, both thyroid hormone content and concentration increased (except for chinook T₃). T₄ and T₃ content in larval yolk stayed constant as yolksac size decreased, resulting in increased thyroid hormone concentration in the yolksac. All of these data suggest that the initial source of thyroid hormones in coho and chinook salmon eggs is maternal, but that by approximately 3–4 weeks after fertilization, the developing embryos begin to produce their own thyroid hormones. After hatching, increases in tissue T₄ and T₃ concentration coupled with constant T₄ and T₃ content in diminishing yolksacs suggest that larvae also produce their own thyroid hormones; yolksac content then may reflect both the original maternal hormones and the larva-producted hormones.     

Changes in fertility of rainbow trout eggs retained in coelom

ABSTRACT:    Effects of prolonged retention time of ovulated eggs in the parental coelom on fertilization success were studied in rainbow trout Oncorhynchus mykiss using cryopreserved sperm with a uniform fertilizing ability. Proportions of successful fertilization, eyed eggs and hatched alevins were examined at different time periods up to a retention time of 14 days beyond the ordinary stripping time, and were compared with eggs incubated in artificial coelomic fluids (ACF). Eggs that were retained longer in the coelom showed gradual decreases in all the proportions of successful fertilization, eyed eggs and hatched alevins. The progress of cleavage after fertilization slowed with prolonged retention times. Eggs incubated in ACF lost their fertilizing ability much sooner than those retained in the coelom. The hatching rate of eggs retained for 2 weeks in coelom was 36%, while it was 1% in those eggs incubated for 4 days in ACF. Thus, eggs retained in the coelom showed higher fertilization success than those incubated in ACF.     

Broken eggs as a cause of infertility of coho salmon gametes

Egg content originating from ruptured ova progressively reduced the success of fertilization in coho salmon (Oncorhynchus kisutch) eggs. The salmon egg differs in ionic composition from the ovarian fluid in exhibiting higher K⁺ and Mg²⁺ and lower Na⁺ concentrations. Normal ovarian fluid induced spermatozoan motility whereas ovarian fluid contaminated with egg content progressively lost the ability to induce motility. Loss of motility was related to decreasing $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ ratios in the ovarian fluid, indicating that reduced fertility may be caused by inactivation of sperm cells in the insemination medium. Removal of contaminated ovarian fluid by rinsing ova in isotonic media induced spermatozoan motility and restored fertility completely. The use of an isotonic NaHCO₃ solution for rinsing of ova is suggested as a routine procedure for artificial insemination in salmonids where the presence of broken eggs is suspected.     

Swelling of wolffish, Anarhichas lupus L., eggs and prevention of their adhesiveness

Wolffish, Anarhichas lupus L. has internal fertilization. The eggs are released into water before the beginning of cleavage and stick together in clutches. To develop methods for separating eggs for subsequent incubation, changes during egg swelling and egg shell hardening were observed. Uninseminated eggs did not swell in ovarian fluid, but a perivitelline space formed in sea water because of a partial cortical reaction. In inseminated eggs kept in ovarian fluid, a cortical reaction took place and swelling was caused by elevation of the yolk membrane. Shrinkage of the yolk and absorption of water were observed after inseminated eggs were placed in sea water. Eggs swelled in ovarian fluid diluted by less than 30% sea water showed abnormal cleavage during subsequent development. An effective method for preventing egg stickiness after fertilization was to distribute them in trays with stagnant sea water for at least 5-6 h. Milk was effective for loss of egg adhesiveness at 50% concentration, but some eggs cleaved abnormally and subsequent mortality was high. The influences of ovarian fluid and milk on initial egg development with respect to peculiarities of egg swelling are discussed.     

