The bacterial flora of rainbow trout, Salmo gairdneri Richardson, and brown trout, Salmo trutta L., eggs and its relationship to developmental success

Abstract. The bacterial flora of rainbow, Salmo gairdneri Richardson, and brown trout, Salmo trutta L., eggs was studied during incubation. Few bacteria were considered to exist internally, although Pseudomonas species and Aeromonas hydrophila were isolated. During incubation, large numbers of bacteria approaching 500 colony forming units/mm² accumulated around egg surfaces. Both rainbow and brown trout eggs placed in a through-flow system were colonized mainly by Pseudomonas sp. and A. hydrophila , whilst rainbow trout eggs in a separate recycling system were dominated by a Cytophaga species. Glass beads were also incubated (as an inert surface) and found to support significantly lower numbers of bacteria. Egg mortalities were recorded daily and found to differ significantly between both fish species and water conditions. Statistical analysis indicated a potentially significant correlation between hatching success and numbers of surface bacteria.     

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.     

Optimal conditions for egg storage, incubation and post-hatching growth for the freshwater turtle, Chelodina rugosa: Science in support of an indigenous enterprise

Incubation of northern snake-necked turtle (Chelodina rugosa) eggs and subsequent sale of hatchlings for the pet industry has the potential to provide culturally suitable employment for indigenous communities in northern Australia. Developmental arrest in response to egg inundation is unique to C. rugosa. Eggs can be stored under water for up to 10 weeks without appreciable impact on egg or embryo survival, allowing the transport and sale of eggs into niche markets without high levels of mortality, and permitting eggs to accumulate in diapause until there are sufficient numbers to incubate as batches. Eggs that are not inundated or inundated for short periods experience similar survival rates to eggs inundated for lengthy periods. Incubation temperature influences embryo survival and development period in C. rugosa. Embryonic survival is greatest at 26 °C, steadily declining as temperature increases to 32 °C. A similar increase in incubation temperature decreases incubation period by approximately 40 days, however almost half of this variation is attributed to the increase in incubation temperature from 26 to 28 °C. Hatchling growth in C. rugosa is characterized by two phases. There is an initial phase of relatively slow growth under the partial influence of initial egg size and incubation duration, followed by a second phase of relatively rapid growth under the partial influence of water temperature and mass at hatching. Post-hatching survival is negatively correlated with duration of egg inundation and water temperature. Evidence suggests that inundation of C. rugosa eggs for 6 weeks, incubation of embryos at 28 °C and raising hatchlings in 28 °C water will yield the best overall outcomes.     

Experimental infection of eggs and yolk sac larvae of halibut, Hippoglossus hippoglossus L.

Abstract Scanning electron micrographs of halibut eggs with an epiflora dominated by Flexibacter sp., showed ulcerations colonized by large numbers of bacteria. The chorion was dissolved in most ulcerations and the zona radiata was severely damaged. Infection experiments showed that exposure to these bacteria caused high mortalities at the late egg stage, hatching and the early yolk sac stage. Eggs exposed to three different Vibrio spp. showed different mortality patterns, with low mortality at hatching, followed by a continuous high mortality throughout the yolk sac stage. Mortality in the uninfected control group was low throughout the experiment. Transmission electron microscopy of the Vibrio -infected larvae showed bacteria present in the gill, heart and frontal yolk sac regions.     

Morphological characterization of Arctic char, Salvelinus alpinus, eggs subjected to rapid post-ovulatory aging at 7 °C

Egg quality in Arctic char, Salvelinus alpinus, kept indoors at 7 °C during spawning season was morphologically classified. Four egg categories based on lipid droplet distributions and egg diameter were characterized. Eggs with homogenous lipid vesicle distributions and a uniform size were classified as Good. Eggs with some yolk lipid droplets coalesced toward one pole but were homogeneous in size were classified as Fair. Eggs with lipid vesicles that were usually coalesced at one or two poles with were classified as Poor. The fourth egg category was Heterogeneous, in which lipid vesicle distribution and egg size were inhomogeneous. This distribution pattern of the lipid vesicle had a strong relationship with the percentage of fertilized and eyed eggs. Percentages of fertilized and eyed eggs in Good and Fair eggs were 85.5 and 83.4; 30.2 and 28.2, respectively. With Poor and Heterogeneous eggs, the percentages of fertilized and eyed eggs were zero. Egg diameter and absolute weight was increased in Poor eggs than in other egg categories. Relative weight increase during hardening after 30, 45 and 60 min were higher in Good and Fair eggs than in Poor and Heterogeneous eggs. Ovarian fluid collected from egg batches with low rates of fertilized and eyed eggs (Heterogeneous and Poor) had a lower pH and higher protein and aspartate amino-transferase enzyme than ovarian fluid from eggs batches classified as Good or Fair, while the ovarian fluid osmolality did not significantly differ. ATP contents of Poor and Heterogeneous eggs were lower than for Good and Fair eggs. In Heterogeneous eggs, both ATP and acid phosphatase contents were very variable.     