Fertilization rate,motility, lipid peroxidation and pH changes after chilled storage of rainbow trout (Oncorhynchus mykiss) eggs and spermatozoa by a RMPI medium

The aim of this study was to evaluate the effects of Roswell Park Memorial Institute (RPMI) 1,640 medium on chilled storage of eggs and spermatozoa of rainbow trout (Oncorhynchus mykiss). After 3 days of storage, eggs in RPMI 1,640 media (pH 8.2, 9 and 10), Cortland medium and coelomic fluid were inseminated with fresh spermatozoa (Experiment 1). Eggs in RPMI 1,640 medium at pH 8.2 shown the lowest thiobarbituric acid‐reactive substances (TBARS, 0.053 ± 0.003 nmol/ml) and pH changes (from 8.20 ± 0.01 to 8.18 ± 0.01), the highest fertilization rate (82 ± 3%). Undiluted and diluted spermatozoa at ratios of 1:2 and 1:9 with RPMI 1,640 media (pH 8.2, 9 and 10) and Cortland medium were inseminated with fresh eggs (Experiment 2). Spermatozoa in RPMI 1,640 medium at pH 9 (1:9) caused the lowest TBARS content (0.037 ± 0.002 nmol/ml) and pH changes (from 9.00 ± 0.01 to 8.98 ± 0.01), the highest fertilization rate (77 ± 2%) and motility parameters. Based on Experiments 1 and 2, eggs and spermatozoa were stored for another 3 days in RPMI 1,640 medium at pH 8.2 and 9 (1:9) respectively (Experiment 3). Fertilization rate of storage eggs and spermatozoa in Experiment 3 was 79 ± 5%, showing successfully storage of rainbow trout gametes with the same medium.     

Chemical treatment of lobster eggs against epibiotic bacteria

Eggs of the European lobster, Homarus gammarus (L.), were exposed to malachite green (5, 10, 15 mg 1^–1: 10 min), glutaraldehyde (50, 100, 150 mg 1^–1: 3 min) and iodine as Buffodine^TM (50, 100, 150 mg 1^–1: 10 min). The efficiency of the treatments was tested by incubating eggs individually in wells of multiwell dishes with TSB agar for 14 days after exposure. In order to find any effect on viability, batches of 30 eggs from each of three females were incubated artificially in a recirculation system for 19 days and repeatedly exposed to the disinfectants. Iodine as Buffodine (150 mg 1^–1) was the only treatment that resulted in a significant decrease of the bacterial growth on lobster eggs, but the treatment also resulted in inhibited hatching compared with the control group. Thus, our results indicate that treatment with 150mg^–1 iodine as Buffodine could be a strategy for reducing bacterial growth on lobster eggs when massive egg mortality due to bacteria is otherwise unavoidable. The treatment could, however, lead to decreased viability of larvae due to inhibited hatching.     

Development of germ cells and reproductive biology in the sipunculid Phascolosoma esculenta

Sipuncula are of increasing interest for fisheries and aquaculture in China. Sustainable harvests will rely on a better knowledge of reproductive characteristics and stock enhancement. Here, we investigated the structural characteristics of and seasonal changes in germ cell development of the sipunculid Phascolosoma esculenta from the south-eastern coast of Zhejiang, China. An annual survey of egg numbers in the coelom (body cavity) fluid by light and electron microscopy of the females indicates that P. esculenta is dioecious. No defined gonad but dissociated germ cells were found in the coelomic cavity during the 1-year observation. The germ cells showed multiplication and development in the coelomic cavity. Reproduction took place from May to September, with a peak in July and August. The oogenesis can be divided into four phases: cell proliferation, pre-vitellogenesis, vitellogenesis and egg envelope formation and maturation. The process of spermatogenesis can also be divided into four phases: cell multiplication, cell growth, cell maturation and metamorphosis. Monthly changes in the relative number of eggs in each stage indicate that P. esculenta lays eggs in batches. The sperm thrives in the coelomic fluid in the form of cell groups with patterns of genesis and release similar to those of the eggs. Eggs of P. esculenta were fertilized only when reaching the nephridium. The sex ratio was about 1:1 throughout the year.     