Bacterial destruction of the egg shell of common wolffish during incubation

Mortality of artificially fertilized, laboratory incubated eggs of common wolffish,Anarhichas lupus L., was 100% within 2–3 weeks, at the gastrulation step. The eggs from batches with a high proportion of normally cleavaged eggs died later than the eggs from batches with a low proportion of such eggs. Light micrographs of egg shells showed ulceration of the outer layer and subsequent destruction of the inner layer of zona radiata, caused byFlexibacter sp. The bacterial infection provoked premature hatching at later stages of embryonal development. Treating eggs with glutaraldehyde at a concentration of 600 mg l^–1 every third to fifth day during incubation prevented the mortality caused by bacteria.     

Effect of Low-Temperature Incubation of Channel Catfish Ictalurus punctatus Eggs on Development, Survival, and Growth

To determine whether the embryonic period of channel catfish lctalurus punctatus could be extended at low temperatures, fertilized channel catfish eggs were incubated at five constant water temperatures: 4, 11. 16. 21, and 26 C. Low-temperature incubation of catfish eggs extended the embryonic period at 16 (244%) and 21 C (56%) when compared to the control hatchery incubation temperature of 26 C. All eggs incubated at 4 and 11 C died within 24–48 h. Developmental stage had a significant ( P > 0.05) effect on percent hatch at 16, 21, and 26 C. Eggs held at 16 C prior to embryonic axis formation died within 48 h. Larvae from eggs hatched at 16 C were incompletely developed and died upon acclimation to 26 C for growth tests. Growth of fry reared at 26 C, following egg incubation at 21 C. paralleled that of fry from eggs incubated at 26 C. The underdevelopment of fry at 16 C combined with the significant effect of egg stage on survival at this temperature suggests that 16 C is below the lower thermal tolerance limit for normal development in this species. The period prior to the formation of the embryonic axis may be considered a vulnerable stage in channel catfish development. Increasing the embryonic period through low temperature incubation would increase the duration of juvenile availability for researchers and commercial operations.     

Preventive efficacy of sodium hypochlorite against water mold infection on eggs of chum salmon Oncorhynchus keta

ABSTRACT:   To protect chum salmon eggs from water mold infection during incubation, the eggs were treated daily with sodium hypochlorite solution (NaOCl) at 10 mg/L residual chlorine concentration for 15 min during their developmental period from fertilization to eyed stages. The number of infected eggs and number of eyed eggs were observed on day 23 of incubation. The percentage of infected eggs to total eggs was significantly lower with NaOCl treatment (1.8 : 33.4%) than in the control (11.3 : 59.3%, P  < 0.01). The percentage of eyed eggs to total eggs was significantly higher with NaOCl (85.9 : 98.6%) than in the control (66.1 : 97.5%, P  < 0.01). The antifungal activity of NaOCl resulted in improving egg survival. Accordingly, NaOCl is a useful antifungal agent against water mold infection on chum salmon eggs.     

Bacteria associated with early life stages of halibut, Hippoglossus hippoglossus L., inhibit growth of a pathogenic Vibrio sp.

Abstract Bacteria capable of inhibiting growth of a pathogenic Vibrio sp. were isolated from the gastrointestinal tract of cultured halibut larvae during the first feeding and weaning stages. No such bacteria were found among isolates from the surface of eggs or the gastrointestinal tract of yolk sac larvae. The fraction of pathogen-inhibitors among the total number of isolates ranged between 0–100% (first feeding) and 0–66% (weaning). All pathogen-inhibitors were Gram-negative rods, and 95% were oxidase and catalase positive fermentative isolates, capable of producing acid aerobically from a varying range of carbohydrates. These isolates possessed the characteristics of the Vibrio/Aeromonas -group, but only 19% were sensitive to the vibriostatic agent 0/129. Isolates from eggs and yolk sac larvae were dominated by bacteria belonging to the Cytophaga/Flexibacter/Flavobacterium - group. The high fraction of isolates with the ability to inhibit growth of the pathogenic Vibrio sp. among the total number of isolates indicates that pathogen inhibition may be an important mutualistic role of the intestinal flora of early life stages of halibut.     

The effect of temperature and duration of incubation on the hatching of diapause eggs of Centropages hamatus (Lilljeborg) (Copepoda, Calanoida)

Diapause eggs of Centropages hamatus were used to investigate the effect of temperature and duration of incubation on egg hatching. Eggs were incubated for 10, 12, 14, 16, 20, 24, 28, 32, 36 and 40 h at 15°C and 14L–10D. After incubation for the designated period, eggs were transferred to 25°C and monitored periodically to determine egg hatching. Control eggs were incubated solely at 15°C and monitored for egg hatching. The greatest daily hatching success of eggs occurred within 1 or 2 days after transfer from 15°C to 25°C, while the controls required 3–4 days. The cumulative hatching success of eggs was significantly lower than the control, with the exception of eggs held for at least 36 h at 15°C before transfer to 25°C. These results indicate that overall time to hatching of diapause eggs of C. hamatus can be reduced by transferring the eggs to a higher temperature, for example, 25°C, following a minimum period of time (36 h) at reduced temperature, for example, 15°C. Exposure to 15°C for only 10 h does not appear to be sufficient to result in any subsequent hatching at higher temperature.     

蛭弧茵裂解作用的初步研究

蛭弧菌是一种寄生性细菌,广泛分布干土壤及备种水域之中,对诸多革兰氏阴性菌尤其是水产动物病原菌具有吞噬裂解活性.本文选取七株细菌为宿主菌,对蛭弧菌的裂解作用进行了初步研究.结果表明:除金黄色葡萄球菌外.蛭弧菌对其他六株菌均具有不同程度的裂解活性,其中以对红色假单胞菌的裂解能力更强.     

蛭弧菌裂解作用的初步研究

蛭弧菌是一种寄生性细菌．广泛分布干土壤及各种水域之中．对诸多革兰氏阴性菌尤其是水产动物病厚菌具有吞噬裂解活性。本文选取七株细菌为宿主菌．对蛭弧菌的裂解作用进行了初步研究。结果表明：除金黄色葡萄球菌外，蛭弧菌对其他六株菌均具有不同程度的裂解活性．其中以对红色假单胞菌的裂解能力更强。     

Strain-specific vital rates in four Acartia tonsa cultures, I: Strain origin, genetic differentiation and egg survivorship

Successful development of planktonic copepods for use as live feed in marine aquaculture relies on optimization of environmental conditions for population growth and egg storage. This study examined strain-specific differences in egg survivorship during cold-storage-induced quiescence in four cultures of the widespread marine calanoid Acartia tonsa Dana, 1849. Experimental cultures were obtained from Øresund, Denmark (DIFRES), Kiel, Germany (KIEL), Turkey Point, Florida, USA (FL), and Mobile Bay, Alabama, USA (AL), and were shown to derive from three highly distinct mitochondrial clades. Eggs from Gulf of Mexico strains had low tolerance for cold storage, and showed very low hatching success after 10 days. In contrast, Baltic Sea strains produced eggs able to tolerate up to 150 days of cold storage, with the DIFRES strain showing the highest egg survivorship during the experiment. Eggs from the Kiel strain showed an increase in hatching over time, indicating the presence of dormant eggs. Parental rearing temperature was also found to affect egg survivorship during quiescence in the DIFRES strain, with lower hatching success observed among eggs produced at 25 °C than at 17 °C. The DIFRES strain is recommended as the optimal strain for use in aquaculture, and conditions for cold storage of eggs are discussed.     

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.     

Adenosine triphosphate levels of chinook salmon (Oncorhynchus tshawytscha) eggs following in vitro maintenance and activation/fertilization

To further examine the concept of egg quality and the physiology of stored salmonid eggs, we investigated the effects of different oxygen tensions on the adenosine triphosphate (ATP) levels of unfertilized, activated, and fertilized chinook salmon (Oncorhynchus tshawytscha) eggs. The ATP levels of unfertilized chinook salmon eggs were 2.61±0.14 nmol ATP per egg (17.6±0.9 mol l^–1 relative to cell water) and ranged from 1.98 to 3.63 nmol ATP per egg. The ATP content of unfertilized eggs maintained at 10 °C under 100% O₂, 21% O₂, and 100% N₂ remained unaltered throughout a 120 h storage period. Storing eggs under identical conditions at 20 °C (in an effort to speed egg metabolism and ATP turnover) resulted in significant O₂-independent decreases in ATP levels. However, ATP levels of unfertilized eggs exposed to 1 mmol l^–1 potassium cyanide (a potent inhibitor of oxidative phosphorylation) at 10 °C were significantly decreased after 24 h and continued to decline throughout the 120 h maintenance period to about 30% of time=0 values. Maintenance with exogenous nutrients (5 mmol l^–1 acetate plus 5 mmol l^–1 pyruvate) over 120 h at 10 °C did not alter the ATP content of unfertilized eggs. Eggs activated by exposing them to 10 °C water for a few minutes showed a rapid decrease in ATP values, regardless of whether the eggs were fertilized or not. Following an initial 25% drop after fertilization, the ATP levels remained stable for the remainder (5 d) of the incubation period in eggs maintained in 10 °C water. Therefore, unfertilized chinook salmon egg ATP levels appear to be relatively stable and maintained by a low, cyanide-inhibitable metabolism. The stability of egg ATP levels may be one reason that salmonid eggs can be stored for several days while eggs from other fishes cannot.     