Surface disinfection of Pacific threadfin, Polydactylus sexfilis, and amberjack, Seriola rivoliana, eggs

The effects of exposing the eggs of Pacific threadfin and amberjack eggs (AEs) to different concentrations of hydrogen peroxide for 5 min on hatch rate and survival were assessed in a series of experiments using a petri dish model rearing system. Despite significant inter‐batch variation in hatch rate, it was shown that eggs of both species could be safely exposed to up to 11 340 mg L⁻¹ H₂O₂ for 5 min. Exposure to 34 230 mg L⁻¹ H₂O₂ for 5 min was shown to be lethal to AEs at a late stage of development. In two further experiments, it was demonstrated that Pacific threadfin eggs were resistant to all tested concentrations of a range of polyvinylpyrrolidone iodine (PVP‐I) concentrations and contact times (up to 1000 mg L⁻¹ PVP‐I for 10 min). The level of bacteria adhering to the eggs of both species was highly variable. Where eggs were heavily colonized (>10⁴ cfu egg⁻¹), hydrogen peroxide concentrations of at least 11 340 mg L⁻¹, or PVP‐I concentrations higher than 500 mg L⁻¹ for 10 min, were required for effective sterilization. In less colonized batches, rinsing in sterile seawater or exposure to lower (550 mg L⁻¹) concentrations of H₂O₂ was sufficient to result in high apparent levels of surface sterility (<1 cfu egg⁻¹).     

哲罗鲑卵子和卵腔液的生化组成与其发眼率的相关性

为了解哲罗鲑(Hucho taimen)卵子及卵腔液的生化组成及其与发眼率的关系,采用生物化学的方法对雌性哲罗鲑Ⅴ期卵子及卵腔液的酶、蛋白质﹑氨基酸﹑维生素和矿物质等组成和含量进行了测定,并且将各组份与哲罗鲑受精卵的发眼率进行了回归分析。结果表明:哲罗鲑Ⅴ期卵子中含有碱性磷酸酶、酸性磷酸酶、谷草转氨酶、琥珀酸脱氢酶﹑Mg2+-ATP酶﹑磷﹑钙离子﹑铁离子﹑维生素C和维生素E。卵子中Mg2+-ATP酶活力≥8.3×10-5μmol(Pi)/(min.mg prot)、磷含量在12.97~23.0 3μmol/g(卵)之间及氨基酸含量在574.89~1195.40μmol/g(卵)之间,哲罗鲑卵的发眼率均≥80%。哲罗鲑的卵腔液中含有碱性磷酸酶、酸性磷酸酶、谷草转氨酶、琥珀酸脱氢酶、磷、钙离子、铁离子、维生素C和维生素E,当谷草转氨酶酶活力≥18.14μmol/(min·L),哲罗鲑卵的发眼率≥80%。哲罗鲑Ⅴ期卵子中Mg2+-ATP酶活力、磷含量﹑氨基酸含量以及卵腔液中谷草转氨酶酶活力与其发眼率存在着显著相关性。     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

A Lactobacillus sp. from diseased female rainbow trout, Salmo gairdneri Richardson, in Newfoundland, Canada

Abstract. Post-spawning mortalities of 2- to 3-year old female rainbow trout, Salmo gairdneri Richardson, at a Newfoundland hatchery were studied. The fish were found to have a mixed bacterial infection involving primarily a Lactobacillus sp. (a Gram-positive, chain-forming coccobacillus) and to a lesser degree Enterobacteriaceae, Aeromonas hydrophila and Pseudomonas fluorescens. The bacteria were isolated from the visceral organs, kidney, and ascitic fluid present in the coelomic cavity. The fish had extensive degeneration and necrosis of the liver, spleen and kidney, and sloughing of intestinal epithelium, but whether these were caused directly by the bacterial infection was not established because the fish appeared to have received substantial stress from the mechanical stripping and from the retention of dead, unshed eggs in the coelomic cavity. Tissue sections suggested that substantial growth of the Lactobacillus sp. was occurring within these unshed eggs. The present study describes this unusual observation and compares the Lactobacillus sp. to two other Lactobacillus isolates from cultured trout.     