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).     

Effects of stripping time on the success of the artificial incubation of white-clawed crayfish, Austropotamobius pallipes (Lereboullet), eggs

One of the main limiting factors in the use of artificial incubation techniques on crayfish farms has been the widespread belief that eggs should be stripped from females in the late stages of embryogenesis. In order to disprove this idea, three different times for egg removal were tested in white-clawed crayfish, Austropotamobius pallipes (Lereboullet): (1) just before the gastrulation process (mean degree days= 335, 34 days after spawning); (2) when the embryo was between the closing of the blastopore and the appearance of mandibular rudiments (mean degree days= 524, 56 days after spawning); and (3) when the embryo had thoracic appendage rudiments (mean degree days= 810, 92 days after spawning). The results showed that it is possible to attain acceptable survival rates up to juvenile stage 2 (51%), even when eggs have been detached at the earliest time (34 days after spawning), in such a way that artificial incubation is used for more than three-quarters (75.7%) of the total duration of the embryonic development. Factors such as the incubation device, water quality and incubation conditions have a major influence on the success of the process. Finally, a critical period was observed during the last stages of development in the present study, particularly between the eyed stage and juvenile stage 2, with mortality rates of between 26.7% and 56%.     

The first spontaneous spawning of European hake Merluccius merluccius L.: characteristics of eggs and early larval stages

For the first time, a spontaneous spawning of hake was recorded in Spain in April 2009. The spawn was obtained from broodstock kept in captivity for two years at the facilities of the Spanish Institute of Oceanography in Vigo (NW Spain). Eggs were transparent, spherical and had an average diameter of 1.067 ± 0.024 mm; yolk occupied the majority of egg volume. The oil droplet had a diameter of 0.27 ± 0.03 mm. The incubation period of the eggs lasted for 4 days at 14°C and the duration from hatching to the total absorption of the yolk sac was between 5–7 days after hatching, at the same temperature. Newly hatched larvae had an average total length of 3.20 ± 0.13 mm and began feeding 6 days after hatching; a daily growth rate of 0.158 mm day‐1 was observed from hatching to yolk sac consumption. This paper describes the daily evolution of biometric and morphological characteristics of the different stages of embryos and larvae of European hake up to the age of 19 days.     

白鱚卵各胚胎发生阶段对高温突变、落下冲击和紫外线照射的感受性

本研究将白鱚卵的胚胎发生过程严格区分为27个阶段,并就各不同发生阶段的卵对高温突变、落下冲击和紫外线照射的感受性进行了详细的比较研究,得出了卵裂早期和原肠晚期至胚孔封闭期的卵对高温突变的感受性高;卵裂晚期至胚体出现期,对落下冲击的感受性高;对紫外线照射的感受性,则以胚体出现期为最高。文中同时分析了这种不同环境因素作用于相同鱼卵的同一发生阶段,产生不同影响的原因;建议在鱼类资源保护和鱼苗生产的孵化管理上,应尽量避免感受性较高的相应环境因素变化对鱼卵的影响。     

Egg cortisol response to stress at early stages of development in Persian sturgeon Acipenser persicus

One of the most important periods in artificial breeding of sturgeon is incubation. In general, little is known about ontogeny of the stress response in early developmental stages of sturgeon. In this study, cortisol content was measured by radioimmunoassay for the first time in eggs of Persian sturgeon Acipenser persicus to elicit at which times during early developmental stages, that they were particularly sensitive to stressful events. In this study, fertilized eggs of mature Persian sturgeons were placed in special boxes (as replicates) that were put in incubation units of Yushchenko. To assess pre-stress samples (resting cortisol levels), 5 eggs were selected randomly from each box in 7 important stages of incubation (2-cell division, blastula, end of blastula, mid of gastrula, neurula, eyed stage and heart beaten). In another experiment, eggs were exposed to an acute stress (10 min out of water) in 7 developmental stages. For determination of post-stress cortisol, eggs were collected 2 and 6 h after the stress. The results indicated significant difference in various stages of incubation but cortisol levels revealed no differences in whole-body levels between stressed samples (2 and 6 h post-stress) and unstressed samples (P > 0.05). The highest percentage of hatching rate for post-stressed eggs was recorded in 2-cell division stage while this percentage declined steadily in the heart beaten stage when a statistically significant difference was revealed with regard to other stages (P = 0.000). These findings suggested that sturgeon eggs were not so much sensitive in their early developmental stages and relatively resistant to stress. However, significant decrease in survival rate in heart beaten stage showed a sensitivity of this stage to stress. Lack of an increase in cortisol after acute stress indicated that hypothalamus-pituitary-interrenal (HPI) axis for cortisol synthesis was not yet functional. However, further studies seem necessary to clarify when HPI axis is activated in this species.