A method for collecting eggs of Pseudocapillaria tomentosa (Nematoda: Capillariidae) from zebrafish Danio rerio and efficacy of heat and chlorine for killing the nematode's eggs

Pseudocapillaria tomentosa is a common pathogen of zebrafish (Danio rerio) in research facilities. We developed a method to collect and concentrate the nematode eggs using a modified sugar centrifugation method and documented their normal development. Embryonating stages with blastomere formation followed by elongation of the embryo prior to larva formation cumulated in developed larvae inside the eggs and hatching after 5–10 day. We then evaluated the efficacy of heat and chlorine to kill them based on a larva development assay. Eggs were exposed to 40, 50, 60 °C for 30 min and 1 h. Chlorine treatment was performed at 100, 250, 500, 1000, 3000 and 6000 ppm for 10 min. Samples exposed to 40 °C for 30 min or 1 h showed incidences of larvated eggs similar to controls. In contrast, no larvation occurred with eggs exposed to either 50 or 60 °C for 30 min or 1 h. Remarkably, in repeated assays, samples exposed to low doses of chlorine (100, 250, 500 and 1000 ppm for 10 min) showed significantly higher incidence of larvation than controls. Eggs treated with 3000 ppm for 10 min did not develop larvae, and no eggs were found after 6000 ppm treatment.     

Surface disinfection of eggs from marine fish: evaluation of four chemicals

For surface disinfection of marine fish eggs Buffodine (1.06% free iodine), glutaraldehyde, chloramine-T and sodium hypochlorite (5% free chlorine) were tested using plaice (Pleuronectes platessa L.) as the main species for evaluation. Glutaraldehyde was the most promising candidate of the four chemicals tested. Good bactericidal effects without any documented negative effects on eggs and larvae were obtained at concentrations of 400–600 mg l^–1 and contact times of 5–10 min. Replicated experiments under identical disinfection conditions revealed a clear correlation between the degree of successful surface disinfection and the initial bacterial load of the egg batch.     

Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), hatcheries: studies on broodstock, eggs, fry and environment

The occurrence of Flavobacterium psychrophilum at four rainbow trout hatcheries was investigated to provide more knowledge about the reservoirs and transmission of this bacterium. Broodstock were sampled at stripping (including both unfertilized and fertilized eggs), and the offspring were then sampled at the eyed egg and fry stages. Water and surface samples (e.g. hatchery trays) were also sampled. Flavobacterium psychrophilum was found in ovarian fluid and milt, indicating that broodstock may serve as a reservoir and are latent carriers of the pathogen. Flavobacterium psychrophilum was not found on or inside eggs, but further egg studies will be necessary to elucidate the possibility of vertical transmission of the pathogen. Flavobacterium psychrophilum was isolated from water samples, but only from water that had been in close contact with farmed rainbow trout or eggs. Flavobacterium psychrophilum isolates were characterized and compared with well-characterized strains, using degradation of elastin, serotype and ribotype profiles. Different ribotypes of F. psychrophilum were found between hatcheries, but a common ribotype A was found at all four hatcheries. Different ribotypes were found in broodstock without clinical disease, whereas only a few ribotypes (mostly ribotype A) were found in diseased fry. The same ribotype A was found in broodstock, in water samples from hatchery trays and in fry, which suggests the possibility of transmission of F. psychrophilum between broodstock and offspring.     

The Use of Formalin and Iodine to Control Saprolegnia Infections on Channel Catfish,Ictalurus punctatus,Eggs

Formalin administered twice daily as a flush at concentrations of 100, 200, or 400 mg/L increased percent hatch of channel catfish, "Ictalurus punctatus," eggs relative to non-treated eggs (P < 0.05). Formalin at 400 mg/L was most effective in controling "Saprolegnia" sp. infections, and the resultant percent hatch was 93.7%. Iodine administered twice daily as a flush at concentrations of 50, 100, or 200 mg/L increased the hatching rate of channel catfish eggs relative to non-treated eggs (P < 0.05). Iodine at 200 mg/L increased percent hatch by 28% over that of the controls. "Saprolegnia" sp. infections were apparent on eggs exposed to all levels of iodine. Formalin was more effective in controlling "Saprolegnia" than iodine and consideration should be given to extending its label to include the treatment of channel catfish eggs.